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

Construction of the full length and N-terminal fragment of serum response factor over-expressing lentiviral plasmid and its impacts on cardiac stem cell differentiation

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。

Purpose To analyze the effects of full length and N-terminal fragment of serum response factor （ SRF-Fu11 and SRF-N） on TGF-131-induced differentiation in c-Kit ＋ cardiac stem cells （CSC）. Methods Rat SRF-Full and SRF-N （ 1-254 aa） coding sequences were obtained from eDNA library and cloned into the linearized lentviral vector GV358 （Ubi-MCS- 3FLAG-SV40-EGFP-IRES-puromycin） to generate the recombi- nant vectors, and then positive clones were selected and sequenced after transducing the competent cells with recombinant vectors. The recombinant lentvirus were packaged through transfecting the HEK293T cells with SRF-FuI1, SRF-N overexpress- ing plasmids and viral packaging plasmids. Neonatal SD rat c- Kit ＋ CSCs were isolated via magnetic activated cell sorting, and TGF-β1-induced differentiation in SRF-Full and SRF-N overexpression virus-infected CSCs was assessed by quantitative PCR. Results SRF-Full and SRF-N coding sequences were successfully obtained and properly cloned into the linearized GV358. The positive clones were selected and further confirmed by sequencing. With the help of packaging plasmids, the SRF- Full and SRF-N overexpressing plasmids-transfected HEK293T cells successfully produced the lentiviral particles with the titer of 2 × 10 8 TU/mL, and the SRF-Full-Flag and SRF-N-Flag fusion protein were detected by Western blot in virus-infected HEK293T cells. Addition of TGF-I31 significantly induced upregulated mRNAs in cardiomyocyte markers （Nkx2. 5, Gata4, cTnI） and smooth muscle cell marker （SM22α） but not the epithelia cell marker （vWF） in CSCs. Overexpression of SRF-Full facilitated TGF-β1-triggered cardiomyocyte differentiation. However, SRF-N exerted anti-differentiation effects in TGF-β1-treated cells. Conclusion The SRF-Full and SRF-N overexpressing recombinant lentiviral vectors are successfully constructed. SRF-Full facilitates while SRF-N suppresses TGF-β1-induced cardiomyocyte differentiation in c-Kit ＋ CSCs.