透骨消痛胶囊含药血清对软骨细胞Cyclin D1 mRNA表达的影响

Study on the effect of TOUGU XIAOTONG capsule medicated serum on the expression of Cyclin D1 mRNA in Chondrocytes

目的:观察透骨消痛胶囊含药血清对软骨细胞CyclinD1 mRNA表达的影响,探讨透骨消痛胶囊治疗骨性关节炎的可能作用机制。方法:采用抽签法将16只12周龄雄性新西兰大白兔随机分为空白组、低剂量组、中剂量组、高剂量组,每组4只。低剂量组按5 mL.kg-1体质量以5 mg.mL-1透骨消痛胶囊溶液灌胃,中剂量组按5 mL.kg-1体质量以10 mg.mL-1透骨消痛胶囊溶液灌胃,高剂量组按5 mL.kg-1体质量以20 mg.mL-1透骨消痛胶囊溶液灌胃,空白组按5 mL.kg-1体质量以生理盐水灌胃。每天灌胃2次,连续7 d,最后1 d连续2次灌胃,中间间隔2 h。末次灌胃后3 h提取各组实验兔含药血清低温保存备用。将6只4周龄雄性新西兰兔处死,取其膝关节软骨,置入盛有Ⅱ型胶原酶的培养皿中进行消化,建立软骨细胞体外培养体系,并采用甲苯胺蓝染色法鉴定细胞功能。将体外培养的第3代软骨细胞随机分为空白组、低剂量组、中剂量组、高剂量组,并分别加入含空白血清、低剂量中药血清、中剂量中药血清、高剂量中药血清的DMEM培养液中继续培养72 h后,采用MTT法检测软骨细胞的增殖情况,采用RT-PCR法检测CyclinD1 mRNA的表达,并在透射电子显微镜下观察细胞形态。结果:Ⅱ型胶原酶消化法成功建立了软骨细胞的体外培养体系,甲苯胺蓝染色可见软骨细胞内呈紫红色异染颗粒。干预72 h后,各组软骨细胞光密度值比较,差异有统计学意义(F=6.209,P=0.004);中剂量组、高剂量组软骨细胞光密度值高于空白组(P=0.005,P=0.002);中剂量组、高剂量组软骨细胞光密度值高于低剂量组(P=0.034,P=0.011)。各组软骨细胞CyclinD1 mRNA表达量比较,差异有统计学意义(F=8.262,P=0.001);中剂量组、高剂量组软骨细胞CyclinD1 mRNA表达量高于空白组(P=0.002,P=0.001);中剂量组、高剂量组软骨细胞CyclinD1 mRNA表达量高于低剂量组(P=0.012,P=0.004);中剂量组、高剂量组可见较多处于分裂期的细胞。结论:透骨消痛胶囊的含药血清能促进软骨细胞G1期正性调节因子CyclinD1 mRNA的表达,从而促进软骨细胞增殖,这可能是透骨消痛胶囊在临床上治疗骨性关节炎具有较好疗效的部分机理,而有关透骨消痛胶囊治疗骨性关节炎的具体作用机制还需作更进一步的深入研究,以便为临床应用提供理论依据。

Objective：To observe the effect of TOUGU XIAOTONG capsule medicated serum on the expression of Cyclin D1 mRNA in chondrocytes,and explore the possible mechanism of TOUGU XIAOTONG capsule in the treatment of osteoarthritis（OA）.Methods：Sixteen male New Zealand white rabbits of 12 weeks old were randomly divided into blank group,low-dose group,middle-dose group and high-dose group by drawing lots,4 cases in each group.Rabbits in the low-dose group were intragastric administrated with 5 mg·mL-1 TOUGU XIAOTONG solution（5 mL·kg-1）,rabbits in the middle-dose group were intragastric administrated with 10 mg·mL-1 TOUGU XIAOTONG solution（5 mL·kg-1）,rabbits in the high-dose group were intragastric administrated with 20 mg·mL-1 TOUGU XIAOTONG solution（5 mL·kg-1）,and rabbits in the blank group were intragastric administrated with normal saline（5 mL·kg-1）.All the rabbits were intragastric administrated twice a day for 7 consecutive days,and they were intragastric administrated continuous 2 times with interval of 2 hours on-the last day.Three hours after the last administration,medicated serums of experimental rabbits in every group were extracted and preserved in low temperature.Six male New Zealand white rabbits of 4 weeks old were executed and their knee articular cartilages were fetched and placed in the Petri dish with typeⅡcollagenase for digestion.The culture system of cartilage cell in vitro was built,meantime,cell function was evaluated through toluidine blue staining method.The third-generation cartilage cell cultured in vitro were randomly divided into blank group,low-dose group,middle-dose group and high-dose group,and they were placed in the DMEM culture fluid respectively added with blank serum,low-dose traditional Chinese medicine（TCM） serum,middle-dose TCM serum and high-dose TCM serum for further culture. Seventy-two hours later,the cartilage cell proliferation was detected by MTT method,Cyclin D1 mRNA expression was inspected through RT-PCR method,and the cell shape was observed under transmission electron microscope.Results： Culture system of cartilage cell in vitro was successfully built through collagenaseⅡdigestion and purple metachromatic granules were found in cartilage cell through toluidine blue staining method.There were statistical differences in optical density（OD） of cartilage cell among the 4 groups after 72 hours＇ intervention（F = 6.209,P = 0.004）;the OD of cartilage cell in middle-dose group and high-dose group were higher than that of blank group（P = 0.005,P = 0.002）;the OD of cartilage cell in middle-dose group and high-dose group were higher than that of low-dose group（P = 0.034, P = 0.011）.There were statistical differences in protein expression of CyclinD1 mRNA in cartilage cell among the 4 groups（F = 8.262, P = 0.001）;CyclinD1 mRNA in cartilage cell of middle-dose group and high-dose group were higher than that of blank group（P = 0.002, P = 0.001） and low-dose group（P = 0.012,P = 0.004）;more chondrocytes in dividing phase were found in middle-dose group and highdose group.Conclusion： TOUGU XIAOTONG capsule medicated serum can promote the proliferation of chondrocytes through promoting the expression of positive regulator CyclinD1 mRNA in G1 period,and it may be partial mechanisms of the better curative effect of TOUGU XIAOTONG capsule in the treatment of OA.Howerer,further research is needed to determin the concrete mechanism of action of TOUGU XIAOTONG capsule on purpose of providing theoretical basis for the clinic.