兔间充质干细胞分离培养和体外向软骨细胞分化的实验研究
发布时间：2018-10-29 20:36
【摘要】： 目的 1.建立一种分离培养兔骨髓间充质干细胞(bone marrow derived mesenchymalstem cells,BMSCs)的有效方法,并观察BMSCs在特定培养条件下向软骨细胞分化的情况。 2.探讨使用G-CSF动员兔外周血,分离培养外周血间充质干细胞(peripheralblood mesenchymal stem cells,PBMSCs)的方法。 方法 1.以4月龄新西兰大耳白兔为实验对象,采用密度梯度离心法、全血培养法、氯化胺红细胞溶解法,分离培养BMSCs,对其细胞存活率、细胞克隆率、首次传代时间、15d后细胞数及扩增成功率进行比较。 2.用含有骨形态发生蛋白(bone morphogenetic proteins,BMP)-2的条件培养液诱导BMSCs在体外单层培养模式下向软骨细胞分化。采用甲苯胺蓝染色、Masson染色、免疫组织化学染色、阿利新蓝比色法测定糖胺聚糖(glycosaminoglycan,GAG)的含量来检测BMSCs分化情况。统计学分析比较第2、4、7代(P_2、P_4、P_7)BMSCs诱导后增殖能力及向软骨细胞分化能力的变化。 3.给新西兰兔皮下注射粒细胞集落刺激因子30μg/(kg·d)4d,抽取动员前后兔外周血采用密度梯度离心法进行分离培养。 结果 1.对于细胞存活率而言,密度梯度离心法和全血培养法均优于氯化胺红细胞溶解法。在其它指标比较中,密度梯度离心法)全血培养法)氯化胺红细胞溶解法。 2.BMSCs定向诱导后表现出软骨细胞的特性,P_2、P_4间的细胞增殖能力和成软骨细胞分化能力比较差异无统计学意义(P＞0.05);P_7与P_2、P_4相比,细胞增殖能力和成软骨细胞分化能力均降低,差异有统计学意义(P＜0.01)。 3.未动员的外周血中未发现有梭形细胞贴壁;动员后的外周血中发现有梭形细胞贴壁,而且这些细胞可以形成小的集落,但是增殖缓慢,增殖能力有限,不能大量扩增。 结论 1.利用成年兔骨髓,体外成功分离出骨髓间充质干细胞,同时发现密度梯度离心法优于全血培养法和氯化胺红细胞溶解法。 2.兔BMSCs能在BMP-2为主要诱导因子的单层培养模式下在体外向软骨细胞分化;细胞传代对BMSCs增殖和向软骨细胞分化有重要影响。 3.从动员后的外周血中,发现有梭形细胞但它们增殖能力有限,还不能说它们就是间充质干细胞。PBMSCs分离培养有待于进一步研究。
[Abstract]:Objective 1. To establish an effective method for isolation and culture of rabbit bone marrow mesenchymal stem cells (bone marrow derived mesenchymalstem cells,BMSCs) and to observe the differentiation of BMSCs into chondrocytes under specific culture conditions. 2. To explore the method of using G-CSF to mobilize rabbit peripheral blood and isolate and culture peripheral blood mesenchymal stem cells (peripheralblood mesenchymal stem cells,PBMSCs). Method 1. Four month old New Zealand white rabbits were isolated and cultured with BMSCs, by density gradient centrifugation, whole blood culture, erythrocytolysis of chloramines, cell viability, cell clone rate and first passage time. After 15 days, the number of cells and the success rate of amplification were compared. 2. BMSCs was induced to differentiate into chondrocytes in monolayer culture with conditioned medium containing bone morphogenetic protein (bone morphogenetic proteins,BMP)-2. Toluidine blue staining, Masson staining, immunohistochemical staining and alisin blue colorimetry were used to determine the content of glycosaminoglycan (glycosaminoglycan,GAG) to detect the differentiation of BMSCs. The ability of proliferation and differentiation into chondrocytes were compared after BMSCs induction. 3. Granulocyte colony stimulating factor 30 渭 g / (kg d) was injected subcutaneously into New Zealand rabbits for 4 days. The peripheral blood was isolated and cultured by density gradient centrifugation before and after mobilization. Result 1. For cell survival, density gradient centrifugation and whole blood culture were superior to chloramines in erythrocyte lysis. Among other indexes, density gradient centrifugation method) whole blood culture method) chloramine erythrocyte lysis method. 2.BMSCs showed the characteristics of chondrocytes after directed induction. There was no significant difference in cell proliferation and chondroblast differentiation among the four groups (P > 0. 05). The ability of proliferation and differentiation of chondroblasts decreased significantly (P < 0.01). 3. There were no fusiform cells adherent to the unmobilized peripheral blood, and fusiform cells adherent to the mobilized peripheral blood, and these cells could form small colonies, but the proliferation was slow and the ability of proliferation was limited and could not be expanded in large numbers. Conclusion 1. Bone marrow mesenchymal stem cells were successfully isolated from adult rabbit bone marrow in vitro. It was found that the density gradient centrifugation method was superior to the whole blood culture method and the chloride amine erythrocyte lysis method. 2. Rabbit BMSCs could differentiate into chondrocytes in vitro in the monolayer culture model with BMP-2 as the main inducer, and cell passage had an important effect on the proliferation and differentiation of BMSCs into chondrocytes. 3. From the mobilized peripheral blood, fusiform cells were found, but their proliferative ability was limited, which can not be said to be mesenchymal stem cells. The isolation and culture of PBMSCs need further study.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R329
本文编号：2298747
[Abstract]:Objective 1. To establish an effective method for isolation and culture of rabbit bone marrow mesenchymal stem cells (bone marrow derived mesenchymalstem cells,BMSCs) and to observe the differentiation of BMSCs into chondrocytes under specific culture conditions. 2. To explore the method of using G-CSF to mobilize rabbit peripheral blood and isolate and culture peripheral blood mesenchymal stem cells (peripheralblood mesenchymal stem cells,PBMSCs). Method 1. Four month old New Zealand white rabbits were isolated and cultured with BMSCs, by density gradient centrifugation, whole blood culture, erythrocytolysis of chloramines, cell viability, cell clone rate and first passage time. After 15 days, the number of cells and the success rate of amplification were compared. 2. BMSCs was induced to differentiate into chondrocytes in monolayer culture with conditioned medium containing bone morphogenetic protein (bone morphogenetic proteins,BMP)-2. Toluidine blue staining, Masson staining, immunohistochemical staining and alisin blue colorimetry were used to determine the content of glycosaminoglycan (glycosaminoglycan,GAG) to detect the differentiation of BMSCs. The ability of proliferation and differentiation into chondrocytes were compared after BMSCs induction. 3. Granulocyte colony stimulating factor 30 渭 g / (kg d) was injected subcutaneously into New Zealand rabbits for 4 days. The peripheral blood was isolated and cultured by density gradient centrifugation before and after mobilization. Result 1. For cell survival, density gradient centrifugation and whole blood culture were superior to chloramines in erythrocyte lysis. Among other indexes, density gradient centrifugation method) whole blood culture method) chloramine erythrocyte lysis method. 2.BMSCs showed the characteristics of chondrocytes after directed induction. There was no significant difference in cell proliferation and chondroblast differentiation among the four groups (P > 0. 05). The ability of proliferation and differentiation of chondroblasts decreased significantly (P < 0.01). 3. There were no fusiform cells adherent to the unmobilized peripheral blood, and fusiform cells adherent to the mobilized peripheral blood, and these cells could form small colonies, but the proliferation was slow and the ability of proliferation was limited and could not be expanded in large numbers. Conclusion 1. Bone marrow mesenchymal stem cells were successfully isolated from adult rabbit bone marrow in vitro. It was found that the density gradient centrifugation method was superior to the whole blood culture method and the chloride amine erythrocyte lysis method. 2. Rabbit BMSCs could differentiate into chondrocytes in vitro in the monolayer culture model with BMP-2 as the main inducer, and cell passage had an important effect on the proliferation and differentiation of BMSCs into chondrocytes. 3. From the mobilized peripheral blood, fusiform cells were found, but their proliferative ability was limited, which can not be said to be mesenchymal stem cells. The isolation and culture of PBMSCs need further study.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R329
【引证文献】
相关硕士学位论文 前2条
1 和小娥;兔骨髓间充质干细胞分离培养及定向分化的研究[D];河南农业大学;2011年
2 闫颖颖;兔骨髓间充质干细胞向心肌样细胞的体外诱导分化研究[D];西北农林科技大学;2010年
，本文编号：2298747
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