Septin2对大鼠肾小球系膜细胞增殖的影响

【作者】 李晓帆；

【导师】 李荣山； 陈香美；

【作者基本信息】 山西医科大学 ， 内科学， 2010， 硕士

【摘要】 目的：探讨Septin2基因表达变化对大鼠肾小球系膜细胞增殖的影响。方法：培养原代大鼠肾小球系膜细胞,进行以下实验：1)将绿色荧光蛋白表达质粒pEGFP-C1用电穿孔转染的方法导入细胞,24h后荧光显微镜下观察绿色荧光蛋白表达情况。2)质粒电穿孔转染：细胞分组：①pRK5空质粒转染组,②pRK5-Septin2质粒转染组,分别进行电穿孔转染,Western blot检测两组细胞Septin2蛋白质表达水平,流式细胞仪分析Septin2对细胞周期进程的影响,Western blot检测系膜细胞周期调节蛋白(cyclin D1、cyclinE、p21)的表达变化。3)设计合成GAPDH的siRNA,依不同浓度分为①200nmol／L,②400nmol／L,③800nmol／L组,④无关对照转染组(siCon),电穿孔转染后培养48h,Western blot检测GAPDH蛋白质表达变化。4)siRNA电穿孔转染：细胞分组：①无关对照转染组(Sicon);②siSeptin2转染组。收集细胞蛋白,Western blot检测两组细胞Septin2蛋白质表达水平,流式细胞仪分析siSeptin2对细胞周期进程的影响。结果：1)在80μg质粒,340V电压、550μF电容的电穿孔条件下,原代大鼠肾小球系膜细胞的电转染效率可以达到50%以上；2)与pRK5空质粒转染组相比,pRK5-Septin2质粒可使系膜细胞Septin2的蛋白质表达水平明显提高(P<0.05),流式细胞仪检测发现pRK5-Septin2质粒使G0／G1期细胞百分比明显增多而S期细胞百分比显著减少(P<0.05),同时细胞周期调节蛋白cyclin D1、cyclinE表达水平下调,p21的表达水平上调(P<0.05)；3) siGAPDH浓度为800nmol／L时蛋白质水平抑制效果最为显著(P<0.05)；4)与无关对照转染组(siCon)比较,siSeptin2转染组Septin2蛋白质表达水平明显降低,流式细胞仪检测结果显示两组细胞G0／G1期及S期细胞百分比无明显差异。结论：Septin2过表达可使大鼠肾小球系膜细胞由G0／G1期向S期转化的百分比减少,细胞周期正调控蛋白表达减少而负调控蛋白表达增多,提示可能通过调节细胞周期蛋白的表达来抑制系膜细胞增殖,影响细胞周期,有望为系膜增生性肾炎提供新的治疗靶点。更多还原

【Abstract】 Objective:To research the in vitro effects of Septin2 on Mesangial Proliferation in Rats.Methods:Cultured primary rat mesangial cells, processing the following experiment:1)The plasmids pEGFP-C 1 which could express the green fluorescent protein (GFP)were transfected into the cells. The transfection efficiency were determined by observing under fluorescent microscope 24 hours subsequently.2)Transfection of Septin2 plasmids by electroporation: Groups dividing:①pRK5 transfection group;②pRK5-Septin2 group. The plasmids pRK5 and pRK5-Setin2 were transfected into the mesangial cell under the optimal transfection conditions. The expression of Septin2 protein were examined by Western blot, cell cycle profiles were determined by flow cytometric analysis, the expressions of cell cycle regulatory proteins (cyclin D1、cyclin E、p21) were examined by Western blot.3) Design and synthesis the siRNA of GAPDH, according to different concentrations cells were divided into 4 groups:①siCon group,②200 nmol/L group,③400 nmol/L,④800 nmol/L group.48h later, Western blot to detect the expression of GAPDH protein.4) Electransfection of siSeptin2:Groups dividing:①siCon group,②siSeptin2 group. Western-blot detects the expression of Septin2 protein, flow cytometric analysis checks the cell cycle profiles.Results:1)When plasmids is 80μg, voltage is 340V, capacitor is 550μF, the electroporation transfection efficiency in primary rat mesangial cells can reach more than 50%.2)Compared to pRK5 transfection group, plasmids pRK5-Septin2 increased protein expression level of Septin2(P<0.05). Cell cycle analysis showed that the percentage of the cells in S stage were markedly reduced in pRK5-Septin2 group (P<0.05), the percentage of the cells in G0/G1 stage were markedly increased (P<0.05). The expression of cell cycle regulatory protein cyclin D1, cyclinE decreased and P21 reduced(P<0.05).3)When the concentration of siGAPDH was 800nmol/L, the inhibitory effect of protein level was the most significant (P<0.05).4) The synthesized siRNA of Septin2 gene inhibited the Septin2 protein expression levels (P<0.05), flow cytometry analysis didn’t show any difference of significance.Conclusion:Overexpression of Septin2 is able to arrest cell cycle progression in the G0/G1 phase,the expression of positive regulators driving the G0/G1 phase, for example cyclin D1,cyclin E, were downregulated in cells transfected with Septin2, whereas expression of negative regulators p21 were upregulated. This is suggested that Septin2 may inhibite proliferation of mesangial cell by regulating the expression of cell cycle proteins. This may have provided a new target of mesangial proliferative glomerulonephritis therapy.更多还原