肖伟++++++马笃军++++++彭力平++++++王立新++++++余阗++++++廖州伟++++++陈达++++++谭锐泉

[摘要] 目的 觀察牛膝醇提物对兔骨关节炎软骨细胞体外增殖及对糖胺聚糖（GAG）形成的影响。 方法 骨关节炎造模成功后提取、分离兔骨关节炎软骨细胞，用甲苯胺蓝染色确定软骨细胞的存在，将软骨细胞接种于5组：空白对照组、牛膝醇提物低剂量组、牛膝醇提物中剂量组、牛膝醇提物高剂量组、经典软骨增殖组。倒置相差显微镜观察软骨细胞形态，用CCK-8法检测软骨细胞增殖率，免疫细胞学检测Ⅱ型胶原蛋白荧光表达，qRT-PCR检测软骨细胞标记基因Ⅱ型胶原mRNA表达水平，邻联甲苯胺（DMB）染色法检测细胞内GAG含量。 结果 牛膝醇提物高、中、低剂量组与空白对照组比较Ⅱ型胶原蛋白荧光明显阳性表达；牛膝醇提物高、中剂量组与空白对照组比较软骨细胞标记基因Ⅱ型胶原mRNA明显增加（P < 0.05），其中牛膝醇提物高剂量组效果最佳（P < 0.01），与经典软骨增殖组比较差异无统计学意义（P > 0.05）；空白对照组GAG含量与经典软骨增殖组及不同剂量牛膝醇提物组比较，差异有统计学意义（P < 0.05），其中牛膝醇提物高剂量组GAG最多（P < 0.01），牛膝醇提物高剂量组与经典软骨增殖组比较差异无统计学意义（P > 0.05）。 结论 牛膝醇提物中药含药血清可促进体外软骨细胞的增殖和蛋白合成，具体作用机制有待进一步探讨。

[关键词] 牛膝醇提物；软骨细胞；骨关节炎；增殖；糖胺聚糖

[中图分类号] R684.3 [文献标识码] A [文章编号] 1673-7210（2017）02（b）-0020-05

[Abstract] Objective To observe the effects of alcohol extractive of Radix Achyranthis Bidentatae on the proliferation in vitro and glycosaminoglycan （GAG） formation of chondrocytes in rabbits with osteoarthritis. Methods After successful establishment of osteoarthritis model， the chondrocytes in rabbits with osteoarthritis were extracted and separated， the exit of chondrocytes was determined by toluidine blue staining， the chondrocytes were inoculated into five groups： blank control group， low dosage of alcohol extractive of Radix Achyranthis Bidentatae group， median dosage of alcohol extractive of Radix Achyranthis Bidentatae group， high dosage of alcohol extractive of Radix Achyranthis Bidentatae group， classic cartilage proliferation group. The cellular morphology of chondrocytes was observed by inverted phase contrast microscope， the proliferation rates of chondrocytes were detected by CCK-8 method， the fluorescent expression of type Ⅱ collagen was detected by immunocytology， the expression of chondrocyte marker gene type Ⅱ collagen mRNA was detected by qRT-PCR， the contents of intracellular GAG were detected by DMB staining method. Results Compared with the blank group， fluorescent expression of type Ⅱ collagen in high， median， low dosage of alcohol extractive of Radix Achyranthis Bidentatae groups were positive； the expression of chondrocyte marker gene type Ⅱ collagen mRNA in high， median dosage of alcohol extractive of Radix Achyranthis Bidentatae groups were significantly increased compared with that of blank control group （P < 0.05）， among which， the effect of high dosage of alcohol extractive of Radix Achyranthis Bidentatae group was the best （P < 0.01）， which had no significant differences compared with that of classic cartilage proliferation group （P > 0.05）； the content of GAG in blank control group had significant differences compared with those of classic cartilage proliferation group and different dosages of alcohol extractive of Radix Achyranthis Bidentatae groups （P < 0.05）， among which， the content of GAG in high dosage of alcohol extractive of Radix Achyranthis Bidentatae group was at most （P < 0.01）， there was no significant difference between high dosage of alcohol extractive of Radix Achyranthis Bidentatae group and classic cartilage proliferation group （P > 0.05）. Conclusion Serum containing alcohol extractive of Radix Achyranthis Bidentatae can promote the proliferation and protein synthesis of chondrocytes in vitro， the specific mechanism needs to be further studied.

[Key words] Alcohol extractive of Radix Achyranthis Bidentatae； Chondrocyte； Osteoarthritis； Proliferation； Glycosaminoglycan

骨关节炎（osteoarthritis）的核心病理改变是关节软骨的软化、破溃和脱落[1]，给社会带来了巨大经济负担，亟待研究解决。软骨细胞是软骨组织中唯一的细胞成分，是软骨破坏过程中的靶细胞，因此软骨细胞的体外培养是研究关节软骨生理及病理的常用体外实验模型[2]。……