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磷酸钙盐沉淀法可建立质粒双转染细胞模型
Stable transfection of two expression plasmids in cells by calcium phosphate precipitation procedure
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DOI:
作者:
马桢,黄雄,牛玉宏,龚惠,邹云增
MA Zhen,HUANG Xiong,NIU Yu-hong,GONG Hui,ZOU Yun-z
作者单位:
上海市青浦区中心医院(上海复旦大学附属中山医院青浦分院)
Qingpu branch of Zhongshan Hospital,Fudan university, Shanghai
关键词:
磷酸钙盐沉淀法;双转染;质粒;AT1;CaMKII
Calcium phosphate precipitation procedure; Transfect; Plasmid;Angiotensin II receptor type 1;Ca2+/camoduLin-dependent kinase II
摘要:
【摘要】目的:建立表达血管紧张素II受体1型(AT1)和钙/钙调蛋白依赖性激酶II(CaMKII)或DN(CaMKII的活性抑制体)的双质粒转染COS7细胞模型,观察其ERK反应。方法:通过扩增、筛选及抽提得到相关质粒,双酶切法及琼脂糖电泳鉴定目的DNA片段。磷酸盐钙沉淀法将质粒AT1和CaMKII/WT或CaMKII/DN转入COS7细胞内。以免疫荧光法(IF)、免疫印迹法(WB)测定细胞表面表达的AT1(及其所带的HA-tag)、胞浆内表达的CaMKII/WT或CaMKII/DN(及其所带的V5-tag)。给予转染细胞以血管紧张素II(AngII)、AT1受体拮抗剂(ARB)+AngII等刺激,WB测定p-ERK改变。结果:质粒酶切后电泳可见目的长度DNA片段。在转染细胞中,IF呈阳性荧光反应,WB可发现目的蛋白;而未转染细胞中则否。AngII刺激引起转染AT1和CaMKII/WT的细胞产生p-ERK升高反应,可为ARB预处理消除;而未转染细胞及转染AT1和CaMKII/DN的细胞无类似反应。结论:磷酸钙盐沉淀法可成功建立该双转染细胞模型。
[Abstract]Objective: We use calcium phosphate precipitation procedure to stable transfect two expression plasmids (angiotensin II receptor type 1(AT1)and Ca2+/camoduLin-dependent kinase II(CaMKII) or DN(a domain negative mutant of CaMKII))in COS7 cell and observe the phosphorylation of ERK . Methods: The palsmids we used were obtained by amplification、selection and extraction. The DNA fragments were verified by double enzyme digestion and agarose gel electrophoresis. AT1 and CaMKII/WT or CaMKII/DN expression plasmids were transfected into cells using calcium phosphate precipitation method. Immunofluorence(IF)and Western blotting (WB) methods were used to measure the expression of AT1 (and its HA-tag) on cell membrane and expression of CaMKII/WT、CaMKII/DN(and their V5-tag) in the cytoplasm. Transfected cells with or without AT1 blocker(ARB) pre-treatment were stimuLated by angiotensin II (AngII),the level of p-ERK were evaluated by WB after stimuLation for 7 minutes. ResuLts: Target DNA fragments were found in theses plasmids. AT1 and CaMKII proteins were measured in transfected cells but not in the untransfected ones.The transfection efficiency was about 30%. Compared with control group, AngII evoked an increase response in p-ERK of cells transfected with AT1 and CaMKII/WT plasmids(p<0.05) which couLd be abolished in cells pretreated with ARB, while the same reaction wasn’t found in cells untransfected and transfected with AT1 and CaMKII/DN plasmids. Conclusions: Calcium phosphate precipitation procedure couLd effectively transfect two plasmids into cultured cells.
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