骨髓间充质干细胞来源的成骨细胞与血管内皮生长因子基因整合的研究  

Activity study of bone marrow mesenchymal stem cells-derived osteoblast transfected by vascular endothelial growth factor gene

作　　者：郭晓华[1] 颉玉欣[1] 张牧霞[1] 赵驻军[1] 

机构地区：[1]河北医科大学第三医院实验中心,河北石家庄050051

出　　处：《临床荟萃》2010年第24期2129-2131,F0003,共4页Clinical Focus

摘　　要：目的大鼠骨髓间充质干细胞（MSCs）在药物诱导下定向分化为成骨细胞,对其进行血管内皮生长因子（VEGF）基因片段转染,观察其生物学特性的变化。方法选择MSCs体外培养的第2代细胞诱导其向成骨细胞分化,实验分为对照组和地塞米松浓度1×10-8mol/L诱导组。对诱导分化后的成骨细胞进行碱性磷酸酶（ALP）染色和活性测定及I型胶原的免疫组化染色、茜素红钙节结染色的鉴定。对诱导后的成骨细胞进行已构建的VEGF基因片段（ad5-h-VEGF）的人5型腺病毒载体进行转染,并进行免疫组化鉴定。结果诱导培养第3、6、9、12天的ALP活性（A405）测定,地塞米松浓度1×10-8mol/L组测定值（A值）分别为0.144±0.004、0.175±0.003、0.207±0.010、0.224±0.004;对照组分别为0.101±0.003、0.124±0.016、0.133±0.005、0.139±0.005。两组在组间、不同时点以及组间和不同时点的交互作用差异均有统计学意义（P〈0.05）。MSCs经地塞米松的成骨诱导剂诱导7~9天可出现碱性磷酸酶染色阳性,连续培养20天后可出现茜素红钙节结染色阳性。转染后细胞VEGF免疫组化染色阳性。结论对骨髓间充质干细胞来源的成骨细胞作ad5-h-VEGF基因转染,可使诱导的成骨细胞胞浆中VEGF阳性表达。Objective This experiment aims at exploring the best conditions of differentiation from bone marrow mesenchymal stem cells（MSCs） to osteoblast and observing the effects of extra vascular endothelial growth factor（VEGF） gene expression on the regeneration of osteoblast cells in rats.Methods In vitro,MSCs P2 cells were selected to be induced to osteoblasts.The cells were divided into two groups：control group and 1×10-8 mol/L DEX group.The expression of alkaline phosphatase（ALP） and type-Ⅰ collagen of the osteoblast cells were observed.Alizarin red staining was applied to observe the calcium nodules.The extra VEGF gene was transfered into the osteoblast cells in vitro by the type 5 adenovirus vector and analyzed by immunohistochemistry staining.Results ALP activity（A405）：the ALP activity of two groups at the day 3,6,9,12 was measured.The results of 1×10-8 mol/L DEX group were 0.144±0.004,0.175±0.003,0.207±0.010,0.224±0.004.The results of control group were 0.101±0.003,0.124±0.016,0.133±0.005,0.139±0.005.Significant statistical difference of interblock,time dissimilarity and interaction of interblock with time dissimilarity existed between two groups（P0.05）.The cells expressed positive staining of ALP when they were induced for 7-9 days and showed alizarin red positive staining of calcium nodules when they were induced for 20 days.The expression of extra VEGF in the osteoblast cells that derived from MSCs showed positive staining.Conclusion The ad5-h-VEGF gene could be integrated with the osteoblast cells that derived from MSCs and expressed in the cytoplasm.

关 键 词：间充质干细胞 成骨细胞 血管内皮生长因子 基因转染 

分 类 号：R683[医药卫生—骨科学]