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目的探讨柔肝中药对体外培养软骨细胞增殖能力及关节软骨低聚基质蛋白(COMP)分泌的影响。方法采用分阶段酶消化法体外培养兔软骨细胞,以2×10~4/ml密度接种3代内细胞,以Ⅱ型胶原的免疫组织化学研究鉴定细胞。给予家兔临床等效剂量灌胃后,抽取含药血清培养细胞,分别用5%、10%的柔肝方含药血清(分给药后1、3、5h 3个时间点)以及正常兔血清、小牛血清干预7d,采用四甲基偶氮唑蓝(MTF)法观察中药含药血清对体外培养的软骨细胞增殖的影响;体外培养人软骨细胞,采用柔肝复方组及单味柔肝药提取物直接添加体外培养体系3 d,添加终浓度为10 mg/ml。采用酶联免疫吸附试验(ELISA)检测药物对体外培养的人软骨细胞COMP分泌的影响。结果培养细胞经Ⅱ型胶原的免疫组织化学染色后有阳性表现。在对软骨细胞增殖的影响方面,柔肝方含药血清各组均优于兔血清组和小牛血清组,其中含药血清组的3 h时间点总体优于1 h及5 h时间点(P<0.05);在对软骨细胞上清COMP分泌方面,柔肝复方及单方提取物组均有促进细胞分泌COMP的作用(P<0.05),但两组之间差异无统计学意义(P>0.05)。结论柔肝方中药含药血清可以促进体外培养软骨细胞增殖;在药物直接干预的条件下,柔肝复方及单方提取物可以促进软骨细胞分泌COMP。
Objective To investigate the effect of softening liver medicine on the proliferation of chondrocytes cultured in vitro and the secretion of articular cartilage oligomeric matrix protein (COMP). Methods Rabbit chondrocytes were cultured in vitro with a stepwise enzyme digestion method. The 3rd passage cells were inoculated at a density of 2×10 4 /ml. The cells were identified by immunohistochemistry of type II collagen. After the rabbits were given a clinically equivalent dose to the rabbit, drug-containing serum cultured cells were collected, and 5%, 10% Rouganfang-containing serum was used (at 3, 5, and 3 hours after administration) and normal rabbits. Serum and calf serum intervened for 7 days. MTT assay was used to observe the effect of drug-containing serum of Chinese medicine on the proliferation of chondrocytes cultured in vitro. Human chondrocytes were cultured in vitro and Rougan compound group and monofilament soft were used. Liver extracts were directly added to the in vitro culture system for 3 days, and the final concentration was 10 mg/ml. The effect of drugs on the secretion of COMP from human chondrocytes cultured in vitro was detected by enzyme-linked immunosorbent assay (ELISA). Results The cultured cells showed positive expression after immunohistochemical staining of type II collagen. In terms of the effect on the proliferation of chondrocytes, Rouganfang drug-containing serum was superior to rabbit serum and calf serum, and the drug-containing serum group was generally superior to the 1 h and 5 h time points at the 3 h time point ( P<0.05); In the secretion of COMP from chondrocyte supernatants, both Rougan compound and unilateral extract groups promoted the secretion of COMP by cells (P<0.05), but there was no significant difference between the two groups (P>0.05). 0.05). Conclusion Rougan recipe-containing serum can promote the proliferation of cultured chondrocytes in vitro. Under the condition of direct drug intervention, Rougan compound and unilateral extract can promote the secretion of COMP by chondrocytes.