RHL抑制大鼠肝纤维化及LX2细胞增殖的研究

发布时间：2018-01-09 10:20

本文关键词：RHL抑制大鼠肝纤维化及LX2细胞增殖的研究　出处：《华北理工大学》2015年硕士论文　论文类型：学位论文

【摘要】：目的探讨赖氨大黄酸(RHL)对大鼠胆汁淤积性肝纤维化的抑制作用,进一步阐明其作用的可能分子机制;在体内实验基础上体外培养人肝星形细胞(HSC)株,探讨RHL抑制HSC增殖的可能作用机制;为RHL防治胆汁淤积性肝纤维化的研究提供实验依据。方法1体内试验:(1)随机将42只大鼠分为假手术组、模型组、35 mg·kg-1、70 mg·kg-1RHL治疗组、大黄酸组及赖氨酸组,每组7只,采用胆总管结扎(BDL)手术建立胆汁淤积性肝纤维化大鼠模型。(2)使用全自动生化分析仪检测各组大鼠血清中天冬氨酸氨基转移酶(AST)、丙氨酸氨基转移酶(ALT)、总胆汁酸(TBA)、总胆红素(TBIL)水平。(3)HE染色对各组大鼠肝脏组织行病理学检查;Masson三色染色观察各组大鼠肝组织纤维组织含量与分布;使用试剂盒通过酶标仪测定各组大鼠肝组织中羟脯氨酸(Hyp)水平;免疫组织化学染色检测各组大鼠α平滑肌肌动蛋白(α-SMA)的表达部位。(4)RT-PCR检测各组大鼠肝组织中转化生长因子β1(TGF-β1)和α-SMA m RNA表达水平;Western blotting检测各组大鼠肝组织中TGF-β1和α-SMA蛋白相对表达量。2体外实验:(1)体外培养HSC-LX2细胞,实验分成对照组、50μmol·l-1、100μmol·l-1和150μmol·l-1 RHL用药组。(2)采用CCK-8法检测不同浓度的RHL对HSC-LX2细胞活化和增殖的抑制作用。(3)Western blotting检测用不同浓度的RHL处理48小时后HSC-LX2细胞中TGF-β1和α-SMA蛋白相对表达量。结果1体内试验:(1)与假手术组比较,模型组大鼠肝脏质量与体质量比、血清中AST、ALT、TBA和TBIL水平升高,差异有统计学意义(P0.05);肝组织肝小叶结构被破坏,胆管显著增生并伴纤维大量增生;Hyp水平升高,差异有统计学意义(P0.05),免疫组织化学染色显示α-SMA着色于胞膜和胞浆且表达增多;RT-PCR和Western blotting结果显示模型组大鼠TGF-β1、α-SMA m RNA和蛋白表达水平升高,差异有统计学意义(P0.05)。(2)与模型组比较,35、70mg·kg-1RHL治疗组和大黄酸组,大鼠肝脏质量与体质量比、血清中AST、ALT、TBA和TBIL水平降低,差异有统计学意义(P0.05);肝脏病理组织学检查提示35、70 mg·kg-1RHL治疗组和大黄酸组大鼠肝小叶结构尚完整,胆管增生,纤维组织沉积减少;Hyp水平减低,差异有统计学意义(P0.05);RT-PCR和Western blotting结果显示35、70 mg·kg-1RHL治疗组和大黄酸组大鼠TGF-β1、α-SMA m RNA和蛋白表达水平降低,差异有统计学意义(P0.05)。(3)与大黄酸组比较,35、70 mg·kg-1RHL治疗组大鼠肝脏质量与体质量比、血清中AST、ALT、TBA和TBIL水平降低,差异有统计学意义(P0.05);肝脏病理组织学检查提示35、70 mg·kg-1RHL治疗组大鼠肝组织中胆管增生减少,纤维沉积减少;Hyp水平减低,差异有统计学意义(P0.05);RT-PCR和Western blotting结果显示35、70 mg·kg-1RHL治疗组大鼠TGF-β1、α-SMA m RNA和蛋白表达水平降低,差异有统计学意义(P0.05)。2体外实验:(1)CCK-8结果显示,与细胞对照组比较,50μmol·l-1、100μmol·l-1和150μmol·l-1RHL用药组HSC-LX2细胞的增殖明显被抑制,差异具有统计学意义(P0.05)。(2)Western blotting结果显示,与对照组比较,50μmol·l-1、100μmol·l-1、150μmol·l-1 RHL用药组处理HSCLX2细胞48小时后细胞中TGF-β1和α-SMA蛋白表达水平降低,差异有统计学意义(P0.05)。结论1 RHL可减轻胆汁淤积性肝纤维化模型大鼠的肝脏损伤,抑制模型大鼠的肝纤维化进程。2 RHL能下调大鼠细胞因子TGF-β1和α-SMA的表达,这种通过下调TGF-β1抑制肝星形细胞增殖的效应可能是RHL抗肝纤维化的机制。3 RHL能抑制LX2细胞的活化和增殖,其抑制作用可能通过下调TGF-β1表达实现。
[Abstract]:Objective to investigate the Rhein lysinate (RHL) inhibitory effect on cholestasis induced liver fibrosis in rats, may further clarify the molecular mechanism of its function; cultured human hepatic stellate cells in vitro in vivo experiments based on (HSC) strains, investigate the possible mechanism of RHL inhibiting the proliferation of HSC; to provide the experimental basis for the prevention and treatment of RHL in bile cholestatic liver fibrosis. Methods 1 in vivo experiment: (1) a total of 42 rats were divided into sham operation group, model group, 35 mg - kg-1,70 Mg - kg-1RHL treatment group, Rhein group and lysine group, 7 rats in each group, the common bile duct ligation (BDL) operation to establish a model of rat cholestatic liver fibrosis. (2) the use of aspartate aminotransferase automatic biochemical analyzer to detect the serum of rats (AST), alanine aminotransferase (ALT), total bile acid (TBA), total bilirubin (TBIL) level. (3) HE staining of pathological liver tissue of rats for learning Check; rats were observed in liver tissue and fibrous tissue content and distribution of Masson trichrome staining; hydroxyproline kit by eliasa detected in liver of rats (Hyp); immunohistochemical staining for alpha smooth muscle actin were detected (alpha -SMA). The expression sites (4) were detected in rat liver RT-PCR transforming growth factor beta 1 (TGF- beta 1) and alpha -SMA m RNA expression; relative expression of.2 in vitro were detected in rat liver tissue Western blotting TGF- beta 1 and alpha -SMA protein: (1) HSC-LX2 cells cultured in vitro were divided into control group, 50 mol - l-1100 mol L-1 and 150 mol L-1 RHL treatment group (2). CCK-8 method was used to detect the inhibitory effect of different concentration of RHL on HSC-LX2 cell activation and proliferation. (3) Western blotting detection with different concentrations of RHL after 48 hours treatment of HSC-LX2 cells in TGF- beta 1 and alpha -SMA protein. The level of expression. Results 1 in vivo experiment: (1) compared with the sham operation group, model group and body mass ratio, serum AST, ALT, TBA and TBIL levels increased, the difference was statistically significant (P0.05); the hepatic lobule structure was destroyed, and a large number of fibers with significant hyperplasia of bile duct hyperplasia; elevated levels of Hyp, the difference was statistically significant (P0.05), immunohistochemical staining showed that alpha -SMA stained in the cytoplasm and membrane and increase the expression of RT-PCR and Western; blotting results showed that TGF- beta 1 rats in the model group, the expression of a -SMA m RNA and protein levels increased, the difference was statistically significant (P0.05). (2) compared with the model group, 35,70mg kg-1RHL treatment group and rhein group, rat liver and body mass ratio, serum AST, ALT, TBA and TBIL levels decreased, the difference was statistically significant (P0.05); liver histopathological examination suggested that 35,70 mg kg-1RHL treatment group and rhein group Rat hepatic lobule structure is complete, bile duct hyperplasia, fibrous tissue deposition decreased; Hyp levels decreased, the difference was statistically significant (P0.05); RT-PCR and Western blotting showed that 35,70 mg kg-1RHL treatment group and rhein group rats TGF- expression of alpha -SMA beta 1, m RNA and protein level decreased, with statistical significance the difference (P0.05). (3) compared with the Rhein group, 35,70 mg kg-1RHL treatment group rat liver and body mass ratio, serum AST, ALT, TBA and TBIL levels decreased, the difference was statistically significant (P0.05); liver histopathological examination showed 35,70 mg kg-1RHL treatment group of rat liver tissue in the bile duct proliferation decreased, fiber deposition decreased; Hyp levels decreased, the difference was statistically significant (P0.05); RT-PCR and Western blotting showed that 35,70 mg kg-1RHL treatment group rats TGF- expression of alpha -SMA beta 1, m RNA and protein levels decreased, the difference was statistically significant (P0.05) .2 in vitro: (1) the results of CCK-8 showed that compared with the control group cells, 50 mol - l-1100 mol - L-1 and 150 mol l-1RHL treatment group the proliferation of HSC-LX2 cells was inhibited significantly, the difference was statistically significant (P0.05). (2) Western blotting results showed that compared with the control group, 50 mol - l-1100 mol - l-1150 mol - L-1 RHL group HSCLX2 cells 48 hours after cells in TGF- beta 1 and alpha protein expression level of -SMA decreased, the difference was statistically significant (P0.05). Conclusion RHL can relieve liver injury 1 cholestatic liver fibrosis in a rat model of hepatic fibrosis,.2 RHL inhibition of rat model can down regulate the expression of cytokines in rats with TGF- beta 1 and alpha -SMA, the effect of down-regulation of TGF- beta 1 inhibits the proliferation of hepatic stellate cells may be RHL anti hepatic fibrosis mechanism of.3 RHL can inhibit the activation and proliferation of LX2 cells, the inhibition produced by through down-regulation of The expression of TGF- beta 1 was realized.

【学位授予单位】：华北理工大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R575.2

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