pLEGFP—N1-基质细胞衍生因子-1α的构建与目的基因在恒河猴骨髓基质细胞内的稳定表达

Construction and stable expression of pLEGFP-Nl-stromal cell-derived factor-1α in bone marrow stromal cells from rhesus

目的 构建携带人基质细胞衍生因子（SDF）1α基因的反转录病毒真核表达载体pLEGFP-N1-SDF-1α,转染恒河猴骨髓基质细胞（BMSC）,观察外源性SDF-1α和增强型绿色荧光蛋白（EGFP）基因在BMSC中的表达情况.方法 应用基因重组技术,从pBudce4.1-SDF-1α获得SDF-1α基因片段,重组到pLEGFP-N1真核表达载体上.pLEGFP-N1-SDF-1α经病毒包装,转染至恒河猴BMSC,用Western免疫印迹和免疫细胞化学检测表达情况.结果 酶切、PCR和DNA序列鉴定均证实插入基因片段的正确性.BMSC转染pLEGFP-N1-SDF-1α后,在荧光显微镜下发出绿色荧光.western免疫印迹和免疫细胞化学证实SDF-1α在细胞内有效表达.结论 成功构建本研究pLEGFP-N1-SDF-1α,经病毒包装转染至恒河猴BMSC,SDF-1α和EGFP基因在BMSC内有效表达.为BMSC-SDF-1α-EGFP工程细胞自体移植治疗相关疾病提供了依据.

Objective To construct a retrovirus eukaryotic expression vector pLEGFP-N1-stromal cell-derived factor-1α （SDF）- 1α that contains human SDF-1α and transfects bone marrow stromal cells （BMSCs） of rhesus, and to examine the expression of exogenous SDF- 1α and EGFP genes in BMSCs .Methods SDF-1α gene obtained from pBudce4.1-SDF-1α was recombined into pLEGFP-N1 vector to generate pLEGFP-N1-SDF-1α by use of genetic recombination techniques. pLEGFP-N1-SDF-1α packaged in virus was transfected into BMSCs of rhesus. Western blotting and immunocytochemistry were used for detection of its expression. Results The inserted gene was verified by enzyme restriction analysis, PCR and DNA sequencing. After transfected with pLEGFP-N1-SDF-1α, the BMSCs emitted green fluorescence, and expressed SDF- 1α as confirmed by Western blotting and immunocytochemistry. Conclusion After transfected to BMSCs of rhesus in virus, pLEGFP-N 1-SDF-1α may effectively express SDF-1α and EGFP,which provides evidences for auto-grafting of BMSC-SDF-1α-EGFP engineered cells in treatment of certain diseases.