【作者】 王艳梅；

【导师】 庄洪兴； 蒋海越； 杨庆华； 赵延勇；

【作者基本信息】 中国协和医科大学 ， 外科学， 2009， 博士

【摘要】 研究背景:外伤或先天性小耳畸形致耳廓缺损患者在整形外科十分常见,而耳廓软骨几乎无再生修复能力,目前临床上主要的治疗方法为全耳廓再造术,对手术医师技术要求较高,且存在不同程度的并发症,如采集自体肋软骨雕刻耳支架可致胸廓畸形,而支架可能感染、吸收、变形。人工高分子耳支架植入容易引发机体排异反应致使支架外露发生率较高。因此,自体软骨细胞组织工程研究应运而生。尽管已有自体软骨细胞组织工程软骨批准进入临床使用,但仅能修复体积小于1ml缺损。研究发现组织工程软骨厚度超过1mm,中心易发生“空心”现象。而这种“空心”现象的主要原因在于软骨组织缺乏血管,细胞代谢依赖细胞外基质（extracellular mattix,ECM）的渗透,随着外层细胞外基质不断的合成与成熟,超过一定厚度的内层细胞因传导效率降低,发生代谢障碍致组织缺氧和坏死。因此,保持内层细胞增殖、存活,并维持软骨细胞生物特性成为我们实验研究的焦点。血管内皮生长因子（vascular endothelial growth factor,VEGF）,是体内重要的促血管生长因子。对成熟软骨细胞分泌表达蛋白的研究发现,软骨细胞表达一定水平的VEGF以提高软骨细胞有丝分裂活性并促进其再分化,在软骨发生发育过程中,对软骨细胞的存活与增殖起着重要的作用。运用基因工程技术,将VEGF基因导入软骨细胞,构建VEGF强化组织工程软骨,将有利于软骨细胞存活、增殖、分化,改善组织工程软骨内部结构。目的:观察检测残耳软骨细胞体外扩增生物学性能。构建hVEGF₁₆₅基因载体并转染体外培养的残耳软骨细胞,通过检测转染后软骨细胞VEGF及主要软骨ECM蛋白表达,以分析转VEGF基因软骨细胞的生物性能,并对转VEGF基因软骨细胞体内构建的软骨形态结构、ECM蛋白及相关基因表达进行检测分析。方法:1.构建重组腺相关病毒rAAV2-hVEGF₁₆₅-CMV-EGFP载体。2.分离、培养人残耳软骨细胞,检测倍增时间,免疫组化,RT-PCR方法检测其产生软骨ECM主要成分能力,包括蛋白聚糖（aggrecan,AG）,Ⅰ型胶原（collagen typeⅠ,COLⅠ）,Ⅱ型胶原（collagen typeⅡ,COLⅡ）,Ⅹ型胶原（collagen typeⅩ,COLⅩ）。3.rAAV2-hVEGF₁₆₅-CMV-EGFP转染软骨细胞,流式细胞技术检测转染效率,组织学染色、免疫组化、Real-time PCR、Western Blot、ELISA检测VEGF表达规律及转染后软骨细胞分泌软骨基质的变化。4.转染rAAV2-hVEGF₁₆₅-CMV-EGFP的软骨细胞与Pluronic F-127混合,形成细胞材料复合物接种于裸鼠皮下,同时分别设立转染rAAV2-EGFP细胞组及单纯软骨细胞组为对照组,10周后取材,组织学染色、免疫组化比较观察三组软骨组织结构,Real-time POR、Western Blot等方法检测其ECM蛋白及与软骨细胞增殖存活相关基因表达。结果:1.成功构建rAAV2-hVEGF₁₆₅-CMV-EGFP载体。2.残耳软骨细胞在体外扩增过程中,逐渐由多角形变为长梭形,AG、COLⅡ、COLⅩ逐渐减少,第5代时COLⅡ、COLⅩ表达消失,并开始表达COLⅠ。3.rAAV2-hVEGF₁₆₅-CMV-EGFP载体体外成功转染软骨细胞,转染第3天转染效率达20.1±0.62%,并维持稳定。转染后VEGF表达延迟期为14-21天,随VEGF表达增高,软骨细胞AG、COLⅡ、COLⅩ及COLⅠ表达显著增高。4.三组细胞-材料复合物接种裸鼠皮下10周后取材,均形成大小不一的软骨,组织切片染色见VEGF转染细胞实验组形成均一软骨组织,软骨细胞及软骨陷窝分布均匀,糖胺多糖及胶原纤维含量丰富,弹性纤维呈网状分布均匀,而两个对照组软骨组织间见局部空泡样结构,糖胺多糖、胶原纤维含量欠佳,弹性纤维成条索样分割包绕软骨细胞。免疫组化结果见三组样本均有VEGF阳性细胞,实验组分布于软骨组织边缘,对照组散在于组织中,三组Ⅷ因子表达均为阴性。Western Blot结果见实验组VEGF、COLⅡ表达强于对照组,VEGFR-2仅在实验组表达。GAG含量检测实验组显著增高,对照组间无明显差异。Real-time PCR结果显示,实验组较对照组的RunX2、Sox9表达增高,PTH/PTHrR无显著性变化,同时COLX及COLⅡ表达增高,而对照组间RunX2等的表达无明显差异。结论:1.rAAV2-hVEGF₁₆₅-CMV-EGFP能体外转染人残耳软骨细胞,转染第3天转染效率达20.1±0.62%,并维持稳定。2.转染后,VEGF表达提高了软骨细胞功能而具备良好的生物学特性。转染14天的第3代软骨细胞ECM主要蛋白表达增高,细胞功能活跃,适宜做为组织工程软骨种子细胞。3.转VEGF基因软骨细胞作为组织工程的种子细胞与pluronic F-127复合后可于裸鼠体内形成转基因组织工程软骨,与对照组相比,转VEGF基因组织工程软骨具良好的生物学特性,结构均一且与正常软骨组织相似,软骨ECM的GAG、COLⅡ、COLⅩ增多,RunX2、Sox9表达增高,细胞处于增生期的肥大状态,初步分析其原因可能是转染后外源性的VEGF持续表达触发了软骨细胞VEGF自分泌,并通过VEGFR-2作用于软骨细胞,提高了软骨细胞活性,促进其存活与增殖,但未在软骨组织内引起血管内皮细胞的迁移及小血管形成。更多还原

【Abstract】 Background Defection of auricle caused by injury or congenital microtia is a common disease which brings negative affection for patients’ complexion and physiology in plastic surgery field.The major treatment method for these patients is auricular reconstruction surgery because bad regeneration of auricular cartilage.The surgery has many complications,such as defection deformity of donor site,infection,deformation,absorption of rib framework, even though autologous one has no rejection action.Artificial polymers exposed from skin easily because of rejection.Tissue engineering cartilage research brings the hope for patients.Although the use of autologous cartilage tissue engineering cartilage in clinical has been proved,but it only fit the defection less than 1mm.Cartilage is avascularity tissue,and chondrocyte metabolism depended on permeability of extracellular matrix（ECM）.The more mature and thicker of extracellular matrix produced by external cells,the lower conduction efficacy induced dysbolism and lack of oxygen.Central chondrocytes of tissue were necrotic,while the circumjacent cells were survival,then,the "hollow" happened.Therefore,our experiments focus on retain the proliferation and survival of central chondrocytes,and keep them alive.Vascular endothelial growth factor（VEGF） is an important angiogenine. There is accumulating evidence shows that expression of VEGF in mature chondrocyte can promote cell redifferentiation and caryocinesia activity. VEGF plays a key role for chondrocyte to survival and proliferation during the chondrogenesis.We used gene engineering technical transfer VEGF into chondrocytes to construction gene -enhance engineering cartilage,which may benefit to the chondrocytes’ survival,proliferation,differentiation and improve the internal construction in engineering cartilage.Objection Observation and analysis the biologic character of cultured chondrocyte from pediatric residual auricular cartilage of microtia patients.We constructed hVEGF₁₆₅ vector and transfected VEGF gene to chondrocyte in vitro,analyzed the expression of VEGF and evaluated the biologic character of chondrocytes transfected VEGF by ECM analysis.When chondrocytes transfected VEGF formed tissue engineering cartilage vivo,we evaluated its biology characters by evaluation of tissue structure,the expression of ECM proteins and related-genes.Method 1.Construction of recombinant adeno-associate virus vectors rAAV2-hVEGF₁₆₅-CMV-EGFP.2.Isolation and culture of chondrocytes from pediatric residual auricular cartilage.Test of double time. Immunohistochemistry,RT-PCR can be used to analyse extracellular matrix produced by chondrocyte.ECM includes Collagen typeⅡ,Ⅹ,Ⅰand aggrecan. 3.When the transfection is completed,transfection efficiency has been quantities by flow cytometry.The expression pattern of VEGF and the chondrocyte extracellular matrix are examined by histology staining, immunochemistry,Real-time PCR,ELASA,Western Blot methods.4.Nude mouse has been implanted complex made up chondrocytes transfected rAAV2-hVEGF₁₆₅-CMV-EGFP and pluronic-F127,while chondrocyte transfected rAAV2-EGFP and pure chondrocvte also implanted respectively as control group. 10 weeks later,cartilage-like tissues were taken out,histology and immunohistochemical staining has been used to analyze the structure of cartilage.The differences among the 3 groups were compared by test of the tissue structure,ECM protein and the expression of gene about survival and proliferation of chondrocyte by Real-time PCR and Western Blot method.Result 1.We constructed VEGF vector rAAV2-hVEGF₁₆₅-CMV-EGFP successfully. 2.With proliferation of chondrocyte in vitro,the shape of it turned fiber-like shape from polygon.The expression of AG,COLⅡand COLⅹdecreased gradually.The COhⅡ、COLⅹdisappeared,while COLⅠbegin express in passage 5.3.rAAV2-hVEGF₁₆₅-CMV-EGFP tranfected chondrocyte successfully in vitro.The transfection efficiency reach 20.1±0.62%after 3 days and keep stable.Following extend of transfection phase,the expression of VEGF is up first,down later,and up again,as well as the expression of ECM. 4.All cell-materials complex groups produced cartilage with different size on nude mouse subcutaneously.Chondrocyte transfcted VEGF gene Pluronic F-127 complex produced homogeneous tissue,while the control tissues were "hollow".The result of Safranin O and Verhoeff stain showed GAG of the experimental groups was plentiful,chondrocyte and cartilage lacuna was well-distributed and elastic fiber distribution was reticulodromous.The result of immunohistochemistry and Western Blot suggested experimental group have more collagenⅡthan control group.VEGF-positive cell were found in all group,but on boundary of experimental group tissue and all field of control group.ⅧFactor could not be found in all groups.The result of Western Blot showed the expression of VEGF are more abundant than control group,and VEGFR-2 was just be found in experimental group.The result of real-time PCR showed expression of RunX2,Sox9,collagen typeⅡ,Ⅹhigher in experimental group,especially of GAG when compare to control group,but there has no significant change in PTH/PTHrR expression.There are not differences between the control groups.)Conclusion:VEGF gene can be transfer chondrocyte by rAAV2-hVEGF₁₆₅-CMV-EGFP vector successfully in vitro.They share the same pattern between VEGF expression and ECM protein.Cells become ageing when the major protein component of ECM decreased,VEGF expressed down.Moreover,senescence chondrocyte can redifferentiation and increase the expression of AG,COLⅡ, COLⅩand COL,while VEOF express up.The results from vivo showed chondrocytes transfected VEGF can form cartilage tissue.Compared with control group,more COLⅡ,COLⅩand AG and the expression of RunX2、Sox9 appeared in chondrocyte transfected VEGF experimental group.Elastic fibers were well-distributed,and vascular endothelial cell did not immigrate.We suggested that expression of ectogenic VEGF triggered paracrine and autocrine of VEGF of chondrocyte and co-acted with VEGF receptor 2 to enhance permeability of chondrocyte and improve internal construct of engineering cartilage,and prevent vascularize proceed.更多还原