脂多糖对皮肤成纤维细胞生物学特性的影响及与创面愈合的关系

目的探讨脂多糖（lipopolysaccharide，LPS）对皮肤成纤维细胞增殖和胶原合成的影响，以研究细菌内毒素与皮肤创面愈合的关系。方法取正常皮肤按黄勇等方法行成纤维细胞培养后，分为1个对照组及6个实验组。实验组分别与终浓度为0．005、0．010、0．050、0．100、0．500和1．000μg／ml大肠杆菌LPS（E．coli055：B5）培养，对照组为DMEM培养。分别通过MTT比色法及细胞计数法观察各组1～9d吸光度（A）值和细胞数量的变化；于LPS加入后7d细胞处于融合状态时，通过^3H-脯氨酸掺入、胃蛋白酶消化法检测细胞胶原合成量的变化，并绘制细胞生长曲线。结果与对照组比较，0．005～0．500μg／ml组A值增加，于5～9d差异有统计学意义（P〈0．05）；1．000μg／ml组A值降低，于3～9d差异有统计学意义（P〈0．05）。与对照组比较，0．005～0．500μg／ml组促进细胞胶原合成，且0．100μg／ml组作用达高峰（P〈0．05），1．000μg／ml LPS抑制细胞胶原合成，差异有统计学意义（P〈0．05）。与对照组比较，0．005～0．500μg／ml组细胞数量明显增加，分别于3～6d、1～6d、3～6d、2～6d及3～6d时比较，差异均有统计学意义（P〈0．05）；1．000μg／ml组细胞数量明显降低，于2～9d差异有统计学意义（P〈0．05）。结论在一定浓度范围内LPS促进成纤维细胞增殖和胶原蛋白合成，但过高浓度LPS则产生抑制效应。提示一定量的细菌内毒素可能有利于皮肤创面愈合，当内毒素过多时将对创面愈合产生负面作用。

INFLUENCE OF LIPOPOLYSACCHARIDE ON THE BIOLOGICAL CHARACTERISTICS OF SKIN FIBROBLASTS AND ITS POTENTIAL ROLE IN WOUND HEALING

OBJECTIVE: To investigate the influence of lipopolysaccharide (LPS) on the proliferation and collagen synthesis of normal human skin fibroblasts so as to elucidate its relation with skin wound healing. METHODS: Fibroblasts were isolated and cultured in vitro, and then exposed to different doses of LPS(0.005, 0.010, 0.050, 0.100, 0.500, and 1.000 microg/ml) from E. coli055:B5 respectively. Then the absorbance (A) value of fibroblasts was determined with the colorirneteric thiazolylblue (MTT) assay, and the cell number was counted under inverted phase contrast microscope from the 1st day to the 9th day after LPS administration, and collagen synthesis of fibroblasts in culture medium was measured with the method of pepsin digestion after incorporation of 3H-proline into stable, single-layered, confluent fibroblasts at 7 days after LPS administration. RESULTS: Compared with control group, A value increased with the increasing concentration of LPS (0.005 microg/ml-0.500 microg/ml) and LPS of 0.100 microg/ml group had the strongest effect. The difference was remarkable from the 5th day to the 9th day(P<0.05). A value decreased when challenged with the LPS of 1.000 microg/ml and the difference was remarkable from the 3rd day to the 9th day(P<0.05). Cell number increased with the administration of LPS of different concentrations (0.005 microg/ml-0.500 microg/ml) and LPS of 0.100 microg/ml group had the strongest effect. The difference was remarkable from the 1st day to the 6th day(P<0.05). Cell number decreased remarkably when challenged with LPS of 1.000 microg/ml and the difference was remarkable from the 2nd day to the 9th day (P < 0.05). Collagen synthesis increased when challenged with LPS of different concentrations (0.005 microg/ml-0.500 microg/ml) and the 0.100 microg/ml group had the strongest effect. However, when the dose of LPS reached 1.000 microg/ml, it inhibited collagen synthesis. CONCLUSION: LPS could promote the proliferation and collagen synthesis of fibroblasts within a certain range of low doses, but over-high dose of LPS might inhibit the proliferation and collagen synthesis of fibroblasts, suggesting that LPS of certain concentrations might contribute to wound healing, while excessive LPS has negative effect on wound healing.