直接共培养诱导人脐带间充质干细胞向软骨细胞的分化

Differentiation of human umbilical cord mesenchymal stem cells into chondrocytes induced by direct co-culture with chondrocytes

背景:软骨细胞和间充质干细胞都可用于构建组织工程化软骨,但体外培养的软骨细胞容易发生去分化,表型难以维持,以致其临床应用受到限制。目的:探讨人脐带间充质干细胞与人关节软骨细胞直接共培养对人脐带间充质干细胞成软骨诱导分化的影响,以及直接共培养的最佳比例。方法:分离培养人脐带间充质干细胞并通过流式细胞仪鉴定表面标志。实验分为6组:直接共培养组按照人脐带间充质干细胞与人关节软骨细胞比例为1∶1,3∶1及5∶1(人脐带间充质干细胞∶人关节软骨细胞)进行共培养;阳性对照组用转化生长因子β1细胞因子诱导;人关节软骨细胞空白对照组和人脐带间充质干细胞空白对照组。免疫荧光细胞化学检测Ⅱ型胶原(COL2A1)的表达水平;Western blot检测细胞SOX9Ⅰ型胶原、Ⅱ型胶原蛋白表达水平;实时荧光定量qRT-PCR检测SOX9、Col1a1、Col2a1 m RNA表达。结果与结论:(1)成功分离并鉴定人脐带间充质干细胞;(2)人脐带间充质干细胞与软骨细胞共培养28 d后,共培养组及单独药物诱导的阳性对照组Ⅱ型胶原免疫荧光检测均呈阳性;(3)阳性对照组的Col2a1 mRNA表达水平高于共培养组,但Ⅱ型胶原蛋白表达总量低于共培养组,1∶1共培养组的Col2a1 mRNA表达水平高于3∶1和5∶1共培养组。阳性对照组和共培养组的Col1a1 m RNA表达水平和Ⅰ型胶原蛋白表达水平均低于人关节软骨细胞空白对照组。(4)结果说明,人脐带间充质干细胞和人关节软骨细胞直接共培养可明显促进人脐带间充质干细胞向软骨样细胞诱导分化,并有效抑制细胞纤维化,从缩减软骨细胞用量方面看,直接共培养体系的适宜比例为5∶1。采用直接共培养诱导人脐带间充质干细胞向软骨细胞分化的成本较低,是一种较为经济合理的诱导方法。

BACKGROUND： Both chondrocytes and mesenchymal stem cells （MSCs） can be used to construct tissue-engineered cartilage Chondrocytes cultured in vitro however are prone to dedifferentiation and difficult to maintain phenotypes, and accordingly, their clinical application is limited, OBJECTIVE： To explore the effect of human articular ohondrocytes and human umbilical cord mesenchymal stem cells （hUC-MSCs） co-culture in vitro on the chondrogenic differentiation of hUC-MSCs and to optimize the co-cuRure ratio. METHODS： The hUC-MSCs surface marker was identified by flow cytometry. Human articular chondrocytes and hUC-MSCs were co-cultured at the ratio of 1：1, 3：1 and 5：1. The hUC-MSCs induced by transforming growth factor-beta 1 were set as a positive control group. Human articular chondrocytes and hUC-MSCs cultured alone were set as two negative control groups. The expression level of type II collagen （COL2） was analyzed by immunofluorescence staining. The protein expression of SRY-box9 （SOX9）, type I collagen （COL1） and COL2 were determined by western blot. The mRNA levels of SOX9, Collal and Col2al were detected by quantitative real-time PCR. RESULTS AND CONCLUSION： The hUC-MSCs were isolated from the human umbilical cord and identified with flow cytometry. After 28 days of culture, both the co-culture group and the positive control group were observed with positive staining under the immunofluorescence microscope. The Col2al mRNA expression level of the positive control group was higher than that of the co-culture group, but the total COL2 protein expression was lower. The Col2al mRNA expression level of the co-culture group in 1：1 was higher than that of the co-culture group in 3：1 or 5：1. Collal mRNA and COL1 protein expression levels of the positive control group and co-culture group were lower than those of human articular chondrocyte negative control group. To conclude, the co-culture of hUC-MSCs and human articular chondrocytes significantly induces the hUC-MSC differentiation into chondrocytes and effectively restrains cell fibrosis. The optimal cell ratio in the co-culture system is demonstrated to be 5：1 （hUC-MSCs：chondrocytes）. Therefore, the direct co-culture can be an economic way for inducing hUC-MSC differentiation into chondrooytes, which provides reliable seeding cells for cartilage tissue engineering. Subject headings： Stem Cells; Umbilical Cord; Mesenchymal Stem Cells; Chondrocytes; Tissue Engineering Funding： the National Natural Science Foundation of China, No. 81572198, 81260161, 81000460; the Natural Science Foundation of Guangdong Province, No. 2015A030313772; the Tackle Key Project of Shenzhen Science and Technology Innovation Commission, No. JSGG2014051905550503; the International Scientific Cooperation Project of Shenzhen Science and Technology Innovation Commission, No. GJHZ20130412159306739; the Promotion Project of Shenzhen Key Laboratories, No. CXB201104220049A; Shenzhen Science and Technology Research Project, No. JCYJ20140414470821200, JCYJ20140414170821160： China Postdoctoral Science Foundation. No. 2013M530385