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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 the traditional Chinese medicine of the traditional Chinese medicine TouJiaoJiaoTong Capsule on the expression of CyclinD1 mRNA in chondrocytes and to explore the possible mechanism of the treatment of osteoarthritis by traditional Chinese medicine. Methods: Sixteen New Zealand white rabbits aged 12 weeks were randomly divided into blank group, low dose group, middle dose group and high dose group by lottery. Low dose group according to 5 mL.kg-1 body weight to 5 mg.mL-1 through the bone decoction capsule solution gavage, medium dose group according to 5 mL.kg-1 body weight to 10 mg.mL-1 transdermal Pain capsule solution gavage, high-dose group according to 5 mL.kg-1 body weight 20 mg.mL-1 through the bone decoction capsule solution gavage, blank group according to 5 mL.kg-1 body weight with saline . Gavage 2 times a day for 7 days, the last 1 d 2 consecutive gavage, the middle interval of 2 h. 3 h after the last gavage, each group of experimental rabbits was extracted and stored at low temperature. Six male New Zealand rabbits, 4 weeks old, were sacrificed and their articular cartilages were removed. The cartilage was removed and placed in a petri dish containing type Ⅱ collagenase for digestion. Chondrocytes were cultured in vitro and cell function was evaluated by toluidine blue staining. The third generation of chondrocytes cultured in vitro were randomly divided into blank group, low dose group, medium dose group, high dose group, and were added with serum containing serum, low dose of traditional Chinese medicine serum, medium dose of serum, high dose of Chinese medicine serum DMEM After cultured for 72 h in culture medium, the proliferation of chondrocytes was detected by MTT assay. The expression of CyclinD1 mRNA was detected by RT-PCR and the cell morphology was observed under transmission electron microscope. Results: The in vitro culture system of chondrocytes was established successfully by type Ⅱ collagenase digestion. Toluidine blue staining showed that the chondrocytes had purple-red heterochromatic granules. The optical density of chondrocytes in each group was significantly higher than that in the blank group (F = 6.209, P = 0.004) P = 0.002). The optical density of chondrocytes in medium dose group and high dose group was higher than that in low dose group (P = 0.034, P = 0.011). The expression of CyclinD1 mRNA in chondrocytes in each group was significantly higher than that in the blank group (P = 0.002, P = 0.001) ). The expression of CyclinD1 mRNA in the middle and high dose groups was higher than that in the low dose group (P = 0.012, P = 0.004). CONCLUSION: The traditional Chinese medicine serum of Tonggu JiaoTong Capsule can promote the expression of CyclinD1 mRNA in G1 phase of chondrocytes and promote the proliferation of chondrocytes, which may be the result of the treatment of osteoarthritis by traditional Chinese medicine Good curative effect of part of the mechanism, and the specific mechanism of action for the treatment of osteoarthritis through the bone decoction need further study in order to provide a theoretical basis for clinical application.