【作者】 张向荣；

【导师】 郭光华； 刘德伍；

【作者基本信息】 南昌大学 ， 外科学， 2009， 博士

【摘要】 严重创伤、大面积烧伤、肿瘤摘除、整形术后的创面覆盖一直是困扰临床医生的难题。目前常规的治疗方法有自体、异体或异种皮移植。自体皮肤来源极为有限,而且存在着形成新创面的缺陷;异体或异种皮肤移植解决了自体皮源不足的问题,但由于难以克服的免疫排斥反应,目前只能用作暂时封闭创面的覆盖物。近年来,组织工程皮肤的构建与应用为皮肤缺损的修复提供了极具发展潜力的治疗途径。但现阶段各种组织工程化皮肤移植存活率明显低于自体断层皮片,其首要原因之一是组织工程化皮肤移植后缺乏足够的血管化而导致低灌注和缺血损伤。因此,如何促进移植皮肤的血管化,成为组织工程皮肤临床推广应用研究的关键。研究表明,利用转基因技术,将血管生成调控因子基因导入目的细胞,使其表达某些调控因子,可有效促进血管生成。骨髓间充质干细胞(bone mesenchymal stem cells , MSCs )是一种具有多向分化潜能的成体干细胞,在不同的诱导条件下可向骨细胞、脂肪细胞、神经细胞、肌细胞和内皮细胞等各种细胞系转化,由于骨髓间充质干细胞属未分化的前体干细胞,表型分化尚不成熟,免疫原性小,所以移植后无排斥反应或反应较弱,而且,骨髓间充质干细胞还具有取材方便、体外扩增容易、体外增殖能力强、易于基因操作等独特的优点,逐渐成为基因治疗适宜的细胞载体。血管内皮细胞生长因子(Vascular endothelial growth factor ,VEGF)是目前已知的最强的促血管再生因子,近年来应用血管内皮细胞生长因子165基因(VEGF165)转染骨髓间充质干细胞,用于心肌缺血或肢体缺血性疾病的实验及临床研究已取得了令人瞩目的成就,但将其应用于组织工程皮肤构建的研究尚未见报道,因此,采用基因转移技术将hVEGF165基因转入骨髓间充质干细胞中,使其有效表达目的基因和蛋白,构建活性组织工程皮肤,植入皮肤创面后使骨髓间充质干细胞在局部短期内分泌VEGF蛋白,从而促进组织工程皮肤的早期血管化,有可能为解决组织工程皮肤血管化这一问题提供新的方法与思路。本研究采用脂质体介导hVEGF165基因转染人骨髓间充质干细胞,观察VEGF165基因mRNA转录、蛋白的表达情况及转染后对骨髓间充质干细胞的影响,并接种转染VEGF165基因的骨髓间充质干细胞于同种异体脱细胞真皮支架上构建VEGF基因修饰的活性皮肤替代物,移植修复动物全层皮肤缺损创面,观察其在皮肤结构和功能重建中的作用及其转归,比较缺损创面移植皮肤的血管化情况,为加速组织工程皮肤移植后血管化、提高其临床移植成功率探索新的途径。基于这种认识,我们开展了以下一系列研究:第一部分人骨髓间充质干细胞的分离、培养和鉴定目的:建立一种分离纯化和培养人骨髓间充质干细胞的方法,为利用骨髓间充质干细胞进行基因治疗提供实验基础。方法密度梯度离心和贴壁培养相结合,分离纯化人骨髓间充质干细胞。倒置相差显微镜下观察人骨髓间充质干细胞形态学变化;流式细胞仪检测人骨髓间充质干细胞的表面标记以及细胞周期;绘制人骨髓间充质干细胞的生长曲线;透射电镜观察人骨髓间充质干细胞的超微结构。结果:密度梯度离心结合贴壁法能分离培养出纯度较高的人骨髓间充质干细胞。流式细胞仪检测人骨髓间充质干细胞CD90、CD29、CD44、CD34、CD45阳性表达细胞比率分别为98.48%、98.74%、97.41%、0.36%、0.64%。人骨髓间充质干细胞的生长曲线呈S形。透射电镜示细胞核浆比大,可见部分细胞器和蛋白分泌物。结论采用密度梯度离心和贴壁培养相结合,可获得纯度较高和活性骨髓MSC,是简便有效实用可行的方法。第二部分血管内皮细胞生长因子165基因转染人骨髓间充质干细胞的实验研究目的:观察人骨髓间充质干细胞转染VEGF165基因后外源性基因及其蛋白的表达,探讨其促进组织工程皮肤移植后血管化的可能性。方法体外分离、培养人骨髓间充质干细胞,采用脂质体介导方法将pShuttle-CMV/VEGF165质粒转染人骨髓间充质干细胞,并设立空质粒对照组(转染pShuttle-CMV载体)、脂质体对照组(转脂质体)和正常对照组(无特殊处理),用RT-PCR、ELISA和Western Blot检测人骨髓间充质干细胞VEGF165 mRNA及其蛋白的表达情况,MTT法及流式细胞仪检测VEGF质粒转染对骨髓间充质干细胞活性的影响。结果:RT-PCR检测结果显示:质粒转染实验组、空质粒对照组、脂质体对照组和正常对照组VEGF165基因mRNA表达量分别为0.89±0.03、0.34±0.04、0.40±0.03、0.30±0.03。质粒转染实验组骨髓间充质干细胞mRNA转录较三个对照组均显著上调(P <0.01)。ELISA检测质粒转染实验组、空质粒对照组、脂质体对照组和正常对照组处理后24h细胞上清hVEGF的浓度分别为778.39±35.21 pg/ml、543.55±24.32pg/ml、561.45±28.08pg/ml、571.53±22.73pg/ml,质粒转染实验组与其他对照组相比具有统计学差异(P<0.01)。质粒转染后第7天表达达到最高,以后再逐渐降低。Western Blot检测结果显示转染组条带明显增强(P<0.01)。MTT法及凋亡检测结果显示VEGF质粒转染对骨髓间充质干细胞增殖无影响。结论实现了VEGF165基因对人骨髓间充质干细胞的成功转染,并有效表达目的基因及其蛋白,具有促进组织工程皮肤移植后血管化的应用前景。第三部分血管内皮细胞生长因子165修饰骨髓间充质干细胞复合脱细胞真皮基质构建活性组织工程皮肤目的:以VEGF修饰的骨髓间充质干细胞复合脱细胞真皮基质构建活性组织工程皮肤,为进一步临床应用奠定基础。方法:体外培养人骨髓间充质干细胞,以脂质体介导pShuttle-CMV/ VEGF165质粒转染骨髓间充质干细胞后,将其接种于制备好的脱细胞真皮支架上,构建组织工程皮肤,观察细胞生长情况及其与支架材料的相容性。结果:脱细胞真皮基质去细胞完全,组织相容性好,转基因骨髓间充质干细胞在脱细胞真皮基质中生长良好,可体外构建组织工程皮肤。结论:利用VEGF修饰骨髓间充质干细胞及制备的脱细胞真皮基质可以体外联合构建活性组织工程皮肤。第四部分含基因骨髓间充质干细胞的活性皮肤移植促进组织工程皮肤血管化的初步研究目的:探讨含基因骨髓间充质干细胞的活性皮肤移植促进组织工程皮肤血管化的基因治疗的可行性。方法:新西兰白兔26只,于兔背部两侧常规全层皮肤缺损创面4个,随机分为四组:A组(以含VEGF基因骨髓间充质干细胞复合脱细胞真皮构建的组织工程皮肤修复创面,hVEGF165-MSCs+ADM实验组),B组(以骨髓间充质干细胞复合脱细胞真皮构建的组织工程皮肤修复创面,MSCs+ADM对照组),C组(以不含细胞的脱细胞真皮修复创面,单纯ADM对照组)和D组(创面不做处理),观察局部组织微血管密度变化及创面愈合情况。结果:移植术后一周, hVEGF165-MSCs+ADM实验组创面的毛细血管密度较MSCs+ADM对照组、单纯ADM对照组明显增高(P < 0.01),两周时三组中hVEGF165-MSCs+ADM实验组的血管密度仍最高,但三组数据无统计学差别。术后2周、3周hVEGF165-MSCs+ADM实验组移植物成活率较MSCs+ADM对照组、单纯ADM对照组高(P < 0.01),hVEGF165-MSCs+ADM实验组创面收缩率较MSCs+ADM对照组、单纯ADM对照组高低(P < 0.01)。结论:含基因骨髓间充质干细胞的活性皮肤移植可有效促进组织工程皮肤的早期血管化及改善创面愈合。更多还原

【Abstract】 Severe trauma, extensive burns, tumor removal, plastic surgery wound coverage has been plagued clinicians problems. At present, conventional treatment methods have autologous, allogeneic or xenogeneic skin transplantation,Source of autologous skin is extremely limited, and there are defects in the formation of a new wound; Allogeneic or xenogeneic skin graft resolve the problem of inadequate sources, but because it is difficult to overcome immune rejection, could only be used for temporary closure of wound coverings. Thus to carry out search for wound healing of skin substitutes research, have important clinical significance. In recent years, tissue engineered skin substitutes provide an alternative to skin autografts for treatment of nonhealing wounds that often develop in patients with extensive burns and with vascular insufficiency such as venous stasis or diabetes or in the elderly. In present tissue engineered skin has been a hot spot in repair of wounds. While skin substitutes such as Apligraf promote healing, they usually do not stably engraft and significant numbers of patients receiving bioengineered skin substitutes require additional interventions as a result of graft failure. Various obstacles have delayed the widespread use of composite skin substitutes. Insufficient vascularization has been proposed as the most likely reason for their unreliable survival. So how to stimulating vascularization of tissue - engineered skins grafting becomes a critical question and a urgent problem need to address either.Study shows that transgenic technology can import angiogenesis regulatory factor gene into target cells to express certain regulatory factor protein and contribute to angiogenesis.Bone mesenchymal stem cells ( MSCs ) are isolated from bone marrow mononuclear cells and can be expanded ex vivo. Under appropriate culture conditions, human bone marrow mesenchymal stem cells have the capacity to differentiate into cells such as bone, cartilage, adipocytes, myocytes, and even cardiomyocytes. Bone marrow mesenchymal stem cells also have immunomodulatory and anti-inflammatory effects, but only evoke little immune reactivity. Moreover, bone marrow mesenchymal stem cells are amenable to genetic manipulation, thus, these cells are currently being tested for their potential use in cell and gene therapy . Over the past two decades as in-depth study of biological materials and tissue engineering technology, constant improvement of the skin tissue engineering research and applications for clinical repair of various skin defects providing a new way. Vascular endothelial growth factor (VEGF) is the strongest known angiogenesis-promoting factor, In recent years the application of VEGF165 gene transfection of bone marrow mesenchymal stem cells for myocardial ischemia or limb ischemic disease of experimental and clinical studies have achieved initial success. So far, the research about construction tissue - engineered skins by transfection hVEGF165 gene into human bone marrow mesenchymal stem cells has not been reported. Therefore, the use of gene transfer technology to transfected VEGF gene into into bone marrow mesenchymal stem cells and to construct tissue - engineered skin with VEGF- modified human bone marrow mesenchymal stem cells, and covered the skin wound by constructed tissue - engineered skin, so the bone marrow mesenchymal stem cells could endocrine VEGF protein in the local in short-term, thereby contributing to tissue-engineered skin early neovascularization , it is possible to provide a novel gene therapentic strategy for neovascularization which can possibly apply for clinic usage.This study is intended to transfect hVEGF165 gene into human bone marrow mesenchymal stem cells with liposome, Observe VEGF expression in vitro and detect the effect of transfection on human bone marrow mesenchymal stem cells and to construct tissue - engineered skin with VEGF- modified hMSCs as well as acellular dermal and then graft tissue - engineered skin to full thickness dermal wounds of animals, Observed its role in the reconstruction of skin structure and function and its prognosis, compared vascularization of wound defect, to speed up tissue-engineered blood vessels after transplantation of skin, to explore new avenues to accelerate neovascularization of tissue - engineered skin and improve its success rate of clinical transplantation.Therefore, a series of experiment studies were performed as follows: Part I Isolation, cultivation and biological identification of human bone marrow mesenchymal stem cellsOBJECTIVE:The aim of this study is to research on the method to isolate and purify human bone marrow mesenchymal stem cells and provides basis for the amendment of tissue defects by tissue engineering.METHODS:Human bone marrow mesenchymal stem cells were isolated by combining density gradient centrifugation with plastic adherence . Morphological observations were performed with phase contrast microscope; growth curves of the cells were drawn by cytometry method; cell phenotype and generation cycle were detected by flow cytometry; cell ultrastructures were determined by transmission electron microscope.RESULTS:Higher purity of hBMSCs could be achieved by density gradient centrifugation. The positive expression rates of cell phenotypes were various as followings respectively: CD90,98.48%; CD29,98.74%;CD44,97.41%; CD34,0.36%; CD45,0.64%. The growth curve of human bone marrow mesenchymal stem cells was“S”shaped. The ratio of cells in S+G2+M stage of the5th passages was 15.26%. The ratio of nucleus to cytoplasm was large and some organelle and protein secretion could be seen in cytoplasm.CONCLUSION:Higher purity of hBMSCs can be isolated and cultured by combining density gradient centrifugation with plastic adherence. Part II Experimental Study of the transfection of Vascular endothelial growth factor 165 gene into human bone mesenchymal stem cells in vitroOBJECTIVE:To observe the expression of exogenous gene and protein after human bone marrow mesenchymal stem cells transfected with human vascular endothelial growth factor 165 (VEGF165) gene, and to investigate the feasible of promotion tissue-engineered skin early revascularization.METHODS:Bone marrow mesenchymal stem cells were separated and purified by combining density gradient centrifugation with adhering method. The vector pShuttle-CMV/VEGF165 was transfected into bone marrow mesenchymal stem cells by liposome mediated and the bone marrow mesenchymal stem cells were divided into four groups : plasmid group,the empty plasmid group , liposome group and the control group, The cells in the plasmid group were transfected with pShuttle-CMV/VEGF165 ,while cells in the empty plasmid group were transfected with pShuttle-CMV and cells in the liposome group were transfected with Lipofectamine 2000 and cells in the control group received no special treatment. Human VEGF expression in vitro was assessed by RT-PCR、ELISA and Western Blot. MTT method was used to detect the effect of transfection on bone marrow mesenchymal stem cells . Apoptosis was detected by flow cytometry.RESULTS:The expressions of VEGF165 mRNA in each group by RT-PCR : the plasmid group、empty lasmid group、liposome group and the control group the VEGF165mRNA expression were 0.89±0.03、0. 34±0.04、0.40±0.03、0.30±0.03, Compared with other three groups, the VEGF165mRNA transcripts of bone mesenchymal stem cells in the plasmid group were significantly raised (P <0.01).The expression of VEGF165 protein in each group by ELLISA: The content of VEGF in the plasmid group、empty lasmid group、liposome group and the control were 778.39±35.21 pg/ml、543.55±24.32pg/ml、561.45±28.08pg/ml、571.53±22.73pg/ml, Compared with other three groups, the expression of VEGF165 protein in the plasmid group were significantly raised (P <0.01), and in the plasmid group , VEGF expression level gradually increased to the highest on day 7, gradually decreased later. Western Blot showed that the intensity of the plasmid group band increased(P <0.01). It wasn’ t found that VEGF transfection had a significantly inhibitory effect on MSCs by MTT method and flow cytometry .CONCLUSION:Bone marrow mesenchymal stem cells were transfected with hVEGF165 gene successfully and deffectively express target gene and protein , provide a novel gene therapentic strategy for neovascularization which can possibly apply for clinic usage. Part III Construction of tissue - engineered skin with vascular endothelial growth factor - modified bone marrow mesenchymal stem cells and acellular dermal matrixOBJECTIVE: To construct tissue - engineered skin with VEGF- modified hBMSCs as well as acellular dermal matrix.METHODS:Bone marrow mesenchymal stem cells were separated and cultured in vitro.The vector pShuttle-CMV/VEGF165 was transfected into bone marrow mesenchymal stem cells by liposome mediated at the time of 80%- 90% confluence and the VEGF- modified bone marrow mesenchymal stem cells were seeded onto the surface of acellular dermal matrix. It was cultured absolutely inside liquid medium for about 2d. In this way, tissue - engineered skin was expected to be constructed in vitro. Observation the cells seeded to scaffold material and its compatibility.RESULTS:With the VEGF- modified cells cultured in the acellular dermal matrix, tissue - engineered skin could be construted in virto.CONCLUSION: Constuction of tissue - engineered skin with VEGF- modified bone marrow mesenchymal stem cells as well as acellular dermal matrix in vitro is feasible. Part IV Experimental study of the transplanted human bone mesenchymal stem cells transfected with geneaccelerate the neovascularization of tissue - engineered skin OBJECTIVE:To investigate the feasibility of transplanting VEGF- modified tissue - engineered skin for accelerating neovascularization of tissue - engineered skin .METHODS:Twenty-six New Zealand white rabbits with four full thickness skin defects on the two sides of the back were separately covered with hVEGF165- MSCs+ADM, MSCs+ADM , ADM or none. Examine wound healing of local tissue and count it’s MVD by CD34 immunocytochemistry staining.RESULTS:1 week after operation the capillary density of wound tissue of VEGF gene transfection treatment group was significantly higher than those of MSCs+ADM treatment group and ADM treatment group ( P < 0.01). 2 weeks after operation the capillary density of wound tissue of VEGF gene transfection treatment group was highest in those three groups, However, among the three groups no significant difference. At the second week and third week after operation, There was significant difference in the survival rate and contraction rate between the three groups, Compared with the two groups, the graft survival rate of VEGF gene transfection treatment group was highest and the contraction rate was lowest ( P < 0.01).CONCLUSION:Transplantation tissue-engineered skin constructed by human bone mesenchymal stem cells transfected with gene VEGF and acellular dermal matrix can promote tissue-engineered skin early revascularization and wound healing effectively.更多还原