本文關鍵詞：RHL抑制大鼠肝纖維化及LX2細胞增殖的研究　出處：《華北理工大學》2015年碩士論文　論文類型：學位論文

  更多相關文章： 賴氨大黃酸 膽汁淤積 肝纖維化 肝星形細胞 TGF-β1 α-SMA

【摘要】：目的探討賴氨大黃酸(RHL)對大鼠膽汁淤積性肝纖維化的抑制作用,進一步闡明其作用的可能分子機制;在體內(nèi)實驗基礎上體外培養(yǎng)人肝星形細胞(HSC)株,探討RHL抑制HSC增殖的可能作用機制;為RHL防治膽汁淤積性肝纖維化的研究提供實驗依據(jù)。方法1體內(nèi)試驗:(1)隨機將42只大鼠分為假手術組、模型組、35 mg·kg-1、70 mg·kg-1RHL治療組、大黃酸組及賴氨酸組,每組7只,采用膽總管結扎(BDL)手術建立膽汁淤積性肝纖維化大鼠模型。(2)使用全自動生化分析儀檢測各組大鼠血清中天冬氨酸氨基轉移酶(AST)、丙氨酸氨基轉移酶(ALT)、總膽汁酸(TBA)、總膽紅素(TBIL)水平。(3)HE染色對各組大鼠肝臟組織行病理學檢查;Masson三色染色觀察各組大鼠肝組織纖維組織含量與分布;使用試劑盒通過酶標儀測定各組大鼠肝組織中羥脯氨酸(Hyp)水平;免疫組織化學染色檢測各組大鼠α平滑肌肌動蛋白(α-SMA)的表達部位。(4)RT-PCR檢測各組大鼠肝組織中轉化生長因子β1(TGF-β1)和α-SMA m RNA表達水平;Western blotting檢測各組大鼠肝組織中TGF-β1和α-SMA蛋白相對表達量。2體外實驗:(1)體外培養(yǎng)HSC-LX2細胞,實驗分成對照組、50μmol·l-1、100μmol·l-1和150μmol·l-1 RHL用藥組。(2)采用CCK-8法檢測不同濃度的RHL對HSC-LX2細胞活化和增殖的抑制作用。(3)Western blotting檢測用不同濃度的RHL處理48小時后HSC-LX2細胞中TGF-β1和α-SMA蛋白相對表達量。結果1體內(nèi)試驗:(1)與假手術組比較,模型組大鼠肝臟質(zhì)量與體質(zhì)量比、血清中AST、ALT、TBA和TBIL水平升高,差異有統(tǒng)計學意義(P0.05);肝組織肝小葉結構被破壞,膽管顯著增生并伴纖維大量增生;Hyp水平升高,差異有統(tǒng)計學意義(P0.05),免疫組織化學染色顯示α-SMA著色于胞膜和胞漿且表達增多;RT-PCR和Western blotting結果顯示模型組大鼠TGF-β1、α-SMA m RNA和蛋白表達水平升高,差異有統(tǒng)計學意義(P0.05)。(2)與模型組比較,35、70mg·kg-1RHL治療組和大黃酸組,大鼠肝臟質(zhì)量與體質(zhì)量比、血清中AST、ALT、TBA和TBIL水平降低,差異有統(tǒng)計學意義(P0.05);肝臟病理組織學檢查提示35、70 mg·kg-1RHL治療組和大黃酸組大鼠肝小葉結構尚完整,膽管增生,纖維組織沉積減少;Hyp水平減低,差異有統(tǒng)計學意義(P0.05);RT-PCR和Western blotting結果顯示35、70 mg·kg-1RHL治療組和大黃酸組大鼠TGF-β1、α-SMA m RNA和蛋白表達水平降低,差異有統(tǒng)計學意義(P0.05)。(3)與大黃酸組比較,35、70 mg·kg-1RHL治療組大鼠肝臟質(zhì)量與體質(zhì)量比、血清中AST、ALT、TBA和TBIL水平降低,差異有統(tǒng)計學意義(P0.05);肝臟病理組織學檢查提示35、70 mg·kg-1RHL治療組大鼠肝組織中膽管增生減少,纖維沉積減少;Hyp水平減低,差異有統(tǒng)計學意義(P0.05);RT-PCR和Western blotting結果顯示35、70 mg·kg-1RHL治療組大鼠TGF-β1、α-SMA m RNA和蛋白表達水平降低,差異有統(tǒng)計學意義(P0.05)。2體外實驗:(1)CCK-8結果顯示,與細胞對照組比較,50μmol·l-1、100μmol·l-1和150μmol·l-1RHL用藥組HSC-LX2細胞的增殖明顯被抑制,差異具有統(tǒng)計學意義(P0.05)。(2)Western blotting結果顯示,與對照組比較,50μmol·l-1、100μmol·l-1、150μmol·l-1 RHL用藥組處理HSCLX2細胞48小時后細胞中TGF-β1和α-SMA蛋白表達水平降低,差異有統(tǒng)計學意義(P0.05)。結論1 RHL可減輕膽汁淤積性肝纖維化模型大鼠的肝臟損傷,抑制模型大鼠的肝纖維化進程。2 RHL能下調(diào)大鼠細胞因子TGF-β1和α-SMA的表達,這種通過下調(diào)TGF-β1抑制肝星形細胞增殖的效應可能是RHL抗肝纖維化的機制。3 RHL能抑制LX2細胞的活化和增殖,其抑制作用可能通過下調(diào)TGF-β1表達實現(xiàn)。
[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.

【學位授予單位】：華北理工大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：R575.2

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