氯通道参与氧化应激诱导晶状体上皮细胞容积变化的相关机制研究

发布时间：2018-08-03 10:39

【摘要】：目的：以过氧化氢损伤HLE B-3细胞为氧化应激损伤的模型，观察氯通道阻断剂DIDS和NPPB对氧化应激诱导的晶状体上皮细胞HLE-B3损伤的影响,并通过细胞容积调节的角度初步探讨氯通道阻断剂对氧化应激下HLE B-3保护的作用途径及其相关的分子机制。材料和方法： 1. Image-Pro Plus6.0图像分析软件控制下实现时滞拍摄固定视野内活细胞图像分析HLE B-3在低渗刺激下细胞容积的改变，并通过细胞外溶液氯离子置换和氯通道阻断剂干预的方式阐明氯通道在RVD中所起到的作用。 2. HLE-B3氧化应激损伤模型的建立及氯通道阻断剂保护作用的观察，外源性500μmol/L过氧化氢（H2O2）作用于HLE B-3细胞，细胞外溶液氯离子置换及氯通道阻断剂（NPPB、DIDS）梯度浓度作用于氧化应激下HLE B-3细胞，通过光镜和吖啶橙/溴化乙锭（AO/EB）荧光染色观察细胞形态学及和核形态改变、MTT法检测细胞生存力、流式细胞术检测细胞凋亡率。 3.观察氧化应激下HLE B-3细胞凋亡及凋亡性细胞回缩的变化和氯通道阻断剂（NPPB、DIDS）干预的影响。在500μmol/L H2O2作用于HLE B-3细胞的6h内，通过MTT法检测细胞生存力，Caspase-3活性检测及Western Blot法检测Caspase-3表达水平、DNA琼脂糖电泳分析、和Image-Pro Plus6.0图像分析软件控制下实现时滞拍摄固定视野内活细胞图像，分析细胞凋亡、Caspase-3表达变化及细胞容积的动态变化。结果： 1.在低渗细胞外灌流液作用下，HLE B-3细胞的容积胀大且呈渗透压相关性，同时调节性容积回缩（RVD）能力增强。通过硝酸根离子取代灌流液中的氯离子方式耗竭细胞内氯离子，这时观察到RVD消失，而用氯通道阻断剂也可以得到相似结果。 2.采用不同浓度的氯通道阻断剂NPPB或DIDS干预下，细胞存活率呈药物剂量依赖性升高，其中100μmol/L，200μmol/L这两个浓度NPPB和DIDS干预组细胞存活率与H2O2处理组相比较，差异存在显著性（n＝5，P0.01）。氯通道阻断剂NPPB(100mol/L)及DIDS(100mol/L)皆可显著抑制H2O2诱导的HLE-B3细胞凋亡及细胞容积减少，氧化应激诱导细胞6h后，测得细胞凋亡率为（72.06士2.73）%，加用NPPB及DIDS后，，凋亡率则将为（20.78士2.62）%和（15.83士2.57）%。 3.氧化应激诱导的细胞凋亡早期即可观察到细胞容积减小。在连续观察的6h中，细胞容积逐渐减小，6h末细胞容积减少(26.98士2.35)%。氯通道阻断剂NPPB(100moL/L)及DIDS(100moL/L)显著抑制氧化应激诱导的凋亡相关性容积减小，分别为(6.81士0.57)%和(7.03士0.67)％。结论： 1. Cl是HLE B-3细胞调节性容积回缩（RVD）的关键性离子，Cl分泌是实现RVD的关键因素，氯通道阻断剂阻断氯通道，几乎完全或大部分阻断RVD，表明氯通道开放是HLE B-3细胞分泌Cl，实现RVD的关键途径。 2.氯通道阻断剂NPPB和DIDS对氧化应激诱导的HLE-B3细胞凋亡具有明显的保护作用，其机制可能与抑制细胞凋亡过程中的异常的容积回缩有关。 3.氯通道介导的凋亡相关性细胞容积回缩发生是一个早于Caspase-3活性增强及DNA断裂的细胞凋亡事件。氯通道参与H2O2诱导的HLE B-3细胞凋亡，机制与其介导凋亡性容积回缩有关。
[Abstract]:Objective:
The effects of chlorine channel blocker DIDS and NPPB on the HLE-B3 damage of lens epithelial cells induced by oxidative stress were observed with the damage of hydrogen peroxide damaged HLE B-3 cells as oxidative stress. The pathway of the action of chlorine channel blocker to HLE B-3 protection under oxidative stress and the related points were preliminarily discussed. Submechanism.
Materials and methods:
Under the control of 1. Image-Pro Plus6.0 image analysis software, the changes in the cell volume of HLE B-3 under the low permeability stimulation were realized by the live cell image analysis in the fixed field of vision with time delay shooting, and the role of the chloride channel in RVD was clarified by the way of chlorine ion replacement and the intervention of chlorine channel blockers.
2. HLE-B3 oxidative stress damage model and the observation of the protective effect of chloride channel blocker, exogenous 500 mol/L hydrogen peroxide (H2O2) acts on HLE B-3 cells, chlorine ion replacement and chloride channel blocker (NPPB, DIDS) gradient concentration on HLE B-3 cells under oxidative stress, through light microscopy and acridine orange / ethidium bromide (AO/E). B) Cell morphology and nuclear morphology were observed by fluorescence staining, cell viability was detected by MTT assay, and apoptosis rate was detected by flow cytometry.
3. the changes of apoptosis and apoptotic cell retracting of HLE B-3 cells and the effect of NPPB (DIDS) were observed under oxidative stress. Cell viability was detected by MTT method in 6h of HLE B-3 cells in 500 mu mol/L H2O2, and Caspase-3 activity detection and Western assay were used to detect the expression level. Agarose electrophoresis analysis was used. Under the control of the Image-Pro Plus6.0 image analysis software, the living cell image in the fixed field of vision was photographed, and the apoptosis, the change of Caspase-3 expression and the dynamic change of cell volume were analyzed.
Result:
1. under the action of hypotonic external perfusion fluid, the volume of HLE B-3 cells is swollen and osmotic pressure is related, and the capacity of regulatory volume retracting (RVD) is enhanced. The chloride ion in the cells is depleted by the substitution of nitrate ions in the perfusion solution, then the RVD disappears, and the chlorine channel blocker can also be used to obtain similar results.
2. under the intervention of different concentration of chloride channel blockers NPPB or DIDS, the cell survival rate was increased in dose dependence, including 100 mol/L and 200 mol/L, the two concentrations of NPPB and DIDS intervention group were compared with H2O2 treatment group, the difference was significant (n = 5, P0.01). Chlorine channel blocker NPPB (100mol/L) and DIDS ( The apoptosis of HLE-B3 cells induced by H2O2 and the decrease of cell volume were significantly inhibited. The apoptosis rate was (72.06 2.73)% after oxidative stress induced 6h, and the rate of apoptosis was (20.78 and 2.62)% and (15.83 SW 2.57) after NPPB and DIDS were added.
Cell volume decreased at the early stage of apoptosis induced by 3. oxidative stress. Cell volume decreased gradually and the volume of terminal 6h cells decreased (26.98 2.35) in the continuous observation of 6h. Chloride channel blocker NPPB (100moL/L) and DIDS (100moL/L) significantly inhibited the apoptosis related volume of oxidative stress induced by (6.81 and 0.57)%, respectively. (7.03 and 0.67).
Conclusion:
1. Cl is the key ion of regulatory volume retracting (RVD) in HLE B-3 cells. Cl secretion is the key factor for the realization of RVD. Chlorine channel blockers block the chloride channel, almost completely or most of the blocking of RVD, indicating that the opening of the chloride channel is the key way for HLE B-3 cells to secrete Cl and realize RVD.
2. chloride channel blockers, NPPB and DIDS, have obvious protective effects on oxidative stress induced HLE-B3 cell apoptosis, and the mechanism may be related to the abnormal volume retraction in the process of inhibiting apoptosis.
The apoptosis related cell volume retraction mediated by 3. chloride channel is a cell apoptosis event earlier than Caspase-3 activity enhancement and DNA fracture. The chloride channel is involved in the apoptosis of HLE B-3 cells induced by H2O2, which is related to the apoptosis induced volume retraction.
【学位授予单位】：福建医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R776

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