【摘要】：目的：(1) 分離兔骨MSCs，體外培養(yǎng)擴(kuò)增，觀察其生物學(xué)性狀及條件培養(yǎng)下成骨分化情況。(2) 提取hBMP-2 cDNA，構(gòu)建其真核表達(dá)載體pIRES2-EGFP-hBMP2。(3) 將pIRES2-EGFP-hBMP2轉(zhuǎn)染兔MSCs，觀察hBMP-2 cDNA在兔MSCs的表達(dá)情況及其誘導(dǎo)成骨情況，，探討其作用機(jī)制。 方法：(1) 抽取兔髂骨及脛骨的骨髓液，密度梯度離心法分離出MSCs，體外純化鑒定培養(yǎng)擴(kuò)增，相差顯微鏡下觀察細(xì)胞的生物學(xué)性狀。取第2代MSCs在條件培養(yǎng)基下成骨誘導(dǎo)，測定其ALP活性及成骨礦化情況。(2) RT-PCR方法獲取人骨肉瘤細(xì)胞中的hBMP-2 cDNA，將此片段重組到pMD18-T克隆載體中，轉(zhuǎn)化大腸桿菌DH5 α篩選擴(kuò)增，并測序目的基因；酶切pMD18T-BMP2中的hBMP-2 cDNA，定向克隆到pIRES2-EGFP真核表達(dá)載體中，篩選擴(kuò)增并鑒定重組質(zhì)粒。(3) 用電穿孔法將pIRES2-EGFP-hBMP2轉(zhuǎn)染至兔MSCs中，熒光顯微鏡下及流式細(xì)胞學(xué)檢查觀察轉(zhuǎn)染效果、檢測轉(zhuǎn)染效率，定量RT-PCR方法觀察hBMP-2 cDNA在兔MSCs中的轉(zhuǎn)錄情況，免疫組織化學(xué)及western blot方法檢測MSCs中hBMP-2蛋白表達(dá)情況。連續(xù)培養(yǎng)轉(zhuǎn)染后的兔MSCs，檢測ALP活性及成骨礦化情況，觀察表達(dá)的hBMP-2蛋白功能情況。 結(jié)果：(1) 兔MSCs為均一的成纖維形細(xì)胞，漩渦狀貼壁生長，增殖成力強(qiáng)。在條件培養(yǎng)基中培養(yǎng)，兔MSCs具有向成骨細(xì)胞表型分化能力，細(xì)胞內(nèi)ALP活性明顯增強(qiáng)，可在兩周時(shí)形成鈣化結(jié)節(jié)。(2) 從人骨肉瘤細(xì)胞中提取到hBMP-2 cDNA，經(jīng)測序后證明為hBMP-2基因全編碼序列，并成功地構(gòu)建hBMP-2 cDNA的真核表達(dá)載體。(3) 電穿孔方法可以將質(zhì)粒pIRES2-EGFP-hBMP2轉(zhuǎn)染至兔MSCs中，熒光顯微鏡下觀察到強(qiáng)綠色熒光蛋白表達(dá)，流式細(xì)胞儀檢測轉(zhuǎn)染效率為(42.7±2.1)％。在轉(zhuǎn)染24小時(shí)后定量RT-PCR方法可以檢測到hBMP-2 cDNA在兔MSC中的逆轉(zhuǎn)錄片
[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 偽 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 鹵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.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2006
【分類號(hào)】：R329.2

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本文編號(hào)：2179693