ADAMs相關(guān)基因在肝癌細胞中的表達及功能分析
發(fā)布時間：2018-08-18 16:12
【摘要】：背景ADAMs家族成員屬于I型跨膜蛋白,具有相似的基本結(jié)構(gòu),都含有去整合素和金屬蛋白酶功能結(jié)構(gòu)域,與細胞外基質(zhì)的降解,細胞-細胞的黏連,細胞-基質(zhì)的黏連及信號轉(zhuǎn)導等多種細胞功能有關(guān)。雖然對ADAMs家族的功能已經(jīng)有很多了解,但是由于其結(jié)構(gòu)復雜,成員眾多且組織分布具有特異性,仍需要進一步研究。目的構(gòu)建去分化處理的常氏肝癌細胞(一種人源肝癌細胞)的c DNA文庫,免疫篩選并克隆與分化相關(guān)的ADAMs基因,并初步研究其功能。方法1.Western blot方法檢測DM(去分化培養(yǎng)基)處理的常氏肝癌細胞中蛋白表達的變化。2.構(gòu)建DM處理的常氏肝癌細胞c DNA文庫。3.用通用抗體對文庫進行免疫篩選,克隆基因全長并測序分析。4.構(gòu)建ARP1的原核表達載體,用SDS-PAGE和Western blot方法分析ARP1的功能。5.用蛋白水解酶復性電泳及SDS-PAGE分析大鼠的包括肝在內(nèi)的17種組織器官的ADAMs的組成及蛋白圖譜和蛋白水解酶譜。結(jié)果1.與未經(jīng)處理的常氏肝癌細胞相比,DM處理的常氏肝癌細胞出現(xiàn)了30k D、50k D和105k D三種ADAMs。其中30k D和50k D ADAMs為DM所誘導。2.經(jīng)檢測,成功構(gòu)建了DM處理的常氏肝癌細胞的c DNA文庫。3.用ADAMs通用抗體對文庫進行了免疫篩選和基因全長克隆,測序分析并注冊了4個物種特異性的與ADAMs相關(guān)的基因。4.成功構(gòu)建了ADAM相關(guān)基因1(ARP1)的原核表達載體,用活性電泳、Western blot方法分析發(fā)現(xiàn),ARP1表達產(chǎn)物具有偏堿性蛋白水解酶活性,可降解明膠,并且可以和ADAMs通用抗體結(jié)合。5.通過分析不同組織器官的ADAMs的分布及蛋白圖譜和蛋白水解酶譜,結(jié)果顯示ADAMs的分布及酶活具有很大差異。結(jié)論本研究通過DM處理常氏肝癌細胞,構(gòu)建c DNA文庫并進行表達文庫免疫篩選等方法,初步驗證了DM處理可以誘導常氏肝癌細胞合成30k D和50k D ADAMs;構(gòu)建的c DNA文庫質(zhì)量達標,可用性強;建立了切實可行的表達文庫免疫篩選方法;注冊了4個物種特異性的與ADAMs相關(guān)基因;構(gòu)建了ADAMs相關(guān)基因1(ARP1)的原核表達載體,表達產(chǎn)物具有偏堿性蛋白水解酶活性,可以和ADAMs通用抗體結(jié)合。在不同組織器官中,ADAMs的分布及酶活具有很大差異。以上結(jié)果為進一步研究ADAMs及其相關(guān)基因的功能提供了重要資料。
[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.
【學位授予單位】：新鄉(xiāng)醫(yī)學院
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：R735.7
本文編號：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.
【學位授予單位】：新鄉(xiāng)醫(yī)學院
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：R735.7
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