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分泌的影响  

In vitro study of different Chinese herbs on the proliferation and COMP expression of chondrocyte

作　　者：曹月龙[1] 冯伟 王翔[1] 徐宇[1] 詹红生[1] 石印玉[1] 

机构地区：[1]上海中医药大学骨伤研究所,201203 [2]上海市骨伤科研究所

出　　处：《中华风湿病学杂志》2007年第8期469-472,513,共5页Chinese Journal of Rheumatology

基　　金：国家自然科学基金(30300459;30472222);上海市自然科学基金(04ZR14128);教育部新世纪优秀人才计划基金(NCET-04-0438);上海市重点学科基金(T0303);上海市曙光计划基金(035SG1029);上海市科委重点基金(03DZ19530);上海市启明星计划基金(04QMX1434);上海市卫生局基金(034065)

摘　　要：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、5h3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3d，添加终浓度为10mg／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3h时间点总体优于1h及5h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 different Chinese herbs on cell proliferation and cartilage oligomeric matrix protein （COMP） expression in chondrocyte culture. Methods Chondrocytes isolated from rabbit knee cartilage were cultured for 3 generations with the density of 2×10^4/cm^2 and were verified by collagen Ⅱ immunohistochemical staining. Rabbit sera containing herbs were obtained after animals orally administrated herbs at the dosage equivalent to human, At 5% and 10% serum density, cells were cultured in the medium that contained liver-softening herbal compound sera. Subgroups setting at 1, 3 and 5 hours after herb intervention were observed. Rabbit and bovine sera were control groups, Seven days after intervention, chon- drocytes proliferation was observed using the MTlr assay kit. For the study of COMP expression, cbondrocytes were isolated from human knee cartilage supernatant, Supernatant COMP level was tested by enzyme-linked immunoabsorbent assays （ELISA） after directly adding compound and extract from liver-softening herbs to the culture at the final concentration of 10 mg/ml for 3 days, Results Liver-softening herbal compound group had significant effect on cell proliferation compared to control, of which, 3-hour subgroup was more significant than 1- and 5-hour subgroups （P〈0.05）, COMP level in both extract and compound liver-softening herb groups was higher than control （P〈0.05）, but no significant difference was demonstrated between extract and compound groups. Conclusion The finding suggests that serum containing liver-softening herbs can stimulate cell proliferation, When added directly to cultures, both extract and compound liver-softening herb can promote COMP expression of chondrocyte in vitro.

关 键 词：骨关节炎 细胞增殖 关节软骨低聚基质蛋白 

分 类 号：R285.5[医药卫生—中药学]