摘要
目的制备重组BIG-3(BMP-2 induced gene 3 Kb,BIG-3)逆转录病毒并初步探讨其对人骨髓基质细胞生物学功能的影响。方法将pGEX 4T-2-BIG-3和pMSCVpuro双酶切,回收凝胶,行连接反应,连接产物转化DH5α感受态细胞,提取质粒,采用菌液PCR方法鉴定所提质粒,以此质粒转染PT67细胞制备重组BIG-3逆转录病毒并用PCR法鉴定;培养人骨髓基质细胞,待其达60%汇合时加入含重组BIG-3逆转录病毒的细胞培养上清,嘌罗霉素筛选,筛选持续10d后所得细胞行碱性磷酸酶染色和钙结节染色。结果重组质粒pMSCVpuro-BIG-3的菌液PCR法和重组BIG-3逆转录病毒的PCR法电泳图片均在1Kb处有清晰的单一条带,符合预期目的,碱性磷酸酶染色和钙结节染色均呈强阳性。结论成功制备BIG-3逆转录病毒,BIG-3可使人骨髓基质细胞向成骨细胞方向分化。
Objective To prepare the recombinant retrovirus BIG-3 and evaluate its effect on biological function of human bone marrow stromal cells （hBMSC）. Methods The pEGX4T-2-BIG-3 and pMSCVpuro were digested by enzymes and pMSCVpuro-BIG-3 plasmid was constructed by the linked reaction. The PT67 cell was transfected with pMSCVpuro-BIG-3 plasmld to produce the recombinant retrovirus BIG-3. The pMSCVpuro-BIG-3 plasmid and the retrovirus were identified by PCR. , after 60% fusion of hBMSCs was achieved, the cells were infected with recombinant BIG-3 retrovirus. Seven days later, the DMEM with 5~g/ml puromycin was used to select the cells. Through 10 day＇s consecutive treatment by puromycin, the positive cells were selected and stained with alkaline phosphatase and Von Kossa. Results Polyacrylamide gel electrophoresis demonstrated the existence of the special 1Kb band both in pMSCVpuro-BIG-3 plasmid and recombinant retrovirus BIG-3 by PCR, and the staining of ALP and Von kossa was positive. Conclusions We successfully developed the recombinant retrovirus BIG-3 which may induce hBMSCs to differentiate into osteoblasts.
出处
《中国骨肿瘤骨病》
2007年第6期328-331,共4页
Chinse Journal Of Bone Tumor And Bone Disease
