中文摘要：

目的构建起搏基因质粒pIRES2-EGFP-mHCN2并转染到大鼠骨髓间充质干细胞（MSCs）,检测其在MSCs中核酸和蛋白水平是否表达。方法采用密度梯度离心法分离获得MSCs。EcoRⅠ和BamHⅠ双酶切质粒pGH-mHCN2和pIRES2-EGFP,回收目的片段,T4DNA连接酶连接。转化筛选阳性菌落,酶切和测序鉴定mHCN2是否插入pIRES2-EGFP中。脂质体转染质粒pIRES2-EGFP-mHCN2至MSCs,荧光显微镜下鉴定,并采用RT-PCR方法和Western blot方法分别检测转染质粒pIRES2-EGFP-mHCN2对mHCN2 mRNA和蛋白表达的影响。结果酶切鉴定和测序结果均证明mHCN2片段插入质粒pIRES2-EGFP。荧光显微镜下可见转染了质粒的MSCs发出绿色荧光。已转染质粒MSCs的mHCN2 mRNA的平均相对表达量是未转染MSCs的5.31倍（P〈0.05）,mHCN2蛋白是未转染MSCs的7.55倍（P〈0.05）。结论成功构建质粒pIRES2-EGFP-mHCN2,证明了目的基因mHCN2在MSCs中mRNA和蛋白均有表达,为进一步研究生物起搏器奠定了基础。

英文摘要：

Objective To construct plasmid expressing pacemaker gene pIRES2-EGFP-mHCN2 and transfect the plasmid to rat mesenchymal stem cells （MSCs）, and to detect the expression of mHCN2 mRNA and protein in transfected MSCs. Methods MSCs were obtained by density gradient centrifugation method. The plasmid pGH-mHCN2 and plasmid pIRES2- EGFP were digested by EcoR Ⅰ and BamH Ⅰ. The objective fragments were reclaimed and linked by T4 DNA ligase. Then the recombinant plasmid was transformed to the competent cells. We chose the masculine colony the next day. Restriction enzyme and sequencing method were used to proof that mHCN2 was inserted to pIRES2-EGFP. The objective gene was transfected with Lipofectamine 2000 into MSCs. The transfecting results were observed under fluorescence microscope. The expression of mHCN2 mRNA and protein in the transfected cells was detected by RT-PCR and Western blot. Results The identification using restriction enzyme and sequencing indicated that the mHCN2 was inserted to the pIRES2-EGFP. The green fluorescence could be seen in transfected MSCs after 24 to 48 h. The mHCN2 mRNA and protein in transfected MSCs was 5.31 and 7.55 times of the MSCs respectively （both P〈0.05）. Conclusion We successfully construct the plasmid pIRES2-EGFP-mHCN2, and mHCN2 mRNA and protein can be expressed in MSCs, which provides the foundation for studying the biologic pacemakers deeply.