血清反应因子全长及N端片段过表达慢病毒载体的构建及其对心脏干细胞分化的影响

发布时间：2018-06-20 20:34

本文选题：血清反应因子 + 血清反应因子N端片段　；参考：《临床与实验病理学杂志》2017年10期

【摘要】：目的探讨血清反应因子全长(SRF-Full)及N端片段(SRF-N,1-254aa)对TGF-β1诱导c-Kit+心脏干细胞(cardiac stem cell,CSC)分化的影响。方法从c DNA文库扩增大鼠SRF-Full及SRF-N表达序列并克隆入酶切线性化慢病毒载体GV358(Ubi-MCS-3FLAG-SV40-EGFP-IRES-puromycin),转化感受态细胞筛选出阳性克隆并测序鉴定。将SRF-Full及SRF-N过表达质粒与病毒包装辅助质粒共转染工具细胞HEK293T,包装慢病毒。磁激活细胞分选法分离乳SD大鼠c-Kit+CSC,将SRF-Full及SRF-N过表达慢病毒感染CSC并经TGF-β1诱导,定量PCR分析下游分化基因mRNA水平的变化。结果成功扩增出SRF-Full及SRF-N编码序列并正确连接入线性化载体,获得的阳性转化子经测序无误。将转化子质粒与病毒包装辅助质粒共转染HEK293T后,获得滴度为2×108TU/m L的慢病毒颗粒,Western blot检测到预期Flag融合蛋白。重组慢病毒感染CSC后细胞核中见e GFP信号。TGF-β1诱导CSC出现心肌细胞分化基因(Nkx2.5、Gata4、c Tn I)及平滑肌细胞分化基因(SM22α)的表达,内皮细胞分化(v WF)无影响,SRF-Full促进CSC的心肌细胞分化,而SRF-N则抑制这种分化。结论成功构建SRFFull及SRF-N过表达重组慢病毒质粒。SRF-Full促进而SRF-N减弱TGF-β1诱导CSC的心肌细胞分化。
[Abstract]:Objective to investigate the effects of SRF-Full and N-terminal SRF-NU (1-254 aa) on the differentiation of c-Kit cardiac stem cells induced by TGF- 尾 _ 1. Methods Rat SRF-Full and SRF-N expression sequences were amplified from the cDNA library and cloned into the vector GV358 Ubi-MCS-3FLAG-SV40-EGFP-IRES-puromycinin. The positive clones were screened and sequenced. SRF-Full and SRF-N overexpression plasmids were co-transfected with viral packaging assistant plasmid HEK293T to package lentivirus. The c-Kit CSCs of SD rats were isolated by magnetic activated cell sorting method. SRF-Full and SRF-N overexpression lentiviruses were infected with CSC and induced by TGF- 尾 1. The mRNA levels of downstream differentiation genes were analyzed by quantitative PCR. Results the SRF-Full and SRF-N coding sequences were successfully amplified and correctly ligated into the linearized vector. The obtained positive transformants were sequenced correctly. After the transformant plasmid was co-transfected into HEK293T with virus packaging assistant plasmid, the expected Flag fusion protein was detected by Western blot with a titer of 2 脳 108TU / mL lentivirus particles. E GFP signal. TGF- 尾 1 induced the expression of differentiation gene Nkx2. 5 and smooth muscle cell differentiation gene SM22 伪 in the nucleus of recombinant lentivirus infected with CSC, but endothelial cell differentiation v WF did not affect SRF-Full in promoting the differentiation of CSC cardiomyocytes. SRF-N inhibits this differentiation. Conclusion SRFFull and SRF-N overexpression recombinant lentivirus plasmids. SRF-Full promoted SRF-Full and SRF-N attenuated TGF- 尾 1 induced myocardial differentiation of CSC.
【作者单位】：广东医科大学基础医学院病理学系;广东医科大学基础医学院病理生理学教研室;海南医学院第一附属医院心血管研究所;
【基金】：国家自然科学基金(81460042) 广东省教育厅科技创新项目基金(KJCX20130088) 广东省“扬帆计划”高层次人才项目(4YF16007G) 2015年国家留学人员科技活动择优资助项目
【分类号】：R54

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