破骨细胞转基因永生化问题的初步探讨

A Study on immortalization of osteoclasts

目的 通过转基因技术建立破骨细胞永生化细胞系。方法 经 1 ,2 5(OH) 2 D3 诱导 ,获得小鼠骨髓来源的破骨前体细胞。脂质体法 (Fugene 6)将猿猴病毒 40 (SV40 )和绿色荧光蛋白(GFP)质粒分别转染入破骨前体细胞 ,G41 8筛选抗性克隆 ;同时将GFP转染入逆转录病毒包装细胞(PT67)中 ,作为方法对照。继而 ,用含有GFP的逆转录病毒感染破骨前体细胞 ,G41 8筛选抗性克隆。结果 获得小鼠骨髓来源的破骨前体细胞。Fugene 6转染SV40和GFP到破骨前体细胞后 ,G41 8筛选未获得阳性克隆 ,但GFP转染PT67细胞后获得阳性克隆和含有GFP的逆转录病毒。将含有GFP的逆转录病毒感染破骨前体细胞 ,G41 8筛选未获得阳性克隆。结论 通过破骨细胞转基因永生化 ,建立破骨或破骨前体细胞系的方法是一个非常有意义的研究课题 ,但是难度较大。可以初步认为

Objective\ To build an immortalized osteoclast cell line by the method of transgene.Methods\ The osteoclast precursors were derived from the mouse bone marrow cells in the presence of 1,25(OH) 2 D 3 in vitro. Transfection of two plasmids(SV40 and GFP)into the above cells respectively was performed using liposome(Fugene 6).G418 was used to select the resistant cell clones.At the same time,the GFP plasmid was introduced into the retrovirus packing cells(PT67) as a contrast.Then the osteoclast precursors were infected using the retrovirus containing GFP.G418 was used to select the resistant cell clones.Results\ Firstly,no G418 resistant cell cloned after the transfection of the GFP and SV40 into osteoclast proecursors by Fugene 6.But the resistant cell cloned after transfection of GFP into PT67 cells by Fugene 6.Secondly,no positive infected cell cloned in the presence of the retrovirus containing GFP.Conclusions\ The cell immortalization by means of transgene is a plausible way to obtain the osteoclast cell lines although it is challenging.Liposome and retrovirus vector are not the best choice for osteoclasts to transgene.\;