The vacuolated effect of Helicobacter (H. pylori) and its relationship to vacuolated cytotoxin antigen (VacA) were investigated by the method of cytotoxic test and SDS-pobyacrylamide gel electrophoresis (SDS-PAGE). Of the 62 clinical isolates, the broth culture filter (BCF) of 43 strains caused the Vero cell intracytoplasmically vacuolated. H. pylori strains were divided into H. pylori (Toxin+) group with vacuolated effect and H. pylori (Toxin−) group without vacuolated effect. The analysis of the BCF of H. pylori (Toxin+) and that of H. pylori (Toxin−) was studied by SDS-PAGE and Scan reader. A kind of protein with 87 ku molecular weight was recognized in the BCF of 30.23% (13/43) H. pylori (Toxin+) strains but in none of that of H. pylori (Toxin−) strains, the difference was statistically significant (P<0.05). There was a significant and concordant relationship between OD of the protein band with 87 ku molecular weight and titer of vacuolated activity of H. pylori (Toxin+) (r=0.67 andP<0.05 by linear regression analysis). H. pylori strains were divided into H. pylori (Toxin+) group with vacuolated effect and H. pylori (Toxin−) group without vacuolated effect. The vacuolated effect of H. pylori (Toxin+) was caused by the protein with 87 ku molecular weight (VacA).
In order to regulate the apoptosis induced by Fas-FasL system, a soluble isoform of mouse Fas was cloned from thymocytes of immature mice with the primers designed according to the full-length Fas cDNA sequence in the GeneBank. It was directionally inserted into the intermedium vector pUC19. DNA sequencing proved that it was consistent with the expected sequence. Then it was subcloned into the eukaryotic expression vector pCA13, which was used to construct the recombinant vector pCA13-FasC. By lipofectamine (LF2000)-mediated transfection, pCA13-FasC was transfected into the 293 cells. RT-PCR and Western blot indicated that the murine soluble Fas C protein was expressed in the 293 cells. Apoptosis inducing test showed that the expression of this murine Fas C could block the Fas-induced apoptosis, which confirmed the biological activity of the recombinant Fas C.
To investigate the inducement of cytotoxic T lymphocytes (CTLs) by antigen peptides mixture from different leukemia cells and the cross-reaction of the mixtures from different cell lines, antigen peptides mixtures were prepared from different leukemia cell lines respectively and then bound with Hsp70in vitro. Activation and proliferation of PBMC were observed after stimulation with different Hsp70-peptide complexes. The ratio of CD8+ in proliferative cells was analyzed by flow cytometry. The cytotoxicity of the activated PBMC to different target cells was assayed. The results showed that the antigen peptides from different leukemia cell lines, bound with Hsp70, could activate PBMC effectively, and stimulate the activated PBMC to proliferate. The proliferative PBMC had specific cytotoxicity to corresponding leukemia cells. CD8+ cells, accounting for a high proportion in proliferative cells, had a specific cytotoxicity to leukemia cells from which antigen peptides were prepared, suggesting that these CD8+ cells were CTLs specific to leukemia cells. CTLs activated by Hut78-peptides or Molt4-peptides had a significantly stronger cytotoxicity to Hut78 cells, Molt4 cells and Jurkat cells than that of CTLs activated by HL-60-peptides (P<0.05). And the cytotoxicity of CTLs activated by Hut78/Molt4-peptides to Jurkat cells was significantly stronger than that of CTLs activated by either Hut78-peptides or Molt4-peptides alone (P<0.05). It is concluded that antigen peptides mixtures from leukemia cells can induce specific antitumor CTLs. There exists cross-reactivity among antigen peptides mixtures from different cell lines of the same type leukemia and more cross-reactive antigen peptides could be obtained from more cell lines, suggesting that antigen peptides mixture with broad antigenic spectrum could be prepared by using multiple leukemia cell lines.
Effect of antiatherogenic high density lipoprotein (HDL) and apolipoprotein AI (apoAI) on production of prostaglandin E2(PGE2) by human monocyte-derived macrophages was investigated. Macrophages were loaded with acetylated low density lipoprotein followed by incubation with HDL3 or apoAI. PGE2 produced and secreted in culture supernatant was quantified by enzyme immunoassay. HDL3 induced production of PGE2 by macrophages in a time-dependent manner. 24 h after incubation, PGE2 production by HDL3-treated macrophages increased 3. 7-fold of that by control cells. ApoAI also induced PGE2 secretion to 2. 1-fold, which was significantly less than HDL3. The data indicate that both HDL3 and lipid-free apoAI enhance PGE2 synthesis and secretion by human macrophages and this may further contribute to the protection from atherosclerosis.
By study on the effect of anisodamine on lipopolysaccharide-induced expression of tissue factor (TF) in vascular endothelial cells (EC), the mechanism of anisodamine antithrombosis, as well as in the treatment of bacteraemic shock was investigated. Human umbilical vein endothelial cells (HUVECs) were cultured by trypsin digestion method. TF activity was measured in the lysates of HUVEC by using a single step clotting assay. Specific mRNA expression was detected by Northern blotting. In order to evaluate a possible contribution of the nuclear factor (NF)-κB pathway on the effects observed, electrophoretic mobility shift assays (EMSA) were performed using nuclear extracts from HUVECs and NF-κB-binding oligonucleotides. The results showed that treatment of HUVEC with LPS resulted in a significant increase in TF activity. Anisodamine dose-dependently inhibited LPS-induced upregulation of TF. These effects was also confirmed on the level of specific TF mRNA expression by Northern blotting. Furthermore, EMSA showed that anisodamine completely abolished LPS-induced NF-κB DNA binding activity in nuclear extracts from HUVECs treated with LPS together with anisodamine. The results suggest that anisodamine counteracts endothelial cell activation by inhibiting LPS-induced TF expression in these cells. Its interference with the NF-κB pathway might—at least in part—contribute to this effect. The ability of anisodamine to counteract LPS effect on endothelial cells might be one underlying mechanism explaining its antithrombosis and efficacy in the treatment of bacteraemic shock.
To set up a method of amplification for the whole CagA gene ofHelicobacter pylori and its fingerprinting by restriction fragment length polymorphism (RFLP), nested PCR was employed in combination with TD-PCR to amplify the gene and EcoRI and Hind III were used to generate the RFLP fingerprinting. Target DNA fragments from 13 of 20 samples were successfully amplified and the relevant RFLP fingerprintings were obtained. It is concluded that the method can be used to amplify the whole CagA gene and CagA gene has apparent diversity of RFLP profile.
The effects of Arecoline (Are) on calcium mobilization were investigated. In isolated single ventricular myocyte of guinea pig, patch clamp whole cell recording techniques were used to record the current of L-type calcium channel and cytosolic Ca2+ level (Ca2+i) labeled with fluorescence probe Fluo-3/AM was measured under a laser scanning confocal microscope. Results revealed that Are (3–100 µmol/L) could inhibit L-type calcium current in a concentration-dependent manner and the value of IC50 was 33.73 µmol/L (n=5). In the absence of extracellular calcium, the resting levels of [Ca2+]i was not affected by Are (n=6,P>0.05), but pretreatment with Are (30 µmol/L) could significantly inhibit the [Ca2+]i elevation induced by caffeine (10 mmol/L,n=6,P<0.01). It was concluded that Are could inhibit not only calcium influx through L-type calcium channel but also calcium release from sarcoplasmic reticulum.
The aim of the present study was to confirm observations on the vertical transmission ofSchistosoma japonicum in the rabbit.S. japonicum-infected pregnant rabbits were used in this study. Perfusion of mother rabbits was done 9 weeks after infection in order to obtain worm burdens in relation to their initial cercarial dose. Anti-schistosoma specific IgM antibodies in serum samples collected from rabbit kittens were detected by ELISA. Our results showed that gestation period lasted the normal 29–31 days. All the exposed mother rabbits became infected withS. japonicum. Positive IgM antibody OD values were detected in 12 out of the 60 kittens examined (20.0%). In group C and A, 40.0% and 17.9% of the kitten were congenitally infected, respectively. 18.1% of the kittens born to mothers infected with a single dose of 200 cercariae per rabbit were positives; this is not significantly different from that obtained for the 600 dose group (22.2%). Three randomly selected IgM+ kittens harbored between one and two adult worms. The livers of these kittens displayed granulomatous lesions. It is concluded that congenitalS. japonicum infection does occur in the rabbit and is affected by the mother stage of pregnancy and to a lesser extent by its infection load.
The synthesis of CCK-4 (H-Trp-Met-Asp-Phe-NH2) by using enzymes exclusively was described. As protection group for the amino group we used the Phenylacetyl group (Phac) which had been cleaved at the end of the synthesis with Penicillin G Amidase (PGA) without affecting the peptide bonds. Thus, beginning with Phac-Trp-OH we had successfully synthesized the target peptide with following 4 enzymes,α-Chymotrypsin, Papain, Thermolysin and PGA in four reaction steps. All reactions were carried out in aqueous buffer in reasonable yields (>65%). FAB-MS or FD-MS verified the correct molecular mass of all peptides.
Primary rat hepatocytes were cultured using differentin vitro models and the enzyme leakage, albumin secretion, and cytochrome P450 1A (CYP 1A) activity were observed. The results showed that the level of LDH was decreased over time in culture. However, on day 5, LDH showed a significant increase in monolayer culture (MC) while after day 8 no LDH was detectable in sandwich culture (SC). The levels of AST and ALT did not change significantly over the investigated time. The CYP 1A activity was gradually decreased in a time-dependent manner in MC and SC. The decline of CYP 1A was faster in MC than in SC. This effect was partially reversed by using cytochrome P450 (CYP450) inducer such as Omeprazol and 3-methylcholanthrene (3-MC) and the CYP 1A induction was always higher in MC than in SC. In bioreactor basic CYP 1A activity was preserved over 2 weeks and the highest albumin production was observed in bioreactor followed by SC and MC. Taken together, it was indicated each investigated model had its advantages and disadvantages. It was also underlined that variousin vitro models may address different questions.
In order to investigate the regulative function of telomerase and phosphorylated (activated) extracelluar regulated protein kinase (ERK) 1 and 2 in the leukemic cell lines HL-60 and K562 proliferation inhibition and apoptosis, three chemotherapeutic drugs Harringtonine (HRT), Vincristine(VCR)and Etoposide (Vp16)were selected as inducers. The proliferation inhibition rate was detected by MTT method, the cell cycle and cell apoptosis was analyzed by flow cytometry and the telomerase activity was detected by the telomeric repeat amplification protocol (TRAP) assay and bioluminescence analysis method. The phosphorylated ERK1/2 protein expression was detected by western blot method. The results showed that HRT, VCR and Vp16 could inhibit cell proliferation, induce apoptosis, inhibit telomerase activity and down-regulate the protein expression of phosphorylated ERK. It was suggested that ERK signal transduction pathway was involved in the down-regulation of telomerase activity and the onset of apoptosis in the leukemic cells treated by HRT, VCR and Vp16.
To investigate whether the Bcl-2 gene family is involved in modulating mechanism of apoptosis and change of cell cycle protein induced by curcumin in acute myeloid leukemia HL-60 cell line and primary acute myelogenous leukemic cells, the Bcl-2 family member Mcl-1, Bax and Bak and cell cycle proteins including P27kipl, P21wafl, cyclin D3 and pRbp− were selected and their expression detected by SABC immuno-histochemical stain method. The attitude of sub-G1 peak in DNA histogram was determined by FCM. The TUNEL positive cell percentage was identified by terminal deoxynucleotidyl transferase (TdT)-mediated Biotin dUNP end labeling technique. It was found that when HL-60 cells were treated with 25 µmol/L curcumin for 24 h, the expression level of Mcl-1 was down-regulated, but that of Bax and Bak up-regulated time-dependently. There was significant difference in the expression level of Mcl-1, Bax and Bak between the curcumin-treated groups and control group (P<0.05−0.01). At the same time, curcumin had no effect on progress of cell cycle in primaty acute myelogenous leukemia at newly diagnosis, but could increase the peak of Sub-G1(P<0.05), and down-regulate the expression of Mcl-1 and up-regulate the expression of Bax and Bak with the difference being statistically significant. The expression of P27kipl, P21wafl and pRbp− were elevated and that of cyclin D3 decreased in the presence of curcumin. These findings suggested that the Bcl-2 gene family indeed participated in the regulatory process of apoptosis induced by curcumin in HL-60 cells and AML cells. Curcumin can induce apoptosis of primary acute myelogenous leukemic cells and disturb cell cycle progression of HL-60 cells. The mechanism appeared to be mediated by perturbing G0/G1 phases checkpoints which associated with up-regulation of P27kipl, P21wafl and pRbp− expression, and down-regulation of cyclin D3.
To investigate the expression of the HO-1 gene in PC12 cells in hypoxic environment and gain further insight to the role of HO-1 in cerebral ischemia, PC12 cells were exposed to hypoxia environment (95 % N2, 5 % CO2) for 0.5 h, 1 h, 4 h, 8 h, 12 h, 24 h respectively. The level of HO-1 mRNA was examined by reverse transcriptase polymerase chain reaction (RT-PCR); the volume of COHb in the media were measured spectrophotometrically and the cGMP concentration of PC12 cell extracts was determined by radioimmunoassay. We found that after exposure to hypoxia for 1 h, 4 h, 8 h, 12 h, 24 h, HO-1 mRNA increased by 3 %, 4 %, 17 %, 31 %, 36 % as compared with that in control group respectively (P<0.01 orP<0.05); the COHb increased by 12 %, 29 %, 59 %, 88 %, 94 % as compared with that in control group respectively (P<0.01 orP<0.05), and the cGMP concentration were 2.2, 3.4, 5.2, 8.1, 10.9-fold as that of the control group (P<0.01). We are led to conclude that hypoxia induced the expression of HO-1 gene, the production of endogenous CO, and the concentration of cGMP was elevated as well.
Chronic treatment with Salvia Miltiorrhiza preventing left ventricular hypertrophy (LVH) and its possible mechanism—inhibiting the action of cardiac aldosterone in spontaneously hypertensive rats (SHR) were investigated. Normotensive Wistar-kyoto (WKY) rats and SHRs were used. Part of SHRs was treated with Salvia Miltiorrhiza for 12 weeks. Systolic blood pressure (SBP) and left ventricular mass index were measured. Sections of heart tissue were stained with HE method and VanGieson method. Collagen volume fraction was determined in the left ventricle by automatically quantitative morphometry. Cardiac aldosterone concentration was measured by radioimmunoassay. The results indicated that compared with WKY rats, SHRs exhibited higher SBP, left ventricular collagen volume fraction, and aldosterone concentration (allP<0.05). After the treatment with Salvia Miltiorrhiza, SBP, left ventricular collagen volume fraction, and aldosterone concentration in SHR were decreased as compared with control group (P<0.05) except SBP. It was concluded that chronic treatment with Salvia Miltiorrhiza could prevent left ventricular hypertrophy in SHR, significantly inhibit collagen compositions in left ventricle. The mechanism was probably related with the inhibition of the cardiac aldosterone action.
To observe the effect of ginsenoside Re on cardiomyocyte apoptosis and Bcl-2/Bax gene expression after ischemia (30 min) and reperfusion (6 h) in rats and to elucidate the possible mechanisms of ginsenoside Re on inhibition of cardiomyocyte apoptosis, the ischemia/reperfusion heart model was established by ligating the left anterior descending branch of coronary artery in Wistar rats. The apoptotic cardiomyocytes were confirmed by transmission electron microscopy and counted byin situ nick end labeling (TUNEL) method and light microscopy. The mRNA and protein expression of Bcl-2 and Bax genes were studied byin situ hybridization and immunohistochemical staining. Mean optical density (OD) value of the positive fields of mRNA and protein expression was quantitatively examined by image analysis system. The results were as follows: (1) The apoptotic cardiomyocytes were found in ischemic fields in the ischemia/reperfusion group and weren’t observed in the sham-operation group by transmission electron microscopy; (2) The numbers of the apoptotic cells were 134.45±45.61/field in the ischemia/reperfusion group, and 90.66±19.22/field in the ginsenoside Re-treated group. The differences was significant between two groups (P<0.01); (3) Gene expression of Bcl-2 and Bax were increased significantly in the ischemia/reperfusion group and ginsenoside Re-treated group when compared with the sham-operation group. There was no significant difference in the gene expression of Bcl-2 between the ginsenoside Re-treated group and ischemia/reperfusion group (P>0.05), but gene expression of Bax was decreased significantly in the ginsenoside Re-treated group as compared with the ischemia/reperfusion group (P<0.01). The ratio of Bcl-2/Bax was increased significantly in the ginsenoside Re-treated group when compared with the ischemia/reperfusion group and sham-operation group. These findings suggest that myocardial ischemia-reperfusion can induce cardiomyocyte apoptosis, and ginsenoside Re can significantly inhibit cardiomyocyte apoptosis induced by ischemia-reperfusion in rats. It is concluded that ginsenoside Re inhibits cardiomyocyte apoptosis by inhibiting expression of pro-apoptotic Bax gene and raising the ratio of Bcl-2/Bax.
The effects of Shenmai injection (SMI) and aminophylline on apoptosis of small airway smooth muscle cells (SASMC) and the Fas/FasL expression in rats with papain-induced emphysema were investigated. Rat emphysema model was established by a single intratracheal instillation of papain. Apoptosis and Fas/FasL expression of SASMC were detected by immunohistochemistry SABC and TUNEL assay at day 1, 3, 5, 7, 15, 30 after modeling, and the effect of SMI and aminophylline on them were observed. The results indicated that the Fas/FasL expression positive rate in SASMC was 2.31±0.55/1.28±0.47 respectively. After a single intratracheal instillation of papain, the expression of Fas/FasL positive rate in the placebo group was increased in a time-dependent manner. SMI could inhibit the expression of Fas/FasL, but aminophylline couldn’t. The positive rate of apoptosis in the control group was 0.87±0.32. After a single intratracheal instillation of papain, the SASMC apoptosis positive rate in the placebo group was increased in a time-dependent manner. The SASMC apoptosis rate in all groups was declined after treatment with SMI, but the effect of aminophylline was not obvious. It was demonstrated that in the pathogenesis of emphysema Fas/FasL played an important role in the regulation of SASMC apoptosis. SMI influenced the expression of Fas/FasL and declined SASMC apoptosis by inhibiting the releasing of inflammatory media and played an important role in the therapy of emphysema.
To study the regulatory effect of acute and chronic insulin treatment on insulin post-receptor signaling transduction pathway in a human hepatoma cell line (Hep G2), Hep G2 cells were incubated in the presence or absence of insulin with different concentrations in serum free media for 16 h and then stimulated with 100 nmol/L insulin for 1 min. Protein levels of insulin receptor β-subunit (IRβ), insulin receptor substrate-1 (IRS-1) and p85 subunit of phosphatidylinositol 3-kinase (PI 3-kinase) were determined in total cell lysates by Western-immunoblot. Phosphorylated proteins IRβ, IRS-1 and interaction of PI 3-kinase with IRS-1 were determined by immunoprecipitation. Results showed that 1-min insulin stimulation rapidly induced tyrosine phosphorylation of IRβ and IRS-1, which in turn, resulting in association of PI 3-kinase with IRS-1. 1–100 nmol/L chronic insulin treatment induced a dose-dependent decrease in the protein level of IRβ and a slight decrease in the protein level of IRS-1. There was a more marked reduction in the phosphorylation of IRβ, IRS-1, reaching a nadir of 22 % (P<0.01) and 15 % (P<0.01) of control levels, respectively, after 16 h treatment with 100 nmol/L insulin. The association between IRS-1 and PI 3-kinase was decreased by 66 % (P<0.01). There was no significant change in PI 3-kinase protein levels. These data suggest that chronic insulin treatment can induce alterations of IRβ, IRS-1 and PI 3-kinase three early steps in insulin action, which contributes significantly to insulin resistance, and may account for desensitization of insulin action.
By means of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay, the association between vitamine D receptor (VDR) genotypes and bone mineral density (BMD) in the patients receiving long-term glucocorticoid therapy was studied. The clinical data and blood of 71 patients with rheumatosis who received long-term glucocorticoid therapy were collected. BMD was measured by dual-energy X-ray absorptimometry. VDR gene fragment (about 185 bp) was amplified by PCR from the extracted genomic DNA, then digested with restriction endonuclease Bsm I. The genotypes were evaluated based on the fragment length following endonuclease digestion and the association between genotypes and BMD or Z-score values was analyzed. Among the 71 cases, the detected genotypes were Bb and bb with the distribution frequency being 11.3 % and 88.7 % respectively. The distribution frequency of the alleles was in agreement with the Hardy-Weinberg equilibrium. There was no significant difference between the two genotypes in age, gender, body mass index (BMI), disease duration, disease types, time of glucocorticoid administration and cumulative dosage (P>0.05). Osteoporosis rate of the patients with Bb or bb genotype was 37.5 % and 33.3 % respectively, with the difference being not significant (χ2=0.05,P=0.8). The BMD and Z-score values at lumbar spine and femur in two genotypes were not similar, but the difference had no significant (P>0.05). The distribution frequency of bb type of VDR genotypes in Han populations of China was more prevalent, followed by Bb and bb types in turn. In the patients receiving long-term glucocorticoid therapy, there was no significant difference in BMD between Bb and bb genotypes. The data suggest that the VDR genotypes may not be means of identifying patients at greater risk of glucocorticoid-induced osteoporosis, which await to be further confirmed by a large sample size.
The method of gene transfer into corneal endothelium was investigated to provide a foundation for the study of TGF-β1 gene transfer to inhibit corneal graft rejection. Two days after direct injection of pMAM TGF-β1 mediated by liposome into the anterior chamber of rabbits, one half of corneas were made into paraffin slides and the endothelial layer was carefully torn from the other half to make a single layer slide of endothelia. By means of immunohistochemical technique, the plasmid pMAM TGF-β1 expression product TGF-β1 in the endothelia was detected. Specific TGF-β1 expression was positive in the endothelia on both the paraffin slide and the single layer slide. The results showed that by direct injection into the anterior chamber, foreign plasmid DNA could be transferred into the endothelia and its expression was obtained. This may provide a foundation for further study on TGF-β1 participating in local induction of corneal immune tolerance.
To evaluate the effects of adenovirus (Ad)-mediated transfer of p53 and p16 on human bladder cancer cells EJ, EJ were transfected with Ad-p53 and Ad-p16. Cell growth, morphological change, cell cycle, apoptosis were measured using MTT assay, flow cytometry, cloning formation, immunocytochemical assays. Ad-p16 or Ad-p53 alone could inhibit the proliferating activity of EJ cellsin vitro. Ad-p53 could induce apoptosis of partial EJ cells. G1 arrest was observed 72 h after infection with Ad-p16, but apoptosis was not obvious. The transfer of Ad-p16 and Ad-p53 could significantly inhibit the growth of EJ cells, decrease the cloning formation rate and induce apoptosis of large number of EJ cells. The occurrence time of subcutaneous tumor was delayed and the tumor volume in 4 weeks was diminished by using Ad-p53 combined with Ad-p16 and the difference was significant compared with using Ad-p53 or Ad-p16 alone. It was suggested that the transfer of wild-type p53 and p16 could significantly inhibit the growth of human bladder cancerin vitro andin vivo.
To explore a novel strategy for antisense gene therapy of cancer, the coding sequence of human proliferating cell nuclear antigen (PCNA) cDNA was reversely inserted into the eukaryotic vector pLXSN by molecular cloning techniques and transferred into bladder cancer EJ cells with liposome. The PCNA expression in transferred cells was dynamically detected by immunofluorescence and RT-PCR techniques. Changes of proliferation activities of cancer cells were assayed by MTT colorimetric and cloning formation methods. In the experiment, the antisense eukaryotic vector was successfully constructed and named as pLAPSN. After transfection with it for 1–7 days, PCNA protein and mRNA levels in cancer cells were blocked by 16.74 % – 84.21 % (P<0.05) and 23.27 % – 86.15 % (P<0.05) respectively. The proliferation activities of transferred cells were inhibited by 27.91 % – 62.07 % (P<0.01), with cloning formation abilities being decreased by 50.81% (P<0.01). It was concluded that thein vitro proliferation activities of cancer cells could be effectively inhibited by blocking PCNA expression with antisense technique, which could serve as an ideal strategy for gene therapy of bladder cancer.
The contribution of angiogenesis inhibitor TNP-470 to the growth and metastasis of ACHN renal cell carcinoma (RCC) was studied. TNP-470 (40 mg/kg, every two days) was administrated to BABL/c nude mice bearing ACHN RCC. The mice were sacrificed after a treatment duration of 31 days and the weight and volume of subcutaneous tumors as well as foci of lung metastasis were measured. The microvascular density (MVD) of the tumor as well as the PCNA index and apoptotic index of the tumor cells were evaluated immunohistochemically. Result showed that the growth of ACHN RCC was suppressed significantly and none metastasis was observed in TNP-470-treated mice. Compared with the control group, the MVD was decreased markedly (P<0.01) and the apoptotic index was increased significantly (P<0.01) in the treated group. The tumor volume was positively correlated to the MVD (r=0.7144,P<0.01) and inversely correlated to the apoptotic index (r=−0.8607,P<0.01), and MVD was conversely correlated to the apoptotic index. It was determined that TNP-470 could effectively inhibit angiogenesis of ACHN RCC, which resulting in ischemia and hypoxia, leading to increased apoptosis, thus obviously suppressing the growth and metastasis of ACHN RCC in nude mice.
In order to investigate the effect of ligustrazine (Lig) i.p. on peritoneal permeability in peritoneal dialysis and its side effects, creatinine was given intravenously and continuously to maintain the high plasma creatinine level. All the rabbits were divided into three groups: normal control group (group A), group B treated with 0.12 % Lig and group C treated with 0.24 % Lig. The peritoneal dialysis of all rabbits lasted 2 h. The plasma and dialysate levels of glucose, protein and creatinine were observed immediate, 30 min, 60 min, 90 min, 120 min after dialysis. Creastinine dialysate/plasma ratio (D/P), protein D/P ratio, glucose D/Do at different time points after dialysis and creatinine mass transfer area coefficient (MTAC) at 120 min were calculated. The structures of peritoneum were observed under optical microscope and electron microscope after continuously intraperitoneal injection of Lig for 14 days. The results showed that the 90-min and 120-min creatinine D/P ratios in the group C were higher than in the group A. The 120-min creatinine MATC in the group C was higher than in the group A. The rabbits treated with Lig did not show significant structure changes of peritoneum and signs of peritoneal irritation. It was suggested that Lig could increase mass transfer ability of peritoneum without significant side effects.
The insulin sensitivity in hypertensive patients with normal glucose tolerance (NGT), impaired glucose tolerance (IGT) and type 2 diabetes mellitus (DM) and the insulin resistance (IR) under the disorder of glucose metabolism and hypertension were studied. By glucose tolerance test and insulin release test, insulin sensitivity index (ISI) and the ratio of area under glucose tolerance curve (AUCG) to area under insulin release curve (AUC1) were calculated and analyzed. The results showed that ISI was decreased to varying degrees in the patients with hypertension, the mildest in the group of NGT with hypertension, followed by the group of IGT without hypertension, the group of IGT with hypertension and DM (P=0). There was very significant difference in the ratio of AUCG/AUC1 between the hypertensive patients with NGT and controls (P=0). It was concluded that a significant IR existed during the development of IGT both in hypertension and nonhypertension. The increase of total insulin secretion (AUC1) was associated with nonhypertension simultaneously. IR of the hypertensive patients even existed in NGT and was worsened with the deterioration of glucose metabolism disorder, but the AUC1 in the HT group changed slightly. A relative deficiency of insulin secretion or dysfunction of β-cell of islet existed in IGT and DM of the hypertensive patients.
To study the resistant mechanism and clinical significance of pseudomonas aeruginosa toβ-lactam antibiotics, the outer membrane permeability rate of 30 P. aeruginosa strains to 5β-lactam antibiotics was measured and their production ofβ-lactamase and theβ-lactamase genes they carried detected. Furthermore, the relationship between the permeability,β-lactamase and the clinical effects ofβ-lactam antibiotics was observed. By using14C-penicillin and liquid-scintillant isotope assay, the affinity of penicillin binding proteins (PBPs) was measured and their roles in the resistant mechanism studied. It was revealed that the permeability rate was higher in sensitive strains than in resistant ones (P<0.05). All strains harbored 1–4β-lactamase genes and producedβ-lactamase. Higher permeability rate and higher degree of stability toβ-lactamase indicated better clinical therapeutic effects. The affinity of PBPs changed little without regard to the permeability andβ-lactamase. These results suggested that the permeability of outer membrane andβ-lactamase, but not PBPs, played important roles in the resistant mechanism of P. aeruginosa toβ-lactam antibiotics and affected the clinical therapeutic effectiveness of some patients.
To explore immune intervention effects of the combined use of cycloporin A (CsA) and 1, 25-dihydroxyvitamin D3[1,25(OH)2D3] at low doses on experimental autoimmune thyroiditis (EAT), porcine thyroglobulin (pTG) was injected into a CBA mouse at the dose of 100µg on day 0 and day 14 to establish the model of EAT. The immune prevention group from day 0 to day 28, and treatment group from day 10 to day 38 were daily administered CsA (10 mg/kg) intragastrically and/or 1,25(OH)2D3(0.2µg/kg) ip. After immunized by pTG, the mice were sacrificed on day 28 and day 38 to examine their thyroid gland pathologically, and to check the levels of serum porcine thyroglobulin antibodies (pTGAb), porcine thyromicrosomal antibodies (pTMAb). The incidences of EAT in the immune prevention group and treatment group, with administration of low dose of CsA and 1,25(OH)2D3, were decreased respectively by 44.44% and 37.50%. Those of severe disease in the two groups were decreased respectively by 71.43% and 60.32%. The levels of serum pTGAb and pTMAb in the immune prevention group were lower than those of the positive control group. It was concluded that combined use of CsA and 1,25(OH)2D3 at low doses could effectively prevent EAT with a synergic effect.
The clinical values of coils emboliztion in the treatment of pulmonary arteriovenous malformations (PAVM) and related complications were investigated. Eleven patients with PAVMs verified by pulmonary arterial angiography were treated by transcatheter coils embolization. Chest X-ray (11 cases), computer tomography (7 cases) and/or magnetic resonance imaging (2 cases) were performed before embolization. Blood-gas analysis was done in 5 cases before and after embolization. The follow-up materials of 8 patients were collected to evaluate the effect of embolization with coils. The clinical manifestations included cerebral embolus, hemoptysis and decreased oxygenation in 9 patients and the remaining 2 had no symptoms. 9/11 cases were found by chest X-ray and 8 were diagnosed definitely. 7/7, 2/2 cases were diagnosed by CT or MR and diagnosis was made in all cases. Embolization was performed in 29 vessels. Partial pressure of oxygen in arterial blood of 5 cases changed significantly before and after embolization. Slight complications occurred in 6 patients, such as low fever, chest pain, pleurisy. The follow-up results showed that 7 cases were cured effectively. No primary and secondary device migration, and no medical paradoxical embolization occurred. It was concluded that coils emboliztion is a well-established method for treating PAVMs. It is a minimally invasive lung preserving treatment with high efficiency and less complication.
To study the effects of 6-hydroxydopamine (6-OHDA) and reduced glutathione (GSH) on the nigral dopaminergic neurons in brain slicesin vitro, immolunohistochemical technique was used to observe the changes of TH-stained neurons, including cell bodies and the dendrites, in the substantia nigra (SN) of midbrain slices of rats after incubation for 1 h in the presence of GSH 15 min before and during the period of incubation with 6-OHDA. The results showed that cell bodies remained intact but dendrites were fragmented and truncated after treatment with 6-OHDA. The antioxidant GSH alone did not significantly affect the dendrites of SN neurons but prevented 6-OHDA-induced damage of dendrites. It was concluded that glutathione may prevent 6-OHDA-induced dopaminergic neurodegeneration and play a protective role in dopaminergic neurons.
Using different models of focal cerebral ischemia, the temporal and spatial rules of metabolism and energy changes in the post-ischemia brain tissue were measured by proton magnetic resonance spectroscopy (1HMRS) to provide valuable information for judging the prognosis of acute focal cerebral ischemia and carrying out effective therapy. Nine healthy Sprague-Dawly rats (both sexes) were randomly divided into two groups: The rats in the group A (n=4) were occluded with self-thrombus for 1 h; The rats in the group B (n=5) were occluded with thread-emboli for 1 h. The1H MRS at 30, 40, 50, 60 min respectively was examined and the metabolic changes of NAA, Cho and Lac in the regions of interest were semiquantitatively analyzed. The spectrum intregral calculus area ratio of NAA, Cho, Lac to Pcr+Cr was set as the criterion. The values of NAA · Cho in the regions of interest were declined gradually within 1 h after ischemia, especially, the ratio of Cho/(Pcr+Cr), NAA/(Pcr+Cr) at 60 min had significant difference with that at 50 min (P<0.05). The ratio of Lac/(Pcr+Cr) began to decrease at 40 min from initial increase of Lac in both A and B groups. MR proton spectrum analysis was a non-invasive, direct and comprehensive tool for the study of cellular metabolism and the status of the biochemical energy in acute ischemia stroke.
A non-invasive acoustical system was developed for the measurement of transmission properties of acoustic waves in the hip joints. The instrumentation consisted of three sub-systems. An excitation system employed a vibratory force at the sacrum of the test subjects. A transduction system included a pair of identical microphones installed in the tubes of two stethoscopes, which were placed at the greater trochanters on both sides for picking up the acoustical signals transmitted across the hip joints. The data acquisition and analysis system was a portable signal analyzer with a program of dual channel digital filter for measuring the power of acoustical signal in 1/3-octave frequency bands. 27 normal adults, 20 normal pre-school children and 40 normal neonates were randomly selected for testing. Coherence function (CF) and discrepancy (D) was measured during the testing. Results from the three groups showed that there was a high coherence of the signals (CF>0.9) and a small discrepancy (D<3 dB) between bilateral hips in the frequency range of 200–315 Hz. For normal neonates, there was a wider frequency range of 160–315 Hz in which the acoustical signals maintained a high coherence (CF>0.93) and a smaller discrepancy (D<2 dB) was observed. This study showed that the development of the acoustical technique provided a practical method with objective parameters. The results obtained in this study can offer a baseline for further investigation of hip disorders particularly those related to structural abnormalities of the hip.
The features of the symptoms, laboratory tests and pathological characteristics of adrenal cortical and medullary hyperplasia were studied. In 6 cases of hypercatecholaminenia, plasma norepinephrine (NE), epinephrine (E), catecholamine (CA) and 24-h urinary vanillylmandelic acid (VMA), 17-hydroxycorticosteroid (OHCS) and 17-ketosteroid (KS) were determined. Adrenal glands were examined by CT scan and131I-MIBG imaging. Pathological examination was performed after operation. The results showed that in 6 cases of hypercatecholaminenia (3 men and 3 women) aged from 34–50 years, the clinical features were just like “pheochromocytoma”, for example, episodic headache, perspiration, palpitation, pallor, apprehension, nausea, tremor, anxiety and so on. Plasma levels of CA, NE and E were elevated in all 6 cases. 24-h urinary samples obtained at the onset revealed elevated VMA in 1 case. 24-h urinary cortisol was obviously elevated in all 6 cases. 24-h urinary 17-OHCS, 17-KS was normal. B-type ultrasound, CT, MRI and131I-MIBG revealed 9 lateral adrenal gland diffuse or nodular enlargement in 6 cases. Pathologic examination showed adrenal cortical and medullary hyperplasia. Clinically, adrenal cortical and medullary hyperplasia resembled “pheochromocytoma”. The most significant feature of this disease was both elevated plasma CA and 24-h urinary cortisol obviously. Pathologic examination showed adrenal cortex nodular hyperplasia and medullar diffuse or limit hyperplasia. Whether it is an independent disease or symptoms of the other disease has not final conclusion up till now
Orbscan- II anterior system was used for early diagnosis of keratoconus. 48 Eyes of 24 patients with suspicious keratoconus were examined by Orbscan- II anterior system from Dec. 1999 to Dec. 2000 and followed up. The values of Diff and anterior chamber depth (ACD) were recorded. Results indicated that values of Diff and ACD were increased in 4 eyes of 2 patients with keratoconus trend during follow-up. Taking advantage of Orbscan- II anterior system to observe the values of Diff and ACD can early diagnose the sub-clinical keratoconus. The values of Diff and ACD can sensitively report the progression of keratoconus.
To explore the role of tenascin (TN) and fibronectin (FN) in the pathophysiology of nasal polyps (NP), the expression of TN and FN in NP from 34 patients and inferior turbinates from 20 patients with deviation of nasal septum was immunohistochemically studied. In patients with NP, the relations between expression and histopathological features, eosinophils (EOS) infiltration, clinical staging and the size of NP were analyzed. Our study showed that the gray score of TN and FN expression was 163.10±10.54 and 163.24±11.52 in NP respectively, whereas it was 175.49±9.29 and 173.93±7.92 in inferior turbinates respectively. The difference between two groups was significant (P<0.01). The expression of TN and FN in edematous type was significantly stronger than that in cystic and glandular type and fibrous type (P<0.05). The association between FN expression and EOS infiltration was significant (r=−0.60,P<0.01). The expression of TN and FN did not correlate with clinical staging and size (P>0.05). It was suggested that abnormal ECM might contribute to proliferation of epithelia, accumulation of EOS and edema formation, thereby causing development of NP.
To assess the curative effects of different reduction techniques on the dislocation of cricoarytenoid joint caused by intubation, indirect laryngoscope (IL) and direct laryngoscope (DL) were utilized for the closed reduction of the displaced arytenoid under local anesthesia. 23 patients who underwent the reduction for dislocated arytenoid under IL or DL from January 1991 to June 2001 were reviewed. The data were collected on the duration of the laryngeal injury, times of receiving reduction, side-effects after the treatment and the period for voice to return to normal. The relationship between the duration of the laryngeal lesion and the period of the voice rehabilitation was examined. 13 patients received the reduction under IL and 10 patients under DL. Except the times of the reduction, which showed significant difference, no differences were found between IL group and DL group in the course and the period of voice rehabilitation, as well as sore throat after the manipulation. The patients’ voice recovery was positively related to their course of disease in both IL and DL group. It is concluded that the recovery of normal voice is obviously affected by the duration of arytenoid dislocation. The reduction under IL is as effective as under DL in the treatment of arytenoid dislocation. Reduction by DL is better suit the patients with long time course of disease.
The health check up flow of digital hospital can be consulted with the assembly line of industry factory. Because they have the following same features: highly specialized workstation, closeness and continuance, rhythm, balanced production, continuous production. The essential prerequisites are as the follows: The inspecting items and methods should be stable; advanced product mix and stable production design; standardized raw material, consumption, procedure, inspection method; there are lots of request for health inspection; the customers move at the least unit; the space arrangement should be reasonable; the time arrangement should be proportion. With the computer net, the digital inspection can achieves the raw material controlling accurately. The basis of check up line concerns about equipment, net and software, data collection, and personnel. The group technology is used in the health inspection flow design of the digital hospital in the field of tiems customers and zone redivided. The digital assembly linemic health inspect has the following stages: member registering, notice, check in, arrange order, time control, report, feedback and analysis. The assembly linemic has following advantages: increasing the productivity, the space utility, satisfaction of customer, fund returning, lowering the cost and ensuring the quality.
Based on a survey of community health service organization in several cities, community health service model based on the family clinic was compared with state-owned community health service model, and status quo, advantages and problems of family community health service organization were analyzed. Furthermore, policies for the management of community health service organization based on the family clinic were put forward.