几丁糖抑制人成纤维细胞增殖的实验研究

目的通过研究几丁糖对人成纤维细胞的细胞周期和Ki-67蛋白表达的影响,探讨几丁糖预防手术后组织粘连的机制。方法分别用含0、0.01、0.1、1.0、10.0mg/ml几丁糖的培养液作用于人成纤维细胞48h后,用流式细胞仪检测细胞周期。制作成纤维细胞的细胞爬片,用以上各浓度的含几丁糖培养液作用24h后,行细胞核的Ki-67蛋白免疫组织化学染色,观察细胞核中Ki-67蛋白的表达情况。结果在几丁糖作用下,成纤维细胞的生长在形态上受到明显抑制。几丁糖浓度为1.0、10.0mg/ml时,处于增殖期的成纤维细胞百分率分别为32.3%±5.2%、14.7%±2.9%,均显著低于几丁糖浓度为0时的百分率41.9%±5.8%,具有统计学意义(P<0.05);几丁糖浓度为0.01、0.1mg/ml时,处于增殖期的成纤维细胞百分率分别为39.0%±6.0%、35.5%±3.4%,虽低于几丁糖浓度为0时,但差异无统计学意义(P>0.05)。几丁糖浓度为0.1、1.0、10.0mg/ml时,Ki-67阳性细胞百分率分别为37.3%±3.4%、30.5%±6.2%、17.8%±3.0%,均显著低于几丁糖浓度为0时的57.6%±8.9%,具有统计学意义(P<0.05);几丁糖浓度为0.01mg/ml时,Ki-67阳性细胞百分率为54.1%±8.0%,低于几丁糖浓度为0时,无统计学意义(P>0.05)。结论几丁糖能抑制人成纤维细胞增殖,使处于静止期细胞数量增多,这可能是其预防手术后组织粘连的机制之一。

INHIBITIVE EFFECTS OF CHITOSAN ON PROLIFERATION OF FIBROBLASTS IN VITRO

OBJECTIVE: To investigate the effects of chitosan on the cell cycle of the human fibroblasts and on the Ki-67 antigen expression in vitro and to investigate the mechanism of chitosan preventing the postoperative tissue adhesion. METHODS: The cultured fibroblasts were treated for 48 hours with 0, 0.01, 0.1, 1.0, 10.0 mg/ml of chitosan, respectively; then, the cell cycle of the fibroblasts was measured by the flow cytometry. The cultured fibroblasts were treated for 24 hours with the chitiosan at the above concentrations; then, the Ki-67 antigen in the cell nucleus was detected with the immunohistochemical staining to observe its expression. RESULTS: The growth of the fibroblasts was obviously suppressed by chitosan, especially in the cell morphology. When the concentrations of chitosan were 1.0 mg/ ml and 10.0 mg/ml, the percentages of the fibroblasts in the proliferation stage were 32.3%+/- 5.2% and 14.7% +/- 2.9%, respectively, which were significantly smaller than the percentage of the fibroblasts when the concentration of chitosan was 0 mg/ml (the control group) (41.9% +/-5.8%, P<005). When the concentrations were 0. 01 mg/ml and 0. 1 mg/ml, the percentages of the fibroblasts in the proliferation stage were 39.0%+/-6.0% and 35.5%+/- 3.4%, respectively, which were smaller than that of the control, but not significantly different from that of the control (P> 0.05). When the concentrations of chitosan were 0.1 mg/ml, 1.0 mg/ml and 10.0 mg/ml, the percentages of the fibroblasts that had the positive Ki-67 antigen were 37.3% +/- 3.4%, 30.5% +/- 6.2% and 17.8% +/- 3.0%, respectively, which were significantly smaller than that of the control (57.6% +/-8.9%, P<0.05). When the concentration was 0. 01 mg/ml, the percentage of the fibroblasts that had the positive Ki-67 antigen was 54.1% +/- 8.0%, which was smaller than that of the control, but not significantly different from that of the control (P>0.05). CONCLUSION: Chitosan can inhibit the proliferation of the fibroblasts and increase the percentage of the fibroblasts in the quiescent stage, which can be considered as one of the mechanisms that chitosan can prevent the postoperative tissue adhesion.