本文選題：CD147 + CD98�。� 參考：《第四軍醫(yī)大學(xué)》2017年博士論文

【摘要】：原發(fā)性肝癌俗稱“癌中之王”,惡性進(jìn)展快,治療難度大,病死率高,是一種非常兇險(xiǎn)的疾病,世界范圍內(nèi)腫瘤致死率排名第三位,國內(nèi)腫瘤致死率的排名第二位,對居民生命健康危害極大。CD147是早期我們科室篩選鑒定的腫瘤藥靶分子,CD147在包括肝癌在內(nèi)多種人類惡性腫瘤中表達(dá),且其高表達(dá)是臨床不良預(yù)后的指標(biāo),CD147既可以作為誘導(dǎo)因素,又可以作為效應(yīng)分子通過不同的機(jī)制促進(jìn)腫瘤細(xì)胞的生長代謝,浸潤轉(zhuǎn)移,促血管生成,耐藥以及死亡抵抗等惡性行為,導(dǎo)致腫瘤進(jìn)展。生理病理?xiàng)l件下,以跨膜形式定位于細(xì)胞膜表面的CD147可以內(nèi)化進(jìn)入胞漿而后重新循環(huán)到細(xì)胞膜上,內(nèi)化到胞漿的CD147以囊泡形式存在;此外,CD147還可以外泌體的形式或者胞外段被水解后以游離形式(shedding)釋放到細(xì)胞外基質(zhì)中。近年來發(fā)現(xiàn),內(nèi)化循環(huán)異常是腫瘤惡性進(jìn)展過程中獲得的表型,這一特點(diǎn)已經(jīng)逐漸被認(rèn)可,但是CD147內(nèi)化循環(huán)對于腫瘤進(jìn)展具有什么意義?同時(shí),CD147水解脫落胞外段之后剩余部分的去向以及有沒有生理意義?這些問題的解答有助于我們進(jìn)一步揭開CD147在腫瘤中發(fā)揮重要意義的神秘面紗,并為以CD147為靶點(diǎn)的藥物研發(fā)提供理論依據(jù)�；诖�,我們展開下面三部分實(shí)驗(yàn):研究第一部分,我們從CD98與肝癌不良預(yù)后密切相關(guān)出發(fā),首先證實(shí)了細(xì)胞膜表達(dá)的CD98通過β1-integrin及其下游信號通路在肝癌細(xì)胞的鋪展和成瘤方面具有重要作用;然后基于CD147正向調(diào)節(jié)CD98的細(xì)胞膜表達(dá)水平的流式結(jié)果,以及證實(shí)CD147與CD98相互作用的共定位、pull-down和SPR等實(shí)驗(yàn)結(jié)果,推測CD147能夠調(diào)節(jié)CD98的細(xì)胞膜定位;進(jìn)而我們采用K7721細(xì)胞過表達(dá)熒光蛋白標(biāo)記的CD98,激光共聚焦結(jié)果發(fā)現(xiàn)在缺失CD147的情況下,CD98阻滯在內(nèi)質(zhì)網(wǎng)以及Rab5陽性的早期內(nèi)體,而恢復(fù)CD147之后,CD98又可以重新定位到細(xì)胞膜上;而進(jìn)一步實(shí)驗(yàn)證實(shí),CD147和CD98的內(nèi)化步驟,也就是囊泡循環(huán)的步驟都是由Flotilin-1介導(dǎo)的,而同時(shí)也都受Arf6分子的調(diào)節(jié)。于是,CD147通過與CD98胞外段直接相互作用實(shí)現(xiàn)共轉(zhuǎn)運(yùn),呈現(xiàn)協(xié)同膜表達(dá)的現(xiàn)象,而后促進(jìn)β1-integrin及其下游信號通路的活化,在肝癌細(xì)胞鋪展和成瘤方面發(fā)揮重要作用。因此,CD147能夠調(diào)節(jié)CD98的細(xì)胞膜定位和囊泡循環(huán)機(jī)制解釋了CD147和CD98的協(xié)同作用,并在肝癌進(jìn)展中具有重要意義。研究第二部分,我們從CD147分子的內(nèi)化現(xiàn)象出發(fā),使用膽固醇抑制劑MβCD在阻斷CD147內(nèi)化之后,流式結(jié)果卻發(fā)現(xiàn)CD147在細(xì)胞膜上的表達(dá)水平反而下降;通過進(jìn)一步研究,我們發(fā)現(xiàn)細(xì)胞膜膽固醇水平下降之后,ADAM10對CD147的膜外切割作用(shedding)增強(qiáng),而使用HAb 18抗體能夠阻斷這一過程;在分別阻斷蛋白酶體降解途徑和溶酶體降解途徑之后,我們發(fā)現(xiàn)CD147胞外切割之后剩余的片段會進(jìn)一步走向溶酶體處理。于是,CD147的胞外切割以及隨后的溶酶體降解可能組成了一種CD147的降解途徑。但是,也有可能CD147的胞內(nèi)段在經(jīng)歷貫續(xù)水解之后發(fā)揮更為重要的生理意義。研究第三部分,我們首先分析發(fā)現(xiàn)CD147胞內(nèi)段存在一個(gè)經(jīng)典的核定位信號序列,且CD147胞內(nèi)段能夠指引融合的熒光蛋白定位到細(xì)胞核;然后,我們通過構(gòu)建GFP-CD147-mCherry載體并轉(zhuǎn)染入HEK293T細(xì)胞,發(fā)現(xiàn)由于CD147的貫續(xù)水解,CD147胞內(nèi)段能夠指引融合的mCherry定位到細(xì)胞核;通過自噬誘導(dǎo)、自噬阻斷實(shí)驗(yàn),我們發(fā)現(xiàn)CD147胞內(nèi)段的產(chǎn)生很可能與肝癌細(xì)胞自噬過程相關(guān),過表達(dá)CD147胞內(nèi)段之后,肝癌細(xì)胞的自噬過程進(jìn)一步增強(qiáng);通過熒光報(bào)告基因篩選、轉(zhuǎn)錄組基因芯片分析以及RT-PCR和Western blot驗(yàn)證,CD147胞內(nèi)段可能抑制NF-κb活性以及NF-κB-TRAIL-caspase8-ATG3通路使肝癌細(xì)胞具有自噬傾向;而具有自噬傾向的肝癌細(xì)胞在一定程度上賦予了肝癌細(xì)胞化療藥物順鉑抵抗的能力;鑒于HAb18在一定程度上阻斷了CD147的貫續(xù)水解,我們發(fā)現(xiàn)順鉑聯(lián)合HAb18能夠顯著抑制肝癌細(xì)胞生長。綜上所述,我們的研究證實(shí)了CD147通過內(nèi)化循環(huán)促進(jìn)了CD98的膜定位表達(dá),以及其下游的integrin信號通路,在肝癌細(xì)胞的粘附以及裸鼠的皮下成瘤方面發(fā)揮了重要作用;通過探尋CD147的貫續(xù)水解現(xiàn)象,我們發(fā)現(xiàn)CD147是在ADAM10和溶酶體的聯(lián)合作用下,實(shí)現(xiàn)了分步切割,這可能是細(xì)胞蛋白降解維持穩(wěn)態(tài)的機(jī)制;最后我們通過體外過表達(dá)CD147胞內(nèi)段分析發(fā)現(xiàn),具有核定位作用的CD147胞內(nèi)段通過自噬促進(jìn)作用增強(qiáng)了肝癌細(xì)胞的順鉑耐藥能力,HAb18通過抑制CD147的貫續(xù)水解,在化療藥物的增敏作用中發(fā)揮顯著的作用。
[Abstract]:Primary liver cancer, commonly known as "the king of cancer", is fast progresses, difficult to treat, and has a high mortality rate. It is a very dangerous disease. The death rate of tumor in the world ranks third, the death rate in domestic tumor is second, and the damage to life and health of the residents.CD147 is a cancer drug target in the early section of our department, CD1 47 expression in a variety of human malignant tumors, including liver cancer, and its high expression is an indicator of poor clinical prognosis. CD147 can be used as an inducer and can be used as an effector to promote the growth and metabolism of tumor cells, infiltration, angiogenesis, resistance and death resistance. In physiological and pathological conditions, the CD147 that is located on the surface of the cell membrane in the form of transmembrane can be internalized into the cytoplasm and then recirculated to the cell membrane, and the CD147 internalized into the cytoplasm exists in the form of vesicles; in addition, CD147 can also be released into the extracellular matrix in the form of Exocyst or the extracellular segment after the hydrolysis of the extracellular segment (shedding). In recent years, it has been found that the characteristics of the malignant progression of the tumor are the phenotype of the tumor, and this characteristic has been recognized, but what is the significance of the CD147 internalization cycle for the progression of the tumor? At the same time, are there any physiological significance of the remaining part after the CD147 hydrolysis off the extracellular segment? These questions help us To further uncover the mystical veil that plays an important role in CD147 and to provide a theoretical basis for the research and development of drugs targeting CD147. Based on this, we launched the following three experiments: Part 1, we start from the close relationship between CD98 and the poor prognosis of liver cancer, and first confirmed that the CD98 of cell membrane expressed through beta 1-integrin and The downstream signal pathway plays an important role in the spreading and tumorigenesis of the hepatoma cells, and then based on the flow results of CD147 forward regulation of the expression level of the cell membrane of CD98, and the co localization of the interaction between CD147 and CD98, pull-down and SPR and other experimental results, we speculate that CD147 can regulate the cell membrane location of CD98; and then we use K77. The 21 cells overexpressed the fluorescent protein labeled CD98, and the laser confocal results showed that in the absence of CD147, CD98 blocked the endoplasmic reticulum and Rab5 positive early endosomes, and after the recovery of CD147, CD98 could be relocated to the cell membrane; and further experiments confirmed that the steps of the internalization of CD147 and CD98, that is, the steps of the vesicle cycle. It is mediated by Flotilin-1 and is also regulated by the Arf6 molecule. Thus, CD147 can be transported by direct interaction with the CD98 extracellular segment, presenting the phenomenon of CO membrane expression, and then promoting the activation of the beta 1-integrin and its downstream signal pathway, and plays an important role in the spreading and tumorigenesis of the hepatoma cells. Therefore, CD147 can be adjusted. The cell membrane localization and vesicular circulation mechanism of CD98 explains the synergy between CD147 and CD98, and is of great significance in the progression of liver cancer. The second part of the study is based on the internalization of CD147 molecules. After the use of the cholesterol inhibitor M beta CD to block the CD147 internalization, the flow results show that the expression level of CD147 on the cell membrane is reversed. After further study, we found that after the decrease of the cholesterol level of the cell membrane, ADAM10 enhanced the extracellular cutting effect of CD147 (shedding), and the use of HAb 18 antibody could block the process. After blocking the proteasome degradation pathway and the lysosome degradation path, we found the remaining fragments after the CD147 extracellular cutting. The exosomal cleavage of CD147 and the subsequent degradation of lysosome may lead to a CD147 degradation pathway. However, it is possible that the intracellular segment of CD147 may play a more important physiological role after the continuous hydrolysis. The third part of the study is that we first found that there is one of the CD147 intracellular segments. The classical nuclear localization signal sequence, and the CD147 intracellular segment can guide the fusion fluorescent protein to the nucleus; then, we construct the GFP-CD147-mCherry vector and transfect into the HEK293T cell, and find that the CD147 intracellular segment can guide the fused mCherry to the nucleus because of the continuous hydrolysis of the CD147; by autophagy induction, autophagy is blocked. We found that the production of CD147 cells is likely to be related to the autophagy process of hepatoma cells. After overexpression of the CD147 intracellular segment, the autophagy process of the hepatoma cells is further enhanced; the fluorescence report gene screening, transcriptional gene chip analysis, and RT-PCR and Western blot verification, the intracellular segment of CD147 may inhibit the activity of NF- kappa B and NF- kappa. B-TRAIL-caspase8-ATG3 pathway makes hepatoma cells autophagic, and autophagic cells with autophagy tend to give liver cancer cell chemotherapeutic drugs cisplatin resistance to some extent. In the light of HAb18 blocking the sequential hydrolysis of CD147 to a certain extent, we found that cisplatin combined with HAb18 can significantly inhibit the growth of hepatoma cells. Our study confirms that CD147 promotes the membrane localization of CD98 through the internalization cycle, as well as the downstream integrin signaling pathway, which plays an important role in the adhesion of hepatoma cells and the subcutaneous tumor formation of nude mice. By exploring the sequential hydrolysis of CD147, we found that CD147 is combined with ADAM10 and lysosomes. In addition, step cutting is realized, which may be the mechanism of cell protein degradation to maintain homeostasis. Finally, through overexpression of CD147 intracellular segment analysis in vitro, we found that the CD147 intracellular segment with nuclear localization enhanced the cisplatin resistance of hepatoma cells through autophagy, and HAb18 was used to inhibit the sequential hydrolysis of CD147 by inhibiting the sequential hydrolysis of the chemotherapeutic drugs. It plays a significant role in the sensitization effect.

【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：R735.7
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本文編號：1820121