本文選題：組織工程 + 骨髓基質(zhì)干細(xì)胞�。� 參考：《瀘州醫(yī)學(xué)院》2011年碩士論文

【摘要】：目的：通過(guò)全骨髓培養(yǎng)法培養(yǎng)、分離、純化骨髓基質(zhì)干細(xì)胞,構(gòu)建攜帶BMP2和VEGF165基因的重組pYr-adshuttle-4質(zhì)粒,并通過(guò)脂質(zhì)體介導(dǎo)將其轉(zhuǎn)染至骨髓基質(zhì)干細(xì)胞。將轉(zhuǎn)染后的BMSCs進(jìn)行體外培養(yǎng),觀察生物學(xué)特性、向成骨細(xì)胞分化的能力,探討B(tài)MP2和VEGF165雙基因修飾大鼠骨髓基質(zhì)干細(xì)胞與單基因修飾誘導(dǎo)成骨能力的差異。方法：1.取小鼠雙側(cè)股骨骨髓并采用全骨髓培養(yǎng)法培養(yǎng)進(jìn)行原代培養(yǎng),通過(guò)換液和傳代培養(yǎng)分離和純化骨髓基質(zhì)干細(xì)胞,傳至第3代用于后續(xù)實(shí)驗(yàn)。2.分別構(gòu)建攜帶BMP2和VEGF165基因的重組pYr-adshuttle-4質(zhì)粒。3.按照未轉(zhuǎn)染組、空載體組、BMP2單基因轉(zhuǎn)染組、VEGF165單基因轉(zhuǎn)染組、BMP2和VEGF165雙基因共同轉(zhuǎn)染組通過(guò)脂質(zhì)體介導(dǎo)分別轉(zhuǎn)染骨髓基質(zhì)干細(xì)胞。4.轉(zhuǎn)染后行常規(guī)細(xì)胞形態(tài)學(xué)觀察7天；48h通過(guò)RT-PCR檢測(cè)BMP2和VEGF165的mRNA含量,Western-Blot檢測(cè)BMP2和VEGF165蛋白表達(dá)變化；轉(zhuǎn)染后一周分別檢測(cè)各組細(xì)胞成骨能力—堿性磷酸酶。結(jié)果：1.全骨髓細(xì)胞培養(yǎng)法成功分離、培養(yǎng)、純化骨髓基質(zhì)干細(xì)胞,骨髓基質(zhì)干細(xì)胞呈長(zhǎng)梭形樣貼壁生長(zhǎng)。2.成功構(gòu)建攜帶骨形態(tài)發(fā)生蛋白-2、血管內(nèi)皮生長(zhǎng)因子-165的重組pYr-adshuttle-4質(zhì)粒3.脂質(zhì)體介導(dǎo)成功將攜帶BMP2和VEGF165基因的重組pYr-adshuttle-4質(zhì)粒轉(zhuǎn)染進(jìn)骨髓基質(zhì)干細(xì)胞,轉(zhuǎn)染率高,無(wú)細(xì)胞毒性作用,轉(zhuǎn)染后細(xì)胞生長(zhǎng)活力增強(qiáng)。4.轉(zhuǎn)染后BMSCs:RT-PCR檢測(cè)顯示BMP2+VEGF165雙基因共同轉(zhuǎn)染組mRNA水平表達(dá)高于骨形態(tài)發(fā)生蛋白-2組和血管內(nèi)皮生長(zhǎng)因子-165組單基因轉(zhuǎn)染。5.Western-Blot檢測(cè)顯示共同轉(zhuǎn)染組中BMP2和VEGF165基因組在蛋白水平表達(dá)量明顯大于單基因組。6.雙轉(zhuǎn)染組中骨髓基質(zhì)干細(xì)胞的ALP活性明顯高于各單轉(zhuǎn)染組(P0.05)結(jié)論：1.全骨髓培養(yǎng)法成功的培養(yǎng)、分離并純化得到骨髓基質(zhì)干細(xì)胞2.利用脂質(zhì)體介導(dǎo)BMP2和VEGF165基因pYr-adshuttle-4質(zhì)粒共同轉(zhuǎn)染BMSCs可在體外長(zhǎng)期高效穩(wěn)定共同表達(dá)BMP2和VEGF165基因,并誘導(dǎo)向成骨細(xì)胞分化。3.BMP-2、VEGF-165雙基因修飾骨髓基質(zhì)干細(xì)胞誘導(dǎo)成骨能力明顯高于單基因修飾骨髓基質(zhì)干細(xì)胞成骨能力。
[Abstract]:Aim: to isolate and purify bone marrow stromal cells by whole bone marrow culture, construct recombinant pYr-adshuttle-4 plasmid carrying BMP2 and VEGF165 genes, and transfect them into bone marrow stromal cells mediated by liposome.After transfection of BMSCs was cultured in vitro, the biological characteristics and the ability to differentiate into osteoblasts were observed, and the difference of osteogenic ability between BMP2 and VEGF165 double gene modified rat bone marrow stromal stem cells and single gene modified bone marrow stromal cells was discussed.Method 1: 1.Bone marrow stromal cells were isolated and purified from bilateral femur bone marrow of mice and cultured with whole bone marrow culture method. The bone marrow stromal cells were isolated and purified by liquid exchange and passage culture, and then transferred to the third passage for further experiment. 2.The recombinant pYr-adshuttle-4 plasmids containing BMP2 and VEGF165 genes were constructed, respectively.Bone marrow mesenchymal stem cells (BMSCs) were transfected by liposome mediated by liposome in the untransfected group and the empty vector group in the single gene transfection group of VEGF165 and the co-transfected group of BMP2 and VEGF165 genes.After transfection, the expression of BMP2 and VEGF165 protein was detected by RT-PCR and Western-Blot, and the osteogenic ability of each group was detected by alkaline phosphatase one week after transfection.The result is 1: 1.Bone marrow mesenchymal stem cells were isolated, cultured and purified successfully by whole bone marrow cell culture method. Bone marrow stromal cells grew as fusiform adherent cells.The recombinant pYr-adshuttle-4 plasmid containing bone morphogenetic protein-2 and vascular endothelial growth factor-165 was successfully constructed.The recombinant pYr-adshuttle-4 plasmid carrying BMP2 and VEGF165 genes was successfully transfected into bone marrow stromal cells by liposome-mediated transfection. The transfection rate was high and there was no cytotoxicity.After transfection, BMSCs:RT-PCR analysis showed that the expression of mRNA in BMP2 VEGF165 double gene cotransfection group was higher than that in bone morphogenetic protein-2 group and vascular endothelial growth factor -165 group. 5. Western-Blot analysis showed that BMP2 and VEGF165 genomes were in protein in co-transfected group.The level of expression was significantly higher than that of single genome.The ALP activity of bone marrow stromal cells in double transfection group was significantly higher than that in each single transfection group (P 0.05) conclusion: 1.Bone marrow stromal cells 2 were isolated and purified by whole bone marrow culture.Cotransfection of BMP2 and VEGF165 gene pYr-adshuttle-4 plasmid with liposome can effectively and stably co-express BMP2 and VEGF165 genes in vitro.The osteogenic ability of bone marrow mesenchymal stem cells modified with VEGF-165 gene was significantly higher than that with single gene modified bone marrow stromal stem cells.
【學(xué)位授予單位】：瀘州醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類(lèi)號(hào)】：R329

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