组织工程化气管构建的实验研究

发布时间：2018-01-24 10:15

  本文关键词： 骨髓 间充质干细胞 原代培养 传代培养 生长曲线 骨髓间充质干细胞 气管粘膜上皮细胞 细胞裂解 射线照射 共培养 家兔气管 环氧化合物 戊二醛 理化性能 体外 细胞毒性　出处：《北京协和医学院》2010年博士论文　论文类型：学位论文

【摘要】：目的本实验通过体外细胞培养的方法,从骨髓中分离、纯化、鉴定骨髓间充质干细胞,研究其体外培养的增殖和生长特性,探讨为组织工程化气管的构建提供种子细胞的可能性。 方法收集成人骨髓血,采用密度梯度离心和贴壁生长的方法分离并提取单个核细胞进行体外培养；收集P2-P4代细胞进行流式细胞仪检测细胞抗原表达；计数并绘制细胞原代培养和传代培养的细胞生长曲线。 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、增殖活跃的骨髓间充质干细胞；该细胞可表达CD29,CD44,CD105,不表达CD54,CD45;原代培养曲线显示,第2d-6d为生长的潜伏期,第7-9d进入对数生长期,10d后进入平台期；传代培养的潜伏期为24h-36h,4d-5d后进入对数增长期,9d左右进入平台期。在对数生长期,细胞倍增时间约为48h,传代培养的细胞在P9-P1o出现衰老现象。 结论1、利用密度梯度离心和贴壁的方法可以有效地分离、纯化骨髓间充质干细胞； 2、在适当的条件下,BMMSCs在体外表现出旺盛的增殖能力,可满足组织工程学种子细胞的需要 3、BMMSCs高表达CD29、CD44、CD105,不表达CD34、CD45。 目的本实验通过体外诱导人骨髓间充质干细胞向呼吸道上皮样细胞的分化并进行形态学的初步鉴定,探讨为组织工程气管提供呼吸道上皮种子细胞的可能性。 方法取P2-P4代生长状态良好的BMMSCs细胞,用含有家兔气管粘膜上皮细胞内容物的培养基进行诱导培养,对诱导、分化的细胞进行形态学观察。 结果BMMSCs细胞在含有家兔气管粘膜上皮细胞内容物的培养基诱导培养下,经过3d时间,细胞形态由长梭形变成多角形,HE染色可见吞饮小泡。 结论骨髓间充质干细胞经体外诱导可以向气管粘膜上皮样细胞分化。 目的采用戊二醛和环氧化合物交联制备家兔气管,评价家兔气管的生物学性能,探讨环氧化合物作为新型制备技术的可行性。 方法新鲜家兔气管12根,随机分为戊二醛组处理组(GA组,n=4)、环氧化合物处理组(PC组,n=4)及新鲜对照组(Fresh组,n=4)。测量各组处理前后的颜色、弹性、管壁厚度的变化。测量并计算各组处理前后的组织含水量,交联固定指数以评价交联特点。通过观察组织断裂强度和断裂延伸率评价其机械性能。采用胶原酶降解并测量降解后的断裂强度以分析组织稳定性。通过气管与BMMSCs共培养、不同浓度交联制备剂对BMMSC存活率的影响这两种方法评价戊二醛和环氧化合物对交联制备家兔气管产生的细胞毒性。 结果PC组外观颜色、管壁弹性、管腔变化更接近天然气管；PC组和GA组的机械强度相当,均明显高于Fresh组,PC组断裂延伸率大于GA组,且两组均低于Fresh组；PC组和GA组固定100小时的固定指数相当；体外Ⅱ型胶原酶的降解结果显示,PC组和GA组的组织稳定性均高于Fresh组,GA组和PC组之间没有明显统计学差异；根据与处理后气管共培养的BMMSCs细胞生长情况所得曲线显示,PC组细胞可继续稳定增长,GA组细胞3d内迅速死亡；原有浓度的PC和GA交联制备剂对细胞均有很强的毒性,处理后不超过15%的细胞能够存活,稀释8倍以后,可以有超过70%的细胞存活。 结论1、戊二醛和环氧化合物均可有效地交联制备家兔气管。 2、环氧化合物交联技术和戊二醛相比,能够改善家兔气管的部分性能(机械性能和细胞毒性效应)。
[Abstract]:Objective To study the proliferation and growth characteristics of bone marrow mesenchymal stem cells ( MSCs ) from bone marrow by means of in vitro cell culture , and to explore the possibility of providing seed cells for the construction of engineered trachea . Methods Adult bone marrow blood was collected by density gradient centrifugation and adherent growth . Single nuclear cells were isolated and cultured in vitro . The expression of cell antigens was detected by flow cytometry in P2 - P4 cells . Cell growth curves of primary culture and subculture were counted and plotted . Results Bone marrow mesenchymal stem cells with certain morphological characteristics , adherent growth and proliferation were obtained by density gradient centrifugation and adherent growth . The cells expressed CD29 , CD44 , CD105 , did not express CD54 , CD54 ; primary culture curves showed that days 2d - 6d were the latent periods of growth . The incubation period of subculture was 24 h - 36h , 4 days - 5 days . Conclusion 1 . Bone marrow mesenchymal stem cells can be purified and purified by using density gradient centrifugation and adherent method . 2 . Under appropriate conditions , BMMSCs exhibit a strong proliferative capacity in vitro , which can meet the needs of tissue engineering seed cells . 3 . The expression of CD29 , CD44 and CD105 in BMMSCs did not express CD34 , CD45 . Objective To explore the possibility of providing airway epithelial seed cells to tracheal epithelial cells in vitro by inducing differentiation of human bone marrow mesenchymal stem cells into respiratory epithelium - like cells and morphology . Methods BMMSCs with good growth status of P2 - P4 were induced and cultured with medium containing the contents of tracheal mucosa epithelial cells in rabbits . Results The cells of BMMSCs were cultured under the culture medium containing the contents of tracheal mucosa epithelial cells in rabbits . After 3d time , the cell morphology changed from long shuttle shape to polygonal shape , HE staining showed that the cells were swallowed . Conclusion In vitro induction of bone marrow mesenchymal stem cells can differentiate into tracheal mucosa epithelioid cells . Objective To evaluate the biological properties of rabbit tracheal and evaluate the feasibility of using epoxy compound as a new preparation technique by cross - linking glutaraldehyde and epoxy compound . Methods 12 rabbits were randomly divided into glutaraldehyde group treated group ( GA group , n = 4 ) , epoxy compound treatment group ( PC group , n = 4 ) and fresh control group ( Fresh group , n = 4 ) . The changes of color , elasticity and wall thickness before and after treatment were measured and calculated . The water content and cross - linking fixation index before and after treatment were measured and calculated to evaluate the cross - linking characteristics . By observing the fracture strength and elongation at break , the mechanical properties were evaluated . The effects of glutaraldehyde and epoxy compound on the survival of BMMSC were evaluated by the co - culture of tracheal and BMMSCs , and the cytotoxicity of glutaraldehyde and epoxy compounds on the tracheal production of rabbits was evaluated . Results The mechanical strength of PC group and GA group was significantly higher than that in fresh group , PC group and GA group were significantly higher than that in fresh group , and that in PC group and GA group were higher than that in fresh group . Conclusion 1 銆，

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