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目的研究丹参酮ⅡA在大鼠膝软骨细胞体外培养中对软骨细胞去分化的影响。方法采用Ⅱ型胶原酶消化法获取2周龄SD大鼠膝关节原代软骨细胞,体外培养传代至P4进行实验。对照组用含10%小牛血清高糖DMEM培养(基础培养液)。实验组有3个浓度:100μg/ml组用基础培养液+100μg/ml丹参酮ⅡA培养,200μg/ml组用基础培养液+200μg/ml丹参酮ⅡA培养,400μg/ml组用基础培养液+400μg/ml丹参酮ⅡA培养。培养72 h后,采用CCK-8法检测各组细胞增值情况,光学显微镜观察软骨细胞形态,阿利新蓝染色观察糖胺聚糖(GAG)含量,实时荧光定量PCR检测Ⅰ型、Ⅱ型、Ⅹ型胶原表达情况。结果 400μg/ml组肉眼可见的细胞密度低于其他各组,且阿利新蓝染色最浅。培养24 h、48 h、72 h,100μg/ml组、200μg/ml组的OD值均大于对照组,而400μg/ml组OD值均小于对照组,差异有统计学意义(P<0.05)。实验组的Ⅰ型、Ⅹ型胶原m RNA含量明显低于对照组,差异有统计学意义(P<0.05)。400μg/ml组Ⅱ型胶原m RNA含量低于对照组,100μg/ml组、200μg/ml组Ⅱ型胶原m RNA含量高于对照组,差异有统计学意义(P<0.05);但100μg/ml组、200μg/ml组差异无统计学意义(P>0.05)。结论不同浓度的丹参酮ⅡA对去分化软骨细胞作用有所区别,100μg/ml、200μg/ml时促进软骨细胞增殖,抑制去分化进程;400μg/ml时抑制软骨生长活性,细胞基质分泌减少。
Objective To study the effect of tanshinone ⅡA on the dedifferentiation of chondrocytes in rat knee chondrocytes cultured in vitro. Methods Primary chondrocytes of knee joints of 2-week-old SD rats were obtained by collagenase digestion method Ⅱ, and passaged to P4 in vitro. The control group was cultured with 10% fetal bovine serum high glucose DMEM (basal medium). The experimental group has three concentrations: 100μg / ml group with basal medium + 100μg / ml tanshinone Ⅱ A culture, 200μg / ml group with basal medium +200μg / ml tanshinone Ⅱ A culture, 400μg / ml group with basal medium + 400μg / ml tanshinone Ⅱ A culture. After cultured for 72 h, CCK-8 method was used to detect the proliferation of cells in each group. The morphological changes of chondrocytes were observed with light microscopy. The contents of GAG were observed with Aliskiren staining. The expressions of type I, type II and type X Collagen expression situation. Results The density of macroscopic cells in 400μg / ml group was lower than that in other groups, and the staining of alitame blue was the lightest. The OD value of cultured group at 24 h, 48 h, 72 h, 100 μg / ml and 200 μg / ml was higher than that of the control group, while OD at 400 μg / ml was less than that of the control group (P <0.05). The contents of m RNA in type I and type X collagen in the experimental group were significantly lower than those in the control group (P <0.05). The content of m RNA in type Ⅱ collagen of 400μg / ml group was lower than that in control group. The content of type Ⅱ collagen in 100μg / ml group and 200μg / ml group was higher than that in control group (P <0.05) Group, 200μg / ml group no significant difference (P> 0.05). CONCLUSION: Tanshinone IIA has different effects on dedifferentiated chondrocytes. 100μg / ml and 200μg / ml can promote chondrocyte proliferation and inhibit the process of dedifferentiation. Inhibition of chondrogenic growth at 400μg / ml and decrease of cell matrix secretion.