| Summary: | 碩士 === 國立嘉義大學 === 生物農業科技學系研究所 === 106 === Liver fibrosis, mostly comes from poisoning or virus infection, is that liver repeated inflammation and generates a lot of extracellular matrix (ECM), causes liver fibrosis, hepatocyte death and liver function loss. If the disease continues to worsen, it will become liver cancer or liver cirrhosis, finally cause cell death. Recently, stem cell therapy has the great potential in disease treatment. Stem cells are undifferentiated cell, it can through self-differentiation or paralysis to ameliorate disease like osteoporosis or leukemia. In the experiments, we used human liver cancer cell HepG2 to treated with thioacetamide (TAA), simulated and induced hepatocyte damage condition like liver fibrosis in vitro. On the other hand, we used bone marrow mesenchymal stem cells (BMSCs) to carry on repair experiment by co-culture method, and investigated the feasibility of BMSCs repairment of damaged HepG2. In the experiments, BMSCs were extracted from FVB TG mouse and co-cultured with HepG2 by trans-well system. After treating 0.02 g/mL TAA to HepG2 cell with four different times, 30, 60, 90 and 120 min, DNA breakage of HepG2 didn`t show by TUNEL staining result; but HepG2 cell membrane appeared fragile by trypan blue staining result; reactive oxygen species content ascended by DCFDA staining result; cell activity reduced by WST-8 measurement. Liver fibrosis related genes analyzed by qPCR, the results showed inflammation gene Tnf expression raised, functional gene Alb declined and ECM related gene Col lαl, Mmp9 decreased, but no effect to Timp1 in 0.02 g/mL TAA treatment after 60 min and 24 hours incubation. Anti-oxidant gene Sod1, Gpx1 and Cat expression were inhibited in 3 hours after 60 min TAA treatment, but raised in 6 and 24 hours incubation. Anti-apoptotic gene Bcl2 expression was raised in 3 and 6 hours incubation. Besides, TAA caused STAT3 protein and its phosphorylation decreased. After co-culture with BMSCs, HepG2 cell activity was improved. Tnf gene expression induced by TAA was decreased and Collαl, Mmp9 and Alb genes expression was improved. Sod1, Gpx1 and Cat gene of HepG2 expression was higher than without co-culture group in 3 hours incubation after TAA treatment, Bcl2 gene expression was higher than without co-culture group in 3 and 6 hours incubation. Besides, the transduction and transcription factor STAT3 protein and its phosphorylation expression of HepG2 was improved under co-cultured with BMSCs. In conclusion, TAA caused HepG2 cell activity decreased, generated ROS damage, induced antioxidant genes expression. Besides, unlike liver fibrosis, TAA did not induced HepG2 generate ECM related genes Timp1, Mmp9 and Collαl expression, on the contrary, inhibit the genes expression. From co-culture experiments, BMSCs helped HepG2 promoted cell activity, restored or upgraded gene expression and increased antioxidant gene expression by paralysis of BMSCs. Co-culture system of BMSCs with TAA treated HepG2 promoted and activated STAT3 protein expression, promoted transcription of anti-apoptotic gene Bcl2.
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