左归丸对间充质干细胞向软骨细胞分化过程中Ⅱ型胶原及蛋白多糖基因表达的影响

Effects of Zuogui Pill on the Gene Expressions of Typetiation of Mesenchymal Stem Cells towards Chondrocytes

目的观察左归丸含药血清对间充质干细胞(mesenchymal stem cells,MSCs)向软骨细胞定向分化过程中MSCs增殖、Ⅱ型胶原及蛋白多糖基因表达的影响。方法分离培养大鼠MSCs,体外诱导其向软骨细胞分化,诱导同时分别以高(57 g/kg)、中(28.5 g/kg)、低(9.5 g/kg)浓度左归丸含药血清及大鼠空白血清刺激细胞。CCK-8法检测各组对MSCs增殖的影响。将第3代MSCs分为空白对照组(加入大鼠空白血清)、诱导对照组(加入诱导液及大鼠空白血清)及左归丸含药血清组(加入诱导液及中浓度左归丸含药血清),采用RT-PCR、免疫组织化学和Real-time PCR检测诱导分化软骨细胞中Ⅱ型胶原和蛋白多糖的表达。结果与空白对照组比较,不同浓度左归丸含药血清均能促进MSCs增殖(P<0.05),其中以中浓度组最为明显(P<0.05)。诱导第21天RT-PCR和免疫组织化学显示诱导对照组和左归丸含药血清组MSCs中均可见Ⅱ型胶原mRNA和蛋白表达,其中左归丸含药血清组中Ⅱ型胶原mRNA表达明显高于诱导对照组。Real-time PCR结果显示,诱导第21天左归丸含药血清组蛋白多糖mRNA相对表达定量分别约是第7、14天表达量的16倍和3倍(P<0.05),并明显高于同期诱导对照组(P<0.05)。结论左归丸含药血清能促进MSCs增殖、Ⅱ型胶原和蛋白多糖基因表达,可能是其软骨保护作用的分子基础之一。

Objective To study the effects of Zuogui Pill（ZP） contained serum on the gene expressions of type-Ⅱ collagen and proteoglycan during the differentiation of mesenchymal stem cells（MSCs） towards chondrocytes.Methods MSCs isolated from rat bone marrow were in vitro induced differentiation towards chondrocytes and stimulated with high-（57 g/kg）,middle-（28.5 g/kg）,and low-dose（9.5 g/kg） ZP contained serums and serum of blank rats.The proliferation of MSCs was analyzed by CCK-8 method.The 3rd-passage MSCs were divided into the blank control group（by adding serum of the blank group rats）,the induction control group（by adding the induction fluid and serum of the blank group rats）,and the ZP contained serum group（by adding the induction fluid and middle-dose ZP contained serum）.The expressions of type-Ⅱ collagen and proteoglycan were determined using reverse transcriptase PCR,Real-time PCR,and immunohistochemistry.Results Compared with the blank control group,the proliferation of MSCs could be promoted by ZP contained serum at different doses（P0.05）,with the most obvious effect shown in the middle-dose ZP contained serum group（P0.05）.The mRNA and protein expressions of type-Ⅱ collagen could be identified in the induction control group and the ZP contained serum group on the 21st day of the induction.Of them,the mRNA expression of type-Ⅱ collagen in ZP contained serum groups was obviously higher than that of the induction control group.Results of Real-time PCR showed that on the 21st day of the induction,the mRNA expression quantitation of proteoglycan in ZP contained serum groups was about 16-fold and 3-fold of the levels on the 7th day and the 14th day（P0.05）,obviously higher than those of the induction control group（P0.05）.Conclusion ZP contained serum could induce MSCs proliferation,the gene expressions of type-Ⅱ collagen and proteoglycan,which might be one of its molecular bases for protecting the cartilage.