背景：血竭为传统医学中生肌方剂的主要成分，长期应用于临床治疗多种难愈性创面，具有确切的疗效，但血竭在创面愈合过程中对正常成纤维细胞分泌胶原的影响，目前未见报道。目的：观察血竭提取物对成纤维细胞增殖及胶原分泌的作用。方法：应用氯仿、乙酸乙酯、乙醇对血竭依次回流提取得到血竭提取液，用DMEM培养液稀释后培养成纤维细胞。将人正常皮肤来源成纤维细胞分别培养于血竭氯仿提取液(氯仿提取组)、血竭乙酸乙酯提取液(乙酸乙酯提取组)、血竭乙醇提取液(乙醇提取组)含1%二甲基亚砜DMEM(含1%二甲基亚砜DMEM组)和正常培养(对照组)条件下。MTT法检测不同血竭提取物在不同质量浓度(0.002，0.02，0.2，2，20 g/L)培养条件下，确定最适合成纤维细胞生长的血竭提取物，并检测在此条件下，成纤维细胞形态、生长曲线、细胞周期、分泌Ⅲ型前胶原水平的变化。结果与结论：血竭乙酸乙酯提取物在0.2-2 g/L范围内，可以促进正常人成纤维细胞增殖，在2 g/L浓度值时促进增殖作用最显著，因此2 g/L乙酸乙酯提取物为最适合成纤维细胞生长的一组血竭提取物。与对照组相比，2 g/L乙酸乙酯提取组成纤维细胞的细胞融合现象增多，细胞浓度增加，S期的细胞比例增加，减少Ⅲ型前胶原表达。提示血竭乙酸乙酯提取物既可促进创面愈合，又不致于过度增生形成病理性瘢痕。%BACKGROUND:Dragon’s blood is the main ingredient of traditional medicine prescription for promoting granulation, which has been used in clinical treatment of a variety of refractory wounds and achieved the exact effects. But the Dragon’s blood effect on colagen secretion from normal fibroblasts has not been reported. OBJECTIVE: To investigate the effects of Dragon’s blood extract on the proliferation and secret function of fibroblastsin vitro. METHODS: Dragon’s blood was extracted by extracts chloroform, acetoacetic ester, and alcohol in turn. Normal human fibroblasts were respectively cultured in Dragon’s blood extracts of chloroform, acetoacetic ester, and alcohol, DMEM containing 1% dimethyl sulfoxide, and normal culture medium. Then, the fibroblasts were cultured in vitro in different media containing gradient dilutions of Dragon’s blood extracts (0.002, 0.02, 0.2, 2, 20 g/L), which was folowed by cel proliferation determination assessed with MTT assay. Under the optimal concentration, the cel growth curves were drawn and the flow cytometry was used to determine the changes of cel cycle. The concentration of procolagen type III in the supernatant of the fibroblast culture systems was measured by radioimmunoassay. RESULTS AND CONCLUSION:0.2 g/L-2 g/L dilution of Dragon’s blood extracted by acetoacetic ester enhanced the proliferation of fibroblasts in a dose-dependent manner. The 2 g/L was the optimal dilution of Dragon’s blood extracted by acetoacetic ester, and it increased the ratio of S cels in cel cycle than control group and decreased procolagen type III. These findings indicate that Dragon’s blood acetoacetic ester extract can improve the proliferation of cultured human fibroblastsin vitro, and decrease the secretion of procolagen type III of fibroblasts, and it can be beneficial to improve wound healing and inhibit hypertrophic scar.
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