绿色荧光蛋白标记基因体内示踪骨髓间充质干细胞的分化  

GREEN FLUORESCENT PROTEIN LABELING GENE TRANSFERRED INTO MESENCHYMAL STEM CELLS TO TRACE THEIR DIFFERENTIATION IN VIVO

作　　者：胡运生[1] 马保安[1] 李文海[2] 张勇[1] 范清宇[1] 

机构地区：[1]第四军医大学唐都医院骨肿瘤研究所暨全军骨科中心,西安710038 [2]第四军医大学唐都医院胸外科

出　　处：《中国修复重建外科杂志》2007年第6期633-637,共5页Chinese Journal of Reparative and Reconstructive Surgery

基　　金：国家自然科学基金资助项目(30330610)~~

摘　　要：目的利用绿色荧光蛋白（green fluorescent protein,GFP）标记技术,观察组织工程骨在体内形成过程中种子细胞的变化与转归。方法Adeno-X^TM-GFP扩增和纯化后,测定病毒滴度,感染新西兰大白兔骨髓间充质干细胞（mesenchymal stem cells,MSCs）,倒置相差显微镜观察细胞形态变化,并在荧光显微镜下观察GFP表达情况。确认标记成功后,将其与未标记的兔MSCs共同成骨诱导培养3周后,接种于磷酸钙骨水泥（calcium phosphate cement,CPC）-纤维蛋白胶（fibrin glue,FG）复合支架材料,构建组织工程骨作为实验组,回植于供体兔皮下。单纯CPC-FG复合支架材料植入对称部位作为对照。术后1、2、3周行碱性磷酸酶（alkaline phosphatase,ALP）活性测定;4周,激光共聚焦显微镜下对GFP进行示踪观察和型胶原、骨钙素（osteocalcin,OC）免疫组织化学检测成骨细胞功能蛋白,Masson染色观察新骨形成。结果Adeno-X^TM-GFP经扩增和纯化后,病毒噬菌斑形成,测得病毒滴度为3×108pfu/ml。Adeno-X^TM-GFP感染MSCs后96h,可见GFP表达,感染率达50%～70%,细胞分裂增殖基本正常。术后1、2、3周,实验组ALP活性分别为12.546±1.091、16.567±0.659和20.443±0.706,对照组分别为0.453±0.113、0.243±0.018和0.308±0.056,差异有统计学意义（P〈0.05）。4周,实验组大体可见组织工程新骨形成,对照组未见新骨形成;组织学显示实验组新生骨小梁围绕材料孔隙形成,CPC-FG复合支架材料部分降解;实验组型胶原、OC免疫组织化学结果阳性,对照组未见阳性染色;实验组可见新生组织内有GFP标记的细胞。结论组织工程骨组织结构与松质骨小梁类似,新生组织表达GFP,提示组织工程骨组织的形成来源于接种的细胞。Objective To observe the tissue engineered bone fabricated with the cultured mesenchymal stem cells （MSCs） by the green fluorescent protein （GFP） gene transfer. Methods The recombinant Adeno-X^TM-GFP expression vector was purified after being packed and proliferated by the HEK293 cells, and then it was used to infect the rabbit＇s MSCs directly after the virus titer was assayed. The cell morphological changes were observed under the inverted phase contrast microscope, and the expression of GFP was observed under the fluorescence microscope to confirm success of the labeling of MSCs. The GPF-labeled MSCs and the pure MSCs were cultured together in the conventional osteogenic supplements for 3 weeks, and then they were seeded onto the compound scaffold of the calcium phosphate cement （CPC） and the fibrin glue （FG） to form a new kind of the tissue engineered bone. It was implanted into the donator rabbit subcutaneously to be used as the experimental group; in contrast, the pure compound scaffold of the CPC-FG without any MSCs was implanted in the same rabbit as a control. The alkline phosphatase （ALP） activity assay was performed respectively at 1, 2 and 3 weeks after operation. GFP was observed under the laser confocal microscope at 4 weeks after operation, and the formed new bone was harvested at 4 weeks and evaluated by the Masson staining, the immunohistochemistry staining of osteocalcin （OC） and collagen type Ⅰ . Results The virus titer was 3×10^8 pfu/ml after proliferation and purification. Expresstion of GFP was confirmed 96 h after MSCs were infected by the Adeno-X^TM-GFP expression vector and the infection rate was proximally 50%-70%. In contrast to MSCs, division and proliferation of the GPF-labeled MSCs were not significantly different. The ALP activity in the experimental group （12. 546± 1. 091, 16. 567±0.659, 20. 443±0.706） was significantly higher than that in the control group （0.453±0.113, 0.243±0.018, 0.308±0.056）, respectively at 1, 2 and 3 weeks after oper

关 键 词：组织工程骨 骨髓间充质干细胞 腺病毒载体 绿色荧光蛋白 兔 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学]