The gene encoding urease subunit A (ureA) of Helicobacter pylori (H. pylori) was cloned from H. pylori isolate by polymerase chain reaction (PCR). Sterile distilled water instead of DNA served as negative control. The nucleotide sequence of the amplified product was determined. Homologous analysis of the ureA against that reported by Clayton CL and the GenBank and SwissProt databases were performed with the BLAST program at the Genome Net through the Internet. 0.8 kb PCR product was amplified from all H. pylori clinical isolators. The nucleotide sequence of the ureA was determined. The nucleotide sequence of the ureA began with ATG as the initiation codon and terminated in TAA as stop codon. The coding regions had a 44% G+C content. The DNA sequence was 98% homologous to that reported by Clayton CL (688 out of 702 residues were identical). The derived amino-acid sequences of the ureA were 99% homologous to that reported by Clayton CL (232 out of 234 residues were identical). The nucleotide sequence and the predicted protein showed significant homology to ureA of H. pylori in the NCBI Entrez database.
To elucidate the mechanism of arrhythmia in healed myocardial infarction (HMI), the changes of action potential duration (APD), transient outward potassium current (1ω), delayed rectifier potassium current (Ik) and inward rectifier potassium current (Iki) of left ventricular myocytes in non-infarcted zone of HMI were investigated. Rabbits were randomly assigned into two groups: HMI group, in which animals were subjected to thoracotomy and ligation of the circumflex coronary and sham-operated group, in which rabbits underwent thoracotomy but no conorary ligation. 3 months after the operation, the whole myocyte patch clamp technique was used to record APD, Ito Ik, and Iki of ventricular myocytes in non-infarcted zone. Our results showed that the membrane capacitance was larger in HMI group than in sham-operated group. Action potential duration was significantly lengthened in HMI group and early afterdepolarization (EAD) appeared in HMI group. The densities of Ito, Kk, tail, and Kki were reduced significantly in HMI group, from 6. 72±0.42 pA/pF, 1.54±0.13 pA/pF and 25.6±2.6 pA/pF in sham-operated group to 4.03±0.33 pA/pF, 1.14±0.11 pA/pF and 17.6±2.3 pA/pF, respectively. It is concluded that the reduced densities of Ito, Ik.tail and Iki in ventricular myocytes of non-infarcted zone in HMI were responsible for the prolongation of APD and the presentation of EAD which played important roles in the development of malignant arrhythmia in HMI.
In order to investigate whether Arg110Gln polymorphism in the coding region of the IL-13 gene is associated with asthma and total plasma IgE level in Han nationality in Hubei Chinese population, the allele frequency of 4257 (g/a) site and Arg110Gln genotype of IL-13 was detected by using restriction fragment length polymorphism in Han nationality in Hubei Chinese population including 43 asthmatic children, 45 asthmatic adults, 31 control children and 46 control adults. Total plasma IgE was measured by Chemiluminescence assay. The results showed that the frequency of allele A at 4257 bp of IL-13 in children and adults was 0.39 and 0.32, respectively. The GlnGln form of Arg110Gln polymorphism of IL-13 gene was associated with susceptibility of asthma and elevated total plasma IgE in children (P=0.030 and 0.0009, respectively), but not with them in adults (P=0.219 and 0.174, respectively). Our results suggest that the Arg110Gln polymorphism of IL-13 gene is associated with susceptibility of asthma and elevated total plasma IgE in Chinese children of Han nationality in Hubei, but not with them in adults.
In order to explore a new special and effective way to prevent, graft versus, host disease (GVHD) after allogenic bone marrow transplantation (alle-BMT), the stem cell antigen-1 (Sea-1) carly hematopoietic cells (EHC) from BALB/c mouse (H-2d) were introduced with exogenous mouse Fas ligand (mFasL) cDNA gene by the retrevirus-mediated gene transfer and expanded for one week, and then they were co-cultured with the spleen mononuclear cells (SMNC) from BAC mouse (H-2d, h) as one way mixed lymphocyte reaction (OWMLR). The cytotoxicity of treated BAC mouse SMNC against Na251CrO4 labeling SMNC from BALB/c mouse was observed. The bone marrow mononuclear cells (BMMNC) from BAC mouse treated by the above methods were transplanted into lethally-irradiated congenic BALB/c mice to observe the occurrence of GVHD. The results showed that the SMNC from BAC mouse after OWMLR with exogenous mFasL cDNA gene-transduced hematopoietic cells (HC) from BALB/c mouse in a ratio of 1 to 5 exhibited an obvious inhibition of the cytotoxicity against the BALB/c mouse spleen cells at different effector/target ratios as compared to the control group (P<0.01). The grade I GVHD or no GVHD and the 80% survival rate at day 60 post-BMT were observed in the BALB/c mouse receiving BAC mouse BMMNC treated with similar way, while the grade II–III GVHD and the 20% survival rate were noted in the control group (P<0.01). It is suggested that the attenuation of GVHD in allo-BMT recipient could be successfully achieved through FasL-Fas pathway in an H-2 haplotype disparate mouse combination.
The expression of intercellular adhesion molecule-1 (ICAM-1) in the pathogenesis of heroin-induced acute lung injury (ALI) in rats was investigated. The model of ALI was established by intravenous injection of heroin into tail vein in rats. Thirty-six rats were randomly divided into heroin-treated groups (1 h, 2 h, 4 h, 6 h and 24 h) and normal control group. Changes in histopathologic morphology and biological markers of ALI were, measured. The expression of ICAM-1 in lung tissue was detected by using immunohistochemistry and RT-PCR. The results showed that the W/D ratio and protein contents in BALF of the heroin-treated groups were significantly higher than that of the control group (P<0.01). The histopathological changes in the lung tissue were more obvious in heroin-treated groups. The ICAM-1 protein and mRNA expression in the lung tissue of heroin-treated groups were significantly increased as compared with that of the control group (P<0.01), and correlated with the ALI parameters in a time-dependent manner. Increasing of ICAM-1 expression was involved in the formation of heroin-induced lung injury. Furthermore, the level of expression was positively correlated with the severity of lung injury.
By using semi-quantitative RT-PCR method, it was found that PD-L1 mRNA but not PD-L2 mRNA was expressed in H22 hepatoma cells and both PD-L1 and PD-L2 mRNAs were expressed in tumor tissues of tumor-bearing mice and upregulated as compared with muscle tissues in normal mice and H22 hepatoma cells. PD-L1 and PD-L2 were also expressed on the surface of the activated T cells. The soluble recombinant sPD-1 expressed from the constructed eukaryotic expression vector could enhance the lysis of tumor cells by lymphocytes stimulated specifically with antigen. The expression of sPD-1 by local gene therapy on the inoculation site of H22 hepatoma cells could inhibit the growth of tumor. The results of this study indicate that expression of soluble receptor of negative costimulatory molecules could reduce the inhibitory effect on T cells in tumor microenvironment and enhance the cytotoxicity of T cells on tumor cells. This possibly provides a new method of improving efficacy of tumor gene therapy.
The aim of this study was to express and purify human histydyl-tRNA synthetase related gene and to prepare its polyantibody. The open reading frame was amplified by PCR, and then recombined into prokaryotic expression vector pQE30 and transformed into E. coli M15 for expression. The expressed products were induced by IPTG after the reconstructed pQE30 was transferred into M15. After purified by Ni affinity chromatography, the product was identified to be a single band by SDS-PAGE. The rabbits were inoculated with purified products. High-titer polyantibody was successfully prepared. Highly-purified expression product and prepared polyantibody may provide a good basis for further study.
To prepare carboxyl terminus truncated human papillomavirus type 58 L1(HPV58L1) protein and evaluate its ability to form virus-like particles, the baculovirus and Sf-9 insect cells was used to express HPV58L1 protein, and pFastBac-Htb containing HPV58L1 gene sequence of carboxyl terminus truncation was generated. Then Sf-9 cells were infected with recombinant baculovirus. After being cultured, the post-infected cells expressing—HPV58L1 protein were harvested and analyzed by SDS-PAGE and Western blot. The ProBond™ purification system was used for protein purification. The bio-activity of purified protein was identified by mouse erythrocyte hemagglutination assay, and the VLP formation was examined with transmission electron microscope. Our results showed that the recombinant baculovirus was generated and the Sf-9 cells was infected with the recombinant baculovirus, and after collecting, total cellular proteins were extracted. Truncated HPV58L1 protein with MW 58KD was revealed by SDS-PAGE and confirmed by Western blot. The purified L1 proteins under native condition could cause mouse erythrocytes to agglutinate and form VLP. It is concluded that HPV58L1 protein with carboxyl terminus truncation could be efficiently expressed. In baculovirus Sf-9 cells expression system, the purified protein could self-assemble into virionsin vitro, and induce agglutination of mouse erythrocytes, indicating that carboxyl terminus truncation does not interfere with the bioactivity of HPV58L1 protein.
To explore the mechanism of interleukin-1beta (IL-1β) in the onset of seizure and the effect of IL-1β on the expression of adenylyl cyclase (AC) in rats with seizure induced by L-glutamate. Experimental rats were first injected with IL-1β and then L-glutamate (a dose under the threshold) was injected into the right lateral ventricle. The rats were sacrificed 4 h after the onset of epileptic activity and examined for changes in behavior, immunohistochemistry and compared with those with seizure induced by L-glutamate alone. It was found that the expression of AC in hippocampal and neocortex of rats with seizure induced by IL-1β and L-glutamate were stronger than that of control group (P<0.05), without significant difference found between the L-glutamate group and IL-1β plus L-glutamate group in the expression of AC, the latent period and the severity of seizure. When IL-ra were given (i.c.v.) first, there was no epileptic activity and the expression of AC did not increase. There were no differences in the expression of AC of rats with IL-1ra and that of control rats. But when 2-methyl-2-(carboxycyclopropyl) glycine (MCCG) was given (i. c. v.) first, the strongest expression of AC, the shortest latent period and the the most serious seizure activities were observed. The results indicated that IL-1β could facilitate the onset of epilepsy induced by L-glutamate through IL-1R, metabotropic glutamate receptors might work with IL-1R and the increased expression of AC might be involved in the process.
To explore the effect of different concentrations of corticosterone (CORT) on primary cultured hippocampal neurons and their Ca2+ CaMK II expression and possible mechanism, the changes of hippocampal neurons were observed in terms of morphology, activity of cells, cell death, concentrations of cytosolic free calcium, and the expression of CaMK II by using MTT assay, flow cytometry, fluorescent labeling of Fura-2/AM and Western blotting after 107. 10−6 and 106 mol/L of CORT was added to culture medium. The evident effect of 10−6 and 105 mol/L of CORT on the morphology of hippocampal neuron was found. Compared with control neurons, the activity of the cells was markedly decreased and [Ca2], increased in the neurons treated with 106 and 105 mol L of CORT, but no change was observed in the neuron treated with 106 mol/L of CORT. The death was either by way of apoptosis or necrosis in the cells treated with 106 and 105 mol/L of CORT respectively. The correlation analysis showed that a reverse correlation existed between [Ca2+], and the expression of CaMK II. Either apoptosis or necrosis occurs in the hippocampal neurons treated with CORT. The increased hippocampal [Ca2+], is both the results of CORT impairing the hippocampal neurons and the cause of the apoptosis of hippocampal neurons and the decreased CaMK II expression.
To comparatively investigate ultrastructural characteristics and expressions of AFP (alpha-fetoprotein) and Tn (Thomsen-Friedenreich-related antigen) protein in AFP negative (AFP−) and AFP positive (AFP+) primary hepatocellular carcinoma. Fourty-three cases of AFP− and AFP+ hepatocellular carcinoma (HCC) tissues and five cases of normal liver tissues were divided into three groups: control group (normal liver tissue,n−5): AFP+HCC group (the serum AFP level was higher than 10 ng/ml.n−22): AFP HCC group (the serum AFP level was lower than 10 ng ml.n 21). The ultrastructural morphology was studied by transmission electron microscopy. the expressions of AFP and Tn protein were detected by immunohistochemistry and cell image analysis, I. The immunohistochemical study showed that (I) the expression intensity and positive rate of Tn protein in AFP—HCC group were markedly higher than that in AFP—HCC group (P 0.01); (2) The expression intensity of AFP in AFP—HCC group was lower than that in AFP—HCC group (P<0.01). 2. The transmission electron microscopy demonstrated that some AFP—HCC cells linked closely with each other. others dispersed loosely just as cultured cells, the remarkable morphologic features in AFP—HCC cells were simple organelles, but they were abundant in the free polyribosones. In AFP—HCC group, all the HCC cells linked closely together and were rich organelles in their cytoplasm. especially the rough endoplasmic reticula. In addition. mitochondria and Golgi complex were obviously observed. (1) The AFP and Tn protein had discrepancy distribution in AFP− and AFP+ HCC tissues. Tn protein may be one of the early diagnostic indicators in AFP HCC: (2) The synthetic locations of the AFP and Tn protein were different in hepatocarcinoma cells by ultrastructural observation.
The effects of 3, 4-Dihydroxyacetophenone (3, 4-DHAP) on cytosolic free calcium [Ca2+]i in pulmonary artery endothelia (PAECs) and smooth muscle cells (PASMCs) during acute hypoxia were studied. Porcine pulmonary artery endothelial and smooth muscle cells (PASMCs) were cultured primarily, and they were divided into 4 groups: groups incubated under normoxia or hypoxia and those with or without treatment with 3, 4-DHAP. The [Ca2+]i of both PAECs and PASMCs was measured by determining the fluorescence of fura 2 AM on spetrofluorometer. Our results showed that hypoxia caused significant elevation of [Ca2+]i, in both PAECs and PASMCs, 3, 4-DHAP could attenuate the hypoxic elevation of [Ca2+]i, only in PASMCs but not in PAECs. It is concluded that 3, 4-DHAP decreases the hypoxic elevation of [Ca2+]i in PASMCs. This might contribute to its inhibitory effect on hypoxic pulmonary vasoconstriction.
The purpose of this study was to evaluate the ergonomic risk factors associated with manual lifting tasks using surface electromyography (EMG). 13 volunteers lifted loads of 6 and 13 kg at two speeds and at two horizontal distances in 3 different postures and three boxes of different sizes, from floor to knuckle height, performing 72 lifting tasks. For each lift, the surface electro-myography signals from the erector spinae muscles, bilaterally at T10 and L3, was recorded. The ergonomic risk factors associated with manual lifting tasks were evaluated by comparing the average amplitude of EMG signals from the erector spinae muscles. The EMG average amplitude for lifting the load of 13 kg was 14.3% greater than that for lifting the load of 6 kg (t=−10.93,P<0.01). The EMG average amplitude at the site of L3 was 10.3% greater than that at the site of T10 (t=−7.98,P<0.01). The EMG average amplitude when performing “fast” lift was 5.9% greater than the “slow” lift (t=−4.63,P<0.01). The posture of lifting affected the EMG average amplitude. It was lowest with semi-squat posture and greatest with squat posture (F=27. 76,P<0.01). The result of multiple stepwise regression analysis showed that the loads of lifting, the size of box, horizontal distance, posture of lifting, the site of the spine subjected to force, lifting speed were the factors affecting the EMG average amplitude. The most significant factor was the loads of lifting, followed by the site of the spine subjected to force and the lifting speed in terms of risk. The ergonomic risk factors associated with manual lifting tasks includes the loads, posture, lifting speed, horizontal distance, the site of the spine subjected to force etc. The results of signal amplitude of EMG from the erector spinae muscles showed that semi-squat posture is the best posture for lifting tasks.
This study was designed to determine the effects of the recombinant adeno-associated virus vector containing sense CD151 gene (rAAV-CD151) and antisense CD151 gene (rAAV-antiCD151) on the migration of Tca8113 cell. Functional fragment of CD151 gene was amplified by RT-PCR, and inserted into the vector pAAV in the sense direction and antisense direction, respectively. The rAAV-CD151 and rAAV-antiCD151 were produced and the titers were determined by dot blot. The CD151, at protein level, was detected by Western blot. The Transwell chamber was used to detect the effects of the rAAV-CD151 and rAAV-antiCD151 on the tumor cell migration. The titers of the rAAV-CD151 and rAAV-antiCD151 were 2×1011 pfu/ml and 1.0×1011 pfu/ml, respectively. The expression of CD151 was increased by 108% in the cells transfected with rAAV-CD151 and decreased by 79% in the cells transfected with rAAV-antiCD151, as compared with non-transfected cells, respectively. The number of the migrating cells was significantly increased in the cells transfected with rAAV-CD151 (93.56±11.59) and decreased in the cells transfected with rAAV-antiCD151 (24.00±4.36) as compared with non-transfected and rAAV-GFP transfected cells (53.00±6.56 and 46.00±7.00,P<0.05). It is an important molecular mechanism of the tumor metastasis that the overexpression of CD151 promotes the migration of the tumor cells. The rAAV-antiCD151 is a novel tool, which can reduce the expression of CD151 and inhibit the migration of the tumor cells, and brings us a new approach of anti-sene gene therapy targeted at CD151 in human carcinoma.
To explore the relation between human heat shock protein 70 (hsp70) and TLR4 in human monocytesin vitro, human monocytes were stimulated with various concentrations of HSP70, and TNF-α production in supernatants was measured by ELISA. Pre-incubated with or without anti-TLR4 mAb, and stimulated with hsp70 (5.0 μg/ml), NF-κB p65 of human monocytes in different time points were detected by immunohistochemistry and monocyte surface expression of TLR4 was measured by flow cytometry. After the human monocytes were pre-incubated with various concentrations of anti-TLR4 and stimulated with hsp70 (5.0 μg/ml), TNF-α production in supernatants was measured. The results showed that hsp70 enhanced NF-κB activation, which was clearly inhibited by anti-TLR4, with the positive cell ratios being 67.44%, 39.17%, 31.56% and 28.05%, respectively. TLR4 was rapidly down-regulated in the presence of hsp70. MFI of TLR4 on monocytes in different time points were 87.77±5.38, 78.16±6.01 and 45.17±4.97 (P<0.05), 26.98±5.83 (P<0.01), respectively. Moreover, hsp70-induced TNF-α production by human monocytes was inhibited by anti-TLR4. It is suggested that TLR4 is involved in the hsp70-mediated activation of innate immunity.
To investigate the effects of leptin on expression of acyl-coenzymeA: cholesterol acyltransferases-1 (ACAT-1) in monocyte macrophage differentiation, human monocytic cells (THP-1) were cultured in RPMI 1640 and made to differentiate into macrophages under the incubation with phorbol myristate acetate (PMA) for 18 h. The cells were divided into 1 groups according to different intervention factors as follows: MCs cultured in RPMI1640 medium with 10% FBS for 48 h served as MC group (control group). MCs cultured in medium with serum-free RPMI1640 containing 54r BSA. 100 nmol L PMA for 18 h as MP group. MCs cultured in RPMI1640 medium with 10%, FBS, 10 μmol/ml leptin for 18 h as leptin-MC group, and MCs cultured in medium with serum-free RPMI1640 containing 5% BSA. 100 nmol L PMA, and 10 μmol/ml leptin for 48 h as leptin MP group. Immunocytochemistry, reverse transcription polymerase chain reaction (RT-PCR) and Western blot were performed, respectively, to observe the effects of leptin on expression of ACAT-1 in the monocyte macrophage differentiation. Our results showed that expression of ACAT-1 protein and mRNA in MP-group is two times that in MC group (P<0.05), and the expression of ACAT 1 protein and mRNA increased by up to 1 folds in leptin-MP group as compared with that of MC group (P<0.01). Thus, our results support the idea that expression of ACAT-1 increases more in cultured human macrophages than in monocytes, and leptin can significantly promote ACAT 1 expression. It was concluded that high expression of ACAT-1 may accelerate the development of human atherogenesis, and leptin might participate in atherogenesis by increasing expression of ACAT 1.
To study the angiogenic potency of hypoxia-prestimulated bone marrow stromal cells (BMSCs) when transplanted into acute myocardial infarction models of rats. BMSCs were cultured under hypoxia condition for 24 h. Their expression of VEGF was investigated. The rat acute myocardial infarction models were made by coronary artery ligation and divided into 3 groups at random. In normoxia group, twice-passaged BMSCs were labeled with Bromodeoxyuridine (BrdU) and then implanted into the infarction regions and ischemic border of the recipients in 4 weeks. The rats in hypoxia group were implanted with hypoxia-prestimulated BMSCs. In control group, the model rats received only DMEM medium injection. Six—weeks after AMI, the infarction regions were examined to identify the angiogenesis and the expression of the VEGF. Our results showed that viable cells labeled with BrdU could be identified in the host hearts. The infarction regions in normoxia and hypoxia groups had a greater capillary density and increased VEGF expression than the regions in control group. The capillary density and VEGF expression in hypoxia group were higher than in normoxia group. It is concluded that the enhanced expression of VEGF in BMSCs could be induced byex vivo hypoxia stimulation. BMSCs implantation promoted the angiogenesis in myocardial infarction tissue via supplying exogenic VEGF. Angiogenic potency of bone marrow stromal cells was improved byex vivo hypoxia prestimulation though the enhanced VEGF expression.
To investigate the effect of N-tosyl-L-phenylalanylchloromethyl ketone (TPCK) on tumor necrosis factor-alpha-induced. NF-κB activation and apoptosis in U937 cell line, changes and subcellular localization of NF-κB/p65 and IκB-α were observed by fluorescencemicroscopy and expression and degradation of IκB-α by flow cytometry. The apoptosis of U937 cells was measured by flow cytometry and electrophoresis of DNA. Immunolfluorescence assay showed that NF-κB/p65, IκB-α only localized in cytoplasm. After TNF-α stimulation, p65 was localized only in nuclei, and IκB-α was only localized in cytoplasm and decreased. The changes of TNF-α stimulation were specifically inhibited by TPCK. Flow cytometry also revealed the downregulation of IκB-α protein during TNF α-induced apoptosis and the down-regulation was specifically inhibited by TPCK. Flow cytometry also showed the apoptosis of U937 cells after TNF-α induction. DNA ladder can be detected in cells treated by TNF-α. It is concluded that degradation of IκB-α protein and NF-κB/p65 translocation occur during TNF-α-induced apoptosis of U937 cells, suggesting the activation of NF-κB. TPCK-sensitive protease plays an important role in the degradation of IκB-α protein induced by TNF-α in U937 cells. TPCK sensitive protease also plays an important role in the apoptosis of U937 cells induced by TNF-α.
In order to examine the strong anticancer action and low toxicity of Trichostatin A (TSA), the effect of TSA was examined on the growth inhibition, acetylation of histone H3 and apoptosis in HL-60 cells by employing MTT, immunocytochemical techniques, and Annexin-V-FITC/PI assay. Our results showed that TSA could inhibit proliferation of HL- 60 cells in a time- and dose-dependent manner, and the IC50 at the 36th h was 100 ng/ml. The apoptosis-inducing effect of TSA on HL-60 cells was also time- and dose-dependent. But it didn’t demonstrate apparent apoptosis induction in NPBMNCs within specific dose and time range. Both of the acetylation of histone H3 in HL-60 cells and NPBMNCs increased significantly (P<0.05) after treated with 100 ng/ml TSA for 4 h. However, there was no significant differences between the two groups (P>0.05). It is concluded that TSA can inhibit growth and induce apoptosis of HL-60 cells in a time- and dose-dependent manner, and is able to selectively induce apoptosis in HL-60 cells but does not respond in NPBMNCs under the same conditions. The difference of TSA between HL-60 cells and NPBMNCs cant be explained by the regulation of histone acetylation.
To study the effects of glucocorticoid on the IL 13-induced Muc5ac expression in airways of mice, and investigate its role in mucus secretion of airways, 24 pathogen-free BALB/c mice were randomly divided into 3 groups, IL-13 group received an nasal instillation of 100 μg of recombinant murine IL-13 solution on days 1, 3 and 5. In dexamethasone group, dexamethasone (0. 5 mg/kg) was administered intraperitoneally 24 h before and 1 h before the first instillation of IL-13 and on 4 consecutive days (day 0 to day 5, 6 consecutive days in total), while control group was not treated with IL-13 or dexamethasone. Bronchoalveolar lavage fluid (BALF) was collected and eosinophils were counted, and expression of Muc5ac mRNA and protein in lungs were detected by reverse transcription-polymerase chain reaction (RT-PCR) technology and immunohistochemical assay respectively. Our results showed that the number of mice, with positve Muc5ac protein expression expression of Muc5ac mRNA and cosinophils in BALF after IL-13 treatment were all significantly higher than that of control group (allP<0.01). Despite cosinophils reduced (P<0.01), the number of mice with positive Muc5ac protein expression, expression of Muc5ac mRNA after dexamethasone treatment didn’t decreas significantly as compared with that of IL-13 group. It is concluded that IL-13 can up-regulate the expression of Muc5ac mRNA and protein, which may play a pivotal role in the mucus overproduction of airways. Dexamethasone can suppress IL-13-induced cosinophilic infiltration in lung but cant inhibit the mucus overproduction.
To develop a method for identification of differential gene expression between different cell population, several conveninent techniques of molecular biology, including subtractive hybridization, suppression PCR, T/A cloning and sequencing, were used to identify genes expressed differentially in CD45− and CD45− cells isolated from U266 cell line of multiple myeloma. Our results showed that the levels of abundant genes genes scale down 20 times through subtractive hybridization. Plasmid DNA from CD45 cell clones was hybridized with forward or backward cDNA probes synthesized from CD45 and CD45 cells, respectively. A few of differentially expressed genes reconfirmed by RT-PCR were identified from 500 expressed clones of CD45′ cells. It is concluded that a strategy for gene expression identification developed from conventional molecular biological methods can be used in different laboratories.
To establish stable methods for detecting plasma endotoxin level and endotoxin inactivation capacity in a normal population and general surgical patients and evaluate their perioperative changes, 50 healthy people and 50 patients receiving gastrointestinal operation were enrolled, their plasma endotoxin levels and plasma endotoxin inactivation capacity were assayed. Our results showed that plasma endotoxin levels were 0.044±0.009 EU/ml in the normal population and 0.044±0.023 EU ml in the preoperative patients. Endotoxin level peaked 3 h after the operation (0.223±0.011 EU ml), and then decreased rapidly on the first day after the operation (0.134±0.164 EU ml). Endotoxin inactivation capacity also had the same time course as endotoxin level. Systemic inflammatory response syndrome and infection induced another elevation in the time course. It is concluded that establishing the endotoxin standard curve by using pyrogenic free water is better than by using plasma. Plasma endotoxin inactivation capacity can be used as an indirect indicator of postoperative immune depression. Plasma endotoxin level and endotoxin inactivation capacity peaked shortly after operation, indicating surgical stress is closely related with the changes.
To investigate the expression of KLF6mRNA in primary hepatocellular carcinoma (HCC), nomal liver tissues and the tissues adjacent to the cancers, reverse-transcription polymerase chain reaction (RT-PCR) was employed to investigate the expression of the KLF6 gene in HCC, the corresponding adjacent non-cancerous tissues and normal liver tissue. Our results showed that an amplified fragment of 127 bp DNA was detected in 18 of 19 (94.7%) adjacent non-cancerous tissues and normal liver tissue, and in 12 (85.7%) of 11 HCC. There were no significant differences in the levels of KLF6 mRNA between normal liver and liver tumors (P>0.05). It is concluded that KLF6 mRNA is generally expressed in HCC.
To investigate the expression and clinical significance of p27kip1 protein in primary liver cancer, the expression of p27kip1 protein and the relationship with clinicopathological factors were studied in primary liver cancer by using SABC immunohistochemical staining in specimens of 40 cases of primary liver cancer and 20 cases of liver cirrihosis. Our results showed that positive expression rate of p27kip1 protein in primary liver cancer was 37.5% (15/40), which was lower than that in benign lesion of liver 80.0% (16/20,P<0.01). The expression level of p27kip1 protein in primary liver cancer showed significant differences in tumor size, Edmonson histological grade, portal invasion, lymph node metastasis, TNM stage (P<0.05, for all), but not significantly correlated with patients age and histological types. Log rank test showed that the p27kip1 expression was significantly related with prognosis of the patients (P<0.05), and the prognosis of the patients with p27kip1 positive expression was markedly better than that of those with p27kip1 negative expression. It is concluded that the expression of p27kip1 was significantly related clinicopathological factors of primary liver cancer, p27kip1 protein may be used as a novel tumor marker for primary liver cancer.
The bile was collected from fro patients with biliary infections, with the bacterium isolated to study the sensitivity of each kind of the bacterium to several antibiotics in common use. Except G bacterium, we also found some kinds of G− bacterium in infection bile, G− bacterium were not sensitive to Clindamycin. G− bacterium were sensitive to Ciprofloxacin. Escherichia coli. Xanthomonas maltophilia. Enterobacter cloacae. Pseudomonas aeruginosa were sensitive to Ampicillin, G− bacterium were not sensitive to Azactam. Enterococcus faccalis. Enterococcus faecium. Enterobacter cloacae were not sensitive to Ceftazidime. Enterococcus faccalis. Staphylococcus coagulase negative. Staphylococcus epidermidis. Pseudomonas aeruginosa were not sensitive to Ceftriaxone Sodium. We didn’t found any bacterium resistance Imipenem. The possibility of the existence of G− bacterium as well as drug resistance should be considered n patients with biliary infections. The value of susceptibility test should be respected to avoid drug abuse of antibiotics.
To study the expression of hypoxia inducible factor-1α (HIF-1α) protein in prostate cancer (Pca) and its biological significance, the expression of HIF-1α was assayed by means of immunohistochemical technique in 12 prostate cancer. 12 prostatic intraepithelial neoplasm (PIN) and 9 normal prostate tissue (NP) specimens. Western blot was used to examine the expression of HIF-1α in prostate cancer cell line (PC-3M) induced by different oxygen tension. HIF-1α expression was positive in 33 Pca and 9 PIN specimens, and the positive rate of HIF-1α was higher in distant metastasis patients than in patients without metastasis of prostate cancer (P<0.05), while there was no expression of HIF-1α in NP. The level of HIF-1α in PC-3M significantly increased with the decrease of oxygen tension (P<0.01). Overexpression of HIF-1α is the preliminary event of the formation of Pca, which may induce carcinoma into malignant phenotype. Thus it may serve as an early diagnosis marker and the novel target for Pca treatment.
To study the expression of vascular endothelial growth factor C (VEGF-C) in colorectal carcinoma and its relationship with lymph node metastasis, the expression of VEGF-C protein in colorectal carcinoma tissues obtained from 94 patients who underwent radical resection was immunohistochemically detected. Meanwhile, the expression of VEGF-C mRNA in 4 colorectal carcinoma cell lines was examined by reverse transcription polymerase chain reaction (RT-PCR). VEGF-C protein was found to be expressed in 53.2% of patients. The expression was more frequently detected in tumors with lymph node metastasis than in those without metastasis (P<0.01), and there was significant correlation between its expression and lymphatic invasion, TNM stage (P<0.01). However, no significant correlation was found between its expression and the age, gender, tumor location, depth of invasion and vascular invasion. 2 of the 4 colorectal carcinoma cell lines, including LoVo and LoVo-5FU, expressed VEGF-C mRNA. The expression of VEGF-C is closely related to lymph node metastasis, and it might take part in the tumor lymphangiogenesis.
To investigate therapeutic efficiency of Ad/CMV-hTGF-β1 gene for rabbit intervertebral dise degeneration model. 60 Japanese white rabbits were selected to form the L5-L6 Anterior-Lateral-Anulus-Fibrosus-Incision-Induced model in order to simulate human intervertebral dise degeneration. 36 rabbits, whose corresponding intervertebral discs were injected with 20 μl (10×106 pfu) of Ad CMV-hTGF-β1 gene, constituted the therapy group, 12 were injected with 20 μl (10×106 pfu) of Ad CMV-LacZ gene as comparison group, while 12 were only injected with equivalent capacity of saline for empty comparison group, 3 weeks after injection, examples were taken for investigation of HE staining, MRI. Western Blotting and immunohistochemical research TGF-β1. Wide distribution of TGF-β1 was detected by immunohistochemical research in the degenerated annulus fibrosus after injection. Western Blotting research showed significant increase of TGF-β1 content in intervertebral dises treated with TGF-β1 gene than comparison groups. MRI signal transformed from low to comparatively high and that intervertebral dise pathological degree improved. Ad CMV-hTGF-β1 gene transfection is a potential method to increase TGF-β1 content and reverse intervertebral dise degeneration.
To investigate the effect of thiopental sodium on the release of glutamate and γ-aminobutyric acid (GABA) from synaptosomes in the prefrontal cortex, synaptosomes were male, the spontaneous release and the evoked release by 30 mmol/L KCl or 20 μmol/L veratridine of glutamate and GABA were performed under various concentrations of thiopental sodium (10–300 μmol/L), glutamate and GABA concentrations were determined by reversed-phase high-performance liquid chromatography. Our results showed that spontancous release and evoked relcase of glutamate were significantly inhibited by 30 μmol/L, 100 μmol/L and 300 μmol/L thiopental sodium, IC50 of thiopental sodium was 25.8±2. 3 μmol/L for the spontaneous release, 23.4±2.4 μmol/L for KCl-evoked release, and 24.3±1.8 μmol/L for veratridine-evoked release. But GABA spontaneous release and evoked release were unaffected. The study showed that thiopental sodium with clinically related concentrations could inhibit the release of glutamate, but had no effect on the release of GABA from rats prefrontal cortical synaptosomes.
In this study, we tested the hypothesis that volatile anesthetic enhancement of muscle relaxation is the result of combined drug effects on the nicotinic acetylcholine receptors. The poly A m RNA from muscle by isolation were microinjected into Xenopus oocytes for receptor expression. Concentration-effect curves for the inhibition of Ach-induced currents were established for vecuronium, rocuranium, and isoflurane. Subsequently, inhibitory effects of NDMRs were studied in the presence of the isoflurane at a concentration equivalent to half the concentration producing a 50% inhibition alone. All tested drugs produced rapid and readily reversible concentration-dependent inhibition. The 50 inhibitory concentration values were 889 μmol/L (95% CI: 711–1214 μmol). 33.1 μmol (95 C1: 27.1–11.7 nmol) and 9.2 nmol (95% CI: 7.9–12.3 nmol) for isoflurane, rocuranium and vecuronium, respectively. Coapplication of isoflurane significantly enhanced the inhibitory effects of rocuranium and vecuronium, and it was especially so at low concentration of NMDRs. Isoflurane increases the potency of NDMRs, possibly by enhancing antagonist affinity at the receptor site.
To study reliability and reliable indices of quantitative assessment of right ventricular systolic function by time-intensity curve (TIC) with right ventricular contrast, 5% sonicated human albumin was injected intravenously at a does of 0.08 ml/kg into 10 dogs at baseline status and cardiac insufficiency. Apical four-chamber view was observed for washin and washout of contrast agent from right ventricle. The parameters of TIC were obtained by curve fitting. The differences of parameters were analyzed in different states of cardiac functions. Among the parameters derived from TIC, the time constant (k) was decreased significantly with decline of cardiac function (P<0.001). But half-time of decent of peak intensity (HT) and mean-transit-time (MTT) of washout were increased significantly (P<0.001). The k was strongly related to cardiac output of right ventricle (CO) and ejection fraction (EF) of left ventricle and fractional shortening (FS) of left ventricle. Right ventricular systolic function could be assessed reliably by the parameters derived from TIC with right ventricular contrast echocardiography. The k, HT and MTT are reliable indices for quantitative assessment of right ventricular systolic function.
To study the dynamic changes of CT perfusion parameters during the first 12 h in the embolic cerebral ischemia models. Local cerebral ischemia model were established in 7 New Zealand white rabbits. All CT scans were performed with a GE Lightspeed 16 multislice CT. Following the baseline scan, further CT perfusion scans were performed at the same locations 20 min, 1–6 h and 8, 10 and 12 h after the embolus delivery. Maps of all parameters were obtained by CT perfusion software at each time point. The brains, taken 12 h after the scan, were sliced corresponding to the positions of the CT slices and stained by 2,3,5-triphenyltetrazolium chloride (TTC). On the basis of the TTC results, the ischemic sides were divided into 3 regions: core, penumbra and the relatively normal region. The changes of all parameters were then divided into 3 stages. In the first two hours (the first stage), the CBV dropped more remarkably in the core than in the penumbra but rose slightly in the relatively normal region while the CBF decreased and MTT, TTP extended in all regions to varying degrees. In the 2nd–5th h (the second stage), all the parameters fluctuated slightly around a certain level. In the 5th–12th h (the third stage), the CBV and CBF dropped, and MTT and TTP were prolonged or shortened slightly in the core and penumbra though much notably in the former while the CBV, CBF rose and MTT, TTP were shortened remarkably in the relatively normal region. We experimentally demonstrated that the location and extent of cerebral ischemia could be accurately assessed by CT perfusion imaging. The pathophysiology of the ischemia could be reflected by the CT perfusion to varying degrees.
To evaluate the feasibility of using polyethyleneimine (PEI) coated magnetic iron oxide nanoparticles (polyMAG-1000) as gene vectors. The surface characteristics of the nanoparticles were observed with scanning electron microscopy. The ability of the nanoparticles to combine with and protect DNA was investigated at different PH values after polyMAG-1000 and DNA were combined in different ratios. The nanoparticles were tested as gene vectors with in vitro transfection models. Under the scanning electron microscope the nanoparticles were about 100 nm in diameter. The nanoparticles could bind and condense DNA under acid, neutral and alkaline conditions, and they could transfer genes into cells and express green fluorescent proteins (GFP). The transfection efficiency was highest (51%) when the ratio of nanoparticles to DNA was 1∶1 (v∶w). In that ratio, the difference in transfection efficiency was marked depending on whether a magnetic field was present or not: about 10% when it was absent but 51% when it was present. The magnetic iron oxide nanoparticles coated with PEI may potentially be used as gene vectors.
To investigate the relationship between the insulin resistance (IR) of polycystic ovarian syndrome (PCOS) rat model induced by dehydroeplandrosterone (DHEA) and hormonal changes in the ovarium and the resistin mRNA levels in adipose tissue, 21-day-old female SD rats were divided into two groups in pairs. The rats in group 1 were injected daily (s. c) with DHEA for up to 20 days and the rats in group 2 injected with oil at the same time. Ovarian weight, serum insulin levels and sex hormone levels in rat blood of both groups were determineds. Oral glucose tolerance tests, light microscopic and electronic microscopic examination were performed. The levels of resistin mRNA in adipose tissue were measured by reverse transcription-polymerase chain reaction (RT-PCR). Our results showed that the ovarian weight in group 1 was greater than that in group 2 (P<0.05). The ovaria in group 1 showed multiple follicular cysts. The serum testeosterone and etrasdiol concentration were significantly higher in group 1 than those in group 2 (P<0.001 andP<0.05 respectively), so as the fasting serum glucose (P<0.001) and fasting serum insulin (P<0.05). The value of 1/FINS×FGC was significantly higher in group 1 than that in group 2 (P<0.001). Moreover, the resistin mRNA level of white adipose tissue in the DHEA-induced group was significantly higher than that in the control rats (P<0.05). It is concluded that the DHEA-induced PCOS rat models were similar to those of the patients with PCOS, and the IR was observed. Resistin secreted by adipose tissue may mediate IR in PCOS, and it is likely involved in the pathogenesis and development of PCOS.
To study the expression of Dickkopf-1 (DKK-1) in endometrium of pregnant mice during the peri implantation period and the role of DKK-1 during the embryo implantation in mice. Immunohistochemical technique was employed to determine the location of DKK-1 protein in endometrium, and semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR) was utilized to determine the levels of DKK-1 mRNA. Our results showed that the expressions of DKK1 mRNA and protein were higher in experimental groups than in control group (P<0.01). and it increased significantly on day 3 and reached its peak on day 4. and then decreased gradually on day 5–7. The levels of DKK-1 mRNA and protein on day 1 was significantly higher than those of other groups (P<0.01). It is concluded that DKK-1 probably plays an important role in signal transudation of embryo implantation and its high expression indicates the opening of implantation window.
The aim of the study was to investigate the correlation between prognosis of human cytomegalovirus (HCMV) infection and zinc in mature women. in order to explore the effect of serum zinc on HCMV infection. 900 mature woman were examined for zinc level and serum HCMV specific antibodies (IgG and IgM). 63, HCMV-IgM positive cases were divided into 3 subgroups according to their serum zinc level. and HCMV-IgM was observed for conversion after treatment with a Chinese traditional medicine (Jinyebadu). Our results showed that the mean concentration of zinc in serum was significantly lower in HCMV-IgM(+) group than that in HCMV-IgM(−) group (P<0.001). A positive correlation was found between the response of mature females with HCMV infection to the treatment and the levels of serum zinc (P<0.001). The response of mature women with HCMV is poor when the serum zinc is lower than 0.70.
To explore the roles of tumor necrosis factor-α (TNF-α) and heat shock protein 60 (HSP-60) in women with tubal factor infertility (TFI) associated withChlamydia trachomatis, and to determine the mechanisms of fallopian adhesions inChlamydia trachomatis (CT) infections, the expressions of TNF-α and HSP-60 were quantitatively determined in 60 cases of TFI and 30 controls by immunohistochemical technique. The patients with TFI were further divided into group A and group B according to the CT-DNA of cervical specimens of PCR. The quantitative analysis was conducted by employing computerized image analysis system. It is found that the expressions of TNF-α and HSP-60 were much higher in TFI patients than those of controls. Among CT-HSP responders, a stronger expression was correlated with more severe salpingeal pathology. It is concluded that TNF-α and HSP-60 play very important roles in fallopian tube adhesion and occlusion in TFI due to CT infection.
To study whether cultured bovine trabecluar meshwork cells (BTMC) are capable of expressing tTG in protein and at mRNA level, BTMC were culturedin vitro and passaged three times, then the cells were transferred onto or cultured on sterile cover or submitted to isolation of RNA with Trizol, and the expression of tTG was detected by immunohistochemical technique and reverse transcription polymerase chain reaction (RT-PCR) respectively. Our results showed that tTG immunostaining was positive in the cytoplasm and rarely in the nucleus of cultured BTMC. No immunostaining was seen in the negative control. Moreover, a single RT-PCR amplified product whose sequence and size were in accordance with our known parameters was obtained. The expression of tTG in cultured BTMC was confirmed in protein and at mRNA level. BMTC is available more readily for the investigation of the relationship between tTG and primary open-angle glaucoma.
To investigate the expression of hypoxia inducible factor-1 alpha (HIF-1α) and its relationship to apoptosis and proliferation in laryngeal squamous cell carcinoma (LSCC), immunohistochemical method was used to detect the expression of HIF-1α and PCNA. Tunnel technique was used to detectin situ cell apoptosis in LSCC. Our results showed that the expression of HIF-1α was related to the clinical stages of cancer and lymph node metastasis (P<0.05). The relationship between HIF-1α and PCNA was statistically significant (P<0.05) and no relationship was found between HIF-1α and apoptosis (P>0.05) It is concluded that HIF-1α plays a role in the carcinogenesis of laryngeal carcinoma and is correlated with proliferation, but bears no relationship with the apoptosis of tumor cells in LSCC.
To investigate the expression of Toll-like receptor (TLR) 2 and 4 mRNA in local tissues of model of oropharyngeal candidiasis in mice and to explore the potential role of TLR2 and TLR1 in carlier period of immune response, a murine model of oropharyngeal candidiasis inoculated by cotton wool balls saturated withcandida albicans was established. Mice were sacrificed at the indicated time points and the oropharyngeal tissues were excised. The expression of TLR2 and TLR4 mRNA was detected by RT PCR. The results showed that low level of TLR2. 1 mRNA could be detected in oropharyngeal tissues, but they were markedly up-regulated 6 h after inoculation, peaking after 12−1 24 h. Tissue TLR4 mRNA was gradually down-regulated 21− 48 h, while TLR2 mRNA levels remained high up to the 72nd h. These data suggested that oropharyngeal infection ofCandida albicans could result in up-regulation of TLR2 1 mRNA expression in local tissues, which might play important roles in earlier period of immune response.
To find a fast and efficient way of identifying seven common dermatophytes in clinical practice, we used the techniques of polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism (RFLP) targeting Topoisomerase II gene. The DNA of 7 dermatophytes, along withCandida albicans, Aspergillus terreus andAspergillus flavus were amplified by consensus primer dPsDl. They were then subjected to a second PCR with primers dPsD2 and species-specific primers PsT and PsME separately. 6 of the products generated by dPsD2 were digested with restriction enzyme Hinc II. DNA fragments of 3390 bp and 2380 bp was amplified by using consensus primer dPsD1 and dPsD2 from the genomic DNA of each dermatophyte species separately. By combining the results of the two species-specific primer sets (PsT and PsME), all species of dermatophyte yielded unique sizes-set of PCR products expect forT. mentagrophytes andT. tonsurans. From the restriction profiles of Hinc II, 6 of the 7 dermatophytoses were diagnosed to species level includingT. mentagrophytes andT. tonsurans. By combining the results of the PCR and PCR-RFLP, the 7 common dermatophytes can be identified to species level. It is conclude that the multiplex PCR and PCR-RFLP identification targeting the DNA topoisomerase II gene is rapid and efficient.