IRE1分子通路在内质网预应激条件下对肝细胞缺氧损伤的保护机制

发布时间：2018-05-04 19:35

本文选题：IRE1分子 + 人正常肝细胞　；参考：《第四军医大学》2012年硕士论文

【摘要】：内质网（ER）保持蛋白质内环境相对稳定的这一功能的失衡，是由多种因素引起的。这其中就包含了缺氧和氧化应激。这一功能的失衡就会导致蛋白质折叠能力和蛋白质负载能力之间的失衡。这些因素共同引发了内质网应激（ER stress）以及未折叠蛋白反应（UPR）。未折叠蛋白反应（UPR）最初是作为细胞的一种适应性反应，但是在内质网应激长期存在的情况下，也能诱导细胞凋亡。衣霉素(TM)是一种常用的体外内质网应激的诱导试剂，在一定的浓度和作用时间下，能够诱导细胞产生内质网预应激的反应而对抗缺氧对细胞的损伤作用。通过筛选未折叠蛋白反应的三条分子通路，我们重点研究IRE1通路中的下游蛋白RACK1分子，在内质网预应激的情况下探讨其蛋白和RNA水平表达是否上调。再沉默其表达，通过Western-blot和RT-PCR检测以及对细胞凋亡率的检测和统计学分析，证明RACK1在内质网预应激的条件下，通过IRE1通路，作为一种保护性蛋白存在于肝细胞当中。实验1．将正常人肝脏细胞HL-7702分成5组，分别为A，B，C，D，E组；A组为正常对照组，其余四组正常培养48小时后换液（分别含衣霉素浓度为0.05，0.10,0.20,0.40μg/ml的培养基）培养48小时后换正常培养基，即刻转入缺氧培养箱分别培养2，4，8，16小时后再统一转入正常培养箱培养6小时后观察细胞形态并收集细胞测凋亡值。结论：根据普通光学显微镜和电子显微镜观察结果和流式细胞检测结果显示D、E两组细胞全部死亡；B组细胞与正常对照无差异；C组细胞凋亡率为20%。将衣霉素浓度0.1μg/ml，缺氧培养4小时再复氧培养6小时作为正常肝细胞缺氧模型的标准时限。实验2.根据实验1的模型，将细胞分为4组：正常对照组（control组）；内质网预应激组（ERS组）；缺氧损伤组（H/R组）；内质网预应激+缺氧损伤组（ERS+H/R组）。收集各组细胞提取蛋白质，通过Western-blot检测内质网应激特异性分子伴侣GRP78和UPR三条通路的蛋白ATF6，PERK以及RACK1的表达。结论：在内质网预应激的条件下，GRP78表达均上调，，证明衣霉素成功诱导了肝细胞内质网预应激的表达。UPR三条通路的蛋白分子ATF6，PERK以及RACK1的表达在内质网预应激的情况下有着不同程度的提高。均比正常对照和单纯缺氧损伤组的表达要高。有统计学意义。通过GRP78这一标志性蛋白的提升我们肯定衣霉素对肝细胞内质网预应激的成功诱导，UPR三条通路的蛋白的表达也不同程度提高，本课题重点研究IRE1下游分子RACK1对肝细胞缺氧的保护机制。实验3．根据实验1的模型，再将培养的人肝细胞分为5组：A组：正常对照；B组：内质网应激+缺氧复氧损伤组；C组：单纯内质网应激组；D组：内质网应激+空载转染组；E组：内质网应激+siRNA转染组。收集各组细胞，以流式细胞仪检测细胞凋亡，Western-bloting及RT-PCR检测内质网应激特异蛋白RACK1表达水平，并通过透射电镜观察各组细胞超微结构改变。结论： E组细胞凋亡率明显升高；B C D组中RACK1的RNA和蛋白质均高表达而E组细胞RNA和蛋白质表达均降低。内质网应激预处理对肝细胞缺氧损伤具有明显的保护作用，沉默RACK1在细胞中的表达将大大提高肝细胞缺氧损伤后的凋亡值，内质网应激特异性蛋白RACK1可能在肝细胞缺氧损伤中作为一种关键性的保护蛋白出现。
[Abstract]:The imbalance in the relative stability of the endoplasmic reticulum (ER) is caused by a variety of factors, including hypoxia and oxidative stress. The imbalance of the function leads to the imbalance between protein folding ability and protein load capacity. These factors jointly trigger the endoplasmic reticulum stress (ER stress) and Unfolded protein reaction (UPR). Unfolded protein reaction (UPR) is initially an adaptive response to cells, but it can also induce apoptosis in the presence of endoplasmic reticulum stress. TM is a common inducer to induce endoplasmic reticulum stress in vitro and can induce cell production at a certain concentration and time of action. By screening three molecular pathways of unfolded protein reaction, we focused on the downstream protein RACK1 molecules in the IRE1 pathway by screening the three molecular pathways of the unfolded protein reaction. We studied the up-regulated expression of the protein and the level of the protein and the level of the protein and the level of the RACK1 in the endoplasmic reticulum prestress. Then the expression was reticted and Western-blot was reticted. The detection of RT-PCR and the detection and statistical analysis of the rate of apoptosis show that RACK1 exists in the liver cells as a protective protein under the prestress of endoplasmic reticulum through the IRE1 pathway.
In experiment 1., normal human liver cell HL-7702 was divided into 5 groups, which were A, B, C, D, E group, and the A group was the normal control group. The other four groups were cultured for 48 hours after normal culture. After 48 hours, the culture medium was changed to the normal medium for 48 hours, and then transferred to the hypoxia incubator and then cultured for 2,4,8,16 hours respectively. The cells were then transferred to normal incubator for 6 hours to observe cell morphology and collect apoptotic values.
Conclusion:
According to the results of common optical and electron microscope observation and flow cytometry, all the cells in D, E two were dead, and there was no difference between the group B and the normal control. The apoptosis rate of the group C was 20%., the concentration of ycomycin was 0.1 mu, and the oxygen culture of the hypoxia culture for 4 hours was used as the standard of the normal liver cell hypoxia model. Limit.
Experiment 2. divided the cells into 4 groups: normal control group (group control), endoplasmic reticulum prestress group (group ERS), hypoxia injury group (group H/R) and endoplasmic reticulum prestress + hypoxia injury group (group ERS+H/R). The protein was collected from each group, and GRP78 and UPR three in endoplasmic reticulum stress were detected by Western-blot, and three UPR were detected by Western-blot. The expression of protein ATF6, PERK and RACK1 of the pathway.
Conclusion:
Under the prestress condition of endoplasmic reticulum, the expression of GRP78 was up-regulated. It was proved that the expression of ATF6, PERK and RACK1 in the.UPR three pathway of hepatocyte endoplasmic reticulum prestress was improved in varying degrees in the condition of endoplasmic reticulum prestress, which were higher than those of normal control and simple hypoxia injury groups. It is statistically significant. Through the promotion of the GRP78 marker protein, we affirm the successful induction of prestress on the endoplasmic reticulum of liver cells and the expression of protein in the UPR three pathway in different degrees. This topic focuses on the protection mechanism of RACK1 on the hypoxia of hepatocytes in the lower IRE1 molecule RACK1.
Experiment 3. according to the model of Experiment 1, the cultured human hepatocytes were divided into 5 groups: A group: normal control; group B: endoplasmic reticulum stress + hypoxia reoxygenation injury group; group C: simple endoplasmic reticulum stress group; D group: endoplasmic reticulum stress + transfection group; E group: endoplasmic reticulum stress +siRNA transfection group. Collection of cells in each group by flow cytometry Apoptosis, Western-bloting and RT-PCR were used to detect the expression level of RACK1 in the endoplasmic reticulum stress specific protein, and the ultrastructural changes in each group were observed by transmission electron microscope.
Conclusion:
The apoptosis rate of E group was significantly higher, the RNA and protein of RACK1 in the group of B C D were highly expressed, while the expression of RNA and protein in the E group decreased. The endoplasmic reticulum stress preconditioning had obvious protective effect on the hypoxia injury of liver cells. The expression of silent RACK1 in the cells would greatly increase the apoptosis value of the liver cells after hypoxia injury and endoplasmic reticulum stress. Specific protein RACK1 may be a key protective protein in hypoxic injury of hepatocytes.

【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R363

【共引文献】