ADAM10基因RNA干扰重组慢病毒的转导对H9C2细胞N型钙粘蛋白的解整合素金属蛋白酶10加工途径的影响  

Construction of lentiviral vector of RNA interference of ADAMIO gene and its inhibitive role on the ADAMs cleavage of N-cadherin in cardiac myocyte

作　　者：李小鸥[1] 黄巍[2] 周丽荣[2] 

机构地区：[1]武汉大学人民医院儿科,430030 [2]武汉市医学科学研究所

出　　处：《国际免疫学杂志》2013年第3期221-225,共5页International Journal of Immunology

基　　金：国家自然科学基金（81000094）;武汉大学自主青年基金（4101016）;武汉市卫生局科研项目（WX10B13）

摘　　要：目的构建ADAM10基因RNA干扰重组慢病毒，观察其在N型钙粘蛋白底物加工过程中的重要性，为探求该加工途径在维持心肌组织结构形态和完整性中的作用打下基础。方法筛选确定ADAM10基因RNA干扰有效靶序列，合成靶序列的OligoDNA，与LentiLox3．7载体连接，PCR筛选阳性克隆，测序鉴定。用重组慢病毒转导心肌细胞（H9C2），通过Westernblotting检测心肌细胞N型钙粘蛋白及其C-端的CTFl片段的蛋白表达，流式细胞学实验检测N型钙粘蛋白在心肌细胞表面的表达，利用黏附实验检测转染前后心肌细胞黏附能力的变化。结果成功构建ADAM10shRNA慢病毒载体，包装慢病毒，转导心肌细胞。与阴性对照组相比，转染组心肌细胞N型钙粘蛋白表达提高，CTF1片段表达减少；细胞表面N型钙粘蛋白的表达增多；心肌细胞黏附能力提高。结论初步证明心肌细胞中ADAMIO在N型钙粘蛋白的加工水解过程中发挥重要作用，为进一步明确该水解途径在维持心肌细胞形态结构和完整性方面所起的作用打下了实验基础。Objective To construct a lentiviral vector of RNA interference of ADAM10-gene and ob- serve the role of the ADAMIO in shedding neuronal cadherin （N-cadherin）. Methods The effective sequence of siRNA targeting ADAMIO gene was confirmed. Both sense and antisense Oligo DNA of the targeting sequence was designed, synthesized and coloned into the LentiLox 3.7 vector, and was confirmed by PCR and sequen- cing. After that, ADAMIO shRNA was transfected into H9C2 cells. The expression of N-cadherin and CTF was detected by Western-blot and fluorescence activated cell sorter（FACS）. The adhesive ability of transfected cells was analyzed. Results PCR and DNA sequencing demonstrated that the lentiviral RNAi vector of ADAMIO producing ADAM10 shRNA was constructed successfully. N-cadherin protein level was increased after AD- AM10 siRNA transfected. The accumulation of N-cadherin on the surface of transfected cells was mediated. The adhesive ability of cells transfected was also significantly increased as compared with the control group. Conclusion ADAM10 play a crucial role in shedding N-cadherin, which may contribute to structural cardiac remodeling.

关 键 词：RNA干扰 ADAM10 N型钙粘蛋白 细胞黏附 慢病毒 

分 类 号：R541[医药卫生—心血管疾病]