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中华眼科医学杂志(电子版) ›› 2019, Vol. 09 ›› Issue (06) : 348 -353. doi: 10.3877/cma.j.issn.2095-2007.2019.06.005

论著

二项酶诱导剂对高糖环境下大鼠Müller细胞凋亡的影响
吴晓强1, 何薇2, 黄棋3, 吕红彬4,†()   
  1. 1. 643020　四川省自贡市第三人民医院眼科
    2. 611130　四川省成都市温江区人民医院眼科
    3. 610071　四川省成都市第一人民医院眼科
    4. 646000　泸州，西南医科大学附属医院眼科
  • 收稿日期:2019-03-24 出版日期:2019-12-28
  • 通信作者: 吕红彬
  • 基金资助:
    四川省科技支撑计划项目(2015SZ0086)

Effects of a two-enzyme inducer on apoptosis in rat Müller cells in the high glucose environment

Xiaoqiang Wu1, Wei He2, Qi Huang3, Hongbin Lyn4,†()   

  1. 1. Department of Ophthalmology, Zigong Third People′s Hospital, Zigong 643020, China
    2. Department of Ophthalmology, Chengdu Wenjiang District People′s Hospital, Chengdu 611130, China
    3. Department of Ophthalmology, Chengdu First People′s Hospital, Chengdu 610071, China
    4. Department of Ophthalmology, Affiliated Hospital of Southwest Medical University, Luzhou 646000, China
  • Received:2019-03-24 Published:2019-12-28
  • Corresponding author: Hongbin Lyn
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引用本文：

吴晓强, 何薇, 黄棋, 吕红彬. 二项酶诱导剂对高糖环境下大鼠Müller细胞凋亡的影响[J]. 中华眼科医学杂志(电子版), 2019, 09(06): 348-353.

Xiaoqiang Wu, Wei He, Qi Huang, Hongbin Lyn. Effects of a two-enzyme inducer on apoptosis in rat Müller cells in the high glucose environment[J]. Chinese Journal of Ophthalmologic Medicine(Electronic Edition), 2019, 09(06): 348-353.

目的

探讨二项酶诱导剂5，6-二氢环戊烯-1，2-二硫杂环戊烯-3-硫酮(CPDT)对高糖环境下大鼠Müller细胞凋亡的影响及其与凋亡相关基因B淋巴细胞瘤-2(Bcl-2)和B淋巴细胞瘤-2相关X蛋白(Bax)通路的关系。

方法

体外培养SD大鼠的Müller细胞，采用数字表法随机分为对照组、高糖组和CPDT干预组。对照组给予25 mM DMEM完全培养基培养；高糖组给予65 mM DMEM完全培养基培养；CPDT干预组给予65 mM DMEM完全培养基和60 μM CPDT培养，均培养72 h。采用双染法流式细胞仪检测各组细胞凋亡和细胞周期的情况；采用蛋白免疫印迹杂交技术检测各组细胞Bcl-2和Bax蛋白的表达情况。不同组的组间比较采用单因素方差分析，当差异有统计学意义时，进一步采用LSD法两两比较。

结果

流式细胞仪检测的结果显示，高糖组的细胞凋亡率为(17.6±3.16)%；对照组的细胞凋亡率为(8.25±0.26)%；CPDT干预组的细胞凋亡率为(13.5±2.25)%，且三组细胞凋亡率间，差异有统计学意义(F＝13.48，P<0.05)。蛋白免疫印迹杂交技术检测的结果显示，高糖组Bcl-2蛋白表达量较对照组降低，Bax蛋白表达量较对照组升高，且差异有统计学意义(t＝150.38，234.46；P<0.05)。CPDT干预组Bcl-2蛋白表达量较高糖组升高，Bax蛋白表达量较高糖组降低，且差异有统计学意义(t＝108.03，36.33；P<0.05)。

结论

高糖可通过上调Bax，下调Bcl-2表达，降低Bcl-2/Bax的比值，促进细胞凋亡。CPDT可增加高糖影响下的大鼠Müller细胞活性、Bcl-2的表达及Bcl-2/Bax的比值，抑制Bax的表达和高糖引起的大鼠Müller细胞的凋亡，从而起到保护高糖环境中Müller细胞受损伤的作用。

关键词: 糖尿病性视网膜病变, Müller细胞, CPDT, Bcl-2/Bax, 凋亡
Objective

This aim of this study was to investigate the effect of 5, 6-dihydrocyclopentene-1, 2-dithiol-3-thione, a two-enzyme inducer(CPDT)on the apoptosis of rat Müller cells in the high glucose environment and whether it exerts its role through the apoptosis-related genes Bcl-2/Bax pathways, in order to explore the prospect of CPDT in the prevention and treatment of diabetic retinopathy.

Methods

SD rat Müller cells were cultured in vitro and randomly divided into 3 groups: control group (25 mM DMEM), high glucose group (65 mM DMEM), CPDT intervention group (65 mM DMEM + 60 uM CPDT). These cells were cultured for 72 h. Apoptosis and cell cycle changes were detected by the flow cytometry with double staining. The expressions of Bcl-2 and Bax proteins in each group were detected by Western blotting. Data were collected and one-way ANOVA was used to performstatistically analyzed between groups.

Results

The results of double staining flow cytometry showed that the apoptotic rate of high glucose group was (17.6±3.16%), that of control group was (8.25±0.26%), and that of CPDT intervention group was (13.5%±2.25%). There was statistically significant among them (F=13.48, P<0.05). The results of Western blotting hybridization detection showed that compared with the control group, the expression of Bcl-2 protein in the high glucose group was significantly decreased and the expression of Bax protein was significantly increased (t=150.38, 234.46; P<0.05). Compared with the high glucose group, the expression of Bcl-2 protein in the CPDT intervention group increased, Bax protein expression decreased (t=108.03, 36.33; P<0.05).

Conclusion

High glucose could up-regulate Bax, down-regulate Bcl-2 expression, decrease Bcl-2/Bax ratio, and promote apoptosis. CPDT could increase the activity of rat Müller cells, Bcl-2 expression and Bcl-2/Bax ratio, inhibit the expression of Bax from the pressure of high glucose, thereby inhibiting the apoptosis of rat Müller cells induced by high glucose, and had a protective effect on Müller cells in a high glucose environment.

Key words: Diabetic retinopathy, Müller cells, CPDT, Bcl-2/Bax, Apoptosis
图2 荧光显微镜下各组Müller细胞标志物波形蛋白染色图　图2A示细胞核的蓝色荧光；图2B示细胞质中的波形蛋白红色荧光；图2C示细胞核及胞质中的波形蛋白荧光组合(免疫组织荧光法染色，×40)
图4 各组细胞周期的流式图　图4A示对照组，图4B示高糖组，图4C示5，6-二氢环戊烯-1，2-二硫杂环戊烯-3-硫酮干预组；各组细胞周期未见明显改变
图5 各组细胞中B淋巴细胞瘤-2、B淋巴细胞瘤-2相关X蛋白的相对表达量　1~3分别示对照组、高糖组和5，6-二氢环戊烯-1，2-二硫杂环戊烯-3-硫酮干预组
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