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  本文關鍵詞：肝細胞生長因子基因修飾的骨髓間充質干細胞移植的促血管新生作用　出處：《華中科技大學》2010年博士論文　論文類型：學位論文

  更多相關文章： 肝細胞生長因子 慢病毒載體 基因治療 肝細胞生長因子 間充質干細胞 慢病毒載體 凋亡 血管新生 基因治療 肝細胞生長因子 干細胞 移植 外周動脈疾病

【摘要】： 第一部分人肝細胞生長因子重組慢病毒載體的構建及病毒的制備 目的：構建能表達重組人肝細胞生長因子(rhHGF)基因的第三代慢病毒載體,為實現(xiàn)rhHGF基因在大鼠骨髓間充質干細胞中長期、高效的表達奠定基礎。 方法：(1)通過NheI/XbaI雙酶切將pRNAT-U6.2慢病毒載體質粒中GFP基因敲除,然后將目的基因hHGF連接到該載體質粒上,構建重組載體質粒pRNAT-U6.2-hHGF。通過限制性內(nèi)切酶酶切、菌落PCR和DNA測序進行鑒定。(2)利用TELEVECTORTM載體試劑盒將pRNAT-U6.2-hHGF聯(lián)合輔助質粒pMDLg/pRRE、pMD2G-VSVG、pRSV-Rev共轉染293T細胞,收集并濃縮慢病毒上清,逐孔稀釋法測病毒滴度。 結果：(1)經(jīng)限制性內(nèi)切酶酶切及DNA測序分析表明,目的基因hHGF-cDNA已經(jīng)插入載體質粒,且基因序列與基因庫序列完全一致；(2)成功制備表達hHGF的慢病毒顆粒濃縮液,滴度為4×108Tu/ml。 結論：表達人肝細胞生長因子的第三代重組慢病毒載體pRNAT-U6.2-hHGF構建成功。 第二部分人肝細胞生長因子在大鼠骨髓間充質干細胞中的表達及生物學活性觀察 目的：觀察慢病毒載體介導的人肝細胞生長因子(hHGF)基因在骨髓間充質干細胞(BM-MSCs)中的表達及其生物學活性。 方法：(1)利用差速貼壁法聯(lián)合Percoll密度梯度離心法分離、純化大鼠原代BM-MSCs,流式鑒定MSC的表型,茜素紅染色和油紅O染色鑒定BM-MSCs的成骨、成脂分化潛能。(2)用高滴度表達hHGF (Lenti-HGF)或GFP (Lenti-GFP)的慢病毒液感染第三代的BM-MSCs,通過倒置相差熒光顯微鏡和流式鑒定來評估轉染效率。ELISA檢測細胞培養(yǎng)基上清中的hHGF濃度。(3)分別通過MTT法、Transwell小室法和Hoechst染色法檢測臍靜脈內(nèi)皮細胞(HUVECs)和血管平滑肌細胞(VSMCs)的增殖、遷移及凋亡,評估感染Lenti-HGF的BM-MSCs分泌的hHGF的生物學活性。 結果：(1)通過聯(lián)合差速貼壁法和密度梯度離心法可獲得大量高純度、具有多向分化潛能的BM-MSCs。(2)在轉染Lenti-GFP后72h,即可在熒光顯微鏡下見明亮的綠色熒光,最佳感染復數(shù)(MOI)為50,流式鑒定轉染效率約為75%。ELISA結果顯示,Lenti-HGF感染BM-MSCs的培養(yǎng)上清液中的hHGF可持續(xù)較高水平地表達14d以上,且明顯高于感染Lenti-GFP者(P0.01)。(3)感染Lenti-HGF的MSCs分泌的hHGF在體外顯示出促HUVECs增殖、遷移并抑制其凋亡,且能促進VSMCs遷移的生物學活性。 結論：Lenti-HGF慢病毒能夠高效地介導轉移外源基因hHGF至BM-MSCs表達,并具有生物學活性。 第三部分肝細胞生長因子基因修飾的骨髓間充質干細胞移植的促血管新生作用 目的：比較hHGF基因修飾的BM-MSCs移植與單純BM-MSCs移植在大鼠后肢缺血模型中的促血管新生作用及其機制。 方法：在結扎左股動脈及其分支1天后,SD大鼠隨機分為3組：HGF-MSC組(即HGF基因修飾的MSCs移植,n=8),MSC組(單純的MSCs移植,n=8)以及PBS對照組(注射PBS,n=8)。在術后7天,進行細胞移植。實驗組每只大鼠接受總計5×106個同一來源的HGF-MSCs或MSCs,分成5個點注入大鼠缺血后肢的股部肌肉；對照組注入同等體積的無菌PBS。分別在處理后1周或3周時處死大鼠,比較各組缺血股部肌肉中的毛細血管密度,移植細胞的分化轉歸,T淋巴細胞浸潤以及生長因子HGF、VEGF蛋白表達等。ELISA檢測HGF-MSC移植前和移植3周后大鼠血清HGF的水平。 結果：細胞移植3周后,MSC組和HGF-MSC組缺血肌肉組織的血管新生均明顯增強,以HGF-MSC組的毛細血管密度最高(P0.05)。從移植后1周開始,HGF-MSC組缺血組織中來源于移植細胞的內(nèi)皮細胞數(shù)目明顯高于MSC組(P0.05)。在移植1周后,HGF-MSC和MSC的同種異體移植幾乎不引起T淋巴細胞浸潤,與PBS組相近(P0.05)。Western-blot結果顯示,MSC組和HGF-MSC組缺血肌肉組織中HGF、VEGF蛋白表達均明顯高于PBS對照組,以HGF-MSC組的表達量最高(P0.05)。HGF-MSC移植前和移植后3周,大鼠血清HGF濃度無明顯變化(P0.05)。 結論：與單純的MSC細胞移植療法相比,HGF基因修飾的MSC移植療法具有更強的促進血管新生和側枝血管形成的能力。以干細胞為基礎的血管新生基因治療策略將可能為治療嚴重缺血性心血管病提供新思路。
[Abstract]:The first part of human hepatocyte growth factor recombinant lentivirus vector construction and virus preparation
Objective: to construct the third generation lentiviral vector expressing recombinant human hepatocyte growth factor (rhHGF) gene, and lay a foundation for long-term and efficient expression of rhHGF gene in rat bone marrow mesenchymal stem cells.
Methods: (1) NheI/XbaI by double enzyme digestion of pRNAT-U6.2 lentiviral vector plasmid GFP gene knockout, the target gene was then connected to the hHGF vector, the recombinant plasmid pRNAT-U6.2-hHGF. by restriction endonuclease, colony PCR and DNA sequencing were identified. (2) the pRNAT-U6.2-hHGF helper plasmid pMDLg/pRRE the use of TELEVECTORTM, carrier pMD2G-VSVG kit, pRSV-Rev 293T cells were co transfected with lentivirus supernatant was collected and concentrated by hole dilution method, the titer was measured.
Results: (1) restriction endonuclease digestion and DNA sequencing analysis showed that the target gene hHGF-cDNA had been inserted into the plasmid vector, and the gene sequence was exactly the same with the gene bank sequence. (2) the lentiviral particle concentrate expressing hHGF was successfully prepared, with a titer of 4 * 108Tu/ml..
Conclusion: the third generation recombinant lentivirus vector pRNAT-U6.2-hHGF expressing human hepatocyte growth factor was constructed successfully.
Expression and biological activity of human hepatocyte growth factor in bone marrow mesenchymal stem cells of rats in second parts
Objective: To observe the expression of human hepatocyte growth factor (hHGF) gene mediated by lentivirus vector and its biological activity in bone marrow mesenchymal stem cells (BM-MSCs).
Methods: (1) using the differential adherence method combined with Percoll density gradient centrifugation, purified rat BM-MSCs, MSC phenotype was analyzed by flow cytometry, osteoblast alizarin red staining and oil red O staining BM-MSCs, adipogenic differentiation potential. (2) the expression of hHGF with high titer (Lenti-HGF) or GFP (Lenti-GFP) lentivirus infection liquid third generation BM-MSCs by inverted fluorescent microscopy and flow cytometry to evaluate the concentration of hHGF radicals in the supernatant of.ELISA cells. The transfection efficiency was detected (3) were detected by MTT method, human umbilical vein endothelial cells by Transwell assay and Hoechst staining (HUVECs) and vascular smooth muscle cell (VSMCs) proliferation, migration and apoptosis, biological activity evaluation of BM-MSCs infection Lenti-HGF secretion of hHGF.
Results: (1) through a combination of differential centrifugation and density gradient centrifugation to obtain large quantities of high purity, multilineage differentiation potential of BM-MSCs. (2) in Lenti-GFP after transfection of 72h, can be seen under fluorescence microscope in bright green fluorescence, optimal multiplicity of infection (MOI) was 50. The transfection efficiency is about 75%.ELISA results showed that the level of sustainable high hHGF culture supernatant of Lenti-HGF infection in the BM-MSCs expression of 14d, and was significantly higher than that of Lenti-GFP infection (P0.01). (3) Lenti-HGF infection MSCs secretion of hHGF in vitro showed HUVECs proliferation, migration and apoptosis, and can promote the biological activity of VSMCs migration.
Conclusion: Lenti-HGF lentivirus can efficiently mediate the transfer of exogenous gene hHGF to BM-MSCs, and has biological activity.
The effect of third parts of hepatocyte growth factor gene modified bone marrow mesenchymal stem cell transplantation on angiogenesis
Objective: To compare the angiogenesis and its mechanism of hHGF gene modified BM-MSCs transplantation and simple BM-MSCs transplantation in rat hind limb ischemia model.
Method: after ligation of the left femoral artery and its branches after 1 days, SD rats were randomly divided into 3 groups: group HGF-MSC (MSCs transplantation, HGF gene modified n=8), group MSC (MSCs transplantation, pure n=8) and PBS group (PBS injection, n=8). After 7 days, cells transplantation of experimental group. Each rat received a total of 5 * 106 the same source HGF-MSCs or MSCs, thigh muscle was divided into 5 points into the hind limb ischemia in rats; the control group injected the same volume of sterile PBS. respectively after treatment for 1 weeks or 3 weeks when the rats were sacrificed and the capillary density in the ischemic group the thigh muscles, the outcome of differentiation of the transplanted cells, T lymphocytes and HGF growth factor, VEGF protein expression of.ELISA HGF-MSC detected before and 3 weeks after transplantation of rat serum HGF level.
Results: 3 weeks after cell transplantation, MSC group and HGF-MSC group ischemic muscle tissue angiogenesis was significantly enhanced in HGF-MSC group the highest vascular density (P0.05). From the 1 week after transplantation, the number of transplanted cells to endothelial cells from ischemic tissue in HGF-MSC group was significantly higher than in MSC group (P0.05). 1 weeks after transplantation, allogeneic transplantation of HGF-MSC and MSC almost does not cause T lymphocyte infiltration, similar to those in PBS group (P0.05).Western-blot results showed that MSC group and HGF-MSC group ischemic muscle tissue HGF, VEGF protein expression were significantly higher than those in PBS group, the expression of HGF-MSC group was the highest (P0.05).HGF-MSC for 3 weeks before transplantation and after transplantation, rat serum HGF concentration did not change significantly (P0.05).
Conclusion: compared with MSC cell transplantation therapy alone, HGF gene modified MSC transplantation therapy has a stronger ability to promote angiogenesis and collateral vessel formation. Angiogenesis gene therapy based on stem cells would be for the treatment of severe ischemic cardiovascular disease to provide new ideas.

【學位授予單位】：華中科技大學
【學位級別】：博士
【學位授予年份】：2010
【分類號】：R329

【引證文獻】

相關期刊論文 前1條

1 朱德星;宋莉;;肝細胞生長因子基因修飾骨髓間充質干細胞的研究進展[J];南昌大學學報(醫(yī)學版);2012年04期

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本文編號：1360205