CaMKⅡα/β稳定表达的PC12细胞株的构建及其功能的初步研究

CaMKⅡ α/β stable expression of PC12 cell line and its preliminary function

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。

Aim To establish stable transfection of PC12 cells with pEGFP-N1-CaMK II α/β recombinant plasmids for further study of the physiological functions of the ealcium/calmodulindependent protein kinase CaMK II α and β subtypes. Methods The expression vector pEGFP-CaMK II α/β was transfeeted into PC12 cells with liposome-mediated method, and then the selective medium containing G418 antibiotic was used for longterm screening. Drug-resistant monoclone was picked and cultured and resistant monoclonal cells were collected. The growth curve of the three transformants were determined by MTT assay. The effects of CaMK 11 α/β in PC12 cells on synaptic vesicles associated protein expression were determined by western blot analysis. The different effects of DA release between the two overexpression of CaMK II α/β in PC12 ceils were detected by highperformance liquid chromatography （HPLC）. Results MTT assay showed that pEGFP-N1-CaMK 11 α/β cells grew at a similar rate to the control group. The expression of fusion protein in PC12 cells was identified by Western blot and the resuhs appeared in the PC12-CaMK II α/β group that molecular weight 82/89 ku detected at a great deal of the target gene expression, while in the control corresponding position in the group there were no any bands, indicating that the exogenous gene over-expressed in cells. Synaptic vesicles associated protein detected by Western blot showed that there was no significant difference in the effects of overexpression of different subtypes of CaMK II α/β on the vesicle protein. HPLC analysis showed that there was significant difference in overexpression of CaMK II α/β on the release of DA in PC12 cells. Couclusions Obtained the stable expression of pEGFP-CaMK II α/β in PC12 cells. Preliminary study the different subtypes of the kinases in cell fuction.