BACKGROUND: Adipose tissue-derived mesenchymal stem cells (ADMSCs) have the multilineage differentiation potential, and are relatively easier to be obtained, thus they have attracted more and more attention as a new seed cell for cell engineering.OBJECTIVE: To observe the in vitro culture conditions of ADMSCs isolated from rat's subcutaneous adipose tissue, and identify them using immunohistochemical staining.DESIGN: An animal experiment.SETTING; Department of Cardiology, the General Hospital of Chinese PLA.MATERIALS: One healthy male Wistar rat of clean degree, 4 months old, weighing 200 g, was used. DMEM, fetal bovine serum were from GIBCO; Monoclone antibodies of rabbit-anti-rat CD13, CD34, CD44, CD45, CD105, D-related human leucocyte antigen (HLA-DR), factor-Ⅷ, vov Willebrand factor (VWF), Myosin, SABC kits and DAB staining kit from Wuhan Booster Biological Engineering, Co.,Ltd; Adeno-associated virus encoding green fluorescent protein from Vector Gene Technology Company Limited (Beijing).METHODS: The experiments were carried out in the Department of Internal Medicine, the General Hospital of Chinese PLA in October 2006. ① Cell isolation and culture: 0.3 g adipose tissue was cut from subcutaneous adipose tissue of Wistar rat's groin under aseptic condition, then minced and digested before culture, DMEM was changed at 2-3 days after plenty of fusiform-shap ed attached cells were observed under microscope, and the cell growth was observed. The cell concentration was adjusted to 2×107 L-1, then seeded into 96-well plate, and 100 μL for each well. From the second day, 3 wells were randomly selected every day, the cells were released with tripsin, and counted with blood cell counting chamber under inverted microscope. ② Cell viability assay: ADMSCs of passages 3 to 8 were added to DMSO freeze medium, and thawed after 2-4 weeks, and the cell viability was assessed by trypan blue dye exclusion. ③Immunohistochemical staining and identification: 2 ×107 L -1 cells were seeded to culture plate, then the immunohistochemical (SABC method) identification and Oil red O staining were performed to determine the cell surface antibodies of CD13, CD34, CD44, CD45, CD105, HLA-DR, factor-Ⅷ, HLA-DR and VWF. ④Lineage-specific differentiation and identification: The ADMSCs were plated on multi-well chamber and induced with lineage-specific media supplementation at least two weeks and identified by histologic/immunohistochemical assay of Oil red O for adipogenisis, alkaline phosphatase (ALP) stain for osteogenisis and Myosin monoclonal antibody for myogenisis. ⑤Transfected adenovirus carried green fluorescence protein (AD-GFP) medium: The fourth generation of ADMSCs were seeded on 96-well plate, 3 000 cells for each well, serum-free DMEM was changed after 24 hours, and added by AD-GFP at the same time, then transfected with different multiplicity of infection (MOI) of 1∶50, 1∶100, 1∶150 and 1∶200respectively, and then the transfection was observed.MAIN OUTCOME MEASURES: ① Results of cell isolation and culture; ② Cell viability after freezing and thawing; ③Results of immunohistochemical staining and identification; ④ Results of lineage-specific differentiation and identification;⑤ Results of transfected adenovirus carried AD-GFP.RESULTS: ① About 3.6×105 attached cells were obtained from 0.3 g subcutaneous adipose tissue, and these cells could be subcultured for passages in vitro with stable population doubling time. ② The cells were thawed after freezing for 2-3 weeks, and the trypan blue staining showed that the cell viability was above 90%. ③ The immunocytochemical staining showed that CD13, CD44, CD105 were positive and CD45, factor-Ⅷ, HLA-DR and VWF negative in different generations. ④ From the second generation, a few Oil red O positively stained cells were observed, which were obviously increased after prolonging the refreshing. After lineage-specific differentiation, the cells were all positive by Oil red O staining, ALP staining and Myosin immunohistochemical staining. ⑤ 72 hours after transfection, it was observed under fluorescence microscope that most cells were green fluorescence when the MOI value was 1∶200, the transfection was successful, and it was generally determined that the transfection rate was above 90%.CONCLUSION: A large number of ADMSCs with multilineage differentiation potential can be easily obtained from rat adipose tissue, osteoblast, myoblasts, they can be expanded in large quantity and stored in vitro for long time, AD-GFP were also successfully transfected.

Objective: To investigate the different efficacy of Yinchenhao Decoction (YCHD), a compound traditional Chinese herbal medicine, for liver cirrhosis induced by dimethylnitrosamine (DMN) or carbon tetrachloride (CCl(4)) in rats. Methods: To induce liver fibrosis, 0.5% DMN solution (2mL/kg body weight, i.p.) was given three consecutive days a week to male Wistar rats for 4 weeks. Cirrhotic rats were randomly divided into DMN group, YCHD group, Xiaochaihu decoction group by the end of the fourth week to accomplish a 2-week recipe treatment course. In CCl(4)-induced liver fibrosis model, 50% CCl(4)-olive solution was injected subcutaneously to rats at a dose of 2 mL/kg body weight twice a week to duplicate rat cirrhosis model. After 8 weeks, rats were divided into CCl(4) group, CCl(4) plus YCHD group and Xiaochaihu decoction group. For the YCHD group, YCHD was administered intragastrically once a day for 4 weeks. For DMN or CCl(4) model, by the end of 6 or 12 weeks respectively, rats were sacrificed for sampling to detect liver function, hepatic histological changes, hydroxyproline (Hyp) content and apoptosis-related gene expressions. Results: In DMN liver fibrosis model, hepatic fibrosis was obvious at week 2 and cirrhosis was evident at week 4 in DMN-treated rats. Compared to 6-week DMN group, hepatic pathological changes and liver function were improved significantly and content of Hyp decreased remarkably in YCHD group. In CCl(4)-induced liver fibrosis model, hepatic fibrosis was obvious at 8 weeks and cirrhosis was evident at 12 weeks in CCl(4)-treated rats. Compared to 12-week CCl(4) group, hepatic pathological changes and liver function were not obviously improvement in YCHD group. The results of gene chip showed that YCHD significantly decreased Fas, Bax and caspase-3 gene expressions, and increased Bcl-xL gene expression in the liver of DMN model. However, in the model induced by CCl(4), YCHD did not inhibit hepatocyte apoptosis induced by CCl(4), but increased tyrosine kinase receptor gene expression by 4.8 times. Conclusion: YCHD exerts more significant therapeutic effects on DMN-induced than CCl(4)-induced cirrhosis in rats in Hyp content and pathological change in liver tissue.

To investigate the effects of three classical anti-jaundice formulas Yinchenhao Decoction (YCHD). Yinchen Wuling San (YCWLS) and Zhizi Baipi Decoction (ZZBPD) on liver fibrosis induced by dimethylnitrosamine (DMN) in rats and explore the formula-syndrome relationship.

Objective:To study the genotype distribution of drug-related gene polymorphism of methotrexate (MTX) and leflunomide (LEF) in patients with rheumatoid arthritis (RA).Methods:The genotyping results of RA patients' MTX and LEF related genes(MTHFR677C/T, MTHFR1298A/C, ABCB13435T/C, DHODH19C/A and CYP1A2734C/A) detected in Shanghai Guanghua Hospital from December 2018 to May2019 and drug-related adverse effect were statisticallyanalyzed. The independence of allele distribution was tested by Hardy-Weinberg test. Counting data of genotypes and allele frequencies among the groups were analyzed by the chi-square test. Measurement data were showed as Mean±SD deviation. The network between incidence of adverse events and genotypes of patients was analyzed by cytoscape software. Results:Genotype distribution in 151 patients was consistent with Hardy-Weinberg genetic balance ( P>0.05), and genotype and allele distribution of each gene showed no statistical difference in gender ( P>0.05). The results showed that the most common genotype in RA were that genotypes of the good response with moderate resistance to MTX (MTHFR677CC/MTHFR1298AA/ABCB13435CT) (16 cases, 13.5%) and the good response with moderate side effect risk to LEF(DHODH19CC/CYP1A2734AC) (25 cases, 28.4%). According to the distribution frequency of alleles, the incidence of high side effects caused by MTX combined with LEF was predicted to be 2.9%, which was close to 1.8% of the actual genotypes of patients. The types and proportion of clinical adverse reactions in patients were retrospectively analyzed and the correlation network analysis was conducted with the genotype analysis results. It was found that the incidence rates of adverse reactions were liver injury (35.4%, 35/99), leukopenia (14.1%, 14/99), thrombocytopenia (2.0%, 2/99), and skin rash (1.0%, 1/99) from the top to the bottom. The top two genotypes that were related to the occurence of adverse events were MTHFR677CT/MTHFR1298AA/ABCB13435CT and DHODH19CA/CYP1A2734AC, respectively, which verified the consistency between drug-related genotype and clinical manifestations in RA patients. Conclusion:Our results suggested that genotype in RA patients is closely related to drug efficacy and adverse events. 2.9% of RA patients need to stop taking MTX and LEF due to high MTX resistance and poor MTX response and increased toxicity when combined with LEF, in which the proportion of liver injury is the highest.