發(fā)布時間：2018-10-20 06:45

【摘要】：背景與目的：NRAGE (neurotrophin receptor p75-interacting MAGE homolog)基因是黑色素瘤相關(guān)抗原MAGE家族成員之一,研究發(fā)現(xiàn)NRAGE參與了細胞凋亡、細胞周期調(diào)控及腫瘤的發(fā)生過程,第一部分的預(yù)實驗發(fā)現(xiàn)在人宮頸癌HELA細胞中NRAGE蛋白在細胞核中有較高的分布比例,但近來針對NRAGE蛋白研究都是以NRAGE作為一個胞質(zhì)蛋白為基礎(chǔ)的,所以課題設(shè)計實驗探討以下問題：1.NRAGE蛋白在正常細胞和癌癥細胞差異性定位分析；2.NRAGE蛋白細胞核定位機制的分析；3.核定位的NRAGE蛋白功能分析。 第二部分的預(yù)實驗發(fā)現(xiàn)NRAGE蛋白與溶酶體相關(guān)膜蛋白LAMP2共定位,根據(jù)實驗推測NRAGE蛋白可能參與了溶酶體的某些功能,所以本課題設(shè)計實驗分析：1.NRAGE蛋白是否參與細胞的自噬過程；2.NRAGE蛋白參與的自噬與衰老、炎癥相關(guān)性。 方法：采用免疫熒光的方法檢測NRAGE蛋白在一些正常細胞和癌癥細胞的定位；構(gòu)建亞克隆分析NRAGE蛋白細胞核定位機制；雙氧水刺激實驗分析核定位的NRAGE蛋白功能。 采用D-HANKS液饑餓培養(yǎng)、m IFN-r刺激培養(yǎng)、chloroquine刺激培養(yǎng)檢測LC3B蛋白來分析NRAGE蛋白是否參與自噬；β-半乳糖苷酶衰老檢測NRAGE基因野生型和敲除型小鼠成纖維細胞的衰老；Q-PCR檢測LPS刺激培養(yǎng)NRAGE基因野生型和敲除型小鼠腹腔巨噬細胞的炎癥因子的表達差異。 結(jié)果：NRAGE蛋白在正常細胞HEK-293細胞、C2C12細胞、HSF細胞、MSC細胞主要定位于細胞質(zhì),而在癌癥細胞HELA細胞、HepG2細胞、MDA-MB-231細胞、MCF-7細胞的NRAGE蛋白細胞核內(nèi)有較高比例的分布；構(gòu)建的NRAGE (920aa、834aa、324aa亞型)質(zhì)粒均定位于細胞質(zhì)；NRAGE蛋白在雙氧水濃度梯度刺激下表達增加, NRAGE基因敲除型的小鼠成纖維細胞LC3B本底和刺激條件下表達均比野生型增加；傳代培養(yǎng)至第6代的NRAGE基因敲除型的小鼠成纖維細胞衰老程度較野生型高；NRAGE基因敲除型的小鼠腹腔巨噬細胞對炎性刺激有較高的敏感性。 結(jié)論：NRAGE蛋白在癌癥細胞的核定位可能是由基因的選擇性剪接造成,并且NRAGE的核蛋白亞型可能參與了細胞損傷后修復(fù)的過程,進一步推測NRAGE的核蛋白亞型可能與腫瘤具有相關(guān)性。 NRAGE蛋白參與了溶酶體的自噬的過程,推測可能導(dǎo)致自噬促進衰老的過程,另其參與的自噬的過程可能與炎癥的發(fā)生具有相關(guān)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.
【學(xué)位授予單位】：南京師范大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R329.2

【共引文獻】

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