芦荟多糖对体外培养人表皮细胞增殖的影响

Influence of polysaccharide from Aloe vera on the proliferation of the human epithelial cells cultured in vitro

目的观察芦荟多糖(AP)对体外培养人表皮细胞增殖的影响.方法体外培养人表皮细胞,依据所用DK-SFM培养液中AP含量的不同,将细胞随机分为25、50、100、200和400 mg/LAP组,对照组细胞仅加入等体积的DK-SFM培养液.通过倒置相差显微镜和透射电镜观察细胞形态和超微结构,并计算细胞融合时间.应用噻唑蓝法、氚标记胸腺嘧啶脱氧核苷(3H-TdR)掺入法、细胞计数法和流式细胞技术分别观察细胞存活率、3H-TdR掺入量、生长曲线分布情况及细胞周期,用以反映细胞增殖状况;通过检测乳酸脱氢酶(LDH)漏出率反映细胞损伤程度.结果倒置相差显微镜观察到,各组表皮细胞形态基本相似;透射电镜下观察到,100、400 mg/L AP组细胞增殖活跃,核内以常染色质为主,而对照组和25 mg/L AP组细胞则以异染色质为主.50～400 mg/L AP组细胞融合时间分别为(154±12)、(141±20)、(130±19)、(124±13)h,明显早于对照组(182±8)h(P＜0.01).从生长曲线上可见,100～400 mg/L AP组细胞增殖达峰值的时间较其他组提前1～2 d;25～400 mg/L AP组细胞存活率、3H-TdR掺入量均高于对照组.与对照组比较,25～400 mg/L AP组G0/G1期细胞所占百分比明显减少,G2/M期和S期细胞则明显增加(P＜0.01).200、400 mg/L AP组细胞LDH漏出率低于对照组及25、50 mg/L AP组(P＜0.01).结论高剂量AP对表皮细胞有保护效能,它通过诱导表皮细胞从G0/G1期进入G2/M期和S期促进细胞增殖.

Objective To investigate the influence of polysaecharide from Aloe Vera （AP） on the proliferation of the human epithelial cells cultured in vitro. Methods The human epithelial cells undergo- ing 3 to 4 passages of confluence culture were randomly divided into control and 25, 50, 100, 200 and 400mg/L AP groups according to different dosage of the polysaccharide （AP） added into the culture medi- um. In the control group（C） , equal volume of DK-SFM medium was added to the culturing cells. The conjugation time of epithelial cells, the changes in the cell morphology and ultrastructure were observed under inverted phase contrast microscope and transmission electron microscope, respectively. The cell proliferation was measured by MTT, cell count analysis and [ ^3H-TdR] incorporation. Flow cytometry analysis was employed to detect the cell cycle. The leakage rate of lactate dehydrogenase （LDH） was assayed for the evaluation of the epithelial cell injury. Results There was no significant difference in the morphology of the epithelial ceils among the groups under inverted phase contrast microscope. But under the transmission electron microscope （TEM） , the cells in 100 to 400mg/L AP groups were seen to have proliferated actively,with euchromatin dominant in the nuclei,while heterochromatin was dominant in the cellular nucleus in control and 25mg/L AP groups. The confluence time of epithelial cells in 50,100,200,400 mg/L AP groups （ 154±12, 141±20,130±19,124±13）h preceded noticeably than that in control group （182 ±8） h,（ P 〈0.01）. The cell proliferation in 100, 200, 400 mg/L groups reached the peak on the 5th day after AP treatment, while that in control and other groups was delayed by 1 to 2 days. The survival rate of the ceils in 25 to 400 mg/L AP groups increased dramatically compared with that in control group, with its [^3H ]-TdR incorporation levels significantly increased in a dose dependent manner. The leakage rate of LDH in 200 and 400 mg/ LAP groups was lower than that in control group （ P 〈0.01 ）. The flow cytometric analysis of the cell cycle distribution revealed that the percentage of cell cycle from phase G0/G1 to G2/M and S in 25 to 400mg/L AP groups increased significantly in a dose dependent manner compared with that in control group（ P 〈 0. 01 ） . Conclusion AP might be beneficial to the protection of epithelial cells by promoting cell proliferation.through inducing the progression of epidermal cells from phase G0/G1 into G2/M and S phases.