For a preliminary study of the role of β-catenin/Tcf signaling in squamous differentiation of airway (tracheobronchial) epithelial cells, a stable mutant of β-catenin was transfected into primarily cultured porcine airway epithelial cells. Western blotting revealed that exogenous protein was observed in large quantity in cytoplasm and nucleus. When co-transfected with Tcf luciferase reporter plasmids, β-catenin mutant increased the reporter’s transcriptional activities. However, mRNA expression of a squamous differentiation marker, small proline-rich protein (SPRP), was not elevated, as shown by reverse transcription-polymerase chain reaction. These findings suggest that β-catenin/Tcf signaling may not be directly involved in the squamous differentiation of porcine airway epithelial cells.
To investigate the disposition and tissue distribution of ML12 after intravenous (iv) administration in rats, the compound in plasma or in tissue was extracted into ethyl acetate under basic condition and was determined by HPLC after extracted by dilute sulfuric acid. Excitation wavelength and emission wavelength of fluorescence detection were 278 nm and 307 nm, respectively. The data were processed with the software 3P97 to calculate the main pharmaceutical parameters of ML12. At dose of 5 and 10 mg/kg, the elimination of the drug from plasma was found to be kinetically linear, but when the dosage was 20 mg/kg, a non-linear feature was observed. The highest level of ML12 was found in the kidney. Distribution of ML12 after iv administration was extensive and the concentration-time profile was found to be fitted to an open two-compartment model.
In order to confirm the alteration and significance of cigarette smoke exposure on SP-A in rats, 20 Wistar rats were assigned randomly to two groups: an N group (n=10), and an S group (n=10). The ultra-structural change was observed by electron microscopy. The number of cells positive for SPA was by immunohistochemically measured. The mRNA expression in the lung tissues was determined by reverse transcription polymerase chain reaction (RT-PCR). The number of cells positive for SPA of the S group (0.52 ± 0.05) was lower than that of the N group (0.72±0.06) (P<0.05). The levels of mRNA of SPA in the lung tissues of the S group (0.3522±0.0512) was significantly lower than that of the N group (0.4432±0.05628) (P<0.05). It is concluded that cigarette smoke alone decreased the level of SP-A and that might have an important effect on surfactant metabolism and the host defense functions of surfactant in the peripheral airways, which might play a crucial role in the development of chronic obstructive lung disease.
To compare the difference in tumor immunity and autoimmunity elicited by adenovirus (Ad) encoding human or murine tyrosinase-related protein 2 (AdhTRP2 or AdmTRP2), and to find the most effective way to induce immunity by AdhTRP2 or AdmTRP2, C57BL/6 mice were immunized with AdhTRP2 or AdmTRP2 intramuscularly at different doses of 105, 106, 107 and 108 separately (10 mice for each dose). Two weeks after the immunization, in vivo CTL assay and intracellular staining (ICS) of IFN-γ were carried out to analyze the dose-effect relationship. Tumor growth and vitiligo (as an sign of autoimmunity) were observed until 3 months after challenge with 105 B16F10 tumor cells. The results showed that Ad encoding AdmTrp2 induced weak tumor immune response. Similar immunization with AdhTrp-2 elicited stronger protective immunity. CTL activity and IFN-γ-produced CD8+T cells were directly proportional to dose of AdhTrp2 or AdmTrp2. Moreover, AdhTrp2 group showed tumor rejection in 100% of challenged mice till the end of 3rd month while 60% of mice immunized with AdmTrp2 were protected against tumor. In the whole process of this experiment, no vitiligo was observed in mice immunized either with AdhTrp2 or AdmTrp2. It is concluded that anti-melanoma responses induced by genetic vaccination expressing xenoantigens breaks immune tolerance effectively and is able to elicit strong antigen-specific cytotoxic T cell response without vitiligo.
To investigate the gene expression profiles in acute allograft rejection of renal transplantation, and identify the markers for the early diagnosis of acute rejection, heterotopic kidney transplantation was performed by using F344 or Lewis donors and Lewis recipients. No immunosuppressant was used. Renal grafts were harvested on days 3, 7, and 14. A commercial microarray was used to measure gene expression levels in day-7 grafts. The expression levels of 48 genes were up-regulated in the allograft in comparison with the isograft control, and interferon-γ-induced GTPase gene was most significantly up-regulated in allografts. It is concluded that a variety of pathways are involved in organ transplant rejection which is dynamic and non-balanced. IFN-inducible genes, such as IGTP, may play an important role in the rejection. A lot of important factors involved in acute rejection are unnecessary but sufficient conditions for the rejection. We are led to conclude that it is virtually impossible to make an early diagnosis based on a single gene marker, but it could be achieved on the basis of a set of markers.
In order to investigate the role of Twist gene in the metastasis of hepatocellular carcinoma (HCC), total RNA was respectively extracted from three HCC cell strains with different metastatic potentials, HepG2, MHCC-97L and MHCC-97H. The first strand cDNA was synthesized by reverse transcription, which was then used as template to perform fluorescent quantitative polymerase chain reaction (FQ-PCR). The quantity of Twist gene expression was normalized by that of the housekeeping gene, GAPDH for each sample. ANOVA was used to estimate the relationship between Twist gene and metastasis potential of HCC. The results showed that the normalized initial cDNA concentrations of Twist gene in HepG2, MHCC-97L and MHCC-97H were (9.45±0.25)×10−4, (1.82±0.41)×10−3, (3.06±0.62)×10−3, respectively. FQ-PCR revealed significant differences in the expression level of Twist among HCC cell strains with different metastatic potentials. It was concluded that high expression level of Twist was closely associated with more aggressive behaviors of HCC. Twist provides a novel indicator for HCC metastasis.
To develop a more efficient antithrombotic way after coronary artery bypass grafting (CABG), the anticoagulant effects were compared of human tissue factor pathway inhibitor (TFPI) gene transfection and aspirin oral administration (traditional method) on vein grafts. An eukaryotic expression plasmid pCMV-(Kozak) TFPI was prepared. Animal model of carotid artery bypass grafting was constructed. In operation, endothelial cells of vein grafts in TFPI group and empty plasmid control group were transfected with pCMV-(Kozak) TFPI and empty plasmid pCMV respectively, while no transfection was conducted in aspirin control group. After operation, aspirin (2 mg·kg−1·−1) was administered (i.g.) in aspirin control group. Three days later, grafts (n=10) were harvested for RT-PCR, Western blotting and immunohistochemical analyses of exogenous gene expression and for pathological, scanning electron microscopic observation of thrombus. Thirty days later, the patency rates of remnant grafts (n=10) were recorded by vessel Doppler ultrasonography. Human TFPI gene products were detected in gene transferred vein grafts. Three days later, thrombi were found in 7 animals of aspirin control group and in 8 animals of empty plasmid control group, but in only 1 of TFPI group (P<0.01). Thirty days later, 5 grafts were occluded in empty plasmid control group, but none of grafts was occluded in the other groups (P<0.05). The endothelial surfaces of grafts in both of the control groups were covered with aggregated erythrocytes and platelets, and it were not seen in TFPI group. It was suggested that the anticoagulant effects on vein grafts of human TFPI gene transfection are better than those of aspirin.
In order to identify the differentially expressing gene of bone marrow mesenchymal stem cells (MSCs) stimulated by electromagnetic field (EMF) with osteogenesis microarray analysis, the bone marrow MSCs of SD rats were isolated and cultured in vitro. The third-passage cells were stimulated by EMFs and total RNA was extracted, purified and then used for the synthesis of cDNA and cRNA. The cRNA of stimulated group and the control group was hybridized with the rat oligo osteogenesis microarray respectively. The hybridization signals were acquired by using X-ray film after chemiluminescent detection and the data obtained were analyzed by employing the web-based completely integrated GEArray Expression Analysis Suite. RT-PCR was used to identify the target genes: Bmp1, Bmp7, Egf and Egfr. The results showed that 19 differentially expressing genes were found between the stimulated group and the control group. There were 6 up-regulated genes and 13 down-regulated genes in the stimulated group. Semi-quantitative RT-PCR confirmed that the expressions of Bmp1, Bmp7 mRNA of the stimulated group were up-regulated (P<0.05) and those of Egf, Egfr were down-regulated (P<0.05). It was suggested that the gene expression profiles of osteogenesis of the bone marrow MSCs were changed after EMF treatment. It is concluded that the genes are involved in skeletal development, bone mineral metabolism, cell growth and differentiation, cell adhesion etc.
To find a new source of seed cells for constructing tissue-engineered intervertebral disc, nucleus pulposus (NP) cells and mesenchymal stem cells (MSCs) were isolated from New Zealand white rabbits. The nucleus pulposus cells population was fluorescence-laelled and co-cultured with MSCs with or without direct contact. Morphological changes were observed every 12 h. Semi-quantitative reverse transcriptase-polymerase chain reaction was performed to assess the expression levels of Sox-9, aggreacan and type II collagen every 24 h after the co-culture. MSCs treated with direct contact rounded up and presented a ring-like appearance. The expression of marker genes was significantly increased when cells were co-cultured with direct contact for 24 h. No significant change was found after coculture without direct contact. Co-culture of NP cells and MSCs with direct contact is a reliable method for generating large amount of NP cells used for cell-based tissue engineering therapy.
This study is to investigate the effect of FK506 on expression of hepatocyte growth factor (HGF) in rats’ spinal cord following peripheral nerve injury and to elucidate the mechanisms for neuroprotective property of FK506. Fifty male rats were randomly divided into normal group, injury group and treatment group. Models of peripheral nerve injury were established by bilateral transection of sciatic nerve 0.5 cm distal to piriform muscle. Then the treatment group received subcutaneous injection of FK506 (1 mg/kg) at the back of neck, while the injury group was given 0.9% saline. The L4–6 spinal cords were harvested at various time points after the surgery. Western blotting and immunofluorescent staining were used to detect the level and position of HGF in spinal cord. Immunofluorescent staining showed that HGF-positive neurons were located in anterior horn, intermediate zone and posterior horn of gray matter in normal spinal cord. Western blotting revealed that there was no significant difference in the expressions of HGF between the injury group and the normal group, while the expression of HGF was significantly higher in the treatment group than in the injury group 7 and 14 days after surgery. It is suggested that peripheral nerve injury does not result in up-regulation of the expression of HGF in spinal cord, while FK506 may induce high expression of endogenous HGF after injury thereby protecting neurons and promoting axonal outgrowth.
To study the effect of titanium alloy cage on the treatment of the ischemic necrosis of femoral head in dog, the model of the ischemic necrosis of femoral head was made with the liquid nitrogen in 15 hybrid adult dogs. The titanium alloy cage made of a hollow cylinder was driven into the subchondral bone of necrotic femoral head via central channel. The dogs were divided into 3 groups, each group was sacrificed 3, 6, 12 weeks after the operation respectively. No collapse of femoral head was observed after the operation. The position of the cages was good on radiograph. Microscopically, the cancellous bone of necrotic femoral head rebuilt gradually and grew into cage. After 12 weeks of creeping substitution, the cancellous bone filled up the hollow cavity and holes of the cages. It is concluded that the titanium alloy cage can provide structural support for the subchondral bone and prevent collapse and can be used for the treatment of the ischemic necrosis of femoral head.
To investigate the preventive effect of epimedium-derived phytoestrogen (PE) on osteoporosis induced by ovariectomy (OVX) in rats, 11-month-old female Wistar rats were randomly divided into Sham, OVX and PE groups. One week after OVX, daily oral administration of PE (0.4 g·kg−1·day−1) started in PE group, and rats in Sham and OVX groups were given vehicle accordingly. The administrations lasted for 12 weeks. The biological markers including serum osteocalcin (OC) and urinary deoxypyridinoline (DPD) for bone turnover were evaluated at the end of the 12th week. On the 13th week, all the rats were sacrificed. The right proximal tibiae were removed, subjected to micro CT for determination of trabecular bone structure and then bone histomorphometry was performed to assess bone remodeling. The OVX rats were in a high bone turnover status as evidenced by increased bone formation markers and bone resorption markers. Treatment with PE could suppress the high bone turnover rate in OVX rats. Micro CT data revealed that PE treatment could ameliorate the deterioration of the micro-architecture of proximal tibiae induced by OVX, as demonstrated by greater bone volume, increased trabecular thickness and less trabecular separation in PE group in comparison with OVX group. The static and dynamic parameters of bone histomorphometry indicated that there were significant increases in bone formation variables and significant decreases in bone resorption variables between PE and OVX groups. The findings suggest that PE has a beneficial effect on trabecular bone in OVX rat model and this effect is possibly associated with stimulation of bone formation as well as inhibition of bone resorption.
To investigate the efficacy of a combination therapy on gluteal muscle contracture, 286 definitely diagnosed patients were subjected to surgical treatment, and then functional exercises and physical therapy. The patients with severe symptoms were asked to have a set of specially-designed functional exercises. All the patients were followed up for 3 to 24 months by hospital visit, correspondence or telephone interview. The effective rate was 100%, and the curative rate was up to 94.6%. Few patients developed complications and relapse was rare. It is concluded that the combination therapy, including surgical removal of diseased tissues, functional exercises and physical therapy, is an effective approach for the treatment of severe juvenile gluteal muscle contracture.
The differences in intracellular and extracellular protein expressions between human prostate cancer lines LNCap and DU145 were examined. The proteins of the two cell lines were extracted and condensed by using protein extraction kits. And the intracellular and extracellular proteins were quantitatively detected on a micro-plate reader by using bicinchoninic acid (BCA) method. The proteins in cell culture fluid were qualitatively assayed by SELDI-TOF-MS. The results showed that the intracellular protein contents of LNCap cells were extremely higher than those of DU145 cells. After serum-free culture, both intracellular and extracellular protein contents of LNCap and DU145 were decreased to some extent. And the intracellular proteins were decreased by 5% in LNCap and by 36% in DU145 respectively, while the extracellular proteins were decreased by 89% in LNCap and 96% in DU145 respectively. SELDI assay revealed that there were 5 marker proteins in LNCap and 6 in DU145. Although both LNCap and DU145 cell lines originated from human prostate cancer, they had some differences in protein expression.
To investigate the effects of diethylstilbestrol (DES) in reestablishing spermatogenesis and the mechanism by which estrogen works on spermatogenesis, rats were exposed to 1% 2,5-HD for 5 week. Then 0.1 mL of DES was given (s.c.) at a rate of 0.3 μg/kg, 30 μg/kg, 3 mg/kg every other day for 2 weeks respectively (DES group) while the other rats received ethyldeate only. Plasma testosterone (T) and LH were measured on the 8th week after the treatment. The rats were killed at the 18th week. The left testis was histopathologically examined. In all the rats in the DES groups, spermatogenesis was re-established and the rats in the 30 μg/kg group showed the best results. Serum T was suppressed markedly in rats of 30 μg/kg and 3 mg/kg groups while T was only mildly inhibited in 0.3 g/kg group, without significant difference found in serum LH. It is concluded that the nearly complete testicular atrophy could be reversed by DES treatment in rats. Estrogen plays an important part in spermatogenesis, and the role of estrogen in spermatogenesis is more than suppressing the hypothalamo-pituitary-testis axis.
To investigate the relationship between the expression of RASSF1A protein and promoter hypermethylation of RASSF1A gene, RASSF1A protein expression was measured by Western blotting in 10 specimens of normal bladder tissues and 23 specimens of bladder transitional cell carcinoma (BTCC). The promoter methylation in BTCC and normal bladder tissues was detected by methylation-specific PCR (MSP). The results showed that the expression level of RASSF1A protein was significantly lower in BTCC tissues than that in normal bladder tissues. However, it was not correlated with its clinical stages and pathological grades. The frequency of promoter methylation of RASSF1A gene was higher in BTCC tissues than that in normal bladder tissues. In 14 patients with the aberrant promoter methylation, 13 showed loss or low expression of RASSF1A protein. It is concluded that RASSF1A gene promoter methylation may contribute to the low level or loss of RASSF1A protein expression, the inactivation of RASSF1A gene and the genesis of BTCC. But, it may bear no correlation with its clinical stages and pathological grades.
In order to evaluate the effect of mitofusin-2 gene (mfn2) on proliferation and chemotherapy sensitivity of human breast carcinoma cell line MCF-7 in vitro, pEGFPmfn2 plasmid carrying full length of mitofusin-2 gene was transfected, by using sofast, into MCF-7 cells. Mitofusin-2 gene expression in MCF-7 cells transfected by sofast after 48 h was detected by PCR and Western blotting, and the stable expression of GFP protein in MCF-7 cells by Western blot analysis. The proliferation of MCF-7 cells was assayed by MTT and cell counting. By using PI method, the effects of mfn2 on the cell cycle distribution of MCF-7 were measured. Annexin-V/PI double labeling method was employed to detect the changes in apoptosis induced by chemotherapeutics before and after transfection. The results showed that the MCF-7 cells transfected with mfn2 gene could stably and highly express GFP protein. MTT assay revealed that after transfection of mfn2 cDNA, the proliferation of MCF-7 cells was significantly inhibited. DNA histogram showed that cells arrested in S phase, and the percentage of S phase cells was 42.7, 17.2 and 19.6 in mfn2 cDNA transfection group, blank plasmid transfection group and blank control group, respectively (P<0.05). The apoptosis ratio of the cells transfected with mfn2 gene was increased from 3.56% to 15.95%, that of the cells treated with camptothecin (CAMP) followed by mfn2 gene transfection was 69.6%, and that in blank plasmid transfection group and blank control group was 31.0% and 23.4% respectively (P<0.05). It was suggested that transfection of mfn2 gene could significantly inhibit the proliferation of MCF-7 cells and promote their sensitivity to CAMP with a synergic effect.
Speckle tracking imaging (STI) was employed to investigate the effect of right ventricular (RV) volume and pressure overload on left ventricular (LV) rotation and twist in 35 patients with atrial septal defect (ASD), 18 of which with pulmonary hypertension, and 21 healthy subjects serving as controls. The peak rotations of 6 segments at the basal and apical short-axises and the average peak rotation and interval time of the 6 segments in the opposite direction during early systolic phase were measured respectively. LV twist versus time profile was drawn and the peak twist and time to peak twist were calculated. LV ejection fraction (EF) was measured by Biplane Simpson. Compared to ASD patients without pulmonary hypertension and healthy subjects, the peak rotations of posterior, inferior and postsept walls at the basal level were lower (P<0.05), and the average counterclockwise peak rotation of 6 segments at the basal level during early systolic phase was higher (P<0.05), and the average interval time was delayed (P<0.05). LV peak twist was also lower (P<0.05), and had a significant negative correlation with pulmonary arterial systolic pressure (r=−0.57, P=0.001). No significant differences were found in LVEF among the three groups. It was suggested that although RV volume overload due to ASD has no significant effects on LV rotation and twist, LV peak twist is lower in ASD patients with pulmonary hypertension. Thus LV twist may serve as a new indicator of the presence of pulmonary hypertension in ASD patients.
To assess right ventricular free wall longitudinal myocardium deformation and examine the changes with normal age by speckle tracking imaging (STI), myocardial systolic peak strain (ɛ), systolic peak strain rate (SRs), early diastolic peak strain rate (SRe), late diastolic peak strain rate (SRa), the ratio of SRe/SRa were measured in the basal, middle and apical segments of right ventricular free wall in 75 healthy volunteers (age range: 21–71 y) by STI from the apical 4-chamber view. RV longitudinal strain and strain rate were highest in the basal segment of the free wall. Older subjects had lower early diastolic strain rate (SRe) than younger subjects, but they had higher late diastolic strain rate (SRa). A negative correlation between age and the ratio of SRe/SRa was found in all RV free wall segments (r=−0.466 − −0.614, P<0.01). It is concluded that RV diastolic strain rate changes with age and STI can be used for the study of RV myocardial deformation.
To study the apoptosis induced by cisplatin in cervical cancer cell line HeLa and its mechanism, cell growth inhibition of cisplatin on HeLa cells was analyzed by MTT assay. Cell apoptosis was examined by cytometry and Hoechst33258 staining after treatment with cisplatin. The effects of cisplatin on transcription of E6 were analyzed by RT-PCR. The protein expressions of E6, p53, p21, Bax and Bcl-2 were studied by Western blotting. Cisplatin inhibited proliferation in a time-and dose-dependant manner. Cytometically, sub-G1 peak showed higher apoptosis rates in the experimental group than those in the control. Hoechst33258staining exhibited apoptosis induced by cisplatin. RT-PCR revealed that cisplatin decreased transcription of E6. Western blotting showed that cisplatin decreased protein expression of E6 and increased protein expression of p53, p21 and Bax. It had no effect on protein expression of Bcl-2. It is concluded that cisplatin can induce apoptosis in HeLa cells by suppressing HPV E6 and thereby restoring the function of p53.
The study examined the inhibitory effect of Atractylodes macrocephala (AM) on the uterine contraction during premature delivery and explored its electrophysiological mechanism by studying the effects of AM on the Ca2+-activated K+ currents of pregnant human myometrial smooth muscle cells with or without the treatment with interleukin-6. Single cells were acutely isolated from pregnant human myometrial smooth muscles. Whole-cell Ca2+-activated K+ currents were recorded by using an Axopatch1-D amplifier. The cells were divided into three groups: group A in which AM was added into perfusate, group B, in which interleukin-6 was added into perfusate) and group C in which AM was added into perfusate after addition of interleukin-6. IL-6 10 ng/mL inhibited BKca by 36.9%±13.7% as compared with control (P<0.01). AM at 2 mg/mL raised BKca by 36.7%±22.6% or 45.2%±13.7% with or without the treatment of IL-6, respectively (P<0.01). It is concluded that AM was able to enhance the BKca of pregnant human myometrial smooth muscle cells treated or untreated with interleukin-6 and its effect on the BKca IL-treated cells was stronger that its effect on BKca of untreated cells. Our results suggested that AM can help to maintain the membrane potentials and the resting status of pregnant human myometrial smooth muscle cells.
To assess the relationship between pronuclear scoring and day-3 embryo quality and pregnancy outcome and to determine the clinical value of pronuclear stage scoring system in human in vitro fertilization-embryo transfer (IVF-ET) program, a pronuclear scoring system was used to score zygotes 16–20 h after insemination during conventional IVF or intracytoplasmic sperm injection (ICSI). The embryos were classified into groups Z1, Z2, Z3 and Z4. Comparisons were made of the rates of arrested embryos and excellent embryos on day 3. Comparisons of pregnancy outcome were made only in those patients in whom cohorts of similarly Z-scored embryos were transferred. The results showed that there were less arrested embryos and more excellent embryos on day 3 in groups Z1 and Z2 than those in group Z3 and Z4. More embryos arrested and less excellent embryos developed in group Z4 than group Z3. The clinical pregnancy rates resulting from the transfer of single pronuclear score homologous embryo types were similar among groups Z1, Z2 and Z3. Implantation rates of group Z1 were higher (P<0.05) than that of group Z3. These findings suggests that pronuclear scoring can predict developmental ability on day 3 and implantation potential. A evaluation that combines the Z-score and day 3 embryo morphology is useful in the determination of the most viable embryos and the number of embryos for transfer.
To investigate the effects of hyperoxia on mitochondrial multienzyme complex III (cytochrome, Cytb) and V (ATPase6, 8) in premature newborn rat lung, the 1-day-old preterm SD rats were randomly assigned to hyperoxia group and air group. The rats in hyperoxia group were continuously exposed to 85% oxygen and those in air group to room air. After 1, 4, 7, 10, 14 day(s) of exposure, these rats were killed, total lung RNA was extracted and Cytb, ATPase6, 8 mRNA were detected by reverse transcription polymerase chain reaction (RT-PCR). Western blotting was used to detect the expression of Cytb protein in lung tissue. The results showed that compared with air group, Cytb mRNA expression was significantly increased (P>0.05) after 1, 4 day(s) of exposure. The general tendency decreased after 7 days, and its expression became weak but difference in mRNA expression between the two groups was not significant (P>0.05). ATPase6 mRNA expression was significantly increased 1 day after the exposure (P<0.05) and did not show any significant change 4, 7, 10 days after the exposure (P>0.05). At the 14th day, ATPase6 mRNA expression was significantly increased (P<0.05). ATPase8 mRNA expression did not show any significant change 1, 4, 10 day(s) after the exposure (P>0.05). At the 7th and 14th day, ATPase8 mRNA expression was significantly increased (P<0.05). Western blotting showed that Cytb protein expression was increased 1,4 day(s) after the exposure, but the difference between the two groups was not significant (P>0.05). The general tendency was decreased after 7 days, and its expression became weak but difference was not significant 7, 10 days after the exposure (P>0.05). At day 14 its expression became significantly weak (P<0.05). We are led to conclude that exposure to high concentrations of oxygen can significantly change the expression of Cytb and ATPase6, 8, which results in uncoupling of oxidative phosphorylation in mitochondrial respiration chain, and plays an important role in the mechanism of hyperoxia-induced lung injury.
Three plasmids (pGenesil-P1, pGenesil-P2, pGenesil-P3) with different p27Kip1-shRNA sequences were designed and synthesized. Their effects on the proliferation of bovine corneal endothelial cells (bCEC) were investigated. Plasmid expressing irrelevant shRNA with a random combination was used as negative control (pGenesil-HK). The recombination of four plamids was confirmed by restrictive enzyme digestion and sequence analysis. The expression of mRNA and protein of p27Kip1 was detected by RT-PCR and Western blotting after stable transfection. The expressions of p27Kip1 mRNA and p27Kip1 protein of pGenesil-P1 group, pGenesil-P2 group and pGenesil-P3 group were all lower than those in the pGenesil-HK group and the blank group (non-transfected group). pGenesil-P3 had the strongest inhibitory effect and was selected for the next steps. The proliferation rates of the pGenesil-P3 group, the pGenesil-HK group and the blank group were assessed by MTT. The influence of shRNA-p27Kip1 on bCEC cell cycle was detected by flow cytometry (FCM). Compared with the control groups, the proliferation rate of the pGenesil-P3 group was increased significantly, and the ratio of S-phase also increased. It is concluded that shRNA-p27Kip1 could down-regulate the expression of p27Kip1 effectively and increase the proliferation of bCEC. RNA interference (RNAi) may be an effective means to promote the proliferation of CEC.
To investigate the role of progesterone receptor (PR) expression in malignant melanoma (MM), PR and proliferative cell nuclear antigen (PCNA) expression were immunohistochemistrically evaluated in a series of 35 specimens of MM, and the correlation between the immunohistochemistrical findings and clinicopathological data was also analyzed. PR expression was detected in 25.7% (9/35) of the patients with MM. No PR expression was observed in nevi. PR expression was inversely correlated with PCNA expression (r=−0.353, P=0.026). PR expression was slightly increased in females, subjects aged under 55 y, those with ulceration, non-acral subtype and diagnosis delay longer than 1 y, but the difference was not statistically significant. Selective expression of progesterone receptor in malignant melanoma might be correlated with inhibited tumor growth.
Human leukocyte antigen G (HLA-G) is one of the molecules implicated in immunotolerance. To investigate the role of HLA-G in primary cutaneous malignant melanoma (CMM), a series of 47 skin melanocytic lesions were immunohistochemically evaluated. The correlation between HLA-G expression and CMM clinicohistopathological data and Bcl-2 expression was also analyzed. HLA-G expression was detected in a variety of cell types. No significant difference in HLA-G expression was observed between malignant and non-malignant melanocytic lesions. HLA-G expression was significantly correlated with the inflammatory infiltration and Bcl-2 expression, whereas no significant correlation with ulceration, tumor thickness, clinical stage, histopathological subtypes were observed. HLA-G expression may be the result of host immune reaction in tumor microenvironment rather than a malignant feature of CMM.
In order to investigate peptide mimics of carbohydrate blood group A antigen, a phage display 12-mer peptide library was screened with a monoclonal antibody against blood group A antigen, NaM87-1F6. The antibody-binding properties of the selected phage peptides were evaluated by phage ELISA and phage capture assay. The peptides were co-expressed as glutathione S-transferase (GST) fusion proteins. RBC agglutination inhibition assay was performed to assess the natural blood group A antigen-mimicking ability of the fusion proteins. The results showed that seven phage clones selected bound to NaM87-1F6 specifically, among which, 6 clones bore the same peptide sequence, EYWYCGMNRTGC and another harbored a different one QIWYERTLPFTF. The two peptides were successfully expressed at the N terminal of GST protein. Both of the fusion proteins inhibited the RBC agglutination mediated by anti-A serum in a concentration-dependent manner. These results suggested that the fusion proteins based on the selected peptides could mimic the blood group A antigen and might be used as anti-A antibody-adsorbing materials when immunoabsorption was applied in ABO incompatible transplantation.
To observe the effects of basic fibroblast growth factor (bFGF) on human adenoid cystic carcinoma ACC-2 cell line proliferation and ERK, cyclin D1/p21waf/cip1 signaling pathways, human adenoid cystic carcinoma cells (ACC-2) were cultured and the influence of bFGF of different concentrations on cell proliferation was determined by MTT. Protein was detected by immuno-precipitation and ERK activity by using ERK agent kit. p-ERK1/2 and down-stream cyclin D1, p21waf/cip1 expression were detected by Western blotting and the interfering role of mitogen protein-activated kinase (MEK) suppressor U0126 in the afore-mentioned indicators was examined. MTT demonstrated ACC-2 cell proliferation was substantially enhanced by bFGF, immuno-precipitation displayed ERK activity was up-regulated by bFGF, and immuno-imprinting also showed p-ERK1/2, cyclin D1 expression was greatly enhanced and p21waf/cip1 expression was inhibited by bFGF. U0126 suppressed the effect of bFGF. It is concluded that bFGF can promote the proliferation of human adenoid cystic carcinoma ACC-2 cells, and its pathways are associated with the up-regulated activity and expression of p-ERK1/2, inhibited p21waf/cip1 expression and enhanced cyclin D1 expression.
HIV/AIDS is increasing in prevalence in China and spread of infection from highly risk populations to the general populations was recognized. Despite the fact, there are still only few scientific reviews on quality of life (QOL) for people living with HIV/AIDS (PLWHAs). However, many PLWHAs are struggling with social and psychological influences such as substances abuse, cultural beliefs, depression, stigma, poverty, which can affect their QOL. Public unawareness about infection and disease, willingness to seek medical care and motivation to follow therapy are indirectly influencing health outcome. In 2003 Chinese government has established the so-called the “Four Frees and One Care” policy. The policy was officially implemented from 2004 in some areas, yet to date it is not implemented nationwide. This paper discussed the epidemiology of HIV, underlying psychosocial factors affecting PLWHAs and their impact on QOL. We put forward some recommendations for stakeholders, advocacy groups, non-government organizations and Chinese government.
To clarify the role of TAFI in hypertensive disorders in pregnancy, 22 subjects, including 10 with pre-eclampsia (PE) and 12 with gestational hypertension were examined for the levels of TAFI and thrombin-antithrombin (TAT) complex. Thirty normal pregnant women served as controls. ELISA was employed for the detection. The results showed that the TAFI antigen levels in normal pregnancy group, gestational hypertension group and PE group were (85.35±24.69)%, (99.65±18.27)%, (110.12±23.36)%; (97.06±21.40)%, (114.08±27.76)%, (125.49±24.70)%; (106.6±19.21)%, (129.2±25.07)%, (139.1±30.12)%, in the 1st, 2nd and 3rd trimester respectively. No significant differences were found between the normal pregnancy group and gestational hypertension group but significant difference existed between normal pregnancy group and PE group in each trimester (P<0.05). TAT complexes were significantly higher in patients with PE than that in controls (P<0.05), but no correlation was found between TAT and TAFI. It is concluded that TAFI may contributed to the impairment of fibrinolysis in the patients with PE and may serves as a sensitive indicator for PE, but it may not help in the diagnosis of the gestational hypertension.