【作者】 贾懿；

【导师】 乔群； 裴雪涛；

【作者基本信息】 中国协和医科大学 ， 整形外科， 2009， 博士

【摘要】 干细胞及组织工程研究的深入进行为机体缺损组织的修复治疗提供新的、有效的途径和策略。然而目前研发的组织工程产品在体内应用研究时,其厚度都必须小于2毫米。一旦超过此厚度将直接影响营养物质和氧气向植入细胞的输送,包括组织工程心肌、肝、脂肪以及平滑肌组织的研发和应用,均受限于组织厚度而难以有效发挥生物学功能。较大体积(直径＞1厘米)的复合植入物不仅依靠受体营养物质扩散,细胞功能的正常发挥还必须借助于良好血运的支持。构建组织工程血管尤其是微血管网成为人工组织、器官产品能否有效发挥生物学功能的关键问题之一。人脂肪干细胞(human adipose derived stem cells,hADSCs)已被证实具有向血管内皮细胞在内的多种组织细胞分化的潜能并初步用于组织工程和再生医学应用研究;纤维蛋白凝胶因其良好的生物相容性被视为组织工程的良好支架材料来源。本课题研究拟采用聚乙二醇修饰剂优化纤维蛋白凝胶特性,将hADSCs与修饰后的纤维蛋白凝胶复合,观察体外构建组织工程微血管的可行性,为组织器官的体外构建及生物学功能的发挥提供相关实验依据。研究目的1、建立并优化体外纤维蛋白凝胶复合hADSCs后向血管内皮细胞(BVEC)诱导分化的技术方法,并进行生物学性状鉴定。2、优化PEG对纤维蛋白原的修饰条件并对纤维蛋白凝胶相关特性改变进行鉴定。3、明确聚乙二醇修饰后的纤维蛋白凝胶对于复合的hADSCs向血管内皮细胞诱导分化有无影响。研究方法1、通过吸脂手术得到正常人脂肪组织,经胶原酶消化和贴壁培养方法获得hADSCs。通过形态学观察、表面标志物检测、向脂肪细胞、血管内皮细胞诱导分化等检测和分析生物学特性。2、取P3代hADSCs,同纤维蛋白原溶液混合后加入凝血酶,制成凝胶。加入血管内皮细胞诱导培养基诱导培养。按照不同细胞种植密度和纤维蛋白原浓度分组观察来优化培养条件。通过观察细胞形态改变,了解其生长状态;HE染色观察凝胶内细胞生长状态;扫描电镜和透射电镜观察凝胶内诱导细胞结构。3、甲氧基聚乙二醇琥珀酰亚胺基碳酸酯(mPEG-sc)和聚乙二醇琥珀酰亚胺基碳酸酯(sc-PEG-sc)对于纤维蛋白原的平均修饰度测定。通过TNBS法和SDS-PAGE法测定不同修饰配比度和反应温度下的纤维蛋白原的聚乙二醇化平均修饰度变化。观察修饰后纤维蛋白凝胶性状变化,包括:成胶时间、成胶率、BCA法测定抗酶降解能力(纤维蛋白溶解酶、胶原酶)及电镜下凝胶超微结构。4、取P3代hADSCs,常规胰酶消化,计数后同聚乙二醇修饰后的纤维蛋白原溶液混合后加入凝血酶,制成凝胶。加入血管内皮细胞诱导培养基,每2天更换一次。通过观察细胞生长形态和测定细胞生长曲线了解其生长状态;RT-PCR鉴定诱导后的血管内皮细胞特异性基因表达。实验结果1、hADSCs呈长梭形,旋涡状排列生长。流式细胞仪检测hADSCs表面标志:CD34、CD45(造血干细胞的特异性表面标志)和Flk-1(内皮细胞表面标志)表达阴性;CD29、CD44(粘附分子)、CD90及CD105(转化生长因子受体),表达阳性CD71(转铁蛋白受体)仅有微弱表达。在一定诱导培养条件下,可以向脂肪细胞诱导分化。2、在特定诱导培养条件瞎,hADSCs可以向血管内皮细胞诱导分化:诱导后4d开始发生形态改变,长梭形细胞逐渐缩短转为椭园形:诱导第8天细胞出现多边形,增殖明显。诱导第12天细胞增殖达到高峰,密度大,局部呈现“鹅卵石”样排列细胞。免疫组化和荧光染色证实有vWF、Flk-1、CD31、CD34、等血管内皮细胞特异性标记物表达。RT-PCR结果显示诱导第7天的hADSCs表达血管内皮细胞特异性基因包括CD31、CD34和VE-cadherin。3、纤维蛋白凝胶由纤维蛋白原溶液加入凝血酶作用后形成。其强度同纤维蛋白原浓度呈正相关。脂肪干细胞按照不同细胞密度种植在2mg/ml的纤维蛋白凝胶后向血管内皮细胞持续诱导。细胞在凝胶内呈现立体生长方式,诱导第2天细胞由梭形条索状转为星芒状,诱导第6天开始出现相邻细胞之间接触形成管状结构,诱导第8天细胞生长形成明显管网状结构,诱导第12天管网状结构密度达到高峰。随着种植的细胞密度增加(0.5～2.0×10~5/ml),细胞形成的立体管网密度明显增大,而随着纤维蛋白原浓度增加(2.5mg/ml～10mg/ml),细胞形成的立体管网密度逐渐减少。复合材料经HE染色后可见hADSCs立体诱导培养后在凝胶内形成的管网状结构。通过扫描电镜可见纤维蛋白凝胶内出现一些血管内皮细胞生长所特有的管状结构。透射电镜可见细胞内出现内皮细胞特有的Weible-palade小体和细胞构成的管腔样结构。经TNBS法和SDS-PAGE法测定,mPEG-sc和sc-PEG-sc对于纤维蛋白原的平均修饰率随着修饰剂用量和反应温度上升而逐渐增大。随着mPEG-sc和sc-PEG-sc用量摩尔配比度的上升,纤维蛋白原成胶率逐渐下降,纤维蛋白原成胶时间逐步增加,而凝胶的稳定性逐渐下降。成胶反应温度升高可减少成胶时间,但对于形成的凝胶稳定性无影响。纤维蛋白原在mPEG-sc低配比度条件下(10:1),纤维蛋白凝胶抗胶原酶降解能力增强,但配比度从25增至100时,抗胶原酶降解能力逐渐减弱。在sc-PEG-sc配比度10:1、25:1和50:1条件下,纤维蛋白凝胶抗胶原酶降解能力增强,配比度增至100:1时,抗胶原酶降解能力明显减弱。不同浓度的纤维蛋白原溶液形成的纤维蛋白凝胶,经电镜观察发现20mg/ml浓度的凝胶内纤维蛋白交联后形成的支架孔径小于10mg/ml的凝胶材料。20mg/ml浓度纤维蛋白原溶液经mPEG-sc和sc-PEG-sc按照50:1和100:1修饰后发现相比未修饰的凝胶,支架孔径逐渐缩小,但是随着修饰度的上升,材料内支架密度下降。同样配比度修饰条件下50:1,sc-PEG-sc-Fn材料孔径小于mPEG-sc-Fn材料。4、通过生长曲线观察到hADSCs在聚乙二醇(mPEG-sc和sc-PEG-sc)修饰后的凝胶内增殖速度无差异,三组细胞生长曲线形态相似。向血管内皮细胞诱导观察发现各组细胞生长良好,都出现立体管网状生长,管网密度相似,无明显差别。RT-PCR显示诱导5天后纤维蛋白原凝胶组和sc-PEG-sc组表达CD31、CD34、VE-cadherin和KDR;mPEG-sc修饰组表达CD31、CD34和KDR。结论1、hADSCs在体外可稳定传代,并具有多向分化能力。体外经诱导后可向血管内皮细胞分化,并表达特异性标记。2、hADSCs同纤维蛋白凝胶复合后以三维立体方式向血管内皮细胞诱导,在表达血管内皮细胞特异性标记同时凝胶内出现立体管网状结构。3、证实了聚乙二醇修饰剂对于纤维蛋白原修饰的可行性。优化聚乙二醇修饰反应条件的同时,明确了纤维蛋白凝胶生物特性的改变。纤维蛋白凝胶的聚乙二醇化能有效改善纤维蛋白作为细胞支架材料的生物特性,有利于体外血管构建方法的完善和体内应用研究。4、聚乙二醇化的纤维蛋白凝胶不影响hADSCs诱导、分化及血管内皮细胞网状结构的形成。该方法优化了纤维蛋白凝胶作为组织工程微血管构建材料的部分功能,为今后完善组织工程微血管材料的研制提供了新的选择。更多还原

【Abstract】 BackgroundThe tissue engineering products provide an effective alteration treatment for those patients with tissue defects and organ dysfunction.However,due to the barrier to nutrients and oxygen transport,the dimension of implants is restricting to 2 millimeter. Researchers had applied tissue engineering composites such as cardiac muscle and hepatic cells to restore the organ function.Moreover,the autologous tissue including adipose and smooth muscle also had been implanted for reconstruction purposes.The results were positive and also gave a clear conclusion that the dimension of implants must be controlled carefully,otherwise,the transplanting cells would be dysfunction. In these cases,the nutrients and oxygen for implants must be brought by the capillary network.In this condition,researchers focus on the construction of capillary network. Human adipose derived stem cells（hADSCs） possess the multi-directional differentiation ability and were applied as seed cells in the study of cell inducing. Being natural extracelluar matrix,fibrin gel was applied as an ideal scalffold for tissue engineered products,especially for artificial blood vessels construction in vitro. Pegylation is the process of covalent attachment of poly（ethylene glycol） polymer chains to another molecule,normally a drug or therapeutic protein.The technique of pegylation has noticeably developed sine 1970s.To this day,pegylation help to meet the challenges of improving the safety and efficiency of many therapeutics.In this study,we will incubate PEG with the fibrinogen macromolecule to improve biological function and construct capillary-like network with human adipose-derived stem cell and pegylated fibrin gel in vitro.Objective1.To optimize the process of blood vessel cells-like inducing with hADSCs mixed within fibrin gel.To observe the biological characteristics of these seeding cells and scalfford for tissue engineered capillary-like patch construction.2.To optimize the process of incubation of a reactive derivative of PEG with the fibrinogen macromolecule.To observe the alteration of biological characteristics of pegylated fibrin gel.3.To establish and optimize the process of blood vessel cells-like inducing with hADSCs mixed within pegylated fibrin gel.Methods1.The human fat tissue collected by liposuction was digested and filtrated to isolate hADSCs.The biological characteristics of hADSCs was evaluated via morphology of cell growth,cell phenotype（CD133、CD11b、CD34、CD71、CD29、CD45、CD31、CD90、CD11a、CD44、Bcl2和Flk1） expression and adipose cell-like inducing.2.The hADSCs was induced to differentiate towards blood vessel endothelial cells （BVECs）.The condition of inducing was estimated by the expression of BVECs specific mark at protein level（vWF、Flk-1、CD31、CD34） and gene level（CD31、CD34,VE-cadherin and Flk-1） through immunochemistry,immunofluorescence assay and RT-PCR assay.3.The hADSCs（P₃） were cultured within fibrin gel and subsequently,induced towards BVECs.The process was optimized through screening various cell density and concentration of fibrinogen.The biological characteristics of differentiating cells within fibrin gel such as phenotype,growth structure and cell ultrastructure were estimated by optical microscopy,scanning electron microscope and transmission electron microscope.4.PEG agents including sc-PEG-sc and mPEG-sc were added to fibrinogen in different molar ratios and reaction temperature.The pegylation level of fibrinogen was measured by TNBS and SDS-PAGE.The modified biological characteristics of fibrin gel were measured through clotting time,percentage of clottable protein and degradation rate of plasmin and collagenase.The ultrastructure of pegylated fibrin gel was observed through scanning electron microscope.5.The hADSCs（P₃） were cultured within pegylated fibrin gel and subsequently, induced towards BVECs.The biological characteristics of differentiating cells within fibrin gel such as growth curve,phenotype,growth structure and BVEC gene expression（CD31、CD34,VE-cadherin and Flk-1） were measured by optical microscopy and RT-PCR assay.Results1.The hADSCs isolated from human adipose tissue were cultured under Dulbecco’s modified Eagle’s medium plus 10%fetal bovine serum.After several passaging, the hADSCs presented a kind of fibroblast-like phenotype.Gradually,the shape of hADSCs turned to be irregular shape with increasing intramembranous particles after 8～10 passaging.The cell senescence presented after 15 passaging. The flow cytometry detected hADSCs（P₄） strongly expressed CD29,CD44 and CD90.However,the expression of CD105 and CD71 were weak and CD34 and CD45 were negative.The hADSCs were induced differentiation towards adipose cell and the lipid droplets were observed at 6^th day which were determined by oil red staining.2.Before differentiating to BVECs,the hADSCs presented a fibroblast-like phenotype.At sixth day of inducing,the phenotype turned to be ovoid in shape and to be polygon with increasing proliferation at eighth day.The proliferation of cells reached the summit at 12^th day with high cell density.The cobble-like cells which were typical pattern of endothelial cells were observed as well.The specific mark of BVECs including vWF,Flk-1,CD31 and CD34 were determined at 8th day.The blood vessel endothelial cells specific gene including CD31, CD34 and VE-cadherin were determined in hADSCs which were inducing differentiation after 7 days.Only weak expression of VE-cadherin was observed in hADSCs without inducing.3.Fibrin gel was prepared by adding thrombin into fibrinogen solution.The strength of gel was determined by the concentration of fibrinogen solution.Within fibrin gel,the differentiated hADSCs presented a special growth pattern which was 3-dimenstion.The cells showed a starlike shape after 24hours and formed network structure between adjacent cells at 6^th day.The network structure was obvious at 8^th day and the density reached the summit at 12^th day.With the increasing of cell density（0.5～2.0×10⁵/ml）,the density of network increasing accordingly.However,the network density decreased gradually when the concentration of fibrinogen increased from 2.5mg/ml to 10mg/ml.The cells which formed the network structure showed Weible-palade which was endothelial special mark.4.The pegylation level of fibrinogen increased gradually when molar ratios and reacting temperature raised.However,the clotting time,percentage of clottable protein and stability of fibrin gel decreased with the raised pegylation level.For pegylated with mPEG-sc,the resistance to collagenase degradation increased at molar ration 10,and reduced at molar ration ranged from 25 to 100.For sc-PEG-sc,the resistance to protease increased at molar ration ranged from 10 to 50 and reduced at molar ratio 100.For the study of plasmin degradation rate,no increased resistance was observed at all molar ratios for both mPEG-sc and sc-PEG-sc.The pore size of fibrin gel which was prepared by fibrinogen solution at 20mg/ml was smaller than 10mg/ml.For those pegylated fibrin gel,the pore size decreased gradually with the increasing level of pegylation with both mPEG-sc and sc-PEG-sc.Moreover,the density of fibrin decreased significantly in fibrin gel with high pegylation level.Under pegylated molar ratio 50,the pore size of sc-PEG-sc pegylated fibrin gel was smaller than that of mPEG-sc.5.There was no difference for cell proliferation condition between pegylated and no pegylated fibrin gel.For BVEC differentiated study,hADSCs formed network structure successfully within pegylated fibrin gd and expressed specific gene CD31、CD34、VE-cadherin and KDR in sc-PEG-sc group as well as CD31、CD34 and KDR in mPEG-sc respectively. Conclusion1.The hADSCs which was obtained through human liposuction processing could be applied as ideal seeding cells for differenciated blood vessel endothelial cells associated tissue engineering.2.The culturing pattern of hADSCs within fibrin gel could be applied as a method for construction of capillary-like network structure in vitro.3.Pegylation of fibrinogen modified the biological characteristics of fibrin gel and optimized partial function as being scalfford for tissue engineered capillary-like patch.4.The successful formation of capillary-like network structure in pegylated fibrin gel provided us a new method for the study of artificial microvessel construction.更多还原