牛膝多糖对软骨细胞Ⅱ型胶原及蛋白聚糖表达的影响

Effect of ABPS on the Expressions of Type Ⅱ Collagen and Proteoglycan of Cartilage Cells

目的:探讨牛膝多糖(ABPS)对大鼠膝关节软骨细胞Ⅱ型胶原及蛋白聚糖表达的影响。方法:取4周龄健康SD大鼠10只,分离膝关节,刮下双侧膝关节表面软骨组织,采用机械-Ⅱ型胶原酶消化法获取膝关节软骨细胞。建立软骨细胞体外培养体系,倒置相差显微镜观察细胞形态,采用Ⅱ型胶原免疫组化法鉴定软骨细胞。体外培养到第2代软骨细胞,分别用0,50,100,200μg·m L^(-1)的ABPS对软骨细胞进行干预。干预后,Western blot检测各组Ⅱ型胶原、蛋白聚糖的表达变化,免疫荧光观察空白组与加药组Ⅱ型胶原的表达变化。结果:第2代软骨细胞的形态学表明软骨细胞的典型特征:软骨细胞含有丰富的Ⅱ型胶原。Western blot检测结果显示,ABPS促进软骨细胞Ⅱ型胶原表达,并且在100μg·m L^(-1)时表达最高,差异有统计学意义(P<0.05)。同时100μg·m L^(-1)的ABPS干预软骨细胞后,软骨细胞中的蛋白聚糖表达增加,但差异无统计学意义(P>0.05)。与空白组的Ⅱ型胶原荧光表达相比,ABPS干预后软骨细胞的Ⅱ型胶原荧光表达更强。结论:ABPS促进了软骨细胞Ⅱ型胶原及蛋白聚糖的表达。

Objective：To investigate the effect of Achyranthes bidentata polysaccharides （ ABPS ） on the expressions of type Ⅱ collagen and proteoglycan in cartilage cells.Methods：Ten 4-week-old healthy SD rats were used to isolate the knee joints,scrape cartilaginous tissue off the surface of the knee joint,and get knee joint cartilage cells with the mechanical type Ⅱ collagenase digestion method.An in vitro culture system was established and the cell morphology was observed with an inverted phase contrast microscope.Type Ⅱ immunohistochemistry method was used to identify the ehondrocytes.By the second generation of in-vitro- cultured chondrocytes,ABPS （0,50,100,200 μg · mL^-l respectively ） was used to interfere in the cartilage cells. After intervention,Western blot was used to detect the expression changes of type Ⅱ collagen and proteoglycan of each group,and immunofiuorescence was used to observe the expression changes of type Ⅱ collagen respectively in the blank control group and the dosing group.Results：The morphology of the second generation chondrocytes showed that cartilage cells contained rich type Ⅱ collagen.Western blot showed that ABPS promoted chondrocyte type II collagen and the expression of type Ⅱ collagen was the highest when there was 100 μg·mL^-I ABPS,the difference being statistically significant （ P 〈 0.05 ） .After intervention with 100 μg · mL^-1 ABPS,the expression of proteoglycan in cartilage cells increased,but the difference was not statistically significant （ P 〉 0.05 ） . Compared with the blank control group,the expression of intervened collagen type Ⅱ collagen was stronger.Conclusion：ABPS promotes the expression of type Ⅱ collagen and proteoglycan.