【作者】 彭兆意；

【导师】 袁庆文； 熊吉信；

【作者基本信息】 南昌大学 ， 外科学， 2008， 硕士

【摘要】 目的:在我们已有小鼠胚胎干细胞诱导且经过RT-PCR和免疫组化鉴定血管平滑肌细胞(VSMc)和内皮细胞(EC)种子细胞的基础上,现在探索用诱导的内皮细胞和血管平滑肌细胞,联合种植在经明胶浸润处理的聚丙交酯/乙交酯(PLGA)静电纺丝材料上,进行体外构建小管径组织工程血管。方法:①先将PLGA用1%明胶处理,并测量明胶浸润处理前后PLGA的接触角。②在明胶处理与否的PLGA片上种植血管平滑肌细胞,60分钟后即用甲苯胺蓝染色法观察材料对细胞的亲和力;用MTT、扫描电镜材料观察经培养1天,3天后细胞在材料上的生长情况。③在片状PLGA材料一侧隔日种植血管平滑肌细胞1次, 3天共2次,再于另外一侧种植内皮细胞1次,并进行联合培养2天后,作H&E和免疫组化检查。④将联合种植了血管平滑肌细胞和内皮细胞的PLGA裹成直经约1mm管状包埋于4周龄的裸鼠皮下,分别于3天,1,3周取出进行HE染色和免疫组化检查。⑤同时用放射免疫法测定单纯种植1次血管平滑肌细胞的A组,单纯种植内皮细胞的B组,联合种植了平滑肌和内皮细胞的C组这三组培养液中内皮素含量。结果:①处理前的接触角是88.5°,处理后61.5°。②甲苯胺蓝染色见血管平滑肌细胞在材料上密集、均匀分布;扫描电镜观察到1小时细胞开始延展,1天开始长入孔隙中,3天呈多层分布,无序状生长;MTT检测支架上细胞在3天内的增殖情况等同于培养板。③H&E和免疫组化见材料两侧分别分布着血管平滑肌细胞和内皮。④包埋3周后材料结构仍存在,未被降解吸收。在材料周围包裹结缔组织,管壁外侧有多层血管平滑肌细胞,内侧有内皮细胞。⑤内皮素含量在A组为0.82±0.37 pg/ml,B组5.59±1.23 pg/ml,C组4.51±0.59 pg/ml。结论:①用1%明胶浸润包被处理PLGA后能改善材料的细胞亲和力;②在PLGA上联合种植血管细胞的方法初步构建了具有血管平滑肌细胞和内皮细胞的组织工程血管片;③包埋裸鼠皮下后形成了具有三层细胞结构的组织工程血管。更多还原

【Abstract】 Objective:Since we have the vessel seed cells that Cultured Esc as embryoid body, then use the inductive factors to induce mouse embryonic stem cells to differentiate into smooth muscle cells and endothelial cells, RT-PCR and immunohistochemistry were used to detect. The experiment use the cells that induced mouse embryonic stem cells to differentiate into smooth muscle cells and endothelial cells implanted on poly(lactide-co-glycolide)(PLGA)electrospinning,treated by gelatin infiltration, to construct a small-diameter vascular in vitro.Methods:①Detected the contact angle of the scaffold treated with/without 1%gelatin.②VSMc was seeding onto PLGA treated by gelatin, Culture scaffold 60 min ,the compatibility detected at initial stage by Toluidine Bluestaining (TB),proliferation were observed for the 1d and 3d scaffolds by SEM and MTT.③Implanted smooth muscle cells on one side of the PLGA other day seeding again ,then seeding endothelial cells on other side after 3 days. Culture the PLGA with the sheet of smooth muscle cells and endothelial cells on the surface 2 days,examined with the H&E and. Immunohistochemistry.④4-weeks-old Nude mice were implanted subcutaneously with vascular scaffolds and sacrificed 3 days,1 and 3weeks after implantation. The scaffolds were anayslzd using H&E staining and immunohistochemistry.⑤Endothelin were examined by means of radioimmunology in the culture solution, group A simple seeding vascular smooth muscle cell,groupB merely seeding endothelial cell, and group C seeding combined with vascular smooth muscle cell and endothelial cell, each group cultured 2 days after sending cells.Results:①Untreated group contact angle were 88.5°and treated group contact angle were 61.5°.②The VSMc distributed even dense on the treated PLGA ,SME observed cells extended 1h ,1d cell have grown into the pore ,and much layer cell distribution disorder; MTT find that cells proliferation on the treated PLGA is as well as the culture plate in the primary 3days.③H&E and immunohistochemistry prospected the VSMc and Ec distribution the scaffold each side.④The PLGA still conserved after implanted the Nude mice 3W, did not been completely absorbation or degradation ,the graft closed by the connect tissue, examined much layer VSMc at the lateral and one layer Ec on the tubule inside.⑤The levels of endothelin in the culture solution were 0.82±0.37 pg/mml, 5.59±1.23 pg/mml, 4.51±0.59 pg/mml respectively in the group A ,B and C.Conclusion:①The scaffold treated by gelatin improved the vascular smooth muscle cells adsorbability .②The tissue engineering vessel film have two-layer cells of vascular smooth muscle cells and endothelial cells by union seeding the VSMc and EC.③It harvest the engineering vessel with three- lay cells by implanted the Nude mice.更多还原