MicroRNA-194过表达与抑制表达慢病毒载体的构建及对骨肉瘤细胞转染与筛选的方法学研究

目的：构建针对人microRNA—194过表达及抑制表达的慢病毒表达载体，寻找与探讨感染人骨肉瘤细胞系SO-SP-9607和U2-os的最佳步骤和方法。方法：利用PcR方法调取相应目的基因进行酶切，经电泳回收后与目的基因进行连接，产物转化细菌感受态细胞，对克隆进行PcR鉴定和测序对比分析后，构建相应microRNA—194过表达及抑制表达慢病毒表达载体；在人骨肉瘤细胞系SO-SP-9607和U2—os转染及筛选过程中，根据不同阶段及浓度设定相应实验组，并设相应对照组。倒置显微镜观察转染效率，筛选情况，进行比较。结果：PCR及测序结果证实重组慢病毒表达质粒构建正确。过表达及抑制表达重组慢病毒的滴度分别为15×10^8TU／ml及4×10^8TU／ml。感染复数（multiply of infection，MOI）值测定，实验组及对照组转染效率无明显差异，获得MOI值及感染时间数据。通过新的综合设计，经筛选后获得转染效率满意的目的克隆细胞。结论：成功构建了microRNA—194过表达及抑制表达慢病毒表达载体，并通过新的综合考虑设计，对人骨肉瘤细胞系SO-SP-9607和U2-os进行转染和筛选后，可较快和较高效率获得满意目的细胞。

Construction of recombinant lentivirus vector expressing has - miR - 194 and transfection and selection of human osteosarcoma cell line SO -SP -9607 and U2 -os

Objective：To construct a lentivirus vector expressing microRNA （miRNA） - 194 and discuss the best way and procedure of tansfeeting and selecting human osteosareoma cell line SO - SP - 9607 and U2 - os. Methods： Target gene which was digestied amplified by PC R and then connected with target gene,which was inserted into plenty - GFP vector. The cells were divided into different levels and groups in different stages of transfeetion and selection of human osteosareoma cell line SO - SP - 9607 and U2 - os,which was obserwed and compared under inverted fluores- cence microscope. Results： Restriction analysis and sequencing proved that recombinant lentivirul expression vector was constructed correctly. The titer of obtained overexpression and suppression expression recombinant lentivirus was 1.5 × 10^8TU/ml and 4 × 10^8TU/,Ul. MOI was measured and no obvious difference of was obsmwed in experimental group and control group. Objective cells were successfully obtained through innovative design. Conclusion：The lenti- viral expression vector for mieroRNA - 194 was successfully constructed. Cells of human osteosareoma cell line SO - SP - 9607 and U2 - os after transfeetion and selection were obtained faster and more efficient through comprehensive and innovative design.