【作者】 张明；

【导师】 徐存拴；

【作者基本信息】 河南师范大学 ， 细胞生物学， 2010， 硕士

【摘要】 胞外蛋白酶抑制剂基因expi编码的蛋白与蛋白酶活性的有关,并参与细胞的迁移和凋亡活动。本实验室的大鼠再生肝Rat Genome 230 2.0芯片检测结果表明,expi在大鼠2/3肝切除后6h起始表达,6-72h持续表达上调,并在30h达到表达高峰,是对照的33倍。但目前还没有人研究其在肝再生中的功能,而作为重要生命活动的肝再生也涉及蛋白质降解,细胞迁移和凋亡等生理生化活动,了解该基因在肝再生中的功能,有助于揭示肝再生分子机制。为此,本文挑选出基因expi,以Higgins等建立的大鼠2/3肝切除模型做为研究对象,利用大鼠尾静脉液压转基因技术,通过基因添加和RNA干涉的方法,研究expi在大鼠肝再生中的作用。根据expi的基因序列设计引物并克隆该基因,同时设计其两条RNA干涉片段,然后进行载体的构建。将上述测序正确的基因及其干涉片段连接到不同的载体上,构建三套载体:一套是表达载体pEGFP-N1-expi,用于基因添加实验;另一套是干涉载体,包括pGenesil-1.0-E6和pGenesil-1.0-E147,用于干涉目的基因的实验;最后一套是检验载体,包括pGenesil-1.0-E6-expi,pGenesil-1.0-E147-expi和pGenesil-1.0-HK-expi,用于检验设计的两条干涉片段是否对靶基因产生干涉作用。将构建的三条检验载体分别进行尾静脉液压转基因操作,通过观察转干涉检验载体的绿色荧光蛋白阳性细胞率(转染率)相较对照检验载体是否有明显减少,来判断设计的干涉片段是否对靶位点具有干涉作用。将表达载体和干涉载体分别进行液压转基因操作,分别在转基因后6、12、24、36、48、60、72和84h取材观察其绿色荧光蛋白阳性细胞率、通过统计分析得出各载体在大鼠肝脏组织中表达高峰的时间点,结合expi芯片检测结果,得出2/3肝切除后的最佳转基因时间。根据以上结果,利用2/3肝切除手术制备的大鼠肝再生模型和液压转基因技术展开expi对大鼠肝再生过程影响的研究。将制备好的转基因后的再生肝材料分别于2/3肝切除后72、120和168h取出,用荧光显微技术、统计学方法和常规组织学技术对死亡率、表达动力学、肝指数和组织形态结构等方面结果进行观察与分析。测序和电泳检测结果显示:基因expi克隆成功,其表达载体、干涉载体和检验载体构建成功。荧光观察结果表明,转检验载体pGenesil-1.0-E6-expi和pGenesil-1.0-E147-expi的绿色荧光蛋白阳性细胞率明显低于其对照载体pGenesil-1.0-HK-expi,这说明设计的两干涉片段对靶位点均具有干涉作用。转表达载体pEGFP-N1-expi和干涉载体pGenesil- 1.0-E6与pGenesil-1.0-E147后,统计分析阳性细胞率得出各载体在大鼠肝脏组织中表达高峰的时间点是转基因后12h,结合expi芯片检测结果,得出最佳转基因时间点是2/3肝切除后18h。肝指数检测结果表明,在进行expi基因添加时,肝指数高于对照;进行expi基因干涉时,肝指数则低于对照。总之,本文成功克隆了expi基因,并成功构建了其表达载体、干涉载体和检验载体,根据上述体内检测结果初步推测expi对大鼠肝再生有一定的影响,这为肝再生分子机理研究提供了资料。更多还原

【Abstract】 Proteinase inhibitor gene expi has the protease activity, and involved in cell migration and apoptosis. According to the expression profile of rat regenerating liver detected with rat genome 230 2.0 chip, expi was observed to begin to express at 6h after 2/3 hepatectomy and increased consistently at 6-72h, reaching peak (33 times higher than control) at 30h. Liver regeneration, an important life process, is also involved in protein degradation, cell migration, and apoptosis and so on. Based on the above description, the study on the function of this gene in liver regeneration will be helpful in revealing the molecular mechanism of liver regeneration. However, at present, the function of this gene on liver regeneration has not been researched yet. Therefore, this paper employed the technique of hydrodynamics-based transgene to explore the influence of expi in the process of rat liver regeneration by means of adding (or interfering) this gene.Based on the nucleotide sequence of Expi, primers were designed to amplify this gene. At the same time, the two RNA interference fragments of this gene were designed. Then, 3 sets of the recombinant vectors were established by ligation with the above right fragments, respectively, which includes the expression vector pEGFP-N1-expi for gene addition assay, the interference vectors pGenesil-1.0-E6 and pGenesil-1.0-E147 for gene inference test, and the test vectors pGenesil-1.0-E6-expi, pGenesil-1.0-E147 -expi and pGenesil-1.0-HK-expi for testing whether the two interference fragments have interference effect on the target gene or not.The constructed test vectors were transgened based on hydrodynamics. At 6, 12, 24, 36, 48, 60, 72 and 84h after transgene, the interference effect of interference fragments on the target site were determined by observing whether the GFP-positive cell rates (that’s transfection rate) were decreased more obviously than control. The optimum time for transgene after partial hepatectomy was obtained by statistically measuring the peak time of the hydrodynamics expression of different vectors (including expression vectors and interference vectors) in rat livers, at the same time, expi expression in rat regenerating livers from the chip data must to be considered. Finally, the transgenic-liver samples at 72, 120, and 168h after partial hepatectomy were taken to analyze the mortality, expression kinetics, index of liver regeneration, and morphology structure, etc. by using fluorescence microscopy techniques, statistical methods and conventional histological technique.DNA sequencing and electrophoresis showed: the cloned expi sequence is right; the vector was successfully constructed; the two interference fragments exhibit the more pronounced interference effect. The best time for transgene was 18 hours after partial hepatectomy.Liver index test results show that if adding expi, liver index was higher than control; if interfering expi, lower than control. In short, this study successfully cloned expi and successfully constructed the expression vector, interference vector and test vector. According to the in vivo assays, it is presumed that expi has effect to a extent on rat liver regeneration, which provides the valuable information for elucidating the molecular mechanism of liver regeneration.更多还原