慢病毒介导SOX9基因转染小鼠脂肪干细胞的实验研究

Experimental study of lentiviral vector of SOX9 gene transducing adipose stromal cells

目的构建携带SOX9基因的慢病毒载体，转染小鼠脂肪干细胞，观察SOX9基因在脂肪干细胞中的表达。方法将实验分为3组，体外培养、分化小鼠ADSCs，行免疫荧光鉴定后，使用RT—PCR的方法获取小鼠SOX9基因编码区片段，将该片段克隆入质粒Pwpxl．MOD2中产Pwpxl—MOD2．SOX9，将四质粒Pwpxl—MOD2-SOX9、pRsv—REV、pMDlg—pRRE、pMD2G共转染包装细胞293T，获得目的基因SOX9基因的重组病毒（实验组A）；同时转染Pwpxl-MOD2，pRsv-REV，pMDlg—pRRE，pMD2G进另一组293T细胞包装产生空载体慢病毒作为阴性对照（实验组B）；未转染组为实验组C。收集病毒后，通过基因测序和限制性核酸内切酶酶切的方法对质粒进行鉴定。将包装好的慢病毒Pwpxl．MOD2．SOX9体外转染脂肪干细胞，利用倒置荧光显微镜观察转染是否成功，并通过流式细胞仪测定转染效率。同时，利用RT—PCR和Westernblot检测小鼠SOX9基因的表达。结果成功分离、培养大鼠的脂肪干细胞；酶切、PCR及测序鉴定证实，慢病毒载体质粒Pwpxl—MOD中插人片段为基因SOX9，包装产生的病毒能较为高效转染小鼠脂肪干细胞。RT—PCR和Westernblot检测显示，经SOX9基因转染的小鼠脂肪干细胞表达目的基因产物。结论成功构建SOX9基因的慢病毒载体并感染脂肪干细胞后能够稳定表达SOX9，这为慢病毒及SOX9基因在软骨组织工程学中的进一步研究奠定了基础。

Objective To construct the lentiviral vector containing SOX9 gene and to detect its expression in ADSCs derived from mouse fat and observe the expression of target gene. Methods ADSCs divided into 3 groups were isolated, cultured, and then identified by immunofluorescence assay, mice SOX9 gene coding region fragment was obtained by RT-PCR and then cloned into the plasinid of Pwpxl-MOIY2 to form Pwp：d-MOD2/SOX9. Pwpxl- MOD2/SOX9, pRsv-REV, pMDlg-pRRE and pMD2G were eo-transfected into 293T ceils to obtain recombinant virus containing SOX9 gene （Group A）. Meanwhile Pwpxl-MOD2, pRsv-REV, pMDlg-pRRE and pMD2G were transfect- ed into another group of 293T cells as a control group packing into blank Lentiviral vector （ Group B）, the untrans- fected group as the experiinental group C. Then the packed Lenfiviral vector was transfected into ADSCs which de- rived from mouse fat, and the Lenti-soxg-ADSCs was selected by inversion fluorescence microscope. The expression of SOX9 gene was detected by RT-PCR and Western blot. Results We succeeded in separating and culturing mice ADSCs; the sequencing and restriction analysis showed that SOX9 gene fragment was correctly connected and cloned into the plasmid Pwpxl-MOD in Lentiviral vectors. The expression of Sox9 gene was confirmed by RT-PCR and Western blot. Conclusion Lenti-SOX9-ADSCs has the phenotypes of chondrocytes, and providing reliable genetic basis for the tissue-engineering in plastic applications.