不同浓度葡萄糖对滑膜间充质干细胞成软骨能力调节作用的研究  

Regulation of glucose concentration in expansion period on synovial mensenchymal stem cells chondrogenesis

作　　者：谢金硕 陈松 邵加华 周义钦 符培亮 吴海山 

机构地区：[1]上海市长征医院关节外科,200001

出　　处：《中华关节外科杂志（电子版）》2016年第5期33-41,共9页Chinese Journal of Joint Surgery(Electronic Edition)

基　　金：国家自然科学基金青年基金(81000798)

摘　　要：目的研究分化前阶段不同浓度葡萄糖对滑膜间充质干细胞(SMSCs)成软骨能力的调节作用,并探究其分子调节机制。方法将分化前阶段SMSCs分组置于含有不同浓度葡萄糖的培养基中进行增殖培养:一组采用低浓度(1.0 g/L)葡萄糖培养SMSCs(LGMSMCs);另一组采用高浓度(4.5 g/L)葡萄糖培养SMSCs(HGMSMCs)。增殖培养7 d后,将两组SMSCs同时进行成软骨诱导分化,通过检测软骨标志基因的表达水平来评价两组SMSCs成软骨能力的大小;通过分析转化生长因子-β(TGF-β)和蛋白激酶-C(PKC)信号通路系统来研究其分子调节机制;设置平行实验,在高糖培养的SMSCs中加入PKC抑制剂G6983进一步研究TGF-β和PKC信号通路系统。不同浓度的葡萄糖培养基中分化前DNA总量,分化前、分化阶段的SMCSs成软骨相关基因的表达比较采用成组t检验。结果 SMSCs培养第21天,HGMSMCs组的蛋白聚糖(AGN)、Ⅰ型胶原(ColⅠ)及Ⅱ型胶原(ColⅡ)的表达明显少于LGMSMCs组的(t=-3.69,P<0.05;t=-77.57,P<0.01;t=-34.42,P<0.01);在SMSCs分化前阶段,GMSMCs组的TGF-βⅡ型受体(TGFβ-RⅡ)的表达明显高于LGMSMCs组的(t=4.5,P<0.01);在SMSCs分化阶段,GMSMCs组的PKC磷酸化水平及TGF-βⅡ型受体(TGFβ-RⅡ)的表达明显低于LGMSMCs组的(t=-5.84,P<0.01;t=-4.79,P<0.01)。另外,在平行实验中,SMSCs分化前阶段,往高糖培养的SMSCs中加入PKC抑制剂G6983较未加入抑制剂更能上调SMSCs在分化阶段TGFβ-RⅡ的表达水平(t=-1.03,P<0.05)。结论在SMSCs分化前阶段,低糖培养可以增加SMSCs成软骨能力;其分子机制是葡萄糖对分化前阶段TGF-β和PKC信号通路进行调节,进而调节分化阶段SMSCs成软骨能力。Objective To study the role of glucose concentration in regulating the chondrogenic ability of synovial mesenchymal stem cells （SMSCs） and characterize the underlying mechanism. Methods SMSCs were cultured in the culture medium of different glucose concentrations （ 1.0 g/L and 4. 5 g/L） , and were induced for chondrogenesis in cell pellets. Fibroeartilage-related marker genes were analyzed to determine the degree of chondrogenesis of SMSCs in different groups; transforming growth factor-beta （TGF-β） and protein kinase C （PKC） were also analyzed. Paired t test was adopted to analyze the expression levels of cartilage-realted genes in different glucose concentrations. Results SMSCs differentiating into cartilage in high-glucose culture were less effective than that cultured in low-glucose culture after 21 days （AGN t= -3.69, P〈0.05; Col I t= -77.57, P〈0.01; Col 11 t= -34.42, P 〈 0.01 ）. High glucose concentration increased TGF-13 receptor type Ⅱ expression more than low glucose concentration during expansion period（ t = 4. 5, P 〈 0.01 ）. The SMSCs presented less sensitive response to chondrogensis in high glucose concentration than that in low glucose concentration during differentiation stage （ PKC t = - 5.84, P 〈 0. 01 ; TGFβ- R Ⅱt = - 4.79, P 〈 0. 01 ）. Inhibition of PKC activity during predifferentiation culture could enhance the expression of TGFβ-R Ⅱin SMSCs＂ differentiation stage （t = - 1.03, P 〈 0. 05）. Conclusions The study demonstrated that glucose concentration could modulate the chondrogenic ability of pre-differentiation SMSCs. SMSCs expanded in low-glucose medium may express a higher potential of chodrogenesis than that in high-glucose medium. The essence of the mechanism for glucose concentration regulating the differentiation capability of SMSCs may lie in the modulating of PKC activity and TGF-β signaling pathway.

关 键 词：滑膜 间质干细胞 葡萄糖 成软骨分化 蛋白激酶C 

分 类 号：R684[医药卫生—骨科学]