ADAMs相关基因在肝癌细胞中的表达及功能分析
发布时间：2018-08-18 16:12
【摘要】：背景ADAMs家族成员属于I型跨膜蛋白,具有相似的基本结构,都含有去整合素和金属蛋白酶功能结构域,与细胞外基质的降解,细胞-细胞的黏连,细胞-基质的黏连及信号转导等多种细胞功能有关。虽然对ADAMs家族的功能已经有很多了解,但是由于其结构复杂,成员众多且组织分布具有特异性,仍需要进一步研究。目的构建去分化处理的常氏肝癌细胞(一种人源肝癌细胞)的c DNA文库,免疫筛选并克隆与分化相关的ADAMs基因,并初步研究其功能。方法1.Western blot方法检测DM(去分化培养基)处理的常氏肝癌细胞中蛋白表达的变化。2.构建DM处理的常氏肝癌细胞c DNA文库。3.用通用抗体对文库进行免疫筛选,克隆基因全长并测序分析。4.构建ARP1的原核表达载体,用SDS-PAGE和Western blot方法分析ARP1的功能。5.用蛋白水解酶复性电泳及SDS-PAGE分析大鼠的包括肝在内的17种组织器官的ADAMs的组成及蛋白图谱和蛋白水解酶谱。结果1.与未经处理的常氏肝癌细胞相比,DM处理的常氏肝癌细胞出现了30k D、50k D和105k D三种ADAMs。其中30k D和50k D ADAMs为DM所诱导。2.经检测,成功构建了DM处理的常氏肝癌细胞的c DNA文库。3.用ADAMs通用抗体对文库进行了免疫筛选和基因全长克隆,测序分析并注册了4个物种特异性的与ADAMs相关的基因。4.成功构建了ADAM相关基因1(ARP1)的原核表达载体,用活性电泳、Western blot方法分析发现,ARP1表达产物具有偏碱性蛋白水解酶活性,可降解明胶,并且可以和ADAMs通用抗体结合。5.通过分析不同组织器官的ADAMs的分布及蛋白图谱和蛋白水解酶谱,结果显示ADAMs的分布及酶活具有很大差异。结论本研究通过DM处理常氏肝癌细胞,构建c DNA文库并进行表达文库免疫筛选等方法,初步验证了DM处理可以诱导常氏肝癌细胞合成30k D和50k D ADAMs;构建的c DNA文库质量达标,可用性强;建立了切实可行的表达文库免疫筛选方法;注册了4个物种特异性的与ADAMs相关基因;构建了ADAMs相关基因1(ARP1)的原核表达载体,表达产物具有偏碱性蛋白水解酶活性,可以和ADAMs通用抗体结合。在不同组织器官中,ADAMs的分布及酶活具有很大差异。以上结果为进一步研究ADAMs及其相关基因的功能提供了重要资料。
[Abstract]:Background the members of the ADAMs family belong to type I transmembrane proteins with similar basic structures, including the functional domains of deintegrin and metalloproteinase, degradation of extracellular matrix and cell-cell adhesion. Cell-matrix adhesion and signal transduction are related to many cell functions. Although the function of ADAMs family has been well understood, it needs further study because of its complex structure, large number of members and specific tissue distribution. Objective to construct the c DNA library of dedifferentiated Changs hepatoma cells (a kind of human hepatoma cells), screen and clone the ADAMs gene related to differentiation, and study its function. Methods 1.Western blot assay was used to detect the changes of protein expression in normal hepatoma cells treated with DM (dedifferentiation medium). Construct the c DNA library. 3. The library was screened with universal antibody, the gene was cloned and sequenced. The prokaryotic expression vector of ARP1 was constructed and the function of ARP1 was analyzed by SDS-PAGE and Western blot. Protein hydrolase refolding electrophoresis and SDS-PAGE were used to analyze the composition, protein map and proteolytic enzyme spectrum of ADAMs in 17 tissues and organs of rats, including liver. Result 1. Compared with untreated normal hepatoma cells, 30 kD 50 KD and 105 KD ADAMswere found in normal hepatoma cells treated with DM. 30kD and 50kD ADAMs were induced by DM. After detection, the c DNA library. 3. 3 was successfully constructed. The library was screened and cloned with ADAMs universal antibody. Four species-specific ADAMs related genes .4were sequenced and registered. The prokaryotic expression vector of ADAM related gene 1 (ARP1) was successfully constructed. The expression product of AARP1 was found to have alkaline proteolytic enzyme activity, degradable gelatin, and could bind to the general antibody of ADAMs. By analyzing the distribution of ADAMs, protein map and proteolytic enzyme spectrum of different tissues and organs, the results showed that the distribution and enzyme activity of ADAMs were very different. Conclusion in this study, the c DNA library was constructed by DM treatment, and the expression library was screened by immunoassay. It was preliminarily proved that DM treatment could induce the synthesis of 30 KD and 50 KD ADAMsand the quality of the constructed c DNA library was up to standard. The prokaryotic expression vector of ADAMs related gene 1 (ARP1) was constructed, and the expression product was found to have the activity of alkaline proteolytic enzyme, and four species-specific genes related to ADAMs were registered, and the prokaryotic expression vector of ADAMs related gene 1 (ARP1) was constructed. Can be combined with ADAMs universal antibody. The distribution and enzyme activity of ADAMs were different in different tissues and organs. These results provide important data for further study on the function of ADAMs and its related genes.
【学位授予单位】：新乡医学院
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R735.7
本文编号：2189994
[Abstract]:Background the members of the ADAMs family belong to type I transmembrane proteins with similar basic structures, including the functional domains of deintegrin and metalloproteinase, degradation of extracellular matrix and cell-cell adhesion. Cell-matrix adhesion and signal transduction are related to many cell functions. Although the function of ADAMs family has been well understood, it needs further study because of its complex structure, large number of members and specific tissue distribution. Objective to construct the c DNA library of dedifferentiated Changs hepatoma cells (a kind of human hepatoma cells), screen and clone the ADAMs gene related to differentiation, and study its function. Methods 1.Western blot assay was used to detect the changes of protein expression in normal hepatoma cells treated with DM (dedifferentiation medium). Construct the c DNA library. 3. The library was screened with universal antibody, the gene was cloned and sequenced. The prokaryotic expression vector of ARP1 was constructed and the function of ARP1 was analyzed by SDS-PAGE and Western blot. Protein hydrolase refolding electrophoresis and SDS-PAGE were used to analyze the composition, protein map and proteolytic enzyme spectrum of ADAMs in 17 tissues and organs of rats, including liver. Result 1. Compared with untreated normal hepatoma cells, 30 kD 50 KD and 105 KD ADAMswere found in normal hepatoma cells treated with DM. 30kD and 50kD ADAMs were induced by DM. After detection, the c DNA library. 3. 3 was successfully constructed. The library was screened and cloned with ADAMs universal antibody. Four species-specific ADAMs related genes .4were sequenced and registered. The prokaryotic expression vector of ADAM related gene 1 (ARP1) was successfully constructed. The expression product of AARP1 was found to have alkaline proteolytic enzyme activity, degradable gelatin, and could bind to the general antibody of ADAMs. By analyzing the distribution of ADAMs, protein map and proteolytic enzyme spectrum of different tissues and organs, the results showed that the distribution and enzyme activity of ADAMs were very different. Conclusion in this study, the c DNA library was constructed by DM treatment, and the expression library was screened by immunoassay. It was preliminarily proved that DM treatment could induce the synthesis of 30 KD and 50 KD ADAMsand the quality of the constructed c DNA library was up to standard. The prokaryotic expression vector of ADAMs related gene 1 (ARP1) was constructed, and the expression product was found to have the activity of alkaline proteolytic enzyme, and four species-specific genes related to ADAMs were registered, and the prokaryotic expression vector of ADAMs related gene 1 (ARP1) was constructed. Can be combined with ADAMs universal antibody. The distribution and enzyme activity of ADAMs were different in different tissues and organs. These results provide important data for further study on the function of ADAMs and its related genes.
【学位授予单位】：新乡医学院
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R735.7
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