国际免疫学杂志
國際免疫學雜誌
국제면역학잡지
INTERNATIONAL JOURNAL OF IMMUNOLOGY
2013年
3期
221-225
,共5页
RNA干扰%ADAM10%N型钙粘蛋白%细胞黏附%慢病毒
RNA榦擾%ADAM10%N型鈣粘蛋白%細胞黏附%慢病毒
RNA간우%ADAM10%N형개점단백%세포점부%만병독
RNA interference%ADAM10%N-cadherin%Cell adhesion%Lentivirus
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Oligo DNA,与LentiLox 3.7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Western blotting检测心肌细胞N型钙粘蛋白及其C-端的CTF1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ADAM10 shRNA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ADAM10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.
目的 構建ADAM10基因RNA榦擾重組慢病毒,觀察其在N型鈣粘蛋白底物加工過程中的重要性,為探求該加工途徑在維持心肌組織結構形態和完整性中的作用打下基礎.方法 篩選確定ADAM10基因RNA榦擾有效靶序列,閤成靶序列的Oligo DNA,與LentiLox 3.7載體連接,PCR篩選暘性剋隆,測序鑒定.用重組慢病毒轉導心肌細胞(H9C2),通過Western blotting檢測心肌細胞N型鈣粘蛋白及其C-耑的CTF1片段的蛋白錶達,流式細胞學實驗檢測N型鈣粘蛋白在心肌細胞錶麵的錶達,利用黏附實驗檢測轉染前後心肌細胞黏附能力的變化.結果 成功構建ADAM10 shRNA慢病毒載體,包裝慢病毒,轉導心肌細胞.與陰性對照組相比,轉染組心肌細胞N型鈣粘蛋白錶達提高,CTF1片段錶達減少；細胞錶麵N型鈣粘蛋白的錶達增多；心肌細胞黏附能力提高.結論 初步證明心肌細胞中ADAM10在N型鈣粘蛋白的加工水解過程中髮揮重要作用,為進一步明確該水解途徑在維持心肌細胞形態結構和完整性方麵所起的作用打下瞭實驗基礎.
목적 구건ADAM10기인RNA간우중조만병독,관찰기재N형개점단백저물가공과정중적중요성,위탐구해가공도경재유지심기조직결구형태화완정성중적작용타하기출.방법 사선학정ADAM10기인RNA간우유효파서렬,합성파서렬적Oligo DNA,여LentiLox 3.7재체련접,PCR사선양성극륭,측서감정.용중조만병독전도심기세포(H9C2),통과Western blotting검측심기세포N형개점단백급기C-단적CTF1편단적단백표체,류식세포학실험검측N형개점단백재심기세포표면적표체,이용점부실험검측전염전후심기세포점부능력적변화.결과 성공구건ADAM10 shRNA만병독재체,포장만병독,전도심기세포.여음성대조조상비,전염조심기세포N형개점단백표체제고,CTF1편단표체감소；세포표면N형개점단백적표체증다；심기세포점부능력제고.결론 초보증명심기세포중ADAM10재N형개점단백적가공수해과정중발휘중요작용,위진일보명학해수해도경재유지심기세포형태결구화완정성방면소기적작용타하료실험기출.
Objective To construct a lentiviral vector of RNA interference of ADAM10-gene and observe the role of the ADAM10 in shedding neuronal cadherin (N-cadherin).Methods The effective sequence of siRNA targeting ADAM10 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 sequencing.After that,ADAM10 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 ADAM10 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.
