血竭提取物对成纤维细胞增殖及合成透明质酸的影响

Effects of extracts of Dragon＇s blood on fibroblast proliferation and extracellular matrix hyaluronic acid

目的 研究血竭提取物对成纤维细胞生物学作用的影响.方法 将血竭经过氯仿、乙酸乙酯、乙醇依次回流提取分别得到3种血竭提取液,培养液稀释后行成纤维细胞培养.噻唑蓝法MTT检测浓度分别为0.002、0.02、0.2、2、20 mg/ml的血竭提取物,在0、12、24、36、48、60、72h7个检测点对体外培养成纤维细胞增殖的影响,并绘制最适浓度下细胞生长曲线.流式细胞术FCM分析最适浓度培养下成纤维细胞的细胞周期变化.应用放射免疫分析法检测0、12、24、36、48、60、72 h细胞培养上清液中透明质酸（hyaluronic acid,HA）含量.结果 血竭乙酸乙酯提取物在0.2～2 mg/ml浓度范围内,促进成纤维细胞增殖,且呈浓度依赖性,在2 mg/ml浓度值时促进作用最显著,处于S期的细胞[（25.80±3.10）％]较对照组[（7.50±0.70）％]显著增加（P＜0.01）,在此条件下培养成纤维细胞,12～72 h实验组分泌量逐渐增加,但较对照组偏低,组间比较差异有统计学意义（P＜0.01）.结论 血竭乙酸乙酯提取物具有显著促进成纤维细胞增殖作用,可能是血竭促进创面愈合的机制之一.

Objective To investigate the effects of Dragon＇s blood extract on proliferation and secret extracellular matrix function of fibroblasts in vitro.Methods Dragon ＇s blood was extracted by chloroform,acetoacetic ester,alcohol.Human fibroblast were cultured in vitro in media containing gradient dilutions of Dragon＇s blood extracts （0.002,0.02,0.2,2,20 mg/ml）,which was followed by cell proliferation assessed with MTT assay on 0,12,24,36,48,60,72 h.Under the optimal concentration,the cell growth curves were drawn and the flow cytometry （FCM） was used to determine the changes of cell cycle.On 0,12,24,36,48,60,72 h,the concentration of hyaluronic acid in the supernatant of fibroblast culture was measured by radioimmunoassay.Results 0.2-2 mg/ml Dragon＇s blood extracts enhanced the proliferation of fibroblasts in a dose-dependent manner.2 mg/ml was the optimal dilution of Dragon＇s blood extract,and it increased the ratio of S cells in cell cycle[（25.80 ± 3.10） %] than control group[（7.50 ± 0.70）%,P 〈 0.01].From 12 h to 72 h,in 2 mg/ml Dragon＇s blood group,concentration of Hyaluronic acid secreted by fibroblasts gradually increased,but were less than control（P 〈 0.01）.Conclusions Dragon＇s blood acetoacetic ester extract improved the proliferation of cultured human fibroblasts in vitro,might be beneficial to promote wound healing.