插头/翼状螺旋转录因子C2基因慢病毒载体构建及其在兔BMSCs中的表达

CONSTRUCTION OF LENTIVIRAL VECTOR CONTAINING HOMO SAPIENS FORKHEAD BOX C2 GENE AND ITS EXPRESSION IN BONE MARROW MESENCHYMAL STEM CELLS OF RABBITS

目的构建插头/翼状螺旋转录因子C2(homo sapiens forkhead box C2,Foxc2)基因慢病毒载体并转染兔BMSCs,检测其在BMSCs中的表达,为进一步应用携带Foxc2基因的BMSCs移植治疗股骨头缺血性坏死奠定实验基础。方法通过RT-PCR法获得人Foxc2基因片段,将该片段克隆至包含绿色荧光蛋白(green fluorescent protein,GFP)的慢病毒载体LV-GFP中,重组获得Foxc2慢病毒质粒,将其与pGC-LV载体、pHelper1.0载体、pHelper2.0载体共转染293T细胞,获得Foxc2基因慢病毒载体;检测病毒滴度。分离、培养兔BMSCs,以Foxc2基因慢病毒载体转染第3代BMSCs,通过荧光表达法判定最佳感染复数(multiplicity of infection,MOI),并以最佳MOI进行转染,倒置荧光显微镜观察、Western blot法检测转染1、3、7 d BMSCs中Foxc2的表达;对转染后BMSCs行成骨诱导,茜素红染色法观察矿化物结节形成情况。结果成功构建Foxc2基因慢病毒载体,经酶切及测序鉴定完全正确,能转染293T细胞并表达GFP,病毒滴度为2×108TU/mL。Foxc2基因慢病毒转染BMSCs的最佳MOI为200,转染BMSCs 3 d后经免疫荧光检测84.5%±4.8%的BMSCs可表达Foxc2。Western blot结果显示Foxc2在BMSCs中高表达,且在7 d内表达水平逐渐升高。成骨诱导培养2周后,茜素红染色示细胞质中有大量红色的钙化基质沉积。结论成功构建并包装获得较高滴度的Foxc2基因慢病毒载体,慢病毒可高效并稳定转染BMSCs,Foxc2表达增加,为基因治疗股骨头缺血性坏死奠定了基础。

Objective To construct the lentiviral vector containing homo sapiens forkhead box C2 （Foxc2） gene and to detect its expression in bone marrow mesenchymal stem cells （BMSCs） of rabbits. Methods Human Foxc2 gene coding region fragment was obtained by RT-PCR and then cloned into the plasmid of LV-green fluorescent protein （GFP） to prepare Foxc2 lentiviral plasmid. Foxc2 lentiviral plasmid, pGC-LV, pHelperl.0, and pHelper2.0 were co-transfected into 293T cells to obtain recombinant virus containing Foxc2 gene. The lentiviral titer was detected. BMSCs were isolated from bone marrow of rabbit and infected with Foxc2 recombined lentiviral, then the optimum multiplicity of infection （MOI） was determined by detecting the intensity of fluorescence expression. The expression of Foxc2 in the infected BMSCs was determined at 1, 3, and 7 days after transfection by inverted fluorescence microscope and Western blot. After osteogenic induction, Alizarin red staining was done to observe the formation of mineralized nodule. Results The Foxc2 recombinant lentiviral vector was constructed and was confirmed by restriction enzyme digestion and sequencing analysis. It could efficiently transfect 293T cells and express in 293T cells. The lentiviral titer was 2 × 10s TU/mL. The optimum MOI was 200. The inverted fluorescence microscope observation showed that the Foxc2 gene expressed in 84.5%± 4.8% of infected BMSCs at 3 days after transfection. The expression of Foxc2 in infected BMSCs was stable and high, and increased gradually within 7 days after transfection by Western blot. At 2 weeks after osteogenic induction, Alizarin red staining showed that there were a large number of red calcified matrix deposition in the cytoplasm. Conclusion Foxc2 recombined lentivirus with high viral titer is successfully constructed and packaged, and the Foxc2 gene can be transfected into BMSCs with stable and high expression of Foxc2 in infected cells, and these cells may be applied for gene therapy of avascular necrosis of the femoral head.