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CaMKⅡ的活性抑制体(DN)的双质粒转染COS7细胞模型,观察其磷酸化细胞外调节蛋白激酶(p-ERK)反应。方法通过扩增、筛选及抽提得到相关质粒,采用双酶切法及琼脂糖电泳鉴定目的DNA片段。采用磷酸盐钙沉淀法将质粒AT1和CaMKⅡ野生型(CaMKⅡ/WT)或CaMKⅡ/DN转入COS7细胞内,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)、细胞质内表达的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;而未转染细胞中则无。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。
Objective To transfect two expression plasmids （angiotensin Ⅱ receptor type 1 [AT1] and Ca2＋/calmodulin-dependent kinase ⅡECaMK Ⅱ ] or DN Fa domain negative mutant of CaMK Ⅱ ]） into COS7 cell by calcium phosphate precipitation procedure, and to observe the phosphorylation reaction of extracellular regulated protein kinase （p-ERK）. Methods The plasmids were obtained by amplification, selection and extraction. Target DNA fragments were verified by double enzyme digestion and agarose gel electrophoresis. AT1 and CaMK Ⅱ/WT or CaMK Ⅱ/DN expression plasmids were transfected into 0OS7 cells by calcium phosphate precipitation method. Immunofluorence and Western blotting were used to measure the expression of AT1（and its HA-tag） on cell membrane and CaMK Ⅱ/WT or CaMK Ⅱ/DN （and their Vs-tag） in cytoplasm. Transfected cells with or without AT1 blocker （ARB） pre-treatment were stimulated by angiotensin Ⅱ and the level of p-ERK was evaluated by Western blotting. Results Target DNA fragments were found in these plasmids. AT1 and CaMK Ⅱ proteins were expressed in transfected cells but not in untransfected ones. p-ERK was increased in the cells transfected with ATm and CaMK Ⅱ/WT plasmids after angiotensin Ⅱ stimulation, which could be abolished in the cells pretreated with ARB; while the same reaction could not happen in the cells untransfected and transfected with AT1 and CaMK Ⅱ/DN plasmids. Conclusion Calcium phosphate precipitation can effectively transfect two plasmids into cultured cells. （Shanghai Med J, 2013, 36： 229-232）
出处
《上海医学》
CAS
CSCD
北大核心
2013年第3期229-232,I0002,共5页
Shanghai Medical Journal
基金
国家973项目(2007CB512003)
国家自然科学基金重点项目(30930043/C140401)资助
关键词
磷酸钙盐沉淀法
双转染
质粒
血管紧张素Ⅱ受体1型
钙
钙调蛋白依赖性激酶Ⅱ
Calcium phosphate precipitation procedure
Transfection
Plasmid
Angiotensin Ⅱ receptor type 1
Ca2＋/calmodulin-dependent kinase Ⅱ
