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(P<0.05)；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(P>0.05)；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(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。
Background
Therapeutic angiogenesis promotes the formation of both new blood vessels and collateral circulation by means of transferring heterogeneous angiogenesis inducing factors into ischemic tissue. It mainly contains two strategies. One is using cytokines or cytokine gene carriers, the other is transplanting multi-directional differentiation potent stem cells to ischemic tissue, where they would differentiate into vascular endothelium and produce vascular growth factors to achieve angiogenesis.
Combined gene therapy and stem cell therapy in genetic engineering, the application of genetically modified stem cells to promote angiogenesis is a promising treatment. HGF gene-modified bone marrow mesenchymal stem cells not only transport therapeutic protein HGF to the ischemia organizations but play a important role as therapeutic materials to promote angiogenesis.This study including three parts investigates those questions:(1) To research the separation, in vitro culture and expanding method of mesenchymal stem cells (MSCs) and identifying the MSCs cultured by this mothod.(2) Rabbit bone marrow mesenchymal stem cells (BMSCs) were transduced with hepatocyte growth factor (HGF) by adenovirus vector. Transduction efficiency was detected by flow cytometry, transgene expression of HGF in vitro were evaluated by ELLISA and immunohistochemical methods.(3) To investigate whether a new strategy that combines MSCs transplantation and exvivo hepatocyte growth factor (HGF) gene transferring with adenovirus vector was more therapeutically efficient than MSCs cell therapy alone in a rabbit hindlimb ischemia model.
Part Ⅰ. In vitro culture and identification of bone marrow mesenchymal stem cells
Objective:To research the separation, in vitro culture and expanding method of mesenchymal stem cells (MSCs) and identifying the MSCs cultured by this mothod. Study the biological characteristics of MSCs.
Method:MSCs were isolated and purified from rabbits using density centrifugation and anchoring culture, then cultured in a-MEM supplement with10%fetal bovine serum (FBS) for expanding. Observed the morphous and growth velocity of MSCs, identify the phenotype of MSCs, draw the growth curve of MSCs and determine the mitotic index and cloning efficiency of MSCs. Observed the survival rate and growth state of MSCs underwent freeze thawing. Flow cytometric was used to dentify BMSC.
Result:The cultured MSCs were typical spindle-shaped, grew in whirlpool anchoring the culture plates, arranged gyrately. Through detection by flow cytometry, the expressions of CD44and CD29were positive; the exprssions of CD11b and CD45were negative, which was in accordance with the phenotype of MSCs. The passage MSCs proliferated fast. MSCs were purified at3rd generation. As the passage increases, the cloning efficiency and proliferative ability of MSCs decrease. The survival rates of MSCs thawed after frozen1month was90%.
Conclusion:BMSCs can be harvested using density centrifugation and anchoring culture. In vitro expanding of BMSCs is feasible. Freeze thawing has no obvious influence on BMSCs.
Part Ⅱ. Preparation of BMSCs transduced with Ad-HGF and invitro experiment
Objective:Rabbit bone marrow mesenchymal stem cells (BMSCs) were transduced with hepatocyte growth factor (HGF) by adenovirus vector.Transduction efficiency was detected by flow cytometry, transgene expression of HGF invitro were evaluated by ELISA and immunohistochemical methods.
Method:After digested with pancreatic enzyme, BMSCs were plated and cultivated in6holes plate with a density of1.5×105/per hole.50ul Ad-GFP with6different MOI(multiple of infection) was added into each hole for BMSCs transduction, with3duplicated holes for each MOI. BMSCs were transduced with Ad-hgf at MOI=150.After digested with pancreatic enzyme, BMSCs were added to silicificated slice, and cultivated. Then BMSCs were detected with ELISA and immunohistochemical methods.Also, transwell was used to detect the efficiency of BMSC migration mediated by HGF.
Result:48h after BMSCs transduction with Ad-GFP at different MOI, percentage of GFP-positive cells showed significant difference(P<0.05). Percentage of GFP-positive cells increased as MOI increased, however there was no significant difference (P>0.05) when MOI was bigger than150. When MOI=150, GFP-positive cells were over98%, so MOI=150was optimal for BMSCs transduction. When BMSCs were transduced with Ad-HGF at MOI=150, the results of ELISA and immunohistochemistry showed that Ad-HGF might effectively transducer BMSCs, and that the transduced BMSCs revealed high efficiency of HGF expression.The result also demonstrated HGF promoted the migration of BMSCs.
Conclusion:Ad-GFP successfully transduced BMSCs. As MOI=150, GFP-positive cells were over98%.There were GFP-positive cells just24h after transduction, with peak percentage from2d to7d. These results indicated that adenovirus vector possessed high effieiency transduction and stable gene expression. Results of ELISA and immunohistochemitry demonstrated that Ad-HGF was capable of effectively transducing BMSCs, and that transduced BMSCs possessed high efficiency of HGF gene expression in vitro.
Part Ⅲ. The research of bone marrow-derived mesenchymal stem cells transduced with HGF gene transplantation for treatment of rabbit limb ischemia
Objective:To investigate whether a new strategy that bone marrow-derived mesenchymal stem cells transduced with HGF gene transplantation was more therapeutically efficient than BMSCs transplantation or HGF gene therapy in a rabbit hindlimb ischemia model.
Methods:New Zealand white rabbits hindlimb ischemia model were randomly divided into four groups:(a) Ad-GFP injection (Ad-GFP group,n=10),(b)BMSCs transplantation (BMSC group, n=10),(c) Ad-HGF injection (Ad-HGF group, n=10),(d)BMSCs transduced with HGF (HGF-BMSCs) transplantation (HGF-BMSC group,n=10). The ischemic thigh muscle were analyzed for arteriography,capillary density, transplanted cell differentiation, the expression of HGF、c-met and VEGF protein after treatment. Serum levels of HGF in HGF-BMSC group after transplantation were determined by ELISA.
Results:Three weeks after transplantation, angiogenesis was significantly enhanced by b、c and d group, while capillary density was highest in the HGF-BMSC group (P<0.05). The number of transplanted cell-derived endothelia cells was higher in the HGF-BMSC group than in the BMSC group since one week after treatment (P<0.05). In treated ischemic hindlimb muscles, allogeneic HGF-BMSC and BMSC injection were associated with comparatively few T lymphocyte infiltration in Ad-HGF group(P>0.05). The expression of angiogenic growth factors like HGF and VEGF in local ischemic muscles undergoing intramuscular cell transplantation was more abundant in HGF-BMSC group than the other three groups (P<0.05).Serum levels of HGF in HGF-MSC group one week after transplantation have no significant difference(P>0.05).
Conclusions:The present study shows that BMSC transduced with HGF gene transplantation induced more potent angiogenesis and collateral vessel formation than BMSC transplantation or HGF gene therapy. Stem cell-based angiogenic gene therapy may be a new therapeutic strategy for the treatment of severe ischemic cardiovascular disease.
引文
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