沙苑子多糖促进兔半月板纤维软骨细胞增殖的作用机制研究

Study on the Mechanism of Enhancement Effects of Astragalus complanatus Polysaccharides on the Proliferation of Meniscal Fibrochondrocyte Cells in Rabbits

目的:研究沙苑子多糖促进兔半月板纤维软骨细胞(以下简称"软骨细胞")增殖的作用机制。方法:分离1月龄新西兰大白兔软骨细胞。将软骨细胞分为正常对照组(PBS)、阳性对照组(硫酸氨基葡萄糖,10 mg/mL)和沙苑子多糖高、中、低(40、20、10mg/mL)剂量组,分组给药干预。采用光学显微镜观察软骨细胞的形态学特征;采用MTT法检测软骨细胞增殖抑制率,并用流式细胞术观察细胞周期;采用酶联免疫吸附试验(ELISA)检测软骨细胞中Ⅱ型胶原蛋白(ColⅡ)、碱性磷酸酶蛋白(ALP)的表达水平;采用逆转录-聚合酶链式反应和Western blotting法检测转化生长因子β1(TGF-β1)、骨形态发生蛋白2(BMP-2)mRNA及蛋白的表达水平。结果:软骨细胞培养72 h后,细胞融合成单层,多数呈现细长梭型外观。与正常对照组比较,阳性对照组和沙苑子多糖高、中、低剂量组软骨细胞的增殖抑制率、G1/G0期细胞百分比均显著降低(P<0.05),S期细胞百分比和ColⅡ、ALP蛋白表达水平及TGF-β1、BMP-2 mRNA及蛋白的表达水平均显著升高(P<0.05)。与阳性对照组比较,沙苑子多糖高剂量组软骨细胞的增殖抑制率、G1/G0期细胞百分比均显著降低(P<0.05),S期细胞百分比和ColⅡ、ALP蛋白表达水平及TGF-β1、BMP-2 mRNA及蛋白的表达水平均显著升高(P<0.05);沙苑子多糖低剂量组软骨细胞的增殖抑制率、G1/G0期细胞百分比均显著升高(P<0.05),S期细胞百分比和ColⅡ、ALP蛋白及TGF-β1、BMP-2 m RNA和蛋白的相对表达水平均显著降低(P<0.05);沙苑子多糖中剂量组上述指标差异均无统计学意义。结论:沙苑子多糖可促进软骨细胞增殖,降低G1/G0期细胞百分比,促进细胞向S期转化;其作用机制可能与上调TGF-β1、BMP-2 mRNA及蛋白表达,促进ColⅡ、ALP蛋白表达水平升高有关。

OBJECTIVE:To study the mechanism of enhancement effects of Astragalus complanatus polysaccharides(ACP)on the proliferation of meniscal fibrochondrocytes cells in rabbits.METHODS:The meniscal fibrochondrocytes cells were isolated from 1-month-old New Zealand white rabbits.The meniscal fibrochondrocytes cells were divided into normal control group(PBS),positive control group(glucosamine sulfate,10 mg/mL)and ACP high-dose,medium-dose and low-dose groups(40,20,10 mg/mL).The morphology of meniscal fibrochondrocytes cells were observed under microscope.Cell proliferation rate was detected by MTT assay.Cell cycle was observed with flow cytometry.ELISA assay was used to detect relative expression of medium collagen typeⅡ(ColⅡ)and alkaline phosphatase protein(ALP)in meniscal fibrochondrocytes cells.RT-qPCR and Western blotting assay were adopted to detect m RNA and protein expression of transforming growth factorβ1(TGF-β1)and bone morphogenetic protein 2(BMP-2).RESULTS:After cultured for 72 h,meniscal fibrochondrocytes cells were fused into a single layer,and most of them were slender type in appearance.Compared with normal control group,the proliferation rate of meniscal fibrochondrocytes cells and the percentage of cells at G1/G0 phase were decreased significantly in positive control group and ACP high-dose,medium-dose and low-dose groups(P<0.05);the percentage of cells at S phase,protein expression of ColⅡand ALP,m RNA and protein expression of TGF-β1 and BMP-2 were increased significantly(P<0.05).Compared with positive control group,inhibitory rate of meniscal fibrochondrocytes cells proliferation and the percentage of cells at G1/G0 phase were decreased significantly in ACP high-dose group(P<0.05),while the percentage of cells at S phase,protein expression of ColⅡand ALP,m RNA and protein expression of TGF-β1 and BMP-2 were increased significantly(P<0.05).The inhibitory proliferation rate of meniscal fibrochondrocytes cells and the percentage of cells at G1/G0 phase were increased significantly in ACP low-dose group(P<0.05),while the percentage of cells at S phase,protein expression of ColⅡand ALP,m RNA and protein expression of TGF-β1 and BMP-2 were decreased significantly(P<0.05).There was no statistical significance in above indexes of ACP medium-dose group.CONCLUSIONS:ACP can promote the proliferation of meniscal fibrochondrocytes cells,reduce the percentage of cells at G1/G0 phase,promote cell transformation to S phase;the mechanism of which may be related to up-regulating TGF-β1,BMP-2 mRNA and protein expression,promoting ColⅡand ALP protein expression enhancement.