發(fā)布時間：2018-04-13 18:52

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

【摘要】：目的：通過全骨髓培養(yǎng)法培養(yǎng)、分離、純化骨髓基質(zhì)干細胞,構(gòu)建攜帶BMP2和VEGF165基因的重組pYr-adshuttle-4質(zhì)粒,并通過脂質(zhì)體介導將其轉(zhuǎn)染至骨髓基質(zhì)干細胞。將轉(zhuǎn)染后的BMSCs進行體外培養(yǎng),觀察生物學特性、向成骨細胞分化的能力,探討B(tài)MP2和VEGF165雙基因修飾大鼠骨髓基質(zhì)干細胞與單基因修飾誘導成骨能力的差異。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)進行原代培養(yǎng),通過換液和傳代培養(yǎng)分離和純化骨髓基質(zhì)干細胞,傳至第3代用于后續(xù)實驗。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)染組通過脂質(zhì)體介導分別轉(zhuǎn)染骨髓基質(zhì)干細胞。4.轉(zhuǎn)染后行常規(guī)細胞形態(tài)學觀察7天；48h通過RT-PCR檢測BMP2和VEGF165的mRNA含量,Western-Blot檢測BMP2和VEGF165蛋白表達變化；轉(zhuǎn)染后一周分別檢測各組細胞成骨能力—堿性磷酸酶。結(jié)果：1.全骨髓細胞培養(yǎng)法成功分離、培養(yǎng)、純化骨髓基質(zhì)干細胞,骨髓基質(zhì)干細胞呈長梭形樣貼壁生長。2.成功構(gòu)建攜帶骨形態(tài)發(fā)生蛋白-2、血管內(nèi)皮生長因子-165的重組pYr-adshuttle-4質(zhì)粒3.脂質(zhì)體介導成功將攜帶BMP2和VEGF165基因的重組pYr-adshuttle-4質(zhì)粒轉(zhuǎn)染進骨髓基質(zhì)干細胞,轉(zhuǎn)染率高,無細胞毒性作用,轉(zhuǎn)染后細胞生長活力增強。4.轉(zhuǎn)染后BMSCs:RT-PCR檢測顯示BMP2+VEGF165雙基因共同轉(zhuǎn)染組mRNA水平表達高于骨形態(tài)發(fā)生蛋白-2組和血管內(nèi)皮生長因子-165組單基因轉(zhuǎn)染。5.Western-Blot檢測顯示共同轉(zhuǎn)染組中BMP2和VEGF165基因組在蛋白水平表達量明顯大于單基因組。6.雙轉(zhuǎn)染組中骨髓基質(zhì)干細胞的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)干細胞2.利用脂質(zhì)體介導BMP2和VEGF165基因pYr-adshuttle-4質(zhì)粒共同轉(zhuǎn)染BMSCs可在體外長期高效穩(wěn)定共同表達BMP2和VEGF165基因,并誘導向成骨細胞分化。3.BMP-2、VEGF-165雙基因修飾骨髓基質(zhì)干細胞誘導成骨能力明顯高于單基因修飾骨髓基質(zhì)干細胞成骨能力。
[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.
【學位授予單位】：瀘州醫(yī)學院
【學位級別】：碩士
【學位授予年份】：2011
【分類號】：R329

【參考文獻】

相關期刊論文 前10條

1 晏霆,朱滿洲;基因治療與基因載體[J];安徽醫(yī)藥;2002年04期

2 陳鵬;劉冰;毛天球;;納米羥基磷灰石/膠原/聚乳酸材料復合自體骨髓基質(zhì)細胞修復犬下頜骨節(jié)段性缺損的實驗研究[J];北京口腔醫(yī)學;2009年04期

3 施斌;林李嵩;邱宇;朱小峰;林耿冰;黃立;;快速原型技術在下頜骨個性化重建中的應用[J];福建醫(yī)科大學學報;2009年03期

4 曹建華;孔新秀;陳群蓉;蔡朝民;;年齡和性別與血清肝功能相關酶的關系探討[J];檢驗醫(yī)學與臨床;2010年04期

5 陳濤;李寧毅;;血管化復合組織工程骨的構(gòu)建及在實驗性下頜骨缺損修復中的應用[J];山東醫(yī)藥;2009年07期

6 王茸影,易靜;骨形成蛋白調(diào)控成骨分化的信號機制[J];生命科學;2005年01期

7 鄧洪新,田聆,魏于全;基因治療的發(fā)展現(xiàn)狀、問題和展望[J];生命科學;2005年03期

8 李建鑫;楊亮;王文良;;脂肪間充質(zhì)干細胞在組織工程中的應用[J];中國組織工程研究與臨床康復;2010年07期

9 丁金勇;靳安民;;生物支架材料在骨組織工程中的研究進展[J];中國矯形外科雜志;2006年01期

10 郁衛(wèi)東,秦書儉;骨形態(tài)發(fā)生蛋白-2在骨形成過程中的作用機制[J];中國臨床解剖學雜志;2000年01期

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