【作者】 韩志军；

【导师】 任华；

【作者基本信息】 中国协和医科大学 ， 胸外科学， 2008， 博士

【摘要】 第一部分猪骨髓间充质干细胞获取、培养及体外诱导分化为软骨样细胞和血管样细胞的实验研究目的本实验拟通过体外细胞培养的方法,将猪间充质干细胞自骨髓血中分离出来,进一步在体外的培养,并诱导其向软骨样细胞、上皮样细胞、血管内皮样细胞分化,探讨其作为组织工程化气管种子细胞的可能性。方法收集猪骨髓血,采用密度梯度离心和贴壁生长的方式,提取单个核细胞进行体外培养;观察其生长方式;收集第2～3代（P₂～P₃）细胞用含10ug/L TGF-β1的DMEM-HG培养液诱导、分化为软骨样细胞;用含COL-I（浓度25mg/L）、乙酸（浓度0.1%）的培养液向呼吸道上皮细胞诱导、分化;用含10ng/mL VEGF的培养液诱导、分化为血管内皮样细胞;对所诱导、分化的细胞进行形态学观察和RT-PCR检测、免疫组化以及相关抗原的免疫荧光鉴定。结果通过密度梯度离心和贴壁生长的方法,可以获取具有一定形态特点、贴壁生长、增殖活跃的猪骨髓间充质干细胞;猪BMMSCs在含10ug/L TGF-β1的培养液培养后,细胞形态逐渐发生变化,甲苯胺蓝染色可见紫红色异染出现;RT-PCR检测Ⅱ型胶原mRNA呈阳性表达:免疫组化检查Ⅱ型胶原为阳性;BMMSCs经诱导培养后免疫组化显示细胞角蛋白阴性;BMMSCs在含VEGF（10ng/mL）的培养液中诱导,细胞形态也逐渐发生变化,2w后,外观呈现为鹅卵石样形态,胞浆丰富,免疫表型显示细胞阳性表达vWF。结论1、用密度梯度离心和贴壁的方法可以得到数量较多的猪BMMSCs,可以提供组织工程化气管所需的种子细胞;2、猪BMMSCs可以在体外诱导分化为软骨样细胞和血管内皮样细胞:3、用Coraux的培养体系未能诱导猪BMMSCs分化为呼吸道上皮细胞。第二部分软骨样细胞和胶原包埋PGA支架组织相容性的实验研究目的本研究利用骨髓间充质干细胞所诱导的软骨样细胞种植在胶原包埋PGA支架的膜片上,进行细胞形态学观察和细胞粘附、增殖的测定,进一步评价其组织相容性。方法利用SD大鼠提取鼠尾胶原,包埋PGA构建复合支架;利用骨髓间充质干细胞诱导软骨样细胞;将单纯PGA支架、胶原包埋的PGA支架用打孔器将其制作为直径为6mm的小圆片,铺于96孔板的板底。实验分为胶原包埋PGA组、PGA组、单纯细胞组和空白对照组。种植软骨样细胞,分别进行HE染色、甲苯胺蓝染色、电镜扫描和MTT法检测,评价软骨样细胞和胶原包埋PGA的组织相容性。结果胶原包埋PGA呈灰白色,固定成型,具有一定的弹性:光镜和电镜观察见多微孔结构,胶原成为主要结构基质,PGA纤维具有支撑作用;HE染色见单纯PGA组因细胞脱落无法提供准确信息,胶原包埋PGA组可见软骨样细胞,而且随着时间的推移,细胞呈增加的趋势,表明胶原有利于细胞的贴附和增殖;甲苯胺蓝染色见软骨样细胞被染成浅蓝色,胶原包埋PGA组较单纯PGA组明显增多;电镜观察:胶原包埋PGA组可见到细胞在支架上贴附和生长良好,活性佳。细胞生长曲线可见,三种培养条件下细胞均生长良好,PGA+胶原组、PGA组不比单纯细胞组生长差。结论1.软骨细胞在无支架的培养液中、在单纯PGA支架、在胶原包埋的PGA支架中均生长良好:2.戊二醛交联鼠尾胶原蛋白包埋过的PGA支架与软骨细胞的相容性良好。第三部分初级组织工程气管化体外构建、体内植入气管壁强化目的本实验采用在动物体外构建初级组织工程化气管、体内强化的方法,探讨组织工程技术构建组织工程化气管的可能性,为进一步在具有免疫力的高等动物体内构建组织工程化气管组织并用其修复气管提供参考。方法Brdu标记软骨样细胞,DAPI标记内皮样细胞,观察细胞标记的成功率:软骨样细胞和血管内皮样细胞分层种植于胶原包埋PGA的复合支架表面;将细胞和支架的复合体种植于动物大网膜内,并设立对照组,分别于植入后的第4、6、8周再次麻醉动物,取出植入大网膜内的组织工程化组织,行组织学检查及免疫荧光检查等。结果Brdu和DAPI标记细胞,免疫荧光示标记效果良好:标记有DAPI的内皮细胞种植于支架的表面后,荧光显微镜观察见支架表面均匀排列细胞核呈蓝色荧光的细胞,证实为所种植的内皮细胞;分别于种植后4w、6w、8w取出标本,组织学观察,单纯支架组见管壁结构不清晰;实验组管壁结构比较清晰,细胞数量也已减少,胶原成分增多,管壁可见分层:实验组皮下培养8w Brdu标记细胞的免疫荧光观察见部分细胞核呈现明亮的黄绿色荧光,证明来源于所种植的种子细胞。结论1、体外构建的初级组织工程化气管,以动物的大网膜为生物反应器,采用静态培养的方式可使工程化组织管壁得以强化,从而构建出次级组织工程化气管:2、组织工程技术构建的次级工程化气管组织具有复层结构;3、组织工程化气管壁细胞有所种植的种子细胞生长,并参与了组织工程化气管的重构。第四部分组织工程气管血管化的研究目的本实验采用在动物体外构建初级组织工程化气管软骨的同时加入骨髓间充质干细胞诱导生成的血管内皮样细胞,将其体内植入强化气管壁并观察血管生成的情况,探讨组织工程化气管软骨血管化的可能性。方法Brdu标记内皮样细胞,软骨样细胞和血管内皮样细胞混合种植于胶原包埋PGA的复合支架表面;对照组为单纯种植软骨样细胞,将细胞和支架的复合体种植于动物大网膜内,于植入后的第8周再次麻醉动物,取出植入大网膜内的组织工程化组织,行组织学检查及Brdu免疫组化检查等;数据进行统计分析结果切片HE染色血管内的红细胞被染成红色,据此可观察红细胞;Brdu免疫组化检查可见组织内有染成棕褐色的毛细血管,证实血管壁细胞来源于所种植的种子细胞;实验组和对照组毛细血管数目比较有显著性差异;结论骨髓间充质干细胞来源的血管内皮样细胞参与并促进了组织工程化气管内部毛细血管的生成。更多还原

【Abstract】 PartⅠ:Experimental Research of Porcine BMMSCs Obtained and Differentiated to Chondrocyte and Vascular Endothelium-Like CellsObjective To harvest,isolate,culture bone marrow mesenchymal stem cells(BMMSCs) from porcine sternum.To investigate the induction and differentiation of BMMSCs into chondrocytes,airway epithelial cells and vascular endothelium-like cells in vitro.To explore the possibility as seed cells for tissue engineering trachea.Methods The porcine bone marrow was aspirated from sternum.BMMSCs were isolated and purified by methods of density gradient centrifugation and adhering to the culture plastic.Chondrocytes derived from BMMSCs which were cultured in DMEM-HG with TGF-β1(10ug/L).Induced the BMMSCs in DMEM-HG with COL-I(25mg/L)and acetic acid(0.1%).Vascular endothelium-like cells derived from BMMSCs which were cultured in DMEM-HG with VEGF(10ng/mL).The cells were observed and identified through morphology,reverse transcription polymerase chain reaction (RT-PCR),immunohistochemistry,toluidine blue staining,and immunofluorescence.Results Methods of density gradient centrifugation and adhering to the culture plastic can harvest mononuclear cells with specific characterics.Both of chondrocyte and vascular endothelium-like cells were cultured successfully.Airway epithelial cell was not culture successfully.Morphology of chondrocyte was changed,heterochromia to toluidine blue. Immunohistochemical staining of collagen typeⅡand RT PCR products for mRNA from collagen typeⅡwere positive.Immunohistochemical staining of cytokeratin was negative. VELCs showed cobble stone-like morphology and sufficient cell organs. Immunofluorescence results showed vWF positive of VELCs. Conclusion 1.Methods of density gradient centrifugation and adhering to the culture plastic can harvest higher purified porcine BMMSCs.BMMSCs can provide stem cells for research of tissue engineering trachea.2.Porcine BMMSCs can be differentiated into chondrocyte and vascular endothelium-like cells in vitro.3.Porcine BMMSCs can not be differentiated into airway epithelial cells with the mothode of Coraux. PartⅡ:Experimental Study on Cytocompatibility of Porcine Chondrocyte and PGA/Collagen ScaffoldObjective To study the compatibility of porcine chondrocyte and polyglycolic acid (PGA) coated with cross-linked collagen(PGA/collagen scaffold) by morphological observation,proliferationof the cells and so on.Methods Rat’s tail collagen and PGA coated with cross-linked collagen(PGA/collagen scaffold) was produced and constructed firstly.Chondrocyte derived from BMMSCs were cultured with PGA/collagen scaffold together.There were several experimental groups including PGA/collagen group,simple PGA group,simple cells group and control group. Morphological characteristics were observed by HE staining,toluidine blue staining and scanning electron microscope.Adhesive and proliferating cells were detected by MTT assay.Results The PGA/collagen scaffold has elasticity and tenacity.Histological and ultrastructural observation showed that there were lots of micropores in the scaffold.HE staining,toluidine blue staining,scanning electron microscope and the curve of cell growth from MTT assay showed that chondrocyte seeded on the PGA/collagen scaffold grew as well as simple PGA group and simple cells group.Conclusion 1.Chondrocyte seeded on the PGA/collagen scaffold grew as well as simple PGA group,simple cells group.2.The PGA/collagen scaffold had a good compatibility with VSMLCs. PartⅢ:Construction of Primary Tissue Engineering Trachea in vitro and Strengthening of Trachea Wall in vivoObjective To investigate the possibility of constructing tissue engineering trachea in vitro and strengthening of tracheal wall in vivo.Methods Chondrocytes were labeled by Brdu and vascular endothelium-like cells were labeled by DAPI respectively firstly.The mixture of seed cells and scaffold were produced by separately seeding chondrocytes and vascular endothelium-like cells derived from BMMSCs on PGA/collagen scaffold.The two layers were separated by ECM gel.The cells-scaffold mixture was wrapped around a silicone tube(external diameter=2mm).The mixture was implanted into omentum in cell donor porcines and the porcines were sacrificed in 4,6 and 8 weeks.The tissue engineering tracheas were examined by both gross and histology with HE staining and immunofluorescence.The control group was PGA/collagen scaffold group with no seeded cells.Results Seed cells could be labeled well by Brdu and DAPI.Histological analysis of the tissue engineering tracheal wall revealed two layers.Eight weeks after implantation,the labeled seed cells by Brdu were found in the tracheal wall,suggesting that implanted cells survived and participated the tracheal tissue regeneration.Conclusion 1.The omentum was a good bioreactor to construct tissue engineering trachea.2.The engineering tracheas had two layers.3.Implanted cells participated the tracheal tissue regeneration. PartⅣ:Study on vascularization of the Tissue Engineering TracheaObjective To study the vascularization of the tissue engineering trachea.Methods Chondrocytes and vascular endothelium-like cells derived from BMMSCs, and then the endothelium-like cells were labeled by DAPI.The experimental group:both seed cells were seeded onto PGA/collagen scaffold.The contral group:chondrocytes were seeded onto PGA/collagen scaffold.The cells-scaffold mixture was wrapped around a silicone tube.Both groups were implanted into omentum in cell donor porcines and the porcines were sacrificed in 8 weeks.The tissue engineering tissues were examined by histology with HE staining and immunohistochemistry.Results HE staining showed red cells was red in the capillary and can count the number of the capillary.Immunohistochemical staining of Brdu was positive in the experimental groups,suggesting that implanted vascular endothelium-like cells survived and participated the vascular regeneration.The number of the capillary of the experimental group was significant more than that of the control group(P＜0.001).Conclusion The vascular endothelium-like cells derived from BMMSCs participated the vascular regeneration of tissue engineering trachea.更多还原