LIF基因慢病毒载体构建及其在人脂肪间充质干细胞的表达

Construction of LIF gene lentiviral vector and its expression in human adipose-derived stem cells after transfection

目的构建LIF慢病毒载体,转染人脂肪间充质干细胞,以此获得高表达LIF的人脂肪间充质干细胞,为LIF在人脂肪间充质干细胞介导的免疫调节效应机制研究中奠定基础。方法以PCR扩增得到LIF基因序列,采用质粒重组技术获得载有LIF基因的重组质粒,然后用重组质粒包装慢病毒。用胶原酶消化法分离培养人脂肪间充质干细胞,然后将慢病毒转染人脂肪间充质干细胞,通过荧光显微镜观察绿色荧光蛋白的表达,采用流式细胞技术检测慢病毒转染率,RT-PCR及Western blot技术检测LIF的表达水平,最后经SPSS 13.0软件对数据进行统计学分析,计量资料均以(x±s)表示,组间均数比较采用成对样本t检验,以P<0.05为差异有统计学意义。结果通过PCR技术扩增得到LIF基因序列,并且成功构建带有LIF基因的重组质粒,重组质粒经限制性内切酶双酶切鉴定,得到大小为608 bp左右的条带,大小与LIF基因序列匹配,通过重组质粒转染293T细胞后获得慢病毒颗粒,滴度检测达1×108TU/ml。成功分离得到纯度高的h ASCs,慢病毒转染h ASCs后,流式细胞仪检测慢病毒转染率可达90%。RT-PCR及Western blot结果经统计学分析提示,转染后LIF的表达水平明显升高。结论携带人LIF基因的慢病毒载体可以有效转染h ASCs,并且表达LIF。

Objective To construct leukemia inhibitory factor（ LIF） lentiviral vector and transfect it into human adiposederived stem cells（ h ASCs）,then get a large number of LIF- h ASCs with a high expression of LIF protein,and to lay the foundation for the further study of the mechanisms involved in immune regulation. Methods LIF gene sequence was amplified by PCR,and then the sequence was subcloned into the Lentiviral expression vector,and the lentiviral packaging plasmids were used for the packaging and production of lentiviral particles. The human adipose-derived stem cells（ h ASCs） were isolated with collagenase Ⅰfrom the human adipose tissue,and then transfected by lentiviral particles. The expression of green fluorescent protein was observed by fluorescence microscopy,and the transfection efficiency of the LIF lentiviral vector to h ASCs was detected by flow cytometry. The level of LIF mRNA and LIF were detected by PCR and Western blot respectively. All the data were analyzed by the SPSS 13. 0 software： measurement data were expressed as mean ± standard deviation（ ^-x ± s）,and the groups were compared using paired samples T-test,while P〈0. 05 was considered significant statistically. Results A recombinant plasmid with sequence of LIF was constructed and confirmed by double digest,and a 608 bp fragment was obtained which was matched with LIF gene. The lentiviral particles with a titer of about 1 × 10^8 TU / ml were produced through transfecting the reconstructed plasmid into 293 T cells. The highly purified h ASCs was isolated and transfected with lentiviral particles with a transfection rate of approximate 90% by FCM. PCR and Western blot revealed that the level of LIF was up-regulated in h ASCs after transfection. Conclusion The LIF gene lentivirus vector can transfect h ASCs efficiently and the LIF can expressed in h ASCs stably.