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目的建立稳定表达CaMKⅡα/β的PC12稳定细胞株,以便进一步研究钙/钙调素依赖性蛋白激酶CaMKⅡα/β不同亚型的生理功能。方法通过脂质体介导的方法将构建的表达载体pEGFP-CaMKⅡα/β转染入PC12细胞中,然后用含G418抗生素的选择性培养基进行长期筛选,挑取耐药单克隆,培养并收集耐药的单克隆细胞;通过MTT法检测转染细胞在体外对PC12细胞增殖与活性的影响;Westernblot分析稳定表达的pEGFP-CaMKⅡα/β不同亚型对PC12细胞中相关突触囊泡蛋白表达的影响;并用高效液相色谱法(HPLC)检测过表达的CaMKⅡα/β对PC12细胞神经递质多巴胺DA释放的影响。结果 MTT细胞活性检测结果显示,转染重组质粒pEGFP-N1-CaMKⅡα/β的PC12细胞稳定株的生长活性与对照组相接近。应用Western blot方法对融合蛋白CaMKⅡα/β-GFP在PC12细胞内的表达进行鉴定,结果显示在PC12-CaMKⅡα/β组中,分子量82/89 ku处检测到该目的基因的大量表达,而在对照组的相应位置则没有任何条带,表明外源性目的基因已在细胞内过表达。West-ern blot方法检测突触囊泡蛋白结果显示CaMKⅡα/β不同亚型的过表达对囊泡蛋白的影响并无差异。HPLC检测结果显示过表达的CaMKⅡα/β对PC12细胞中DA的释放差异具有显著性。结论该实验获得稳定表达pEGFP-CaMKⅡα/β的PC12细胞株。初步探讨了该激酶的不同亚型对细胞功能的影响。
Objective To establish a stable PC12 cell line stably expressing CaMKⅡα / β in order to further study the physiological functions of different subtypes of CaMKⅡα / β, a calcium / calmodulin-dependent protein kinase. Methods The constructed expression vector pEGFP-CaMKⅡα / β was transfected into PC12 cells by liposome-mediated method and then screened by selective medium containing G418 antibiotics for a long time. The monoclonal antibody was picked and cultured and collected The effect of transfected cells on the proliferation and activity of PC12 cells in vitro was analyzed by MTT assay. The expression of related synaptic vesicle proteins in PC12 cells was analyzed by Western blot analysis of the different subtypes of stably expressed pEGFP-CaMKⅡα / β Effects of over-expressed CaMKⅡα / β on dopamine DA release in PC12 cells were detected by high performance liquid chromatography (HPLC). Results The results of MTT cell activity showed that the growth activity of PC12 cells transfected with recombinant plasmid pEGFP-N1-CaMKⅡα / β was similar to that of the control group. The expression of fusion protein CaMKⅡα / β-GFP in PC12 cells was identified by Western blot. The results showed that a large amount of the target gene was detected at the molecular weight of 82/89 ku in the PC12-CaMKⅡα / β group, while in the control The corresponding position of the group did not have any band, indicating that the exogenous gene of interest has been overexpressed in the cell. West-ern blot detection of synaptic vesicle protein results showed that CaMK Ⅱ α / β overexpression of the vesicle protein no difference. HPLC results showed that over-expression of CaMKⅡα / β in PC12 cells in the DA release difference was significant. Conclusions The PC12 cell line stably expressing pEGFP-CaMKⅡα / β was obtained in this experiment. The effects of different subtypes of this kinase on cell function were discussed preliminarily.