pEGFP-HSP70重组质粒的构建及其在大鼠神经干细胞中的表达  

Construction of recombinant plasmid pEGFP-HSP70 and its expression in rat neural stem cells

作　　者：舒晓明[1] 逯丹[1] 王珍[1] 张婵娟[1] 赵佳仪 朱丽红[1] 戚仁斌[1] 陆大祥[1] 

机构地区：[1]暨南大学医学院病理生理学系,国家中医药管理局重点实验室,广东广州510632

出　　处：《中国病理生理杂志》2014年第5期842-846,共5页Chinese Journal of Pathophysiology

基　　金：国家重点基础研究发展计划(973计划)(No.2011CB707504);广州市科学与技术重大专项(No.11BppZXaa2070006)

摘　　要：目的:构建重组pEGFP-HSP70质粒并观察其在神经干细胞中的表达。方法:采用RT-PCR方法从SD胎鼠肝组织中获取总RNA,扩增出热休克蛋白70(HSP70)基因的全序列cDNA,并克隆到含有增强型绿色荧光蛋白(EGFP)基因的真核表达载体pEGFP-C2上,经EcoRⅠ、BamHⅠ酶切及测序分析对重组质粒pEGFP-HSP70进行鉴定。采用Nucleofector转染技术将重组质粒pEGFP-HSP70转染至胎鼠神经干细胞中。结果:(1)胎鼠HSP70 cDNA序列被正确地克隆到真核表达载体pEGFP-C2中,成功构建重组大鼠pEGFP-HSP70质粒。荧光强度检测空质粒转染组(pEGFP-C2组)、pEGFP-HSP70组与对照组相比显著升高(P<0.01);pEGFP-HSP70组内24 h的荧光强度与7 d(P<0.05)、14 d(P<0.05)和21 d(P<0.01)相比降低明显。(2)转染后HSP70的表达在1 d(P<0.05)、7 d(P<0.01)和14 d(P<0.01)与对照组相比均明显升高。结论:神经干细胞可直接作为基因靶细胞,能有效地表达重组质粒pEGFP-HSP70,且HSP70在神经干细胞中的表达与转染时间密切相关。AIM： To explore the method of constructing recombinant plasmid pEGFP-HSP70 and its expression in neural stem cells. METHODS： Total RNA was acquired from the fetal liver tissue of SD rat. cDNA complete sequence of heat-shock protein 70 （HSP70） gene was amplified by RT-PCR. and cloned into an eukaryotic expression vector containing the enhanced green fluorescent protein （EGFP） reporter gene. pEGFP-C2. Sequencing analysis was performed to confirm the recombinant plasmid pEGFP-HSP70. The technique of nucleofector transfection was used to transfect the recombinant plasmid pEGFP-HSP70 into neural stem cells. RESULTS： HSP70 cDNA sequence was correctly cloned into the eukaryotic expression vector pEGFP-C2. The recombinant plasmid pEGFP-HSP70 was constructed successfully. Compared with control grouP. the fluorescence intensities in pEGFP-C2 group and pEGFP-HSP70 group were significantly increased. The fluoresce nee intensity in pEGFP-HSP70 group after 24 h of transfection was significantly decreased compared with other time points of 7 d. 14 d and 21 d. The expression level of HSP70 significantly increased 1 d. 7 d and 14 d after transfectioncompared with control group. CONCLUSION： The neural stem cells can be directly used as gene action target cells. The HSP70 expression level in the stem cells is closely related to the time after transfection.

关 键 词：质粒 转染 热休克蛋白70 神经干细胞 

分 类 号：R363[医药卫生—病理学]