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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 ABPS on the expression of type Ⅱ collagen and proteoglycan in rat articular chondrocytes. Methods: Ten healthy 4-week-old Sprague-Dawley rats were used to separate the knee joint and scrape the cartilage on both sides of knee joint. The chondrocytes were harvested by mechanical-type Ⅱ collagenase digestion. The chondrocytes culture system was established in vitro. The morphological changes of cells were observed by inverted phase contrast microscope. The type Ⅱ collagen immunohistochemistry was used to identify the chondrocytes. The second generation of chondrocytes were cultured in vitro. Chondrocytes were treated with ABPS at 0, 50, 100 and 200 μg · L -1, respectively. After intervention, the expression of type Ⅱ collagen and proteoglycan were detected by Western blot, and the expression of type Ⅱ collagen in blank group and dosing group was observed by immunofluorescence. Results: The second generation of chondrocytes showed the typical characteristics of chondrocytes: chondrocytes are rich in type II collagen. Western blot results showed that ABPS promoted the expression of type Ⅱ collagen in chondrocytes, and the expression of type Ⅱ collagen was the highest at 100 μg · m L -1, with statistical significance (P <0.05). At the same time, the expression of proteoglycan in chondrocytes increased after chondrocytes were treated with 100 μg · m L -1 ABPS, but the difference was not statistically significant (P> 0.05). Compared with the control group, collagen type Ⅱ collagen expression in chondrocytes was stronger after ABPS intervention. Conclusion: ABPS promotes the expression of type Ⅱ collagen and proteoglycan in chondrocytes.