【作者】 杨波；

【导师】 高建华；

【作者基本信息】 南方医科大学 ， 整形外科， 2008， 博士

【摘要】 研究背景组织工程是应用细胞生物学和工程学原理,对病损组织结构和功能的修复与重建进行研究开发的一门新兴学科。20世纪80年代组织工程开始应用于医学领域的研究,到目前已经能够成功地将体外大量扩增的细胞与生物材料复合后植入体内以达到恢复、替代、维持或提高组织器官功能的目的。然而,随着研究的进一步深入,学者们发现当组织的体积达到数立方毫米时,其内部的细胞将难以通过渗透作用获得营养和氧分的支持,而必须要求毛细血管的长入才能维持其正常的代谢。对血循环的重建而言,血管内皮细胞生长因子（vascular endothelialgrowth factor,VEGF）作为血管内皮细胞的特异性丝裂原是血管形成最重要的调节因子,在新生血管形成过程中起重要作用,而通过将VEGF编码基因导入细胞的基因修饰已成为获得血循环重建的最具发展潜力的治疗模式之一。国外有些研究机构开始尝试将细胞移植与生长因子联合应用,并获得很好的疗效。但就目前的研究而言,不论是国内或是国外文献,尚未见在组织工程脂肪研究上同时植入由生长因子修饰的脂肪干细胞来改善组织工程脂肪血管形成的研究。研究目的1.研究从成人脂肪抽吸物液体部分中获取的脂肪组织来源干细胞（adipose-derived stem cell,ASCs）作为脂肪组织工程理想种子细胞的优势。2.对ASCs进行腺病毒重组增强型绿色荧光蛋白（recombinedadenovirus-enhanced green fluorescent protein,Ad-EGFP）体外转染和标记,观察其对细胞生物性状的影响,为脂肪干细胞研究寻找理想的细胞标记方法。3.探讨腺病毒为载体对种子细胞进行VEGF₁₆₅基因修饰的可行性。4.利用Ⅰ型胶原蛋白作支架材料和ASCs在体内构建组织工程化脂肪的可行性。5.通过VEGF₁₆₅基因修饰干预组织工程脂肪组织血管形成,评价促进组织工程脂肪血管形成的效果,使再生的脂肪组织内部获得血供能力而保证其长期稳定存活,提高组织工程化脂肪成活水平。材料与方法1.ASCs的分离培养及鉴定取人吸脂术抽吸物的液体部分,使用直接离心过滤法进行原代培养,观察细胞形态及功能变化,细胞传至第4代后供实验用。利用流式细胞仪检测细胞表面部分与干细胞相关的分子,确定细胞类型;台盼蓝拒染法测细胞活性并绘制细胞生长曲线;同时对细胞进行体外定向成脂、成骨、成软骨诱导分化,并以油红0染色、茜素红染色和阿新蓝染色进行鉴定。2.Ad.EGFP转染并标记ASCsAd.EGFP按不同感染复数（multiphcity of infection,MOI）值体外转染ASCs,进行EGFP荧光标记,倒置显微镜及荧光显微镜观察细胞生长情况及传代后荧光强度的变化;测定腺病毒转染ASCs的效率;检测细胞标记后成脂能力。使用台盼蓝拒染法检测细胞活力和绘制细胞生长曲线,同时将培养细胞上清液进行乳酸脱氢酶（LDH）含量测定,检测Ad.EGFP对细胞的毒性。3.Ad.VEGF₁₆₅基因转染ASCs后目的基因表达按上述试验确定的MOI值（50）进行Ad.VEGF₁₆₅体外转染ASCs,通过免疫细胞荧光检测VEGF在细胞内的表达情况;同时利用ELISA测定细胞培养上清液中VEGF浓度水平。4.Ⅰ型胶原构建工程化脂肪的实验研究将脂肪干细胞接种于支架材料形成复合物,将培养细胞上清液进行乳酸脱氢酶（LDH）含量测定,检测材料的细胞毒性;细胞-支架粘附率检测;最后扫描电镜观察细胞与载体的黏附性。在确定细胞和支架材料生物相容性较好的情况下,准备三组植入物（细胞被EGFP标记）:Ⅰ组为空支架,Ⅱ组为ASCs和支架,Ⅲ组为成脂分化的ASCs和支架,分别在不同部位植入同一裸鼠皮下。在8w取新生标本,进行组织工程新生物大体观察和湿重测定后,荧光显微镜观察大体组织标本,最后组织学检测、油红0染色定性。5.VEGF₁₆₅促进工程化脂肪血管新生准备三组植入物:Ⅳ组为成脂分化的ASCs和支架,Ⅴ组为EGFP基因转染的ASCs、成脂分化的ASCs和支架,Ⅵ组为VEGF₁₆₅基因转染的ASCs、成脂分化的ASCs和支架。分别植入同一裸鼠皮下。按2w、12w取新生标本,通过大体观察、HE染色和免疫荧光观察新生组织结构和血管生长情况。结果1.吸脂物液态部分分离培养的ASCs形态类似于成纤维细胞,具有很强的增殖及多向分化能力。在脂肪、成骨及成软骨分化诱导培养基的作用下,分化出成熟的脂肪细胞、成骨细胞和软骨细胞,油红0、茜素红染色和阿新蓝染色阳性。实验检测到细胞表面抗原分子CD29、CD44持续表达,说明ASCs具有干细胞特性。2.Ad.EGFP可成功进入ASCs。24h可见绿色荧光,5d荧光表达强度明显较高,ASCs传代后仍有强荧光表达。转染ASCs后,不影响其增殖活性。MOI为0、10、20、30、50、100时转染率分别为0%,10.3%,26.6%,47.6%,94.7%,96.8%。3.经免疫荧光和ELISA检测证实腺病毒能成功地将人VEGF₁₆₅基因转染至人ASCs中,并获得有效的表达。4.支架与ASCs有良好的相容性及黏附性,对细胞无毒性,不影响细胞增殖。8w时,Ⅰ组植入物已降解,Ⅱ和Ⅲ组均有新生组织形成,Ⅱ组新生物平均湿重为18.83±0.71mg,Ⅲ组新生物平均湿重为21.10±1.16mg;Ⅲ组比Ⅱ组形成的脂肪组织结构更完整及更多。Ⅱ和Ⅲ组相互间均有统计学意义（P＜0.05）。常规病理切片及油红0染色均证实有脂肪组织形成,EGFP荧光显色阳性。5.术后2w,Ⅳ、Ⅴ、Ⅵ组的脂肪组织存活湿重分别为20.85±1.35mg,22.52±0.75mg,22.95±0.79mg。Ⅳ与Ⅵ组,P＜0.01;Ⅳ与Ⅴ组P＜0.05;Ⅴ与Ⅵ组,P＞0.05。血管计数显示:组Ⅳ的血管密度为6.50±2.07个/HPF;组Ⅴ为9.56±2.73个/HPF;组Ⅵ为12.00±2.45个/HPF。术后12w,Ⅳ、Ⅴ、Ⅵ组的脂肪组织存活湿重分别为13.53±1.16mg,21.20±1.00mg,22.80±0.72mg;Ⅳ与Ⅴ、Ⅵ组,P＜0.01;Ⅴ与Ⅵ组,P＜0.05;Ⅵ组与Ⅴ组脂肪组织血管密度有显著差异（P＜0.01）,均高于Ⅳ（P＜0.01）。血管计数显示:11.11±1.94个/HPF;组Ⅴ为15.55±2.77个/HPF;组Ⅵ为18.39±3.73个/HPF。在体内,ASCs这些细胞表达内皮细胞表面标记——CD31,参与血管的形成。讨论种子细胞的大量获取是构建组织工程脂肪核心问题之一。在脂肪组织中已被证实包含具有多向分化潜力的细胞,并有研究已经证实在脂肪基质中包含有多能干细胞。这种干细胞被命名为脂肪组织来源干细胞,缩写为ASCs。本实验从抽脂术液态部分成功获取ASCs。ASCs作为种子细胞与其他细胞相比具有相当的优越性,该细胞来源广泛、取材容易,大量获取,受干扰小。组织工程另一核心内容是寻找具有组织再生潜力的支架材料。在生物材料方面,已开发和研制了适用于不同组织构建的生物支架材料。各类材料都具有各自的优点和缺点,而胶原蛋白在组织工程构建中作为支架材料的效果已被肯定。在这个实验中,显示胶原蛋白作为支架材料与ASCs有良好的相容性及黏附性,对细胞无毒性,不影响细胞增殖。同时,细胞标记一直是困扰组织工程技术修复机制研究的一大难题,其在干细胞来源的种子细胞的缺损修复研究中尤为重要。寻找一种直观、长期稳定,同时不影响细胞组织形成能力的标记方法非常重要。在本实验中,EGFP对ASCs成功进行了标记,且具有上述优点。另外,VEGF应用方式一般可分为直接应用和间接应用。直接应用VEGF有其局限性:①VEGF价格昂贵;②难以在体内保持持续的有效浓度;③不能自我调节。若因子不能持续有效地支持新生血管网的形成,就不能防止血管形成后的退化和塌陷。而间接应用是利用基因转染技术将基因整合进宿主细胞,将一些能产生VEGF的细胞种植在局部组织,使其在较长时间内持续、稳定地产生VEGF。能很好地克服直接应用的缺陷。在这个实验中,我们利用基因转染技术,通过基因载体——腺病毒把VEGF₁₆₅基因成功转入ASCs内,ASCs能大量、稳定且持续较长时间分泌VEGF。本实验结果表明,通过腺病毒介导,将VEGF₁₆₅基因转染到体外培养的人ASCs中,应用脂肪组织工程植入物联合移植裸鼠皮下的动物实验模型,发挥了基因的治疗性血管形成作用,促进了工程化脂肪组织的血管新生,血供的及时重建,进而提高其移植存活水平及存活质量。同时通过大体标本及组织学检测,未发现有血管瘤等病变发生,提示此治疗是安全的。另外,在体内,ASCs这些细胞表达内皮细胞表面标记,具有向内皮细胞分化的潜能,参与血管形成,从而促进组织工程脂肪的快速血管化。使再生的脂肪组织内部获得血供能力而保证其长期稳定存活,从而提高组织工程脂肪成活水平具有可操作性。结论:1.从成人脂肪抽吸物液体部分中获取的ASCs,从采集到培养都具有明显优势,将会为脂肪组织工程提供比较理想的种子细胞。2.腺病毒是较好的基因载体,是一个高效、安全快捷的基因修饰操作系统,EGFP在ASCs中能持续稳定表达,将为基因治疗和细胞示踪提供有效的实验方法。3.转染VEGF₁₆₅基因的ASCs能大量、稳定且持续较长时间分泌VEGF,能为血管形成提供丰富稳定的血管生成因子。4.从抽脂术液态部分获取的ASCs能够满足脂肪组织工程的种子细胞要求,而所选用的Ⅰ型胶原支架能在裸鼠体内降解吸收,在体内能成功构建脂肪组织。5.转染VEGF₁₆₅基因的ASCs具有促进工程化脂肪组织的血管化,具有向内皮细胞分化的潜能,参与血管形成,使得内皮细胞需要的数量相对减少,增加了脂肪组织工程在临床上应用的可能。更多还原

【Abstract】 BACKGROUND:Tissue engineering is a newly emerging subject that provides a new method to repair or regenerate construction and function of tissue defects using principle of cytobiology and engineering.Recently,tissue engineering has been being constructed as a newly emerging biomedical technology to repair or regenerate a body defect by combining cells of high proliferation and differentiation potential with an artificial matrix of cells scaffold and growth factor,including bone,skin,cartilage,vascular, and adipose tissues.However,the only products of tissue engineering that have been used in a clinical setting are of a relatively circumscribed dimension,supplied by diffusion. Because diffusion can supply cells only over a distance of 150μm,this nutrition alone is insufficient for three-dimensional constructs of larger size.The nutrient supply to cells at the center of the graft is inadequate,leading to cell necrosis.To overcome this limitation in the application of cultured engineering tissue,research must focus on accelerating vascularization of the transplanted cells.Especially,fat tissue represents a highly vascularized tissue and the substitution of soft tissue by autologous transplantation of cultivated ASCs requires a large cell volume.Nutrition of these composites by diffusion alone is insufficient.A volume-persistent culture of adipose tissue can be successful only via early vascularization of the composite in order to avoid considerable cell death,especially in the central graft area. VEGF is a kind of the most important angiopoiesis regulatory factor in reconstructing blood circulation as specificity mitogen of vascular endothelial cell, and it is important in process of neovascularization.It is a optima development potentiality therapy mode that transplanting cell imported with VEGF₁₆₅ gene to reconstruct blood circulation in vivo.Studies for combined application of cellular transplant and angiogenic factors in small animal model have been reported recently to enhance the effects of neovascularization.Until now,the research about transplanting human adipose tissue-derived stem cell transfected with VEGF₁₆₅ gene in the adipose tissue engineering for increasing neovascularization and the survival has no report.In this study,we applied this method to the adipose tissue engineering and investigate to see whether autologous ASCs encoding by VEGF₁₆₅ gene are capable of improving neovascularization in the adipose tissue engineering,with potential clinical application for the adipose tissue engineering.OBJECTIVES:1.To find a new method which can be used for isolation and cultivation of ASCs from liposuction,and the ASCs ability differentiated into lipocytes and osteoblast with the foundation of ASCs is the seed cell of adipose tissue engineering。2.To investigate the transfection efficiency and affect of recombinant adenovirus in human adipose tissue-derived stem cell,and to find a better method to label ASCs.3.To establish the foundation of advancing vascularized capability of adipose tissue engineering,by detect the gene expression of ASCs transfected with vascular endothelial growth factor 165(VEGF₁₆₅) mediated by adenovirus.4.To assess the possibility of constructing adipose tissue via the attachment of ASCs to typeⅠcollagen scaffold.5.To investigate the feasibility of transplanting human adipose tissue-derived stem cell（ASCs） transfected with VEGF₁₆₅ gene to the adipose tissue engineering for increasing neovascularization and the survival.METHODS:1.ASCs Isolation and cultivation and identification ASCs were isolated and cultivated from the liquid portion of liposuction aspirates by directly centrifugate for primary culture and observe changes in morphology and function of cells.The cells are used for experiments after the generation four.Flow cytometry was used to detect the molecular expression which is associated with the surface of the stem cells;making cells in vitro to adipogenic, osteogenic,cartilagenic differentiation.Oil Red O staining and Alizarin red staining and Alcian blue staining to identify the success of differentiation.2.Transfection and marker of EGFP in human adipose-derived stem cellsThe ASCs was infected with adenovirus expressing the EGFP gene by different multiphcity of infection（MOI=0,10,20,30,50,100）.The expression of EGFP was detected by fluorscent microsecope.The cytotoxicity of adenovirus to ASCs was evaluated by cell proliferation cell vitality and cell differentiation.3.Expression of adipose-derived stem cell transferred by VEGF₁₆₅ geneHuman adipose tissue were isolated and cultured the cells that the ASCs of human was infected with adenovirus expressing VEGF₁₆₅ gene according to multiphcity of infection equaling to 50.Gene expression of transtected cells was detected by immunofluorescent staining and ELISA analysis in vitro.4.Construction of tissue-engineered adipose using typeⅠcollagen in nude mouseHuman adipose tissue were isolated and cultured the cells from fluid portions of liposuction aspirates.We evaluated the use of a combination of adipose tissue derived adult stem cells and collagen sponge for adipose tissue engineering.Adipogenesis was examined in nude mice imbeded subcutaneously with collagen sponge（Ⅰ）,ASCs attached collagen sponge（Ⅱ）,or adipogenic differentiation of ASCs attached collagen （Ⅲ） cultured in medium for 3 days.After 8 weeks,newly formed adipose tissue was observed by fluorescent microscope,histology and Oil red O.5.Transplanting ASCs transfected with VEGF₁₆₅ in the adipose tissue engineering for increasing neovascularizationASCs was isolated from fluid portions of human liposuction aspirates.After transfection by VEGF₁₆₅ gene,then adipogenesis was examined in nude mice imbeded subcutaneously with adipogenic ASCs with differentiation attached collagen sponge（groupⅣ）,differentiation and indifferentiation with EGFP gene transfection attached collagen sponge（groupⅤ）,differentiation and indifferentiation with VEGF₁₆₅ gene transfection attached collagen sponge（groupⅥ） cultured in medium for 3 days.Its were transplanted to the adipose tissue engineering at the same of a nude mouse.The capillary density of transplanted the engineered adipose tissue was detected by blood vessel counting.RESULTS:1.There was a large amount of ASCs in the liquid portion,the cells growed appearance as fibroblasts-like,but it has strong tendency for high proliferation and multiple differentiation.With the effect of adipogenic and osteogenic and cartilagenic differentiation medium,it is proved to be able to differentiate into mature adipocyte and bone cells and chondrocyte.Adipogenic differentiation of ASCs was assessed by Oil Red O staining after 2 weeks and significant fraction of the cells contained multiple,intracellular lipid-filled droplets that accumulated Oil Red-O.After 2 weeks’ osteogenic induction,cells were positively stained by alizarin red.After 2 weeks’ cartilagenic induction,cells were positively stained by Alcian blue.These kind of cell （ASCs） is also proved to be CD29and CD44 positive expression which are one of the main proof for stem cells.2.Adipose-derived stem cells were successfully infected by adenovirus.EGFP was initially expressed in the adipose tissue-derived stem cell of all transfected groups 24h following infected by Ad.EGFP.The level increased with the incease of MOI and reached peak on 5d.Transfection efficiency of the Expression were 0%（0）; 10.3%（10）;26.6%（20）;47.6%（30）;94.7%（50）;96.8%（100）,respectively.During the period of the culture,cell proliferation,cell vitality and cell differentiation in the transfected and untransfected groups had no difference on 1,3,5,7,9,11d.3.ASCs were successfully infected by vascular endothelial growth factor 165(VEGF₁₆₅) mediated by adenovirus.The expression of human VEGF₁₆₅ in the transfected human ASCs was demonstrated by immunofluorescent and ELISA analysis.The level increased with the incease of time and reached peak on 7d.4.ASCs were successfully cultured in collagen sponge.collagen represent good compatibility and adhesion with ASCs without a little toxicity;newly constructed tissue are found in the experiment.After 8 weeks,newly formed adipose tissue was observed in groupsⅡandⅢ,but groupⅠwas fully reabsorbed.The wet weigh of the groupsⅢwas significantly higher than those of the groupsⅡ（P＜0.05）.HE or Oil red O staining of newly formed tissue showed that there was substantially more tissue regeneration and adipogenic effect in groupⅢthan in groupⅡ.EGFP expressed in the newly formed adipose tissue was observed by fluorescent microscope.5.Two weeks after transfection,transplanted ASCs survived and were incorporated into the capillary networks in the engineered adipose tissue.The wet weight of transplanted tissue of VEGF₁₆₅ gene transfection group was 22.95±0.79mg, significantly higher than that of the groupⅣ（20.85±1.35mg,P＜0.01）,but groupⅤ（22.52±0.75mg,P＞0.05）.The vessels counting of the newly formed adipose tissue for each group were:6.50±2.07/HPF,9.56±2.73/HPF,12.00±2.45/HPF,respectively.The capillary density of transplanted tissue of VEGF₁₆₅ gene transfection group was significantly higher than those of the groupⅣand groupⅤ（P＜0.05）.Twelve weeks after transfection,transplanted ASCs survived and were incorporated into the capillary networks in the engineered adipose tissue.The survival volume of transplanted tissue of VEGF₁₆₅ gene transfection group was 22.80±0.72mg, significantly higher than that of the groupⅤ（21.20±1.00mg,P＜0.05） and groupⅣ（13.53±1.16mg P＜0.01）.The vessels counting of the newly formed adipose tissue for each group were:11.11±1.94/HPF,15.55±2.77/HPF,18.39±3.73/HPF,respectively. The capillary density of transplanted tissue of VEGF₁₆₅ gene transfection group was significantly higher than those of the groupⅣand groupⅤ（P＜0.01）.These kind of cell（ASCs） is also proved to be CD31 positive expression in vivo,and to take part in angiopoiesis.DISCUSSIONS:One of the major challenges facing the emerging field of regenerative medicine is finding a reliable source of cells for tissue repair and regeneration.In experiment, Human ASCs form fluid portions of human liposuction aspirates in vitro meet many of the requirements required of the ’ideal’ cell for tissue engineering.These stem cells can be easily obtained,easily purified,and are readily expanded in culture,using a relatively noninvasive method.Stem cells offer a potentially unlimited source of cells for tissue engineering;thus,research in using stem cells to produce adipose tissue has become increasingly popular.Another of the major challenges facing the emerging field of regenerative medicine is finding a reliable source of scaffold for tissue repair and regeneration.A lot of scaffolds were applied to tissue engineering,but there were much disadvantage. In the experiment,ASCs were successfully cultured in collagen protein.Collagen protein represents good compatibility and adhesion with ASCs without little toxicity; newly constructed tissue are found.The collagen protein was positived about application of engineering adipose tissue.At the same time,one of the major challenges facing the emerging field of repair mechanism of engineering adipose tissue is finding a better method to label ASCs.It is important for us to find a better method to label ASCs.In the experiment, transduction of EGFP gene to ASCs by adenovirus is safe and high efficient,and the target protein can be expressed in cell in vitro.EGFP transfection is a better method to gene therapy and label ASCs.In addition,On molecular level,angiopoiesis was a aggregation of a series of molecular event.Vascular growth factor was the most superlative and multiplicity in angiopoiesis,of the total,VEGF was the most effective in the initial stage.VEGF₁₆₅ is predominant and powerthl secrete modality.In general,applied way of VEGF was divided to direct application and indirect application.In the direct application,there were much disadvantage of expensive price,and hard to maintain effective concentration in vivo,and inability self-adjustment.If the factor was inefficiently to maintain formation of new vasoganglion,it is not to prevent the retrogradation and collapse of vasoformation.However,it is very good for indirect application to overcome disadvantage of direct application.In the experiment,transduction of VEGF₁₆₅ gene to ASCs by adenovirus is safe and high efficient,and the target protein can be expressed in cell and secreted to surrounding at a high level in vitro.It may be used to increase vascularized capability of ASCs in the study of adipose tissue engineering.Eventually,in experimental result,ASCs from fluid portions of human liposuction aspirates in vitro were successfully infected by vascular endothelial growth factor 165(VEGF₁₆₅) mediated by adenovirus,and can increase the engineered adipose tissue neovascularization and the survival,and bring into full play effectiveness of genic remedial angiopoiesis.Meanwhile,the kind of treatment is safe, because we do not find pathological changes of vascular tumor by the detection of specimen and histology.In vivo,ASCs can be differentiated the endothelial cell to construct blood vessel by immunofluorescent staining,which is possible for us to increase application of the adipose tissue engineering in clinical.CONCLUSIONS:1.ASCs was isolated by directly centrifugate and filtrate the fluid portions of autologous liposuction aspirates of human without enzymatic digestion.The way of Isolation and cultivation is convenient and easier to carry out.possessed the capabilities of pluripotential differentiation.The ASCs can rapidly in vitro,and reached the number for implantation in short time.Thus,the use of ASCs as the seed cell of to engineer adipose tissue seems to be more inspiring.2.Transduction of EGFP gene to ASCs by adenovirus is safe and high efficient, and the target protein can be expressed in cell in vitro.EGFP transfection is a better method to gene therapy and label ASCs.3.Transduction of VEGF₁₆₅ gene to ASCs by adenovirus is safe and high efficient,and the target protein can be expressed in cell and secreted to surrounding at a high level in vitro.It may be used to increase vascularized capability of ASCs in the study of adipose tissue engineering.4.This study provides significant evidence that ASCs obtained from fluid portions of liposuction aspirates and collagen can be used in adipose tissue engineering.5.ASCs form fluid portions of human liposuction aspirates in vitro can increase the engineered adipose tissue neovascularization and the survival,and the ability of promoting neovascularization of ASCs transfected with VEGF₁₆₅ gene is more potent than ASCs alone.In vivo,ASCs can be differentiated the endothelial cell to construct blood vessel,which is possible for us to increase application of the adipose tissue engineering in clinical.更多还原