NRAGE蛋白的亚细胞定位及其功能研究进展

发布时间：2018-10-20 06:45

【摘要】：背景与目的：NRAGE (neurotrophin receptor p75-interacting MAGE homolog)基因是黑色素瘤相关抗原MAGE家族成员之一,研究发现NRAGE参与了细胞凋亡、细胞周期调控及肿瘤的发生过程,第一部分的预实验发现在人宫颈癌HELA细胞中NRAGE蛋白在细胞核中有较高的分布比例,但近来针对NRAGE蛋白研究都是以NRAGE作为一个胞质蛋白为基础的,所以课题设计实验探讨以下问题：1.NRAGE蛋白在正常细胞和癌症细胞差异性定位分析；2.NRAGE蛋白细胞核定位机制的分析；3.核定位的NRAGE蛋白功能分析。第二部分的预实验发现NRAGE蛋白与溶酶体相关膜蛋白LAMP2共定位,根据实验推测NRAGE蛋白可能参与了溶酶体的某些功能,所以本课题设计实验分析：1.NRAGE蛋白是否参与细胞的自噬过程；2.NRAGE蛋白参与的自噬与衰老、炎症相关性。方法：采用免疫荧光的方法检测NRAGE蛋白在一些正常细胞和癌症细胞的定位；构建亚克隆分析NRAGE蛋白细胞核定位机制；双氧水刺激实验分析核定位的NRAGE蛋白功能。采用D-HANKS液饥饿培养、m IFN-r刺激培养、chloroquine刺激培养检测LC3B蛋白来分析NRAGE蛋白是否参与自噬；β-半乳糖苷酶衰老检测NRAGE基因野生型和敲除型小鼠成纤维细胞的衰老；Q-PCR检测LPS刺激培养NRAGE基因野生型和敲除型小鼠腹腔巨噬细胞的炎症因子的表达差异。结果：NRAGE蛋白在正常细胞HEK-293细胞、C2C12细胞、HSF细胞、MSC细胞主要定位于细胞质,而在癌症细胞HELA细胞、HepG2细胞、MDA-MB-231细胞、MCF-7细胞的NRAGE蛋白细胞核内有较高比例的分布；构建的NRAGE (920aa、834aa、324aa亚型)质粒均定位于细胞质；NRAGE蛋白在双氧水浓度梯度刺激下表达增加, NRAGE基因敲除型的小鼠成纤维细胞LC3B本底和刺激条件下表达均比野生型增加；传代培养至第6代的NRAGE基因敲除型的小鼠成纤维细胞衰老程度较野生型高；NRAGE基因敲除型的小鼠腹腔巨噬细胞对炎性刺激有较高的敏感性。结论：NRAGE蛋白在癌症细胞的核定位可能是由基因的选择性剪接造成,并且NRAGE的核蛋白亚型可能参与了细胞损伤后修复的过程,进一步推测NRAGE的核蛋白亚型可能与肿瘤具有相关性。 NRAGE蛋白参与了溶酶体的自噬的过程,推测可能导致自噬促进衰老的过程,另其参与的自噬的过程可能与炎症的发生具有相关性。
[Abstract]:Background & objective: NRAGE (neurotrophin receptor p75-interacting MAGE homolog) gene is a member of MAGE family of melanoma associated antigens. It has been found that NRAGE is involved in apoptosis, cell cycle regulation and tumorigenesis. In the first part, we found that there was a high distribution of NRAGE protein in the nucleus of human cervical cancer HELA cells, but the recent studies on NRAGE protein were based on NRAGE as a cytoplasmic protein. So we designed the experiment to discuss the following problems: the differential localization analysis of 1.NRAGE protein in normal cells and cancer cells, the analysis of nuclear localization mechanism of 2.NRAGE protein, and the analysis of nuclear localization mechanism of 2.NRAGE protein. 3. Nuclear localization of NRAGE protein functional analysis. The second part of the pre-experiment found that NRAGE protein and lysosomal associated membrane protein LAMP2 co-localization, according to the experiment speculated that NRAGE protein may participate in some functions of lysosome, so this paper designed experimental analysis: 1.NRAGE protein involved in the process of autophagy; 2.NRAGE protein is involved in autophagy and associated with aging and inflammation. Methods: immunofluorescence was used to detect the localization of NRAGE protein in some normal cells and cancer cells. Subclone was constructed to analyze the nuclear localization mechanism of NRAGE protein. The function of NRAGE protein was analyzed by hydrogen peroxide stimulation experiment. D-HANKS medium starvation culture, m IFN-r stimulation culture, chloroquine stimulation culture were used to detect whether NRAGE protein was involved in autophagy, 尾 -galactosidase senescence was used to detect the senescence of NRAGE gene wild type and knockout type mouse fibroblasts. Q-PCR was used to detect the expression of inflammatory factors in murine peritoneal macrophages stimulated by LPS and cultured in wild type and knockout type of NRAGE gene. Results: NRAGE protein was mainly localized in the cytoplasm of HEK-293 cells, C2C12 cells, HSF cells and MSC cells in normal cells, but in the nucleus of NRAGE protein in HELA cells, HepG2 cells, MDA-MB-231 cells and MCF-7 cells of cancer cells. All of the constructed NRAGE (920aaanhuo 834aaf324aa) plasmids were located in the cytoplasm, the expression of NRAGE protein was increased under the hydrogen peroxide concentration gradient, and the expression of NRAGE gene knockout mouse fibroblasts was higher than that of wild-type fibroblasts under both background and stimulation conditions. The senescence of fibroblasts of NRAGE knockout type was higher than that of wild type, and the peritoneal macrophages of NRAGE knockout type were more sensitive to inflammatory stimulation. Conclusion: the nuclear localization of NRAGE protein in cancer cells may be caused by the selective splicing of genes, and the nuclear protein subtype of NRAGE may be involved in the repair process after cell injury. It is further speculated that the nucleoprotein subtype of NRAGE may be related to tumor. NRAGE protein is involved in the process of lysosomal autophagy, which may lead to autophagy promoting aging. The process of autophagy may be associated with inflammation.
【学位授予单位】：南京师范大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R329.2

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