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目的:研究化浊解毒方含药血清对体外培养的大鼠肝星状细胞增殖、凋亡及细胞外基质和PPARγmRNA表达的影响,以探讨化浊解毒方治疗肝纤维化的机制。方法:选择健康SD大鼠,每天灌服不同剂量(每L分别含有生药30,60,120 g)的化浊解毒方以及α干扰素(1 000 U.mL-1),按1 mL.(100 g)-1大鼠体质量给药,以制备含药血清;常规培养活化的肝星状细胞(HSC-T6),用不同浓度的含药血清进行干预,用MTT法检测HSC增殖、流式细胞术测HSC凋亡,ELISA法及RT-PCR法分别测细胞上清液中I型及Ⅲ型胶原的含量以及细胞中PPARγmRNA基因的表达。结果:与正常血清组比较,化浊解毒方药含药血清各浓度组均呈现出抑制HSC增殖的作用(P<0.05),以作用48 h最为明显,且呈剂量和时间依赖性。与正常血清对照组比较,化浊解毒方药含药血清各个浓度组在作用48 h后,细胞上清液中的I型及Ⅲ型胶原的含量均降低(P<0.05),并上调PPARγmRNA的表达(P<0.05)。结论:化浊解毒方药能有效治疗肝纤维化可能通过上调PPARγ的表达起到抑制肝星状细胞增殖,促进肝星状细胞凋亡,降低肝星状细胞活性有关。
Objective: To study the effects of Huazhuo Jiedu Decoction-containing serum on the proliferation, apoptosis and the expression of extracellular matrix and PPARγmRNA in rat hepatic stellate cells cultured in vitro to explore the mechanism of Huazhuo Jiedu Decoction in treating hepatic fibrosis. Methods: Healthy SD rats were selected and fed with Huazhuo Jiedu Decoction and IFN-α (1 000 U.mL-1) at different doses (30, 60 and 120 g / L respectively) ) -1 was used to prepare rat serum containing HSC-T6. HSC-T6 cells were cultured routinely. HSC-T6 cells were cultured with different concentrations of serum containing HSC. The proliferation of HSCs was detected by MTT assay. Flow cytometry The apoptosis of HSC was measured by enzyme-linked immunosorbent assay (ELISA) and RT-PCR. The contents of type I and type III collagen and the expression of PPARγmRNA in cells were detected by ELISA and RT-PCR respectively. Results: Compared with the normal serum group, the concentrations of Huazhuo Jiedu Decoction serum showed inhibitory effects on the proliferation of HSC (P <0.05), and the effect was most obvious at 48 hours, in a dose-and time-dependent manner. Compared with the normal serum control group, the content of type I and type III collagen in the cell supernatant decreased (P <0.05) and the expression of PPARγmRNA increased (P <0.05). Conclusion: Huazhuo Jiedu decoction can effectively treat liver fibrosis by up-regulating the expression of PPARγ, which can inhibit the proliferation of hepatic stellate cells, promote the apoptosis of hepatic stellate cells and reduce the activity of hepatic stellate cells.