放射损伤细胞中高尔基体弥散现象及其机制的研究

发布时间：2018-03-19 18:18

本文选题：辐射损伤　切入点：高尔基体　出处：《安徽医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：高尔基体是细胞内膜系统中重要的细胞器,参与了细胞内蛋白质的加工、分泌和囊泡运输等过程。近年发现,DNA损伤可以引起高尔基体的重组,使其片段化并分散到整个细胞之中,并认为这种高尔基体结构的变化可能与受照细胞的存活有关。尽管此研究结果发表在著名的“细胞”杂志上,但相关领域的研究报道到目前为止仍然很少。高尔基体的结构变化是否真正参与了细胞存活反应,或仅仅是细胞放射损伤后亚细胞结构发生的一种伴随反应,本文将以此为切入点,探索放射损伤后高尔基体的弥散现象以及对其机制进行探讨。目的:通过观察辐射对高尔基体形态结构的影响,了解高尔基体弥散的机制以及对弥散效应和生物学意义进行初步探讨。方法:采用免疫荧光技术检测放射损伤细胞中高尔基体弥散现象并对弥散面积进行统计分析,流式细胞术检测细胞周期变化,克隆形成实验分析细胞存活率,Western Blot检测高尔基体蛋GOLPH3及DNA断裂损伤标志物γH2AX的表达,采用RT-PCR检测辐射细胞后miRNA分子miR-378e、miR-486-3p表达变化及其靶蛋白GOLPH3L、MYO18A表达变化。结果:免疫荧光检测结果显示,照射后高尔基体的弥散面积增加,具有一定的剂量效应和时间效应关系;具有激活DNA-PKcs的放射损伤防护剂香兰素衍生物VND3207能够减轻γ射线对高尔基体的损伤,其表现为与未加药组相比,VND3207能抑制不同剂量照射后高尔基体弥散面积的增加,同时促进了受照细胞的存活;细胞周期测定结果显示,4Gyγ射线照射后G2/M期阻滞峰值出现在12h左右;免疫印迹结果显示DNA损伤引起的G2/M期阻滞也在12h后解除;而高尔基体弥散在细胞周期阻滞解除后如照射后24h甚至更长时间仍然存在。RT-PCR结果显示,4Gy、10Gy照射后,人脐静脉内皮细胞(HUVEC)中miR378e、miR 486-3p和GOLPH3L及MYO18A m RNA分子有表达上调的趋势,12h趋势明显;免疫荧光检测结果显示,转染miR 378e、miR 486-3p、si GOLPH3L后的细胞中,高尔基体呈现弥散的趋势;细胞周期结果显示,转染siGOLPH3L、siMYO18A能延长细胞照射后G2/M期的阻滞;同时观察到,转染miR 378e、miR486-3p、siGOLPH3L、siMYO18A后,受照细胞的存活率降低。结论:1.γ射线照射后,细胞高尔基体出现弥散现象,且具有一定的剂量效应和时间效应。2.高尔基体的弥散现象与细胞周期G2/M期阻滞相关,但在辐射损伤中的高尔基弥散与周期阻滞没有因果关系。3.高尔基体的弥散与细胞所受损伤程度相关,辐射防药物VND3207能抑制弥散。4.DNA-PKcs抑制剂NU7441抑制了DNA损伤的修复,也抑制了高尔基体弥散。5.高尔基体蛋白GOLPH3,在照射后表达下调,可被DNA-PKcs敲低抑制下调。6.miRNA分子miR-378e、miR-486-3p在高尔基体弥散中起一定作用。
[Abstract]:Golgi apparatus is an important organelle in the endomembrane system, which is involved in the process of protein processing, secretion and vesicle transport. Recently, it has been found that DNA damage can lead to the recombination of Golgi body. It was fragmented and dispersed throughout the cell, and the changes in Golgi structure were thought to be related to the survival of irradiated cells, although the findings were published in the well-known journal Cell. However, there have been few reports about the changes in Golgi body structure, whether the changes in Golgi apparatus are really involved in cell survival response, or is it only a kind of accompanying reaction of subcellular structure after cell radiation injury. In this paper, we will explore the dispersion of Golgi apparatus after radiation injury and its mechanism. Objective: to observe the effect of radiation on the morphological structure of Golgi apparatus. To understand the mechanism of Golgi body dispersion and to explore the diffusion effect and biological significance. Methods: the dispersion of Golgi apparatus in radiation injured cells was detected by immunofluorescence technique and the dispersion area was analyzed statistically. Cell cycle changes were detected by flow cytometry, cell survival rate was analyzed by clone formation assay and the expression of Golgi body egg GOLPH3 and DNA break damage marker 纬 H 2AX was detected by Western Blot. RT-PCR was used to detect the expression of miR-378esimiR-486-3p and the target protein GOLPH3LnMYO18A after irradiation. Results: the results of immunofluorescence showed that the dispersion area of Golgi was increased after irradiation, and there was a dose-effect and time-effect relationship between the expression of MIR-486-3p and the target protein GOLPH3LnMYO18A. Vanillin derivative VND3207, a radiation-protective agent with activated DNA-PKcs, can attenuate the damage of Golgi body induced by 纬 -ray. VND3207 can inhibit the increase of Golgi dispersion area after different doses of irradiation compared with the control group. The cell cycle analysis showed that the peak of G _ 2 / M phase arrest appeared at about 12h after irradiation with 4Gy 纬 -ray, and the G _ 2 / M phase block induced by DNA damage was also released after 12h by Western blot. The results of RT-PCR showed that the expression of miR378eR486-3p, GOLPH3L and MYO18A m RNA in human umbilical vein endothelial cells (HUVECs) was up-regulated for 12 h after the release of Golgi apparatus cell cycle arrest, such as 24 h or longer after irradiation, and the results of RT-PCR showed that the expression of miR378eR486-3p and MYO18A m RNA in HUVECs was significantly up-regulated at 12 h. The results of immunofluorescence assay showed that after transfection of miR 378em miR486-3pnsiGOLPH3L, Golgi showed a diffusing tendency, cell cycle showed that transfection of siGOLPH3L siMYO18A could prolong the arrest of G _ 2 / M phase after irradiation, at the same time, it was observed that after transfection of miR 378eanmiR486-3psiGOLPH3LsiO18A, the cell cycle showed that transfection of siGOLPH3L siMYO18A could prolong the arrest of G _ 2 / M phase after irradiation. The survival rate of irradiated cells was decreased. Conclusion the Golgi body dispersion appears in the cells after irradiation by 1: 1. 纬-ray, and has a dose effect and a time effect. 2. The dispersion phenomenon of Golgi body is related to the arrest of G 2 / M phase of cell cycle. However, there was no causal relationship between Golgi dispersion and cycle arrest in radiation injury. The dispersion of Golgi apparatus was related to the degree of cell damage. Radiation inhibitor VND3207 could inhibit the dispersion. 4. DNA-PKcs inhibitor NU7441 inhibited the repair of DNA damage. Golgi body protein Golgi body protein GOLPH3 was down-regulated after irradiation and down-regulated by DNA-PKcs knockdown. 6. MiRNA molecule miR-378esimiR-486-3p played a certain role in Golgi dispersion.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R730.55

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