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目的:探讨兔骨髓间充质干细胞(BMMSCs)与髓核细胞(NPCs)共培养时BMMSCs的营养效应和类髓核分化效应及其动态变化规律。方法:应用1月龄新西兰大白兔的骨髓和髓核进行BMMSCs及NPCs的分离培养与鉴定,建立3个细胞培养组,BMMSCs与NPCs共培养组(实验组),BMMSCs单独培养组和NPCs单独培养组作为对照组。在培养的不同时间点(3、6、9、12、15、18、21d)分别检测细胞培养上清液中转化生长因子β1(TGFβ1)、血小板衍化生长因子(PDGF)的含量变化及BMMSCs与NPCs的增殖能力,采用RT-PCR法检测培养不同时间点BMMSCs与NPCs的蛋白聚糖Aggrecan及Ⅱ型胶原蛋白mRNA的表达变化。结果:分离培养的两种细胞经鉴定分别为BMMSCs及NPCs。从第3天开始的各个时间点,实验组上清液中TGFβ1、PDGF的含量较两对照组明显增高(P<0.05),且随时间的延长而逐渐增高,第15天达最高,TGFβ1、PDGF分别达815.81±25.69pg/ml和494.28±20.01pg/ml,此后第18d、21d逐渐下降。实验组细胞DNA含量在各个时间点上均较两对照组明显升高(P<0.05)。从第3天开始的各个时间点,实验组NPCs的蛋白聚糖Aggrecan及Ⅱ型胶原mRNA的表达较对照组高(P<0.05);同时从第15天开始至第21天,实验组BMMSCs的蛋白聚糖Aggrecan及Ⅱ型胶原mRNA的表达较对照组高(P<0.05)。结论:BMMSCs与NPCs共培养时BMMSCs可通过表达TGFβ1、PDGF等细胞因子发挥其营养效应,激活NPCs,促进其增殖及细胞外基质的合成;在共培养后期,BMMSCs在髓核局部微环境下,可发挥其类髓核分化效应,开始合成蛋白聚糖Aggrecan及Ⅱ型胶原蛋白。
OBJECTIVE: To investigate the nutritional effect and the differentiation effect of BMMSCs and their dynamic changes during co-culture of rabbit bone marrow mesenchymal stem cells (BMMSCs) and nucleus pulposus cells (NPCs). METHODS: The BMMSCs and NPCs were isolated and cultured from the bone marrow and nucleus pulposus of 1-month-old New Zealand white rabbits. Three cell culture groups, BMMSCs and NPCs co-culture group (experimental group), BMMSCs alone group and NPCs alone culture Group as a control group. At different time points (3, 6, 9, 12, 15, 18 and 21d), the content of TGFβ1 and PDGF in the cell culture supernatant were detected, NPCs proliferation, RT-PCR method was used to detect the expression of aggrecan and collagen type II mRNA in BMMSCs and NPCs cultured at different time points. Results: Two kinds of cells were identified as BMMSCs and NPCs respectively. The levels of TGFβ1 and PDGF in the supernatant of the experimental group were significantly higher than those in the two control groups (P <0.05) at all time points starting from the 3rd day. The levels of TGFβ1, PDGF were 815.81 ± 25.69pg / ml and 494.28 ± 20.01pg / ml respectively, and gradually decreased on the 18th and 21st days. The DNA content of the experimental group at each time point was significantly higher than the two control groups (P <0.05). At each time point from day 3, the expressions of proteoglycan, Aggrecan and type II collagen mRNA in experimental NPCs were higher than those in control group (P <0.05). From the 15th day to the 21st day, The expression of proteoglycan Aggrecan and type II collagen mRNA was higher than that of the control group (P <0.05). CONCLUSION: BMMSCs co-cultured with NPCs can exert their nutritional effects by expressing cytokines such as TGFβ1 and PDGF, activating NPCs and promoting their proliferation and extracellular matrix synthesis. At the late stage of co-culture, Can play its class of nucleus differentiation, began the synthesis of proteoglycan Aggrecan and type Ⅱ collagen.