平滑肌细胞纯化载体构建及在小鼠胚胎干细胞中的表达

Construction of vector SM22α-PAC-IRES2-EGFP used for purification of smooth muscle cells and its expression in mouse embryonic stem cells

背景:胚胎干细胞是平滑肌细胞重要的来源之一,但是胚胎干细胞分化细胞的异质性导致难以获得较纯的平滑肌细胞。目的:为进一步纯化胚胎干细胞来源的平滑肌细胞,拟在体外构建平滑肌特异性SM22α启动子驱动的嘌呤霉素抗性(puromycin acetyltransferase,pac)基因与增强型绿色荧光蛋白(enhanced green fluorescence protein,EGFP)基因双表达载体,即pSM22-PAC-IRES2-EGFP载体,并在胚胎干细胞中检测其有效性及特异性。设计、时间及地点:基因水平细胞观察实验,于2007-05/2008-09在解放军沈阳军区总医院全军心血管病研究所完成。材料:小鼠胚胎干细胞系R1购自美国ATCC公司,编号SCRC-1011TM。pSM22α-EGFP载体由本实验室构建;pIRES2-EGFP载体、pSM2C载体、pSuper.basic载体购自Invitrogen公司。方法:用聚合酶链反应方法从pSM22α-EGFP中扩增SM22α启动子,然后用该启动子替换pIRES2-EGFP载体中的CMV启动子,构建pSM22-IRES2-EGFP。再从pSM2C中用HindⅢ/ClaⅠ酶切获得pac基因,将pac基因片段亚克隆到pSuper.basic中,构建pSuper-PAC。最后BgⅢ/AccⅠ双酶切pSuper-PAC获得pac基因片段,将其插入到pSM22α-IRES2-EGFP,构建成pSM22α-PAC-IRES2-EGFP。将pSM22α-PAC-IRES2-EGFP用脂质体法转染胚胎干细胞,G418筛选阳性克隆。诱导胚胎干细胞阳性克隆分化,RT-PCR扩增pac基因鉴定阳性克隆。对分化细胞行平滑肌细胞标志物SMα-actin免疫荧光染色。主要观察指标:①pSM22α-PAC-IRES2-EGFP测序结果。②pac基因扩增。③荧光显微镜下同时观察分化细胞EGFP的表达及SMα-actin染色情况。结果:HindⅢ/ClaⅠ双酶切得到261bp,664bp,5000bp3个片段,与预期结果一致,测序结果证实pSM22α-PAC-IRES2-EGFP构建成功。Pac基因扩增证实有4株胚胎干细胞克隆转染成功。转染成功的胚胎干细胞被诱导分化后,部分细胞表达EGFP,且这些细胞SMα-actin染色呈阳性。结论:实验成功构建了平滑肌细胞筛选载体pSM22α-PAC-IRES2-EGFP。成功转染这一载体的胚胎干细胞表达pac基因及EGFP基因,且EGFP表达具有平滑肌特异性。

BACKGROUND： Embryonic stem cells （ESCs） serve as a major cell source for smooth muscle cells, but the heterogeneity of cells derived from ESCs result in difficulty to obtain high purity smooth muscle cells. OBJECTIVE： To construct a double expression vector of puromycin resistance （pac） gene and enhanced green fluorescence protein （EGFP） gene driven by smooth muscle specific SM22α promoter （pSM22α-PAC-IRES2-EGFP）, in addition, to detect its availability and specificity in ESCs. DESIGN, TIME AND SETTING： The observational experiment of gene level was performed at the Cardiovascular Institute, General Hospital of Shenyang Military Region from April 2007 to September 2008. MATERIALS： ESCs line R1 with number SCRC-1011TM was purchased from American ATCC Company. The pSM22α-EGFP vector was constructed by our laboratory. And the plRES2-EGFP, pSM2C and pSuper.basic vectors were purchased from Invitrogen Company. METHODS： SM22α promoter was cloned from pSM22α-EGFP by polymerase chain reaction. CMV promoter of plRES2-EGFP vector was replaced by SM22 promoter to establish pSM22α-IRES2-EGFP. Pac gene, excised from pSM2C by HindⅢ/ClaⅠ digestion, was sub-cloned into pSuper.basic to establish pSuper-PAC. After BgⅢ/AccⅠ enzyme digestion of pSuper-PAC, pac gene fragment was obtained, which was further sub-cloned into pSM22α-IRES2-EGFP to produce pSM22α-PAC-IRES2-EGFP. ESCs were transfected with pSM22α-PAC-IRES2-EGFP using lipofectamine. Positive clones were selected by G418 and induced to differentiate and further identified by amplification of pac gene by RT-PCR. Differentiated cells were immunostained by SM α-actin, and expression of SM α-actin and EGFP was observed simultaneously under fluorescence microscope. MAIN OUTCOME MEASURES： Sequencing result of pSM22α-PAC-IRES2-EGFP; Amplification of pac gene; EGFP expression; as well as SM α-actin immunostaining. RESULTS： Three segments of 261 bp, 664 bp, and 5 000 bp were obtained by HindllllC/al digestion, which was coincident with expectation, and the sequencing results showed that pSM22α-PAC-IRES2-EGFP vector was successfully constructed. Amplification of pac gene identified 4 ESCs clones successfully transfected. After induction of differentiation, partial portion of differentiated cells expressed EGFP, accompanied by positively stained by SM α-actin antibody. CONCLUSION： pSM22α-PAC-IRES2-EGFP vector was successfully constructed. ESCs clones transfected with this vector expressed pac gene and EGFP gene, and the expression of EGFP is smooth muscle specific.