Objective To increase the sensitivity of quantitative PCR using DNA polymerase in combination with aptamer 6-10.Methods The quantitative PCR was performed using DNA polymerase mixed with aptamer 6-10 or specific antibody.The low detection limit of each modified method was compared with that of the conventional method.Results The quantitative PCR using aptamer 6-10(200 nmol/L) or specific antibody increased the low detection limit of human death receptor 5(DR5) plasmid from 105 copies/?l to 103 copies/?l.Melting curves showed that each method had minimal nonspecific amplification when the concentration of DR5 plasmid was 105 copies/?l;however,when the concentration of DR5 plasmid was 103 copies/?l,the conventional method had nonspecific amplification,whereas the method using aptamer 6-10 had minimal nonspecific amplification.Conclusions Aptamer 6-10 of DNA polymerase can increase the sensitivity of quantitative PCR.

ObjectiveTo study the effect of allo-human bone marrow mesenchymal stemcells (bMSCs) on the secretion of interleukin(IL)-1, tumor necrosis factor(TNF)-α and transforming grouth factor (TGF)-β of patients with rheumatoid arthritis (RA) in vitro. MethodsBMSCs were isolated from bone marrow of healthy volunteers and purified by density gradient centrifugation and cultured in vitro. The mononuclear cells from the peripheral blood of patients with RA and healthy controls were isolated respectively.bMSCs and mononuclear cells were co-cultured in vitro and the density of IL-1, TNF-α and TGF-β3 in the co-culture system were detected by ELISA. ANOVA and Pearson correlation were used for statistical analysis.ResultsMononuclear cells from peripheral blood of patients with RA were co-cultured with bMSCs for seven days. There were an decreased density ofIL-1[(38.4±0.5) vs(6.2±1.0) ng/L], TNF-α[(29.4±1.3) vs (4.6±1.2) ng/L]and an increased density of TGF-β[(2.6±1.0) vs (22.5±2.2) ng/L]in the co-culture system (P＜0.05). But on the other hand, for healthy volunteers there were no significant change in the density of IL1[(4.4±1.1) ng/L]and TNF-α[(5.0±1.7) ng/L]in the coculture group, as compared with the mononuclear cell group[(4.4±1.3) vs(5.3±1.7) ng/L](P＞0.05). There was an increased density of TGF-β in the coculture system[(4.8±1.4) vs(10.5±1.2) ng/L](P＜0.05). IL-1 was positively correlated to TNF-αt (r=0.896,P=0.000), TNF-β1 was nagative correlation with 1L-1 and TNF-α (r=-0.356,P=0.019; r=-0.380,P=0.000).ConclusionHuman bone marrow MSCs have modulatory effects on main cytokines of patients with RA in vitro. bMSCs could down-regulate the levels of IL-1 and TNF, but up-regulate the density of TGF-β. These immune-modulatory effects are not MHC restricted. The results of this study have provided evidence for the development of effective therapy for RA.

Objective To study the distribution of allogenic bone marrow-derived mesenchymal stem cells (BM-MSCs) on joints of collagen-induced arthritis (CIA) rats and to investigate their repair effects on joint damages. Methods Five Wistar rats were used for extraction of mesenchymal stem cells and 30 adult female Wistar rats were divided into 3 groups: the CIA rats group A (n=10), CIA rats group B (n=10) and normal rats control group C (n=10). BM-MSCs of Wistar rats were isolated, cultured in vitro routinely and the fourth passages was taken for identification of specific surface antigens by flow cytometry, then the cells were labeled with 5-bromodeoxyuridine (5-BrdU) in vitro. The models of CIA rats were established. 5-BrdU labeled BM-MSCs (1.0×107 cells/kg) were imfused from through tail vein to CIA rats group A and control group C. During the first 4 weeks after BM-MSCs transplantation, changes of general condition and left hind paw swelling were examined. At the fourth week, immunohistochemical examination of 5 -BrdU and osteoprotegerin (OPG) were performed to investigate BM-MSCs aggregation around the knee joints. The contribution of BM -MSCs to repairing of joint damages was identified. Comparisons between groups were performed by t-test. Results After BM-MSCs transplantation, left hindpaw swelling of group A were relieved compared with group B (P＜0.05) and the mobility of the joints was significantly improved. At the fourth week, much more implanted cells (5-BrdU positive cells.) were detected in the damaged knee joints than those in normal knee joints. The average grey scale values on synovium of knee joints in the CIA group A (85±9) was significantly lower than that of the normal group C (110±6, P＜0.05). At the same time, OPG expression was increased in damaged knee joints. The average grey scale values on synovium of knee joints in CIA group A (54±4) was significantly lower than that of the CIA group B (77±6, P＜0.05). Conclusion The transplanted allogeneic bone marrow mesenchy-mal stem cells can migrate to sites of damaged tissue in arthritis. They can prevent tissue damage and repair the damaged joints tissue by increasing OPG expression. This study has provided some evidence for developing effective therapy for rheumatoid arthritis.

Objective To isolate and culture bone marrow mesenchymal stem cells (BMSCs) from patients with rheumatoid arthritis (RA) and examine their biological characteristics. Methods MSCs were isolated from bone marrow of RA patients and purified by density gradient centrifugation and cultured in vitro. The morphology, immunophenotype, and proliferative; property of BMSC and colony forming unit-fibroblast (CFU-F) were measured and analyzed. Results The culture expanded cells from RA patients presented a typical fibroblast-like morphology. Ceils were positive for SH2 (CD105), CD71, and CD44, but negative for CD45. Their proliferative capacity and CFU-F number were similar to those of BMSCs from healthy donors. Conclusion In respect to morphology, immuno-phenotype, proliferative property and colony forming unit-fibroblast (CFU-F), MSCs from bone marrow of RA patients are not different from those of MSCs isolated from bone marrow of normal donors. MSCs from the bone marrow of RA patients have the potential for clinical application.

Objective:To discuss the regulation of berberine and dioscin extracted from traditional Chinese medicine in the expression of protein moleculer related with glucose metabolism in trophoblast cells,such as IRS-1,P1-3K,Glut1,PPAR ?. Methods:To culture the chorion trophoblast cell in the early pregnancy,to induce cells to suffer glucose metabolism obstacle with the use of WortmaninnTake advantage of berberine and dioscin extracted from the Chinese traditional medicine with intervention,simultaneously set the troglitazone(T) and dimthyl(R) for the control group. Detect the gene expression with the use of RT-PCT,simutaniously detect the protein expression in molecular level. Examine the expression in the protein level with the technique of Western blot in combination with the technique of LSM for the expression location of protein moleculer related.Results:① With the induction of WT,the glucose metabolism inside the tropholast cells becomes abnormal,compared with normal(P

Patients with systemic lupus erythematosus (SLE)are susceptible to influenza virus infection due to autoimmune disorder,use of glucocorticoids and the immunosuppressive agents.Influenza virus infection affects the disease stability,and the mortality of influenza virus infection in SLE patients is 5 times higher than that in general people infected by influenza virus.Vaccination is the most effective measure to prevent influenza virus infection.There is lack of studies on the influenza immunization in SLE patients in China,so this paper reviews prevention effect and safety of inoculation,the immune response to vaccine as well as the factors influencing immune response in influenza vaccination for SLE patients.

BACKGROUND: Radix astragali has the effect of protecting cells from damage in ischemic reperfusion, whether pre-treatment with radix astragali can protect myocardial eells from apoptosis in ischemic reperfusion ? OBJECTIVE: To investigate the effect of pre-treatment with radix astragali on apoptosis and its relative genes in rats with ischemic myocardial reperfusion DESIGN: A randomized and controlled trial taking Wistar rats as experimental subjects.SETTING: The Basic Medical Department of Chengde Medical College and the Geriatric Department of the Affiliated Hospital.MATERIALS: The experiment was completed in the Imunnohistochemical Laboratory of Basic Medical Institute in Chengde Medical College from February to December in 2004. A total of 30 healthy male Wistar rats were selected, and at random classified as groups of radix astragali pre-treated (radix astragali), ischemic reperfusion and psuedo-operated (control), 10 rats for each group.METHODS: Radix astragali injection was given peritonealy for rats in radix astragali pre-treated group before operation, and the equivalent normai saline was given for those in ischemic reperfusion and psuedo-operated groups. One week later, the model of ischemic reperfusion was set up. After operation the myocardia in marginal zone of ischemic reperfusion were sampled, and the myocardia of the corresponding zone were taken for control group. The method of terminal (TdT)-mediated dUTP-biotin nick end labeling (TUNEL) was used for assay of myocardial apoptosis rate, and the ABC immunohistochemical method was used for assay of myocardial bcl-2 (inhibiting apoptosis gene) and bax (promoting apoptosis gene).MAIN OUTCOME MEASURES: Apoptosis rates, and expression of bcl2 and bax genes of myocardia RESULTS: ① Apoptosis rate of myocardial cells: The rate in radix astragali group was decreased compared with that in ischemic reperfusion group [ (14.06 ±9.97) ％, (19.34±12.30) ％, t = 1.863, P ＜ 0.05].② Expression of bcl-2: There was no significant difference between radix astragali and ischemic reperfusion groups[(9.14±4.46) ％, (8.99±4.54) ％, P ＜ 0.05].③ Expression of bax: The expression in radix astragali group was decreased compared with that in ischemic reperfusion group [(12.65 ±7.23)％,(18.12±7.92) ％, t = 2.096, P ＜ 0.05]CONCLUSION: Pre-treatment with radix astragali can down-regulate the expression of promoting apoptosis gene so as to reduce the rate of myocardial cell apoptosis, hence it can protect the myocardial cells in ischemic reperfusion.

Objective To study the effect of allo-human bone marrow mesenchymal stem cells (BMSCs) on T and B cells from patients with Rheumatoid arthritis (RA) in vitro. Methods BMSCs were isolated from bone marrow samples of healthy volunteers and purified by density gradient centrifugation and cultured in vitro. Peripheral lymphocytes were isolated from patients with RA.Then, BMSCs and lymphpcutes were co-cultured. The modulatory effect of BMSCs on proliferation, activation and maturation of T and B lymphocytes of RA patients stimulated by PHA and SAC respectively was observed. The cell generation cycle and the degree of apoptosis were assessed by flow cytometry with PI/ Annexin V. After co-cultured with or without BMSCs for 72 hours, T cells were harvested, then they were labeled with anti-CD3, anti-CD4, anti-CD8, anti-CD25 antibodies and analyzed by flow cytometry. The density of IgG in the co-culture system was detected by ELISA. Results T and B cells proliferation was significantly suppressed when co-cuhured with bMSCs but did not induce T cell apoptosis. There was a significant decrease in the ratio of CD4+ CD3+ T cells in the co-cuhure group (34±6), as compared with the control group (44±7) (P<0.05). There was a decrease in CD25+ T cells and increase of CD4+ CD25+ cells in BMSCs co-cultured group (P<0.05). IgG was in creased in the cocuhure system. Conclusion Human BMSCs inhibit T and B cell activation and proliferation in patients with RA in vitro. And these immunomodulatory effects are not MHC restricted. The results of this study have provided evidence for the fact that BMSCs has the potential to be an effective treatment for RA.

BACKGROUND:Transient receptor potential channel C6 (TRPC6) is a new and important slit diaphragm-associated protein in podocytes involved in regulating glomerular filter function. Glomerular TRPC6 expression is closely associated with proteinuria in diabetic kidney disease. OBJECTIVE: To investigate the expression of canonical TRPC6 in mouse podocytes induced by high glucose, and to explore the possible mechanism of diabetic kidney disease. METHODS:Mouse podocyte cels were cultured and divided into normal glucose group (5.6 mmol/L D-glucose), normal control group (5.6 mmol/L D-glucose+25 mmol/L mannitol) and experimental groups which were in the environment of high glucose (30 mmol/L). The experimental groups included high glucose group, valsartan treatment groups (10-5 mol/L) and U73122 control group (10μmol/L U73122). After 48 hours, the expressions of mRNA and proteins of TRPC6, nephrin and angiotensin II (AngII) were detected respectively by real-time quantitative PCR and western blot analysis. RESULTS AND CONCLUSION:Compared with the normal control group, the expressions of mRNA and proteins of TRPC6 and angiotensin II were markedly elevated in the high glucose group (P < 0.01), while the expressions of mRNA and proteins of nephrin were decreased (P < 0.01). The mRNA and proteins of TRPC6 and angiotensin II expressions were significantly down-regulated by valsartan (P < 0.05,P < 0.01), while the mRNA and protein expressions of nephrin were effectively up-regulated (P < 0.05). Compared with the high glucose group, the expressions of mRNA and proteins of TRPC6 and angiotensin II were ameliorated in the U73122 control group. The expressions of mRNA and proteins of TRPC6, nephrin and angiotensin II had no statistical significance between the normal control group and normal glucose group (P > 0.05). Angiotensin II-TRPC6 signaling pathway may mediate high glucose-induced podocyte injury, meanwhile it provides a new theoretical basis for the treatment of diabetic kidney disease, by which the angiotensin receptor blockers can protect podocytes in diabetic kidney disease.

Objective To observe homing of mesenchymal stem cells (MSCs) in immune organs and inflammatory joints in collagen induced arthritis(CIA) rats. MethodsRats MSCs were isolated and expanded from bone marrow cells by density gradient centrifugation and adhering to the culture cell walls, and the phenotypes were assessed by flow cytometry. MSCs were labeled by PKH-26 and Brdu. Sixty-four rats were randomly divided into normal group and CIA group. Every 8 rats were sacrificed at 3, 11, 30, 42 days after transplantation of MSCs. At the end of the experiment, the specimens of thymus gland, spleen, ankle joints were exposed, fixed, decalcified, wrapped and cut into slices. Confocal laser scanning microscope and immunohistochemical method were used to observe migration and distribution of MSCs in different organs. Independent samples group t test with SPSS 12.0 software package was used for statistical analysis. ResultsIt was found that allogenic MSCs could stay in spleen, thymus gland and joints of CIA rats for a relatively long period (42days). Forty-two days after transplantation of MSCs, the average grey scale values of spleen and thymus gland in CIA group(37.5±8.8, 29.9±5.9 respectively) were significantly higher than the normal group(16.0±2.3,13.2±4.3 respectively), the average grey scale values of ankle joints in CIA group 78±8 was significantly lower than the normal group 93±14(P<0.05). ConclusionIt has been found that MSCs can stay in the injured tissue and organs preferentially.