本文選題：人骨形態(tài)蛋白-2 + 腺病毒載體　； 參考：《福建醫(yī)科大學(xué)》2011年碩士論文

【摘要】：目的:(1)構(gòu)建人骨形態(tài)蛋白-2(Human bone morphogenetic protein-2,hBMP-2)基因的重組腺病毒載體并鑒定; (2)檢測hBMP-2基因轉(zhuǎn)染對SD大鼠骨髓間充質(zhì)干細(xì)胞(Bone marrow mesenchymal stem cells,BMSCs)的影響。 方法:(1)以人cDNA為模板PCR擴增hBMP-2基因,插入線性化的表達載體pAV-MCMV-EGFP-3FLAG后,轉(zhuǎn)化E.coli感受態(tài)細(xì)胞,經(jīng)菌落PCR鑒定陽性轉(zhuǎn)化子、陽性克隆測序無誤后,擴增、抽提,將帶有目的基因的腺病毒表達載體和攜帶有腺病毒大部分基因的輔助包裝質(zhì)粒共轉(zhuǎn)染293細(xì)胞進行病毒包裝擴增,PCR檢測目的基因、Western Blot檢測目的蛋白及終點稀釋法檢測病毒滴度; (2)分離純化SD大鼠BMSCs并體外擴增,通過腺病毒載體介導(dǎo)hBMP-2基因轉(zhuǎn)染BMSCs,分別通過熒光顯微鏡觀察熒光表達情況、蛋白質(zhì)水平來測定轉(zhuǎn)染后hBMP-2的表達、ALP定量測定鑒定成骨活性及MTT法評估hBMP-2對BMSCs的影響。 結(jié)果:(1)PCR獲得長度為1223bp的hBMP-2目的基因片段,同源重組表達載體經(jīng)陽性克隆PCR及測序鑒定,結(jié)果正確。 (2)293細(xì)胞內(nèi)包裝、擴增,經(jīng)Western blot及PCR鑒定無誤后,獲得病毒滴度為5x1010pfu/ml的重組腺病毒。 (3)從SD大鼠骨髓提取物中分離培養(yǎng)的細(xì)胞形態(tài)為梭形,呈鋪路石狀、漩渦狀生長,經(jīng)流式細(xì)胞儀檢測及多項分化能力鑒定符合BMSCs的特征。 (4)經(jīng)轉(zhuǎn)染hBMP-2基因后,BMSCs表達hBMP-2、ALP。MTT法檢測轉(zhuǎn)染hBMP-2基因后,BMSCs增殖能力明顯增強(P0.05)。 結(jié)論:成功構(gòu)建攜帶有hBMP-2基因的重組腺病毒載體,轉(zhuǎn)染BMSCs后可以表達hBMP-2、ALP,在體外明顯促進BMSCs的增殖。
[Abstract]:Objective to construct and identify the recombinant adenovirus vector of human bone morphogenetic protein human bone morphogenetic protein-2hBMP-2 gene. The effect of hBMP-2 gene transfection on bone marrow mesenchymal stem cells (BMSCs) of bone marrow mesenchymal stem cells (BMSCs) in SD rats was detected. Methods the hBMP-2 gene was amplified by PCR using human cDNA as template, inserted into the linearized expression vector pAV-MCMV-EGFP-3FLAG, then transformed into E.coli receptive cells. The positive transformants were identified by colony PCR. The positive clones were sequenced, amplified and extracted. The adenovirus expression vector with the target gene and the auxiliary packaging plasmid carrying most of the adenovirus gene were co-transfected into 293 cells. The virus packaging amplification was carried out. The target protein was detected by Western Blot and the titer of the virus was detected by end-point dilution method. BMSCs was isolated and purified from SD rats and amplified in vitro. HBMP-2 gene was transfected into BMSCs by adenovirus vector. Fluorescence expression was observed by fluorescence microscope. Protein level was used to determine the expression of hBMP-2 after transfection. The osteogenic activity was evaluated by ALP and the effect of hBMP-2 on BMSCs was evaluated by MTT method. Results the target gene fragment of hBMP-2 with length of 1223bp was obtained by PCR. The homologous recombinant expression vector was identified by positive clone PCR and sequencing, and the results were correct. The recombinant adenovirus with 5x1010pfu/ml titer was obtained by Western blot and PCR. The cells isolated from the bone marrow extracts of SD rats were spindle-shaped with paving stone and whirlpool growth. The results of flow cytometry and multiple differentiation were consistent with the characteristics of BMSCs. (4) after transfection of hBMP-2 gene, hBMP-2ALP.MTT assay was used to detect the proliferative ability of hBMP-2 gene. Conclusion: the recombinant adenovirus vector carrying hBMP-2 gene was successfully constructed, which could express hBMP-2ALP after transfection of BMSCs, and promote the proliferation of BMSCs in vitro.
【學(xué)位授予單位】：福建醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R329

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