Ad-HGF基因转染骨髓间充质干细胞移植治疗兔肢体缺血的实验研究

发布时间：2017-12-30 19:34

本文关键词：Ad-HGF基因转染骨髓间充质干细胞移植治疗兔肢体缺血的实验研究　出处：《山东大学》2012年博士论文　论文类型：学位论文

【摘要】：研究背景目前治疗性血管新生主要有两种方法,一是局部缺血区域注射外源性血管生长因子蛋白或基因,二是局部缺血组织移植具有多向分化潜能的干细胞,干细胞在缺血缺氧状态下分化为血管内皮细胞并产生促血管生长因子来进一步促进血管新生。在基因工程范畴内联合基因治疗和细胞治疗,应用基因修饰的干细胞促进血管新生是一种极具前景的治疗手段。HGF基因修饰的骨髓间充质干细胞可以将治疗性蛋白HGF转入缺血的组织中以满足基因治疗的特定需要,安全性更高；它在体内同时发挥治疗性原材料和持续输送促血管新生的生长因子HGF两种作用,与单纯的干细胞治疗或基因治疗相比更具优越性。本课题分三部分,依次探讨以下问题：(1)研究兔骨髓间充质干细胞的分离、培养及鉴定方法。(2)利用腺病毒载体将肝细胞生长因子转染兔骨髓间充质干细胞,流式细胞仪检测转染效率及转染对细胞生物学特性有无影响,ELISA及免疫组化检测转染后外源基因HGF的表达。(3)制备兔后肢缺血模型,HGF基因修饰的BMSCs移植与单纯BMSCs移植或HGF基因治疗在兔后肢缺血模型中的促血管新生作用及其机制。第一部分：兔骨髓间充质干细胞的体外培养及鉴定目的：研究兔骨髓间充质干细胞的分离、培养及鉴定方法,观察其生物学特性。方法：穿刺股骨采集兔骨髓血,应用淋巴分离液密度梯度离心结合贴壁筛选培养骨髓间充质干细胞。通过观察细胞形态、流式细胞仪检测细胞表面标志物以及对其进行诱导分化培养,对培养的骨髓间充质干细胞进行鉴定。结果：应用淋巴分离液密度梯度离心结合贴壁筛选培养法得到纯化的BMSCs。通过本方法培养得到的BMSCs贴壁生长,呈典型的长梭形,漩涡状排列；流式细胞仪检测细胞表面标志物CD29、CD44表达阳性,CDllb、CD45表达阴性；细胞成骨诱导分化后I型胶原免疫细胞化学染色阳性,以上这些结果符合文献报道的BMSCs特征。结论：密度梯度离心结合贴壁筛选培养可以有效的体外扩增骨髓间充质干细胞,获得高度纯化的骨髓间充质干细胞。第二部分：Ad-HGF转染骨髓间充质干细胞及体外检测目的：利用重组腺病毒载体将肝细胞生长因子转染兔骨髓间充质干细胞,用流式细胞术检测基因转染效率,ELISA及免疫组化检测转染后外源基因HGF的表达。方法：携带HGF基因和绿色荧光蛋白(green fluorescene protein, GFP)的重组腺病毒(adenovirus)载体Ad-HGF、Ad-GFP,均由兰州军区总医院哈小琴教授提供。以不同MOI值(multiple of infection,感染复数)的Ad-GFP病毒液转染BMSCs,流式细胞检测表达GFP阳性的细胞率。以MOI=150的Ad-HGF转染BMSCs,ELISA检测及免疫细胞化学检测HGF表达；流式细胞仪检测HGF-BMSC细胞表面标志物以及诱导分化培养,以确定基因转染后细胞生物学特性有无变化；同时transwell检测HGF介导BMSCs细胞迁移能力。结果：Ad-GFP以不同的MOI转染兔BMSCs48h后,表达GFP的阳性细胞率有显著性差异(P0.05)；MOI=150时表达GFP阳性的细胞率达98%以上,MOI大于150以上时,表达GFP阳性的细胞率无显著性差异(P0.05)；因此选择MOI=150转染BMSCs。将Ad-HGF以MOI=150转染兔BMSCs后,ELISA及免疫细胞化学检测结果显示：细胞培养上清及胞浆内有丰富的HGF蛋白表达,表明Ad-HGF可高效转染BMSCs,BMSCs有HGF蛋白的高表达。流式细胞仪检测及诱导分化培养结果显示：腺病毒感染以及HGF基因修饰不改变BMSC表型和分化潜能。同时实验结果显示HGF对BMSC有极强的趋化作用。结论：Ad-GFP可成功转染骨髓间充质干细胞,以MOI=150转染BMSCs,转染效率最高,可达98%以上,转染24h即可见绿色荧光,2-4天转染率最高；表明重组腺病毒作为载体转染BMSCs具有高转染率与稳定表达。肝细胞生长因子腺病毒表达载体Ad-HGF转染骨髓间充质干细胞不改变BMSCs的固有特性；经ELISA及免疫组化检测,BMSCs可高表达HGF蛋白。HGF对BMSC有明显的趋化作用。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治疗兔肢体缺血目的：比较HGF基因修饰的BMSCs移植与单纯BMSCs移植或HGF基因治疗在兔后肢缺血模型中的促血管新生作用及其机制探讨。方法：后肢缺血的新西兰大白兔模型随机分为四组,每组10只,治疗方法：a.空腺病毒对照组(Ad-GFP组)：缺血部位肌肉多点注射1×109Pfu Ad-GFP。b.骨髓间充质干细胞(BMSC组)组：缺血部位肌肉多点注射1×107个骨髓间充质干细胞。c.HGF基因治疗组(Ad-HGF组)：缺血部位肌肉多点注射1×109Pfu Ad-HGF。d.转染HGF的BMSC治疗组(HGF-BMSC组)：缺血部位肌肉多点注射1×107个转染HGF的BMSC。移植后28天行动脉造影及侧枝血管计数；免疫组织化学检测缺血区域毛细血管密度；免疫荧光检测BMSCs在体内的分布及分化；Western-blot检测组织中HGF、C-met蛋白的表达；ELISA检测移植前后兔血清中HGF的浓度。结果：造影结果显示骨髓干细胞组(BMSC组)、HGF基因治疗组(Ad-HGF组)及HGF-BMSC组血管数明显高于空腺病毒对照组(PO.05)；HGF-BMSC组的侧支血管计数最高。移植后Ad-GFP标记的骨髓间充质干细胞散在分布于各肌纤维的间质中,呈现明亮的绿色荧光。在细胞移植后3周,部分Ad-GFP标记的骨髓间充质干细胞表达内皮细胞标记物CD31。Western blot结果显示,骨髓干细胞组(BMSC组)、HGF基因治疗组(Ad-HGF组)及HGF-BMSC组HGF表达明显高于空腺病毒对照组(P0.05),以HGF-BMSC组的HGF表达最高。ELISA结果显示,HGF-BMSC移植前和移植后1周时,兔血清HGF的浓度无明显变化(P0.05) 结论：与单纯的BMSC细胞移植或HGF基因治疗相比,肝细胞生长因子基因修饰的骨髓间充质干细胞可使HGF获得稳定而有效的表达,具有更强的促进血管新生能力,从而更加有效地改善肢体缺血。
[Abstract]:Research background
At present, therapeutic angiogenesis mainly has two kinds of methods, one is the local ischemia regional injection of exogenous vascular endothelial growth factor gene or protein, two ischemic tissue transplantation of pluripotent stem cells, stem cells in ischemia hypoxia differentiate into vascular endothelial cells and vascular growth factor to promote angiogenesis.
Genetic therapy and gene engineering in cells within the scope of application of gene modified stem cells to promote angiogenesis is the treatment of.HGF gene modified a promising bone marrow mesenchymal stem cells can be therapeutic protein HGF into ischemic tissue in order to meet the specific needs of gene therapy, it has higher safety; in vivo and play therapeutic raw materials and continuous delivery of angiogenic growth factor HGF two, and the pure stem cell therapy and gene therapy superior. The thesis is divided into three parts, in order to discuss the following issues: (1) study on rabbit bone marrow mesenchymal stem cells were separated and cultured. Identification methods. (2) using adenovirus vector HGF transfection of rabbit bone marrow mesenchymal stem cells, flow cytometry was used to detect the gene transfection efficiency and has no effect on the biological characteristics of cells, ELISA and free The expression of exogenous gene HGF was detected by histochemical examination. (3) rabbit hind limb ischemia model was prepared. The angiogenesis promoting effect and mechanism of HGF gene modified BMSCs transplantation combined with simple BMSCs transplantation or HGF gene therapy on hind limb ischemia model in rabbits were studied.
Part 1: in vitro culture and identification of rabbit bone marrow mesenchymal stem cells
Objective: To study the isolation, culture and identification of rabbit bone marrow mesenchymal stem cells (MSCs), and to observe their biological characteristics.
Methods: rabbit bone marrow puncture femoral blood collection, using lymphocyte separating medium density gradient centrifugation and adherent cultured bone marrow mesenchymal stem cells. Cell morphology was observed by flow cytometry, cell surface markers and differentiation culture on the identification of cultured bone marrow mesenchymal stem cells.
Results: the application of lymphocyte separating medium density gradient centrifugation and adherent culture method to obtain the purified BMSCs. cultured by the method to get the BMSCs adherent, spindle whorls; typically, detection of cell surface marker CD29 by flow cytometry, the expression of CD44, CDllb, CD45 negative expression cells; osteogenic differentiation of type I collagen after immunocytochemical staining, these results are consistent with the characteristics of BMSCs reported in the literature.
Conclusion: density gradient centrifugation combined with adherent screening and culture can effectively amplify bone marrow mesenchymal stem cells in vitro and obtain highly purified bone marrow mesenchymal stem cells.
The second part: Ad-HGF transfection of bone marrow mesenchymal stem cells and in vitro detection
Objective: to transfect rabbit bone marrow mesenchymal stem cells with recombinant adenovirus vector, and transfect rabbit bone marrow mesenchymal stem cells. The gene transfection efficiency was detected by flow cytometry, and the expression of HGF gene after transfection was detected by ELISA and immunohistochemistry.
Methods: carrying HGF gene and green fluorescent protein (green fluorescene, protein, GFP) of the recombinant adenovirus vector Ad-HGF (adenovirus), Ad-GFP, were provided by the General Hospital of Lanzhou military region ha xiao-qin2 professor. With different MOI values (multiple of infection Ad-GFP, MOI) virus was transfected by BMSCs, flow cytometry expression GFP positive cell rate. MOI=150 Ad-HGF was transfected into BMSCs, detect the expression of ELISA and immunocytochemistry HGF; flow cytometry HGF-BMSC cell surface markers and differentiation, gene transfer to determine cell biological characteristics after dyeing has no change; at the same time, Transwell detection of HGF mediated BMSCs cell migration.
Results: Ad-GFP with different MOI after BMSCs48h transfection, the expression of GFP positive cells rate has significant difference (P0.05); MOI=150 the expression of GFP positive cell rate of more than 98%, more than 150 MOI, the expression of GFP positive cells was no significant difference (P0.05); MOI=150 BMSCs. will therefore choose transfection Ad-HGF was transfected with MOI=150 after BMSCs, ELISA and immunocytochemical assay showed that the expression of HGF protein in cell culture supernatant and abundant cytoplasm, showed that Ad-HGF can efficiently transfected BMSCs, BMSCs have the high expression of HGF protein. Flow cytometry results showed: culture and induced differentiation of adenovirus infection and HGF gene modification does not change the BMSC phenotype and differentiation potential. The experimental results show that HGF has strong chemotactic effect on BMSC.
Conclusion: Ad-GFP can be successfully transfected into bone marrow mesenchymal stem cells transfected by MOI=150 BMSCs, the highest transfection efficiency can reach above 98%, 24h transfected with green fluorescence could be found in 2-4 days, the highest transfection rate; recombinant adenovirus as a vector transfected BMSCs with high transfection efficiency and stability. The expression of hepatocyte growth factor adenovirus expressing the inherent characteristics vector Ad-HGF was transfected into bone marrow mesenchymal stem cells did not alter BMSCs; by ELISA and immunohistochemistry, BMSCs high expression of HGF protein.HGF has obvious chemotactic effect on BMSC.
Third part: transplantation of bone marrow mesenchymal stem cells modified by hepatocyte growth factor gene in the treatment of limb ischemia in rabbits
Objective: To compare angiogenesis promoting effect of HGF gene modified BMSCs transplantation with BMSCs transplantation or HGF gene therapy in hind limb ischemia model of rabbits and explore its mechanism.
Methods: a model of New Zealand white rabbits were randomly divided into four groups of hind limb ischemia, 10 rats in each group, treatment methods: A. adenovirus control group (Ad-GFP group): ischemic muscle multi-point injection of 1 * 109Pfu Ad-GFP.b. of bone marrow mesenchymal stem cells group (BMSC group): ischemic muscle multi-point injection of 1 x 107 bone marrow mesenchymal stem cells,.C.HGF gene therapy group (Ad-HGF group): BMSC group ischemic muscle multi-point injection of 1 * 109Pfu Ad-HGF.d. transfected with HGF (HGF-BMSC group): BMSC. transplantation ischemic muscle multi-point injection of 1 * 107 HGF transfected 28 days after arterial angiography and collateral vessel count; immunohistochemical detection of ischemic area capillary density; immunofluorescence detection of BMSCs in distribution and differentiation in vivo; detection of Western-blot in HGF, the expression of C-met protein; concentration in rabbit serum HGF before and after transplantation of ELISA detection.
Results: the imaging results showed that bone marrow stem cells group (BMSC group), HGF gene therapy group (Ad-HGF group) and HGF-BMSC group was significantly higher than the number of vessels empty adenovirus control group (PO.05); HGF-BMSC group of collateral vessels after transplantation. The highest count of Ad-GFP labeled bone marrow mesenchymal stem cells scattered in the muscle fiber the interstitial, showed bright green fluorescence. In 3 weeks after transplantation, Ad-GFP labeled bone marrow mesenchymal stem cells expressed endothelial cell markers CD31.Western blot results showed that bone marrow stem cells group (BMSC group), HGF gene therapy group (Ad-HGF group) and HGF-BMSC group the expression of HGF was significantly higher than that of empty glands the virus control group (P0.05), in HGF-BMSC group the highest expression of HGF.ELISA showed that 1 weeks after HGF-BMSC transplantation, rabbit serum HGF concentration had no significant change (P0.05)
Conclusion: compared with simple BMSC cell transplantation or HGF gene therapy, hepatocyte growth factor gene modified bone marrow mesenchymal stem cells can achieve stable and effective expression of HGF, and have stronger angiogenesis promoting ability, thereby improving limb ischemia more effectively.

【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R329

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