人类BMP-2基因在兔骨髓MSCs的表达及其作用机制研究
发布时间：2018-08-12 17:00
【摘要】：目的：(1) 分离兔骨MSCs，体外培养扩增，观察其生物学性状及条件培养下成骨分化情况。(2) 提取hBMP-2 cDNA，构建其真核表达载体pIRES2-EGFP-hBMP2。(3) 将pIRES2-EGFP-hBMP2转染兔MSCs，观察hBMP-2 cDNA在兔MSCs的表达情况及其诱导成骨情况，，探讨其作用机制。 方法：(1) 抽取兔髂骨及胫骨的骨髓液，密度梯度离心法分离出MSCs，体外纯化鉴定培养扩增，相差显微镜下观察细胞的生物学性状。取第2代MSCs在条件培养基下成骨诱导，测定其ALP活性及成骨矿化情况。(2) RT-PCR方法获取人骨肉瘤细胞中的hBMP-2 cDNA，将此片段重组到pMD18-T克隆载体中，转化大肠杆菌DH5 α筛选扩增，并测序目的基因；酶切pMD18T-BMP2中的hBMP-2 cDNA，定向克隆到pIRES2-EGFP真核表达载体中，筛选扩增并鉴定重组质粒。(3) 用电穿孔法将pIRES2-EGFP-hBMP2转染至兔MSCs中，荧光显微镜下及流式细胞学检查观察转染效果、检测转染效率，定量RT-PCR方法观察hBMP-2 cDNA在兔MSCs中的转录情况，免疫组织化学及western blot方法检测MSCs中hBMP-2蛋白表达情况。连续培养转染后的兔MSCs，检测ALP活性及成骨矿化情况，观察表达的hBMP-2蛋白功能情况。 结果：(1) 兔MSCs为均一的成纤维形细胞，漩涡状贴壁生长，增殖成力强。在条件培养基中培养，兔MSCs具有向成骨细胞表型分化能力，细胞内ALP活性明显增强，可在两周时形成钙化结节。(2) 从人骨肉瘤细胞中提取到hBMP-2 cDNA，经测序后证明为hBMP-2基因全编码序列，并成功地构建hBMP-2 cDNA的真核表达载体。(3) 电穿孔方法可以将质粒pIRES2-EGFP-hBMP2转染至兔MSCs中，荧光显微镜下观察到强绿色荧光蛋白表达，流式细胞仪检测转染效率为(42.7±2.1)％。在转染24小时后定量RT-PCR方法可以检测到hBMP-2 cDNA在兔MSC中的逆转录片
[Abstract]:Objective: (1) to isolate and amplify MSCs from rabbit bone in vitro. (2) hBMP-2 cDNAwas extracted and its eukaryotic expression vector pIRES2-EGFP-hBMP2 was constructed. (3) pIRES2-EGFP-hBMP2 was transfected into rabbit MSCs. Methods: (1) Bone marrow fluid from iliac bone and tibia of rabbits were extracted and MSCs were isolated by density gradient centrifugation. The second generation of MSCs was induced by osteogenesis in conditioned medium, and its ALP activity and osteogenesis mineralization were determined. (2) RT-PCR method was used to obtain hBMP-2 cDNA in human osteosarcoma cells, and the fragment was recombined into pMD18-T clone vector and transformed into E. coli DH5 伪 for screening and amplification. The target gene was sequenced, the hBMP-2 cDNA of pMD18T-BMP2 was digested and cloned into the eukaryotic expression vector of pIRES2-EGFP, and the recombinant plasmid was amplified and identified. (3) the pIRES2-EGFP-hBMP2 was transfected into rabbit MSCs by electroporation, and the transfection effect was observed by fluorescence microscope and flow cytometry. The transfection efficiency was detected, the transcription of hBMP-2 cDNA in rabbit MSCs was observed by quantitative RT-PCR method, and the expression of hBMP-2 protein in MSCs was detected by immunohistochemistry and western blot. The ALP activity and osteogenic mineralization were detected and the function of the expressed hBMP-2 protein was observed. Results: (1) MSCs was a homogeneous fibroblast with swirl adherent growth and strong proliferation. Cultured in conditioned medium, rabbit MSCs had the ability to differentiate into osteoblast phenotype, and the activity of ALP in the cells increased obviously. Calcified nodules could be formed in two weeks. (2) hBMP-2 cDNAs were extracted from human osteosarcoma cells and confirmed to be the full coding sequence of hBMP-2 gene, and the eukaryotic expression vector of hBMP-2 cDNA was successfully constructed. (3) the plasmid pIRES2-EGFP-hBMP2 could be transfected into rabbit MSCs by electroporation. The expression of strong green fluorescent protein was observed under fluorescence microscope, and the transfection efficiency was (42.7 卤2.1) by flow cytometry. Reverse transcription of hBMP-2 cDNA in rabbit MSC can be detected by quantitative RT-PCR 24 hours after transfection.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R329.2
[Abstract]:Objective: (1) to isolate and amplify MSCs from rabbit bone in vitro. (2) hBMP-2 cDNAwas extracted and its eukaryotic expression vector pIRES2-EGFP-hBMP2 was constructed. (3) pIRES2-EGFP-hBMP2 was transfected into rabbit MSCs. Methods: (1) Bone marrow fluid from iliac bone and tibia of rabbits were extracted and MSCs were isolated by density gradient centrifugation. The second generation of MSCs was induced by osteogenesis in conditioned medium, and its ALP activity and osteogenesis mineralization were determined. (2) RT-PCR method was used to obtain hBMP-2 cDNA in human osteosarcoma cells, and the fragment was recombined into pMD18-T clone vector and transformed into E. coli DH5 伪 for screening and amplification. The target gene was sequenced, the hBMP-2 cDNA of pMD18T-BMP2 was digested and cloned into the eukaryotic expression vector of pIRES2-EGFP, and the recombinant plasmid was amplified and identified. (3) the pIRES2-EGFP-hBMP2 was transfected into rabbit MSCs by electroporation, and the transfection effect was observed by fluorescence microscope and flow cytometry. The transfection efficiency was detected, the transcription of hBMP-2 cDNA in rabbit MSCs was observed by quantitative RT-PCR method, and the expression of hBMP-2 protein in MSCs was detected by immunohistochemistry and western blot. The ALP activity and osteogenic mineralization were detected and the function of the expressed hBMP-2 protein was observed. Results: (1) MSCs was a homogeneous fibroblast with swirl adherent growth and strong proliferation. Cultured in conditioned medium, rabbit MSCs had the ability to differentiate into osteoblast phenotype, and the activity of ALP in the cells increased obviously. Calcified nodules could be formed in two weeks. (2) hBMP-2 cDNAs were extracted from human osteosarcoma cells and confirmed to be the full coding sequence of hBMP-2 gene, and the eukaryotic expression vector of hBMP-2 cDNA was successfully constructed. (3) the plasmid pIRES2-EGFP-hBMP2 could be transfected into rabbit MSCs by electroporation. The expression of strong green fluorescent protein was observed under fluorescence microscope, and the transfection efficiency was (42.7 卤2.1) by flow cytometry. Reverse transcription of hBMP-2 cDNA in rabbit MSC can be detected by quantitative RT-PCR 24 hours after transfection.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R329.2
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