人脐带间充质干细胞向软骨细胞诱导分化的实验研究
发布时间：2019-01-16 05:24
【摘要】：目的：1.体外传代培养，观察人脐带间充质干细胞(Human umbilical cordmesenchymal stem cells,hUC-MSCs)多向分化能力的改变，探索其体外诱导分化的最佳时间窗。2.寻求一种更佳的，体外无支架培养条件下诱导hUC-MSCs向组织工程软骨细胞分化的方法。 方法： 1.体外传代培养，观察hUC-MSCs多向分化能力的改变——采用胶原酶消化法分离hUC-MSCs，胰酶消化传代并培养。流式细胞术检测不同代细胞表面标志物与细胞周期；MTT法检测不同代细胞增殖能力；对不同代细胞行成骨细胞、成软骨细胞和成脂肪细胞诱导并鉴定；实时定量PCR检测不同代细胞Oct-4、Sox-2、Nanog的mRNA表达水平。 2.hUC-MSCs向组织工程软骨细胞分化的诱导——收集第3代hUC-MSCs，通过两种无支架培养体系，体外诱导其向组织工程软骨细胞分化。HE染色观察离心管聚集成团培养诱导后组织的形态；免疫组织化学法检测离心管聚集成团培养体系诱导后组织细胞II型胶原表达；免疫荧光法检测平面诱导培养体系诱导后组织细胞II型胶原表达；阿利新蓝及甲苯胺蓝染色检测两种培养体系诱导后组织细胞糖胺聚糖(Glycosaminoglycans,GAG)的表达；羟脯氨酸法和阿新蓝法分析两种培养体系诱导前后Ⅱ型胶原与GAG的含量变化；实时定量PCR分析两种培养体系诱导前后Sox-9、Ⅱ型胶原及GAG的mRNA表达水平。 结果： 1.体外传代培养至23代，hUC-MSCs细胞表面标志物表达率及细胞周期无显著差异。细胞生长曲线相似，细胞增殖率无明显下降。细胞均可被诱导成成骨细胞、软骨细胞和脂肪细胞。不同代细胞Oct-4、Sox-2、Nanog的mRNA表达量无显著差异。 2.HE染色结果显示，，离心管聚集成团培养诱导后组织具有软骨组织特有形态。免疫组化、免疫荧光及阿利新蓝染色、甲苯胺蓝染色结果显示，两种培养体系诱导后组织细胞均分别表达Ⅱ型胶原与GAG；Ⅱ型胶原与GAG定量检测(羟脯氨酸法及阿利新蓝法)结果显示，两种培养体系的实验组较各自对照组Ⅱ型胶原与GAG表达量高，离心管聚集成团培养实验组的表达量显著高于平面诱导培养实验组；实时定量PCR结果显示，诱导后细胞较诱导前均高表达GAG、Ⅱ型胶原和Sox-9的mRNA；离心管聚集成团诱导培养与平面诱导培养比较，其GAG和Ⅱ型胶原的mRNA表达量更高，有显著性差异。 结论： 1.体外培养至23代，随着传代次数的增加，hUC-MSCs多向分化能力未受影响。 2.离心管聚集成团培养可显著提高诱导后组织Ⅱ型胶原及GAG的表达水平，其更有利于hUC-MSCs向软骨细胞分化。
[Abstract]:Objective: 1. The multidirectional differentiation of human umbilical cord mesenchymal stem cells (Human umbilical cordmesenchymal stem cells,hUC-MSCs) was observed in vitro, and the optimal time window of differentiation was explored. 2. To find a better way to induce hUC-MSCs to differentiate into tissue engineering chondrocytes in vitro without scaffold culture. Methods: 1. The ability of multidirectional differentiation of hUC-MSCs was observed by in vitro subculture. HUC-MSCs, trypsin was digested and cultured by collagenase digestion. Flow cytometry was used to detect cell surface markers and cell cycle; MTT assay was used to detect the proliferation of different generation cells; osteoblasts, chondroblasts and adipocytes were induced and identified by MTT assay. The mRNA expression of Oct-4,Sox-2,Nanog in different generations of cells was detected by real time quantitative PCR. Induction of 2.hUC-MSCs differentiation into tissue engineered chondrocytes: the third generation of hUC-MSCs, was collected through two scaffolding free culture systems. The cells were induced to differentiate into tissue engineering chondrocytes in vitro. The morphology of the cells was observed by HE staining. Immunohistochemical method was used to detect the expression of II type collagen in tissue cells induced by colony culture system of centrifuge tube, and immunofluorescence method was used to detect the expression of II type collagen in tissue cells induced by plane-induced culture system. The expression of glycosaminoglycan (Glycosaminoglycans,GAG) was detected by alimine blue and toluidine blue staining, and the changes of type 鈪
本文编号：2409509
[Abstract]:Objective: 1. The multidirectional differentiation of human umbilical cord mesenchymal stem cells (Human umbilical cordmesenchymal stem cells,hUC-MSCs) was observed in vitro, and the optimal time window of differentiation was explored. 2. To find a better way to induce hUC-MSCs to differentiate into tissue engineering chondrocytes in vitro without scaffold culture. Methods: 1. The ability of multidirectional differentiation of hUC-MSCs was observed by in vitro subculture. HUC-MSCs, trypsin was digested and cultured by collagenase digestion. Flow cytometry was used to detect cell surface markers and cell cycle; MTT assay was used to detect the proliferation of different generation cells; osteoblasts, chondroblasts and adipocytes were induced and identified by MTT assay. The mRNA expression of Oct-4,Sox-2,Nanog in different generations of cells was detected by real time quantitative PCR. Induction of 2.hUC-MSCs differentiation into tissue engineered chondrocytes: the third generation of hUC-MSCs, was collected through two scaffolding free culture systems. The cells were induced to differentiate into tissue engineering chondrocytes in vitro. The morphology of the cells was observed by HE staining. Immunohistochemical method was used to detect the expression of II type collagen in tissue cells induced by colony culture system of centrifuge tube, and immunofluorescence method was used to detect the expression of II type collagen in tissue cells induced by plane-induced culture system. The expression of glycosaminoglycan (Glycosaminoglycans,GAG) was detected by alimine blue and toluidine blue staining, and the changes of type 鈪
本文编号：2409509
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