汉防己甲素对兔角膜基质细胞和上皮细胞抑制作用的对比研究
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摘要
目的
比较汉防己甲素对离体兔角膜基质细胞和上皮细胞的抑制作用,并与艾氟龙对比,然后初步探讨其对角膜基质细胞和上皮细胞的作用机理,一方面了解汉防己甲素对兔角膜上皮细胞和基质细胞抑制作用是否存在差异,另一方面了解其与艾氟龙的药效作用是否存在差异,以寻求一种能替代皮质类固醇用于防治屈光术后Haze形成的药效强、副作用低的药物。
方法
1、选用原代及传代兔角膜基质细胞,实验组加入含不同质量浓度汉防己甲素(1 ~ 10μg/ml)的培养液,对照组加入等量空白培养液,同时用不同质量浓度艾氟龙(5 ~ 90μg/ml)作比较;选用原代及传代兔角膜上皮细胞,实验组加入含不同质量浓度汉防己甲素(1 ~ 10μg/ml)的培养液,对照组加入等量空白培养液,同时用不同质量浓度艾氟龙(5 ~ 130μg/ml)作比较。分别培养24、48、72 h后,采用MTT比色法检测药物对细胞增殖的抑制作用,计算出抑制率和IC50,并对其进行比较。2、将汉防己甲素作用于基质细胞和上皮细胞48h后,运用流式细胞仪测定细胞周期和细胞凋亡率的变化。3、汉防己甲素作用于基质细胞和上皮细胞48h后,用免疫细胞化学法检测基质细胞和上皮细胞表达P21和E2F1的情况。
结果
1、汉防己甲素在角膜基质细胞各时间段不同浓度组,均能明显抑制基质细胞的增殖,其24、48、72 h的IC50分别为4.14、3.42和2.84μg/ml,并具有明显的时间剂量-效应依赖关系。汉防己甲素在角膜上皮细胞各时间段不同浓度组,均能抑制上皮细胞的增殖,其24、48、72 h的IC50分别为7.38、4.14和3.37μg/ml,也具有明显的时间剂量-效应依赖关系;汉防己甲素作用于基质细胞各时间点IC50均低于上皮细胞。艾氟龙在各时间段不同浓度组,对角膜基质细胞和上皮细胞生长都具有抑制作用,其对角膜基质细胞和上皮细胞各时间点IC50分别为52.81、36.27、33.16μg/ml和69.19、42.01、35.83μg/ml;汉防己甲素作用于基质细胞和上皮细胞各时间点IC50均低于艾氟龙。2、汉防己甲素作用后,流式细胞仪结果显示角膜基质细胞和上皮细胞G0/G1期细胞均增加,并可见到细胞凋亡峰和凋亡率的升高。3、免疫细胞化学结果显示,角膜基质细胞和上皮细胞对照组和实验组均有P21蛋白和E2F1蛋白的表达,但两种细胞实验组中P21蛋白的表达均增强,E2F1蛋白的表达均减弱。
结论
1、汉防己甲素对角膜基质细胞和上皮细胞的增生均有抑制作用,其抑制作用均强于艾氟龙,且汉防己甲素对基质细胞的抑制作用强于对上皮细胞的抑制作用。
2、汉防己甲素可能通过使细胞周期停滞在G0/G1期和诱导细胞凋亡而发挥其对角膜基质细胞和上皮细胞的抗增殖作用。3、汉防己甲素可能是通过调节P21、E2F1使角膜基质细胞和上皮细胞周期停滞在G0/G1期。
Objective To investigate the effect of tetrandrine on inhibition of RCSCs and RCECs and to explore the mechanism. To try hard to search out a strong effect and weak side effect drug which can replace corticosteroid to treat haze after refractive surgery.
Methods 1. The primary cultured and subcultured RCSCs were exposed to different concentrations of tetrandrine(1~10μg/ml) and fluorometholone(5~90μg/ml) in experimental groups and to blank culture media in contrast group. The primary cultured and subcultured RCECs were exposed to different concentrations of tetrandrine(1~10μg/ml) and fluorometholone(5~130μg/ml) in experimental groups and to blank culture media in contrast group. Then RCSCs and RCECs were cultivated for 24 h、48 h、72h. The inhibitive characteristics to the cells’proliferation were measured with methyl thiazolyl tetrazolium (MTT) assay. The antiproliferative rate of cells and the IC50 were calculated and compared. 2. After the effect of tetrandrine on RCSCs and RCECs for 48h, the changes of cells cycles and the apoptosis rate were observed with flow cytometer (FCM). 3. After the effect of tetrandrine on RCSCs and RCECs for 48h, the changes of the expression of P21 and E2F1 were measured by immunocytochemistry.
Results 1. After the treatment of tetrandrine , RCSCs showed the marked inhibition in 24h、48h、72h and showed marked time-dosage -effect dependence. Its IC50 of three time-point were 4.14、3.42 and 2.84μg/ml in RCSCs. After the treatment of tetrandrine , RCECs showed the inhibition in 24h、48h、72h and showed marked time-dosage -effect dependence. Its IC50 of three time-point were 7.38、4.14 and 3.37μg/ml in RCECs. The IC50 of tetrandrine at each time point in RCSCs was lower than that in RCECs. After the treatment of fluorometholone, RCSCs and RCECs showed the inhibition in 24h、48h、72h and showed marked time-dosage -effect dependence. The IC50 of fluorometholone at each time point in RCSCs and RCECs were 52.81、36.27、33.16μg/ml and 69.19、42.01、35.83μg/ml. The IC50 of tetrandrine at each time point in RCSCs and RCECs were lower than that of fluorometholone. 2. After the treatment of tetrandrine, FCM result showed that the percentage of RCSCs and RCECs in G0/G1 phase increased obviously and the apoptosis cusp and rate also increased. 3. Immunocytochemistry showed increased expression of P21 and decreased expression of E2F1 in RCSCs and RCECs after the treatment of tetrandrine.
Conclusion 1. Tetrandrine has inhibitory effect on the proliferation of RCSCs and RCECs. The effect is more dominant in comparison with fluorometholone. The inhibitory effect of tetrandrine on RCSCs is stronger than that on RCECs . 2. Tetrandrine may exert its antiproliferative effect on RCSCs and RCECs by ceasing cell cycle with P21 and E2F1 and by inducing cell apotosis.
比较汉防己甲素对离体兔角膜基质细胞和上皮细胞的抑制作用,并与艾氟龙对比,然后初步探讨其对角膜基质细胞和上皮细胞的作用机理,一方面了解汉防己甲素对兔角膜上皮细胞和基质细胞抑制作用是否存在差异,另一方面了解其与艾氟龙的药效作用是否存在差异,以寻求一种能替代皮质类固醇用于防治屈光术后Haze形成的药效强、副作用低的药物。
方法
1、选用原代及传代兔角膜基质细胞,实验组加入含不同质量浓度汉防己甲素(1 ~ 10μg/ml)的培养液,对照组加入等量空白培养液,同时用不同质量浓度艾氟龙(5 ~ 90μg/ml)作比较;选用原代及传代兔角膜上皮细胞,实验组加入含不同质量浓度汉防己甲素(1 ~ 10μg/ml)的培养液,对照组加入等量空白培养液,同时用不同质量浓度艾氟龙(5 ~ 130μg/ml)作比较。分别培养24、48、72 h后,采用MTT比色法检测药物对细胞增殖的抑制作用,计算出抑制率和IC50,并对其进行比较。2、将汉防己甲素作用于基质细胞和上皮细胞48h后,运用流式细胞仪测定细胞周期和细胞凋亡率的变化。3、汉防己甲素作用于基质细胞和上皮细胞48h后,用免疫细胞化学法检测基质细胞和上皮细胞表达P21和E2F1的情况。
结果
1、汉防己甲素在角膜基质细胞各时间段不同浓度组,均能明显抑制基质细胞的增殖,其24、48、72 h的IC50分别为4.14、3.42和2.84μg/ml,并具有明显的时间剂量-效应依赖关系。汉防己甲素在角膜上皮细胞各时间段不同浓度组,均能抑制上皮细胞的增殖,其24、48、72 h的IC50分别为7.38、4.14和3.37μg/ml,也具有明显的时间剂量-效应依赖关系;汉防己甲素作用于基质细胞各时间点IC50均低于上皮细胞。艾氟龙在各时间段不同浓度组,对角膜基质细胞和上皮细胞生长都具有抑制作用,其对角膜基质细胞和上皮细胞各时间点IC50分别为52.81、36.27、33.16μg/ml和69.19、42.01、35.83μg/ml;汉防己甲素作用于基质细胞和上皮细胞各时间点IC50均低于艾氟龙。2、汉防己甲素作用后,流式细胞仪结果显示角膜基质细胞和上皮细胞G0/G1期细胞均增加,并可见到细胞凋亡峰和凋亡率的升高。3、免疫细胞化学结果显示,角膜基质细胞和上皮细胞对照组和实验组均有P21蛋白和E2F1蛋白的表达,但两种细胞实验组中P21蛋白的表达均增强,E2F1蛋白的表达均减弱。
结论
1、汉防己甲素对角膜基质细胞和上皮细胞的增生均有抑制作用,其抑制作用均强于艾氟龙,且汉防己甲素对基质细胞的抑制作用强于对上皮细胞的抑制作用。
2、汉防己甲素可能通过使细胞周期停滞在G0/G1期和诱导细胞凋亡而发挥其对角膜基质细胞和上皮细胞的抗增殖作用。3、汉防己甲素可能是通过调节P21、E2F1使角膜基质细胞和上皮细胞周期停滞在G0/G1期。
Objective To investigate the effect of tetrandrine on inhibition of RCSCs and RCECs and to explore the mechanism. To try hard to search out a strong effect and weak side effect drug which can replace corticosteroid to treat haze after refractive surgery.
Methods 1. The primary cultured and subcultured RCSCs were exposed to different concentrations of tetrandrine(1~10μg/ml) and fluorometholone(5~90μg/ml) in experimental groups and to blank culture media in contrast group. The primary cultured and subcultured RCECs were exposed to different concentrations of tetrandrine(1~10μg/ml) and fluorometholone(5~130μg/ml) in experimental groups and to blank culture media in contrast group. Then RCSCs and RCECs were cultivated for 24 h、48 h、72h. The inhibitive characteristics to the cells’proliferation were measured with methyl thiazolyl tetrazolium (MTT) assay. The antiproliferative rate of cells and the IC50 were calculated and compared. 2. After the effect of tetrandrine on RCSCs and RCECs for 48h, the changes of cells cycles and the apoptosis rate were observed with flow cytometer (FCM). 3. After the effect of tetrandrine on RCSCs and RCECs for 48h, the changes of the expression of P21 and E2F1 were measured by immunocytochemistry.
Results 1. After the treatment of tetrandrine , RCSCs showed the marked inhibition in 24h、48h、72h and showed marked time-dosage -effect dependence. Its IC50 of three time-point were 4.14、3.42 and 2.84μg/ml in RCSCs. After the treatment of tetrandrine , RCECs showed the inhibition in 24h、48h、72h and showed marked time-dosage -effect dependence. Its IC50 of three time-point were 7.38、4.14 and 3.37μg/ml in RCECs. The IC50 of tetrandrine at each time point in RCSCs was lower than that in RCECs. After the treatment of fluorometholone, RCSCs and RCECs showed the inhibition in 24h、48h、72h and showed marked time-dosage -effect dependence. The IC50 of fluorometholone at each time point in RCSCs and RCECs were 52.81、36.27、33.16μg/ml and 69.19、42.01、35.83μg/ml. The IC50 of tetrandrine at each time point in RCSCs and RCECs were lower than that of fluorometholone. 2. After the treatment of tetrandrine, FCM result showed that the percentage of RCSCs and RCECs in G0/G1 phase increased obviously and the apoptosis cusp and rate also increased. 3. Immunocytochemistry showed increased expression of P21 and decreased expression of E2F1 in RCSCs and RCECs after the treatment of tetrandrine.
Conclusion 1. Tetrandrine has inhibitory effect on the proliferation of RCSCs and RCECs. The effect is more dominant in comparison with fluorometholone. The inhibitory effect of tetrandrine on RCSCs is stronger than that on RCECs . 2. Tetrandrine may exert its antiproliferative effect on RCSCs and RCECs by ceasing cell cycle with P21 and E2F1 and by inducing cell apotosis.
引文
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