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肝再生增强因子对大鼠肾小管上皮细胞转分化影响的研究
【作者】 邓莉莉；
【导师】 张玲；
【作者基本信息】 重庆医科大学 ， 内科学， 2008， 硕士
【摘要】 目的:利用已构建的大鼠肝再生增强因子(rALR)毕赤酵母(pichia pastoris)重组表达质粒pPIC9K-rALR,在酵母菌GS115中诱导表达rALR,并进行纯化和体外生物学活性鉴定,为进一步研究rALR的生物学功能提供基础。方法:经PCR鉴定的重组质粒pPIC9K-rALR酶切线性化后转入宿主菌GS115中,在甲醇诱导下进行表达。表达产物rALR经超滤方法纯化后,用15% SDS-PAGE和Western blot印迹鉴定。采用3H-TdR掺入法,观察rALR在体外对人肝癌细胞株QGY的促增殖作用以鉴定其活性。结果:目的蛋白rALR以外分泌方式在宿主菌GS115中获得高效表达,其分子量约为15KD,与预计理论值相符合,超滤纯化得到的目的蛋白经SDS-PAGE和Western bolt鉴定为单一条带,在体外rALR确能刺激QGY增殖,而且这种刺激作用存在着量效关系。结论:rALR在毕赤酵母菌中被高效表达和成功纯化,在体外rALR确能以剂量依赖关系刺激人肝癌细胞株QGY增殖。目的:探讨重组大鼠肝再生因子(rrALR)对转化生长因子β1(TGF-β1)诱导的肾小管上皮细胞转分化的影响。方法:观察细胞形态学变化,检测α平滑肌肌动蛋白(α-SMA)、E-钙粘蛋白(E-cadherin)和结缔组织生长因子(CTGF)蛋白及mRNA的表达水平。结果:NRK-52E细胞在加入TGF-β1刺激培养后,细胞形态由方形或多角形变为梭形成纤维细胞;α-SMA和CTGF的mRNA及蛋白表达增加,E-cadherin mRNA及蛋白表达明显降低。而TGF-β1和rrALR共培养组,多数细胞保持正常上皮细胞形态,NRK52E细胞转分化程度减轻,α-SMA和CTGF蛋白及mRNA表达增高程度高于正常对照组,但明显低于TGF-β1刺激组(P<O.05),而E-cadherin mRNA及蛋白表达低于正常对照组,明显高于TGF-β1刺激组(P<O.05)。rrALR组未见上述改变(P>O.05)。结论:rrALR能抑制TGF-β1诱导的NRK52E2细胞转分化,减少细胞CTGF表达。
【Abstract】 Objective: To express rALR in GS115 with constructed pichia pastoris expression plasmid of rat augmenter of liver regeneration (rALR) and purify it by ultrafiltration and identify its bioactivity in vitro, for studying its biological functions.Methods: The recombinant plasmid pPIC9K-rALR was transformed into GS115 by electroporation. The rALR was expressed in GS115 under the induction of methanol and purified with ultrafiltration. The purified product was identified by 15% SDS-PAGE and Western blot. The effects of rALR on the proliferations of QGY were evaluated by 3H-TdR methods in vitro.Results: The rALR was efficiently expressed as a secretive protein in GS115. Its molecular weight(1.5×104 dalton) was in correspondance with theoretic values. Single band of rALR was observed in SDS-PAGE electrophoresis and in western blot after ultrafiltration. The rALR promoted the proliferation of QGY by a dose-dependent way in vitro. Conclusion: The rALR was efficiently expressed in GS115 and successfully purified by ultrafiltration. The proliferation of QGY was stimulated by rALR in vitro with a dose-dependent way.Objective: To investigate the effect of recombination rat augmenter of liver regeneration(rrALR) on TGF-β1-induced renal tubular epithelial cells transdifferentiation(TEMT). The cells were cultured for 48 hours. Morphological change of cellsMethods: The cells of normal rat kidney tubular epithelial cell line(NRK-52E) were divided into four groups, normal control cells, rrALR treated cells, TGF-β1 treated cells, TGF-β1 plus rrALR treated cells. was observed and expressions ofα-SMA, CTGF, E-cadherin protein and mRNA were analysed. Results: The results showed that TGF-β1 induced cell morphological change of NRK-52E as the development of an elongated and fusiforill appearance, and up-regulated protein and mRNA expressions ofα-SMA, CTGF and down-regulated E-cadherin expression. Transformation of tubular epithelial cells was inhibited in TGF-β1 plus rrALR group as compared with TGF-β1 treated group. The protein and mRNA expression of E-cadherin was increased and levels ofα-SMA and CTGF were decreased in cells exposed TGF-β1 plus rrALR medium.Conclusion: 1.There was no transdifferentiating effect when NRK52E cells cultured with rALR alone. 2. rrALR may inhibit transdifferentiation of NRK52E cells into myofibroblast-like cells and CTGF protein and mRNA expression induced by TGF-β1.