本文選題：干擾素和維甲酸聯(lián)合應用誘導的細胞凋亡相關(guān)基因-19 + 亞細胞定位��； 參考：《吉林大學》2017年博士論文

【摘要】：干擾素和維甲酸聯(lián)合應用誘導的細胞凋亡相關(guān)基因-19(GRIM-19)被認為是一種新類型的抑癌基因,有研究顯示GRIM-19基因敲除后,腫瘤細胞顯示出對IFN-β/RA引發(fā)細胞凋亡的耐受能力。因此,研究GRIM-19在IFN-β/RA引發(fā)細胞凋亡中的作用機制,對基于該分子機制的腫瘤療法的研究和開發(fā)具有重要意義。最初GRIM-19被認為是一種核內(nèi)蛋白,后又被證實定位于線粒體,是線粒體復合物I的重要組成部分,這前后矛盾的研究結(jié)論值得注意。GRIM-19如在線粒體中可能更多地參與能量代謝相關(guān)過程;如在細胞核中則可能通過激活相關(guān)信號轉(zhuǎn)導通路來參與凋亡調(diào)控。因此,應首先明確GRIM-19的亞細胞定位變化。鑒于過往成像技術(shù)的限制導致難以明確GRIM-19的胞內(nèi)定位,本研究應用3D-SIM超高分辨率成像系統(tǒng)對GRIM-19在IFN-β/RA作用前后的亞細胞定位進行了觀察。結(jié)果顯示,正常狀態(tài)下,GRIM-19主要分布于細胞質(zhì)中,與線粒體具有明顯的共定位現(xiàn)象,而在細胞凋亡過程中,GRIM-19的分布向細胞核內(nèi)移動,細胞質(zhì)中的GRIM-19明顯減少。在明確凋亡過程中GRIM-19向細胞核移動后,接下來探討GRIM-19對凋亡相關(guān)信號轉(zhuǎn)導通路的影響。為保證后續(xù)實驗的順利進行,本研究首先成功構(gòu)建了能表達GRIM-19的真核表達質(zhì)粒——p GRIM-19,經(jīng)RT-PCR和Western blotting等方法的檢測,證明p GRIM-19能夠顯著上調(diào)腫瘤細胞中GRIM-19的表達。然后,本研究檢測了GRIM-19的表達上調(diào)對相關(guān)信號通路的激活情況,研究發(fā)現(xiàn),GRIM-19的上調(diào)能夠抑制STAT3信號通路及VEGF、Bcl-2、cyclin D1的表達。綜上,細胞凋亡過程中,GRIM-19的亞細胞定位發(fā)生從線粒體到細胞核的變化,并能夠引起STAT3相關(guān)信號轉(zhuǎn)導通路的抑制作用。上述結(jié)果為GRIM-19作為腫瘤治療的靶點提供了可能,而已有報道顯示GRIM-19能夠抑制多種腫瘤的生長,但尚無其在口腔鱗癌中的作用研究。因此本研究試圖揭示GRIM-19對口腔鱗癌的增殖、凋亡、轉(zhuǎn)移及侵襲的影響。MTT法和克隆形成實驗結(jié)果顯示,GRIM-19的上調(diào)能夠抑制口腔鱗癌細胞的增殖;吖啶橙染色法和Caspase活性法結(jié)果表明GRIM-19的上調(diào)能夠促進口腔鱗癌細胞的增殖;細胞遷移實驗證明GRIM-19的上調(diào)能夠抑制口腔鱗癌的遷移;Transwell侵襲實驗顯示GRIM-19的上調(diào)能夠抑制口腔鱗癌細胞的侵襲,進一步的侵襲機制證明GRIM-19的上調(diào)能夠降低口腔鱗癌細胞中MMP-2和MMP-9的表達,從而抑制細胞侵襲。在上述體外實驗的基礎(chǔ)上,本研究檢測了GRIM-19對小鼠體內(nèi)口腔鱗癌腫瘤生長的影響�？谇击[癌細胞接種于BALB裸鼠皮下,GRIM-19上調(diào)后腫瘤的重量和體積均明顯下降,同時小鼠脾細胞的增殖也受到明顯抑制。為進一步證明GRIM-19對口腔鱗癌腫瘤的抑制,將腫瘤組織取出后檢測其中細胞的凋亡情況,TUNEL法結(jié)果顯示,GRIM-19上調(diào)后裸鼠模型的腫瘤細胞凋亡明顯得到增強。因此,GRIM-19確實具備成為口腔鱗癌治療靶點的潛力。上面已證明GRIM-19有成為口腔鱗癌治療靶點的可能,但以目前的藥物研發(fā)水平,短時間內(nèi)開發(fā)出GRIM-19的激活劑是十分困難的。因此首先尋求GRIM-19與現(xiàn)有抗腫瘤藥物的聯(lián)合治療可能是當前GRIM-19在臨床應用上更可行的策略。而現(xiàn)有口腔鱗癌治療藥物中,順鉑具有較好的治療效果,但其毒性和耐藥性等問題大大地限制了其進一步臨床應用。已有報道稱順鉑與抑癌基因的聯(lián)合治療能夠克服其耐藥性并增強治療效果。因此,本研究試圖探討GRIM-19的表達上調(diào)對順鉑治療口腔鱗癌效果的影響。首先確定了順鉑的合適使用濃度為10μM,而GRIM-19的上調(diào)能夠增強順鉑對口腔鱗癌細胞增殖和細胞遷移的抑制、并能增強順鉑對細胞凋亡的促進。同時,體內(nèi)實驗結(jié)果也表明,相比于單獨注射順鉑,GRIM-19的上調(diào)和順鉑聯(lián)合治療后口腔鱗癌腫瘤的重量和體積均明顯減小,并能進一步抑制小鼠脾細胞的增殖。因此,GRIM-19的表達上調(diào)能夠增強順鉑對口腔鱗癌的治療效果。綜上,在細胞凋亡過程中GRIM-19的亞細胞定位會發(fā)生變化,部分從線粒體向細胞核移動,并能夠抑制核內(nèi)STAT3相關(guān)信號轉(zhuǎn)導通路的激活。同時,經(jīng)驗證GRIM-19具備成為口腔鱗癌治療靶點的可能,其表達上調(diào)能夠增強順鉑對口腔鱗癌的治療效果。本研究揭示了GRIM-19的亞細胞定位變化及其對相關(guān)信號轉(zhuǎn)導通路的影響,進一步闡述了GRIM-19在細胞凋亡中的作用機制。而關(guān)于GRIM-19在口腔鱗癌中治療效果的研究,擴大了GRIM-19的潛在臨床治療范圍,為GRIM-19相關(guān)的藥物開發(fā)提供了初步的臨床前實驗數(shù)據(jù)。
[Abstract]:The combined use of interferon and retinoic acid to induce apoptosis related gene -19 (GRIM-19) is considered as a new type of tumor suppressor gene. Some studies have shown that after GRIM-19 knockout, the tumor cells show the tolerance to apoptosis induced by IFN- beta /RA. Therefore, the mechanism of GRIM-19 in the apoptosis induced by IFN- beta /RA is studied. The research and development of tumor therapy based on this molecular mechanism is of great significance. Initially, GRIM-19 was considered as an intranuclear protein, and then proved to be located in mitochondria, an important component of the mitochondrial complex I. This contradictory conclusion is worth noting that.GRIM-19 may be more involved in the energy generation of Xie Xiang in mitochondria. The process of closing the process, such as in the nucleus, may be involved in the regulation of apoptosis by activating the related signal transduction pathway. Therefore, the changes in the subcellular localization of GRIM-19 should be defined first. In view of the limitation of the past imaging techniques, the intracellular localization of GRIM-19 is difficult to be clearly defined. This study applied the 3D-SIM ultra high resolution imaging system to the effect of GRIM-19 in the IFN- beta /RA The subcellular localization was observed before and after. The results showed that in the normal state, GRIM-19 was mainly distributed in the cytoplasm, and there was a obvious co localization with the mitochondria. In the process of apoptosis, the distribution of GRIM-19 was moved into the nucleus, and the GRIM-19 in the cytoplasm decreased obviously. In the clear apoptosis process, the GRIM-19 moved to the nucleus. Then, the effect of GRIM-19 on the apoptosis related signal transduction pathway is discussed. In order to ensure the smooth progress of the follow-up experiment, this study first successfully constructed a eukaryotic expression plasmid that can express GRIM-19, P GRIM-19, through the detection of RT-PCR and Western blotting, and proved that P GRIM-19 can significantly increase the table of GRIM-19 in tumor cells. Then, this study detected the activation of GRIM-19 expression on the related signaling pathway. The study found that the up-regulation of GRIM-19 inhibited the STAT3 signaling pathway and the expression of VEGF, Bcl-2, cyclin D1. In the process of apoptosis, the subcellular localization of GRIM-19 occurred from mitochondria to nucleus, and could cause STAT3 correlation. The inhibitory effect of signal transduction pathway. These results provide the possibility of GRIM-19 as a target for cancer treatment, and it has been reported that GRIM-19 can inhibit the growth of a variety of tumors, but there is no study on the role of GRIM-19 in oral squamous cell carcinoma. Therefore, this study attempts to reveal the effect of.M on the proliferation, apoptosis, metastasis and invasion of oral squamous cell carcinoma. The results of TT and clone formation showed that the up-regulation of GRIM-19 could inhibit the proliferation of oral squamous cell carcinoma cells. The results of acridine orange staining and Caspase activity showed that up regulation of GRIM-19 could promote the proliferation of oral squamous cell carcinoma cells. Cell migration experiments showed that the up regulation of GRIM-19 could inhibit the migration of oral squamous cell carcinoma; the Transwell invasion experiment showed that the up regulation of GRIM-19 was significant. The up-regulation of GRIM-19 can inhibit the invasion of oral squamous cell carcinoma cells. Further invasion mechanism shows that up regulation of GRIM-19 can reduce the expression of MMP-2 and MMP-9 in oral squamous cell carcinoma cells and inhibit cell invasion. On the basis of the above in vitro experiments, this study detected the effect of GRIM-19 on the growth of oral squamous cell carcinoma in mice. The tumor cells were inoculated subcutaneously in BALB nude mice. The weight and volume of the tumor were significantly decreased after the GRIM-19 up regulation, and the proliferation of splenocytes in mice was also significantly inhibited. It was further proved that the inhibition of GRIM-19 on oral squamous cell carcinoma and the detection of the cell apoptosis after taking out the tumor tissue. The TUNEL method showed that the GRIM-19 up regulation was up. The tumor cell apoptosis in the nude mouse model is obviously enhanced. Therefore, GRIM-19 has the potential to be the target of oral squamous cell cancer treatment. It has been proved that GRIM-19 has the potential to be the target of oral squamous cell cancer treatment, but it is very difficult to develop GRIM-19 activator in a short time at the current level of drug development. Therefore, the first search for GR Combined treatment of IM-19 with existing antitumor drugs may be a more feasible strategy for the current clinical application of GRIM-19. However, cisplatin has a good therapeutic effect in the existing oral squamous cell carcinoma treatment drugs, but its toxicity and drug resistance have greatly limited its further clinical application. Therefore, this study attempts to explore the effect of up regulation of GRIM-19 on the effect of cisplatin in the treatment of oral squamous cell carcinoma. First, the appropriate concentration of cisplatin is 10 u M, and the up-regulation of GRIM-19 can enhance the inhibition of cisplatin to the proliferation and cell migration of oral squamous cell carcinoma, and enhance the cisplatin. At the same time, the experimental results also showed that the weight and volume of GRIM-19 and cisplatin combined with cisplatin were significantly reduced in weight and volume compared with cisplatin alone, and it could further inhibit the proliferation of murine splenocytes. Therefore, the up-regulation of GRIM-19 can enhance the treatment of cisplatin to oral squamous cell carcinoma. To sum up, the subcellular location of GRIM-19 changes in the process of apoptosis, partly from mitochondria to the nucleus and can inhibit the activation of STAT3 related signal transduction pathway in the nucleus. At the same time, it is proved that GRIM-19 has the potential to be a target for the treatment of oral squamous cell carcinoma. Up regulation of cisplatin can enhance cisplatin to oral squamous cell carcinoma This study revealed the changes in the subcellular localization of GRIM-19 and its effect on the related signal transduction pathway, and further elaborated the mechanism of GRIM-19 in cell apoptosis, and the research on the therapeutic effect of GRIM-19 in oral squamous cell carcinoma expanded the potential clinical scope of GRIM-19 for the development of GRIM-19 related drugs. Preliminary data of preclinical trials were provided.
【學位授予單位】：吉林大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：R739.8

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