hVEGF-165和hBMP-2真核共表达载体的构建及其转染对人骨髓间充质干细胞成骨能力的影响

发布时间：2018-01-09 08:37

本文关键词：hVEGF-165和hBMP-2真核共表达载体的构建及其转染对人骨髓间充质干细胞成骨能力的影响　出处：《中国协和医科大学》2005年博士论文　论文类型：学位论文

【摘要】：颅颌面部骨缺损是平、战时意外事故的常见伤类。骨形态发生蛋白(Bone Morphogenetic Proteins，BMPs)对骨原细胞的分化起主要的决定性作用，是促进成骨的重要因子；血管内皮生长因子(vascular endothelial growth factor，VEGF)是新近发现的在创伤愈合过程中促进血管再生发挥着重要作用的生长因子。本研究在创建颅骨缺损动物模型的基础上，采用免疫组化及原位杂交技术，动态观察了创伤后组织中BMP2、4及VEGF的变化。应用分子克隆和转基因技术，构建了hVEGF165与hBMP2真核细胞共表达载体——pIRES-hBMP2-hVEGF165，并应用组织工程技术进行人骨髓间充质干细胞(bone marrow mesenchymal stem cells，BMSCs)的分离和培养，在传代后采用阳离子脂质体介导的转染方式，将外源VEGF165及hBMP2 cDNA导入BMSCs，使其正常表达，以提高BMSCs的成骨能力，提供改良骨组织工程种子细胞，从而促进骨创伤愈合过程。本实验通过免疫组化、免疫印记、RT-PCR等方法检测基因转染后BMSC中的VEGF165、BMP2蛋白质骨钙素mRNA的表达程度，同时也观察了对BMSCI型胶原表达及碱性磷酸酶活性的影响。结果发现：VEGF在颅骨缺损后骨缺损3天时，骨缺损端内软骨细胞和成骨细胞呈阳性表达，基质内呈弱阳性表达，1周表达逐渐减弱；2周时表达增强，3周时软骨细胞分裂增生，呈团状排列，表达达到高峰，呈强阳性，4周时减弱，5周时仍可见部分细胞呈阳性。BMP2在骨缺损3天后，骨缺损端可见散在分布的未分化间充质细胞及细胞间质呈阳性。1周时原始骨痂成骨细胞、骨端骨细胞、新形成的软骨基质呈阳性。肉芽组织间质呈阳性。2周呈弱阳性，3周时，新形成得软骨细胞出现阳性染色，软骨基质也呈阳性。幼稚软骨细胞、骨痂表面成骨细胞呈阳性，4周时减弱，5周时仍可见部分细胞呈阳性。BMP4 mRNA在缺损后1周，骨折间充质细胞呈阳性，3周时表达明显增强，，4周时减弱，其余时间点未见表达。构建的重组质粒pIRES-hBMP2-hVEGF165经酶切和电泳及测序鉴定后证明构建正确。基因转染后BMSC能检测到BMP2和hVEGF165蛋白分泌量明显升高，促进BMSC合成I型胶原，提高其碱性磷酸酶活性以及骨钙素mRNA的表达程度与对照组相比，非常显著。这说明： 1．VEGF在颅骨缺损创伤后的表达与创伤愈合过程中新生血管形成的时间相似，说明VEGF参与调节了骨创伤愈合过程中的血管再生阶段，对毛细血管的发生有积极促进作
[Abstract]:Craniofacial bone defect is a common injury in wartime accidents. Bone morphogenetic protein (Bone
Morphogenetic Proteins, BMPs) on the differentiation of osteoprogenitor cells play a decisive role in the main, is an important factor in promoting osteogenesis; vascular endothelial growth factor (vascular endothelial, growth factor, VEGF) is to promote angiogenesis plays an important role in the growth factor in wound healing process of newly discovered. In this study the skull defect animal model, immunohistochemistry and in situ hybridization, observed the changes of BMP2,4 and VEGF after trauma in the tissue. The application of molecular cloning and transgenic technology, constructed vector pIRES-hBMP2-hVEGF165 co expression of hVEGF165 and hBMP2 in eukaryotic cells, and the application of tissue engineering of human bone marrow mesenchymal stem cells (bone marrow mesenchymal stem cells, BMSCs) were isolated and cultured, using cationic liposome mediated transfection method in the passage, the exogenous VEGF165 and hBMP2 cDN A into BMSCs, the normal expression, in order to improve the osteogenic ability of BMSCs, provides improved seed cells for bone tissue engineering, so as to promote the process of bone wound healing. This experiment by immunohistochemistry, Western blot and RT-PCR were used to detect gene transfection in BMSC VEGF165. The expression level of BMP2 protein osteocalcin mRNA, at the same time we also observed the effect on the expression of type BMSCI collagen and alkaline phosphatase activity.
The results showed that: VEGF in skull bone defect after 3 days, in the end of chondrocyte bone defect and osteoblasts showed positive expression of stroma showed weak positive expression, the expression decreased gradually 1 weeks; 2 weeks was enhanced at 3 weeks, the proliferated chondrocytes arranged in clusters, reaching a peak expression that was strongly positive, decreased at 4 weeks, 5 weeks still visible part of the cells were.BMP2 positive in bone defect 3 days after the end of bone defect scattered in the distribution of undifferentiated mesenchymal cells and stromal cells were positive for.1 weeks when the primary callus bone cells, bone bone cells, cartilage matrix the formation of granulation tissue stroma was positive. Positive.2 weeks were weakly positive at week 3, the newly formed cartilage cells appeared positive staining of cartilage matrix is also positive. Immature cartilage cells, bone callus surface cells were positive, decreased at 4 weeks, 5 weeks still visible part of the cells were positive for.BMP4 in the mRNA defect After 1 weeks, the mesenchymal cells in the fracture were positive, the expression was obviously enhanced at 3 weeks and decreased at 4 weeks, and no expression was found at the rest of the time.
The recombinant plasmid pIRES-hBMP2-hVEGF165 was verified by enzyme digestion and electrophoresis and sequencing proved correctly constructed. After transfection of BMSC gene can be detected BMP2 and hVEGF165 protein secretion increased significantly, BMSC promote the synthesis of collagen type I, increase the activity of alkaline phosphatase and osteocalcin mRNA expression level compared with the control group that is very significant:
The expression of 1.VEGF in traumatic skull defect is similar to the time of neovascularization during wound healing. It indicates that VEGF is involved in regulating the stage of angiogenesis in the process of bone wound healing, and has a positive effect on the occurrence of capillaries.

【学位授予单位】：中国协和医科大学
【学位级别】：博士
【学位授予年份】：2005
【分类号】：R346

【引证文献】