肝细胞生长因子基因修饰的骨髓间充质干细胞移植的促血管新生作用

发布时间：2017-12-31 14:37

本文关键词：肝细胞生长因子基因修饰的骨髓间充质干细胞移植的促血管新生作用　出处：《华中科技大学》2010年博士论文　论文类型：学位论文

【摘要】： 第一部分人肝细胞生长因子重组慢病毒载体的构建及病毒的制备目的：构建能表达重组人肝细胞生长因子(rhHGF)基因的第三代慢病毒载体,为实现rhHGF基因在大鼠骨髓间充质干细胞中长期、高效的表达奠定基础。方法：(1)通过NheI/XbaI双酶切将pRNAT-U6.2慢病毒载体质粒中GFP基因敲除,然后将目的基因hHGF连接到该载体质粒上,构建重组载体质粒pRNAT-U6.2-hHGF。通过限制性内切酶酶切、菌落PCR和DNA测序进行鉴定。(2)利用TELEVECTORTM载体试剂盒将pRNAT-U6.2-hHGF联合辅助质粒pMDLg/pRRE、pMD2G-VSVG、pRSV-Rev共转染293T细胞,收集并浓缩慢病毒上清,逐孔稀释法测病毒滴度。结果：(1)经限制性内切酶酶切及DNA测序分析表明,目的基因hHGF-cDNA已经插入载体质粒,且基因序列与基因库序列完全一致；(2)成功制备表达hHGF的慢病毒颗粒浓缩液,滴度为4×108Tu/ml。结论：表达人肝细胞生长因子的第三代重组慢病毒载体pRNAT-U6.2-hHGF构建成功。第二部分人肝细胞生长因子在大鼠骨髓间充质干细胞中的表达及生物学活性观察目的：观察慢病毒载体介导的人肝细胞生长因子(hHGF)基因在骨髓间充质干细胞(BM-MSCs)中的表达及其生物学活性。方法：(1)利用差速贴壁法联合Percoll密度梯度离心法分离、纯化大鼠原代BM-MSCs,流式鉴定MSC的表型,茜素红染色和油红O染色鉴定BM-MSCs的成骨、成脂分化潜能。(2)用高滴度表达hHGF (Lenti-HGF)或GFP (Lenti-GFP)的慢病毒液感染第三代的BM-MSCs,通过倒置相差荧光显微镜和流式鉴定来评估转染效率。ELISA检测细胞培养基上清中的hHGF浓度。(3)分别通过MTT法、Transwell小室法和Hoechst染色法检测脐静脉内皮细胞(HUVECs)和血管平滑肌细胞(VSMCs)的增殖、迁移及凋亡,评估感染Lenti-HGF的BM-MSCs分泌的hHGF的生物学活性。结果：(1)通过联合差速贴壁法和密度梯度离心法可获得大量高纯度、具有多向分化潜能的BM-MSCs。(2)在转染Lenti-GFP后72h,即可在荧光显微镜下见明亮的绿色荧光,最佳感染复数(MOI)为50,流式鉴定转染效率约为75%。ELISA结果显示,Lenti-HGF感染BM-MSCs的培养上清液中的hHGF可持续较高水平地表达14d以上,且明显高于感染Lenti-GFP者(P0.01)。(3)感染Lenti-HGF的MSCs分泌的hHGF在体外显示出促HUVECs增殖、迁移并抑制其凋亡,且能促进VSMCs迁移的生物学活性。结论：Lenti-HGF慢病毒能够高效地介导转移外源基因hHGF至BM-MSCs表达,并具有生物学活性。第三部分肝细胞生长因子基因修饰的骨髓间充质干细胞移植的促血管新生作用目的：比较hHGF基因修饰的BM-MSCs移植与单纯BM-MSCs移植在大鼠后肢缺血模型中的促血管新生作用及其机制。方法：在结扎左股动脉及其分支1天后,SD大鼠随机分为3组：HGF-MSC组(即HGF基因修饰的MSCs移植,n=8),MSC组(单纯的MSCs移植,n=8)以及PBS对照组(注射PBS,n=8)。在术后7天,进行细胞移植。实验组每只大鼠接受总计5×106个同一来源的HGF-MSCs或MSCs,分成5个点注入大鼠缺血后肢的股部肌肉；对照组注入同等体积的无菌PBS。分别在处理后1周或3周时处死大鼠,比较各组缺血股部肌肉中的毛细血管密度,移植细胞的分化转归,T淋巴细胞浸润以及生长因子HGF、VEGF蛋白表达等。ELISA检测HGF-MSC移植前和移植3周后大鼠血清HGF的水平。结果：细胞移植3周后,MSC组和HGF-MSC组缺血肌肉组织的血管新生均明显增强,以HGF-MSC组的毛细血管密度最高(P0.05)。从移植后1周开始,HGF-MSC组缺血组织中来源于移植细胞的内皮细胞数目明显高于MSC组(P0.05)。在移植1周后,HGF-MSC和MSC的同种异体移植几乎不引起T淋巴细胞浸润,与PBS组相近(P0.05)。Western-blot结果显示,MSC组和HGF-MSC组缺血肌肉组织中HGF、VEGF蛋白表达均明显高于PBS对照组,以HGF-MSC组的表达量最高(P0.05)。HGF-MSC移植前和移植后3周,大鼠血清HGF浓度无明显变化(P0.05)。结论：与单纯的MSC细胞移植疗法相比,HGF基因修饰的MSC移植疗法具有更强的促进血管新生和侧枝血管形成的能力。以干细胞为基础的血管新生基因治疗策略将可能为治疗严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.

【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R329

【引证文献】