本文選題：RT112-GR細(xì)胞株 + RT112細(xì)胞株�。� 參考：《山東大學(xué)》2016年博士論文

【摘要】：第一部分細(xì)胞自噬對(duì)膀胱癌化療耐藥性的影響研究背景膀胱癌是泌尿系統(tǒng)最常見的惡性腫瘤,在我國(guó)其發(fā)病率位于泌尿系統(tǒng)腫瘤的首位。大約25%的膀胱癌初發(fā)患者是肌層浸潤(rùn)性膀胱癌,盡管另外75%的膀胱癌患者為非肌層浸潤(rùn)性膀胱癌,但大部分也會(huì)出現(xiàn)復(fù)發(fā)或侵犯肌層。因此,早期手術(shù),術(shù)后常規(guī)進(jìn)行膀胱灌注化療是治療膀胱癌的關(guān)鍵,這樣可以有效降低膀胱癌術(shù)后的復(fù)發(fā)率,改善患者的預(yù)后并提高其生存率。但是仍有2/3的患者會(huì)出現(xiàn)復(fù)發(fā),并且復(fù)發(fā)患者中15%～20%會(huì)進(jìn)展為更高的病理分期和/或伴有分級(jí)升高。大部分膀胱癌相關(guān)死亡是因?yàn)榧咏䴘?rùn)性膀胱癌導(dǎo)致的局部浸潤(rùn)或遠(yuǎn)處轉(zhuǎn)移。較高的死亡率亟待泌尿外科醫(yī)師尋找新的更有效的治療方案來(lái)治療膀胱癌。近年來(lái),盡管吉西他濱(Gemcitabine)和順鉑(Cisplatin)的新型GC聯(lián)合方案作為一線晚期膀胱癌的化療措施療效顯著,但耐藥的發(fā)生大大的限制了化療藥物的遠(yuǎn)期療效。吉西他濱廣泛應(yīng)用于胰腺癌、卵巢癌、乳腺癌、膀胱癌、非小細(xì)胞肺癌等腫瘤的化療。然而,越來(lái)越多的泌尿外科醫(yī)師意識(shí)到了吉西他濱耐藥帶來(lái)的危害。盡管膀胱癌患者最初的化療效果顯著,但60～70%有效的患者5年內(nèi)出現(xiàn)復(fù)發(fā),平均生存期僅有12～14月,這要?dú)w因于在治療過(guò)程中出現(xiàn)的耐藥現(xiàn)象。許多研究表明,化療藥物能誘導(dǎo)腫瘤細(xì)胞的凋亡而發(fā)揮治療作用,但是化療藥物也能誘導(dǎo)腫瘤細(xì)胞產(chǎn)生自噬,這種條件下誘導(dǎo)的自噬往作為一種保護(hù)性機(jī)制來(lái)抑制細(xì)胞的凋亡,而這種保護(hù)性機(jī)制又可以使腫瘤細(xì)胞對(duì)抗各種化療藥物治療,從而使腫瘤細(xì)胞具有耐藥性,不利于腫瘤的治療。已有研究表明,腫瘤細(xì)胞在接受化療后,再利用RNAi技術(shù)干擾自噬相關(guān)基因的表達(dá)可以增加其自噬死亡。另外,有學(xué)者把3-甲基腺嘌呤、氯喹等一些自噬抑制劑與抗腫瘤化療藥物聯(lián)用,發(fā)現(xiàn)在自噬抑制劑與化療藥物對(duì)腫瘤細(xì)胞凋亡具有協(xié)同作用。所以,我們假想在腫瘤化療過(guò)程中,我們通過(guò)抑制腫瘤細(xì)胞化療中由自噬引起的耐藥性,那么可能會(huì)激活腫瘤相關(guān)的凋亡活性,可能會(huì)增加細(xì)胞凋亡過(guò)程,從而增加化療藥物的效應(yīng)。如果能通過(guò)某種方式抑制由腫瘤細(xì)胞自噬引起的化療耐藥性,則可以啟動(dòng)凋亡相關(guān)信號(hào)通路,通過(guò)促進(jìn)腫瘤細(xì)胞的凋亡而提高療效。目的探討細(xì)胞自噬(cell autophagy)對(duì)膀胱癌吉西他濱化療耐藥性的影響。方法吉西他濱濃度梯度加藥進(jìn)行反復(fù)篩選、構(gòu)建膀胱癌RT112吉西他濱耐藥株(RT112-GR),并且通過(guò)MTT細(xì)胞毒性實(shí)驗(yàn)檢測(cè)細(xì)胞的耐藥性。利用RT-PCR及Western boltting等方法研究RT112及RT112-GR兩組細(xì)胞株中自噬標(biāo)記蛋白(LC-3)的mRNA水平及蛋白水平的表達(dá)差異；利用電鏡檢測(cè)RT112及RT112-GR兩組細(xì)胞株中自噬小體水平的差異。利用MTT細(xì)胞活力實(shí)驗(yàn)及CCK8細(xì)胞毒性實(shí)驗(yàn)檢測(cè)氯喹(CQ)抑制RT112-GR自噬水平后,RT112-GR細(xì)胞株對(duì)吉西他濱耐藥性的變化。結(jié)果MTT實(shí)驗(yàn)結(jié)果顯示,RT112-GR細(xì)胞株的吉西他濱半數(shù)抑制濃度(IC50)為4.2 umol/L,是其親代細(xì)胞RT1 12的350倍(RT112 IC50值為12 nmol/L),并且對(duì)吉西他濱耐藥的RT112-GR細(xì)胞株對(duì)阿霉素及順鉑并無(wú)交叉耐藥性。當(dāng)加用自噬抑制劑CQ處理耐藥細(xì)胞株后,MTT實(shí)驗(yàn)結(jié)果顯示其IC50值為10.9 nmol/L,與普通膀胱癌細(xì)胞比較,統(tǒng)計(jì)學(xué)無(wú)顯著性差異(NS)。45 nM的吉西他濱處理RT112和RT112-GR兩組細(xì)胞株,RT-PCR結(jié)果顯示RT112-GR組細(xì)胞株的LC-3的mRNA表達(dá)水平明顯高于普通RT112細(xì)胞組(P=0.023)。Western boltting結(jié)果顯示膀胱癌耐藥細(xì)胞株LC3的蛋白表達(dá)明顯高于普通RT112細(xì)胞組(P0.05)。電鏡照片中可見,45 nM吉西他濱處理兩組細(xì)胞株后,RT112-GR胞漿中的自噬小體明顯增多(P0.05)。MTT細(xì)胞毒性實(shí)驗(yàn)檢測(cè),加入CQ后吉西他濱對(duì)耐藥組的毒性恢復(fù)。結(jié)論本研究中,我們成功構(gòu)建了膀胱癌吉西他濱耐藥株(RT112-GR)。等同治療量45 nM的吉西他濱處理RT112和RT112-GR兩組細(xì)胞株后,RT112-GR的自噬水平明顯高于親代RT112細(xì)胞,表明保護(hù)性自噬的產(chǎn)生,而當(dāng)加用自噬抑制劑CQ處理上述吉西他濱耐藥細(xì)胞后,自噬水平降低,且耐藥性缺失。第二部分圓柱瘤基因轉(zhuǎn)染細(xì)胞構(gòu)建及相關(guān)功能學(xué)實(shí)驗(yàn)研究背景腫瘤抑制因子圓柱瘤基因(Cylindromatosis, CYLD)編碼一種去泛素化酶,CYLD基因突變或者缺失導(dǎo)致圓柱瘤,并與多種腫瘤的發(fā)生、發(fā)展密切關(guān)聯(lián)。CYLD通過(guò)自身去泛素化酶活性移除特定底物的K63連接的泛素鏈,并調(diào)控包括NF-κB、JNK及AKT等在內(nèi)的多條信號(hào)通路。CYLD在腫瘤中與自噬的關(guān)系,目前國(guó)內(nèi)外尚無(wú)研究。有研究提出,成年大鼠腦后突觸中,CYLD可能通過(guò)影響lysine48及l(fā)ysine6的關(guān)聯(lián)泛素化,影響細(xì)胞自噬,而自噬已經(jīng)明確與多種腫瘤化療耐藥性有關(guān),CYLD是否通過(guò)調(diào)節(jié)自噬影響膀胱癌生長(zhǎng)、凋亡及膀胱癌化療敏感性的能力亟待我們研究進(jìn)一步明確。我們前期的研究結(jié)果表明：通過(guò)腺病毒感染的方法降低大鼠血管平滑肌細(xì)胞內(nèi)CYLD表達(dá)水平可以提高NF-κB、JNK及Akt傳導(dǎo)通路活化水平,以上三條通路的激活正是自噬激活的關(guān)鍵信號(hào)通路。李丹丹等的研究證實(shí),化療藥物正是通過(guò)激活JNK,磷酸化下游底物c-Jun,上調(diào)Beclinl基因的表達(dá),介導(dǎo)腫瘤細(xì)胞Hep3B、CNE2發(fā)生自噬性死亡。而高俊玲等人的研究則指出：阻斷JNK的傳導(dǎo)通路可以有效的抑制腦創(chuàng)傷后神經(jīng)元的自噬,具有細(xì)胞保護(hù)作用。Wang W等人的研究證實(shí)對(duì)NF-k,B的調(diào)節(jié)可以影響自噬對(duì)細(xì)胞的保護(hù)作用。除此,我們最近的研究結(jié)果表明,使用腺病毒感染的方法上調(diào)大鼠血管平滑肌細(xì)胞內(nèi)CYLD表達(dá)水平后,可以顯著降低自噬蛋白LC3的表達(dá)；而下調(diào)節(jié)大鼠血管平滑肌細(xì)胞內(nèi)CYLD表達(dá)水平后,可以顯著提高自噬蛋白LC3的表達(dá),這說(shuō)明CYLD表達(dá)水平與自噬密切相關(guān)。綜上所述,以往研究已經(jīng)證實(shí)自噬可以影響腫瘤生長(zhǎng)、凋亡及化療耐藥性。而細(xì)胞自噬可能由CYLD調(diào)控,其調(diào)控機(jī)制很可能與NF-κB、JN、Akt傳導(dǎo)通路的活化水平相關(guān)。綜合我們前期的研究結(jié)果,我們提出研究假設(shè)：CYLD在膀胱癌細(xì)胞中表達(dá)上調(diào),影響自噬關(guān)鍵信號(hào)轉(zhuǎn)導(dǎo)通路NF-κB、JNK、Akt傳導(dǎo)通路的活化水平下調(diào),抑制自噬,從而誘導(dǎo)自噬性細(xì)胞死亡或者凋亡,逆轉(zhuǎn)膀胱癌化療敏感性。目的探討圓柱瘤基因(CYLD)與膀胱癌自噬的關(guān)系。方法利用免疫組化技術(shù)檢測(cè),收集我院病例庫(kù)中的膀胱癌患者55例,手術(shù)切取患者的癌組織,以10例良性膀胱組織作為對(duì)照,構(gòu)建組織芯片。應(yīng)用免疫組化染色法對(duì)芯片組織進(jìn)行CYLD免疫組化染色,同時(shí)應(yīng)用免疫組化染色法對(duì)芯片組織進(jìn)行LC-3染色。應(yīng)用Pearson相關(guān)性分析研究LC-3與CYLD表達(dá)之間是否存在相關(guān)性；構(gòu)建敲除CYLD的膀胱癌細(xì)胞,利用Western Blot和RT-PCR技術(shù)檢測(cè)實(shí)驗(yàn)組(敲除CYLD, Ad-Cyld shRNA沉默腺病毒)、CYLD過(guò)表達(dá)組(Ad-Cyld上調(diào)腺病毒)、空病毒轉(zhuǎn)染組及空白對(duì)照組的CYLD表達(dá)；利用Western Blot和RT-PCR技術(shù)檢測(cè)敲除CYLD的膀胱癌細(xì)胞、過(guò)表達(dá)組、空病毒轉(zhuǎn)染組及普通細(xì)胞的LC-3表達(dá)。結(jié)果免疫組化染色結(jié)果提示,與對(duì)照組(正常膀胱組織)相比較,膀胱癌組織中CYLD的表達(dá)明顯降低(P0.05),差異有統(tǒng)計(jì)學(xué)意義。LC-3在膀胱癌組織中的表達(dá)與CYLD的表達(dá)呈負(fù)相關(guān),Pearson相關(guān)系數(shù)為-0.511(P0.05)。實(shí)時(shí)定量PCR的研究結(jié)果表明：與對(duì)照組細(xì)胞(RTl12)和空載病毒轉(zhuǎn)染組相比,實(shí)驗(yàn)組細(xì)胞(敲除CYLD的RT112)內(nèi)的LC-3的基因水平表達(dá)升高,而CYLD的表達(dá)顯著下調(diào),并且差異具有統(tǒng)計(jì)學(xué)意義(P0.05)。結(jié)論敲除CYLD的普通膀胱癌細(xì)胞構(gòu)建成功；CYLD在膀胱癌中的作用可能與自噬相關(guān)。第三部分圓柱瘤對(duì)細(xì)胞自噬的調(diào)節(jié)與膀胱癌化療耐藥性的關(guān)系研究背景CYLD是一種公認(rèn)的抑癌基因,當(dāng)CYLD基因突變或缺失,可能影響腫瘤的發(fā)生及發(fā)展。微管相關(guān)輕鏈蛋白-3 (Microtubule associated protein light chain3,LC-3)是酵母細(xì)胞自噬相關(guān)基因ATG8的同源基因。LC-3在多種腫瘤疾病中呈現(xiàn)異常表達(dá)。有研究發(fā)現(xiàn)LC-3的表達(dá)量與腫瘤的浸潤(rùn)深度、淋巴結(jié)轉(zhuǎn)移、淋巴結(jié)入侵及不良預(yù)后等呈負(fù)相關(guān)。與LC-3低表達(dá)相比,LC-3高表達(dá)的腫瘤患者整體生存期要好,而且LC-3蛋白是一個(gè)較好的自噬相關(guān)性的標(biāo)記物。有研究表明,細(xì)胞自噬可能由CYLD調(diào)控,其調(diào)控機(jī)制很可能與NF-κB、JNK、Akt傳導(dǎo)通路的活化水平相關(guān)。在膀胱癌化療過(guò)程中,CYLD是否與LC-3存在一定的相互作用需要我們進(jìn)一步的研究。CYLD對(duì)膀胱癌自噬的調(diào)節(jié)作用及其機(jī)制,對(duì)我們了解自噬調(diào)控的精確分子生物學(xué)機(jī)制,并藉此設(shè)計(jì)新穎、合理的針對(duì)性化療方案,進(jìn)一步提高我國(guó)膀胱癌的診療水平有重要意義。目的研究CYLD對(duì)自噬的調(diào)節(jié)與膀胱癌吉西他濱化療耐藥性之間的關(guān)系。方法利用實(shí)時(shí)定量PCR技術(shù)檢測(cè)實(shí)驗(yàn)組(敲除CYLD:RT112-CYLD shRNA)、耐藥組(RT112-GR)及普通細(xì)胞組(RT112)中CYLD的基因表達(dá)；CCK8檢測(cè)實(shí)驗(yàn)組(敲除CYLD)與兩組細(xì)胞IC50的差異；應(yīng)用自噬抑制劑CQ作用于實(shí)驗(yàn)組(敲除CYLD)后檢測(cè)IC50與前三組的差異；同等治療量吉西他濱處理三組細(xì)胞后,利用實(shí)時(shí)定量PCR技術(shù)檢測(cè)三組細(xì)胞的LC-3的mRNA表達(dá)及應(yīng)用CCK8實(shí)驗(yàn)進(jìn)行各組細(xì)胞毒性的檢測(cè),實(shí)驗(yàn)組加用CQ,同樣利用RT-PCR技術(shù)對(duì)LC-3的mRNA水平進(jìn)行檢測(cè)及用CCK8實(shí)驗(yàn)對(duì)CQ的細(xì)胞毒性進(jìn)行檢測(cè)。結(jié)果實(shí)時(shí)定量PCR結(jié)果顯示：膀胱癌耐藥組的CYLD呈現(xiàn)類似敲除實(shí)驗(yàn)組的低表達(dá),與普通細(xì)胞組比較差異具有統(tǒng)計(jì)學(xué)意義(P0.05)；敲除CYLD實(shí)驗(yàn)組LC-3表達(dá)呈現(xiàn)類似膀胱癌耐藥組的高表達(dá)；敲除CYLD實(shí)驗(yàn)組呈現(xiàn)出類似耐藥組的吉西他濱耐藥性,耐藥組的吉西他濱IC50為4.2μmol/L,敲除CYLD實(shí)驗(yàn)組IC50約為4.12μmol/L,無(wú)顯著性差異,即IC50與耐藥組比較差異無(wú)統(tǒng)計(jì)學(xué)意義(P0.05)；加入自噬抑制劑CQ后,耐藥性反轉(zhuǎn)即IC50降低與普通細(xì)胞組相比較差異無(wú)統(tǒng)計(jì)學(xué)意義(P0.05)。結(jié)論耐藥細(xì)胞株中CYLD的低表達(dá)所導(dǎo)致的保護(hù)性自噬可能是其耐藥性產(chǎn)生的原因,同時(shí)應(yīng)用自噬抑制劑CQ作用于細(xì)胞后,耐藥株恢復(fù)對(duì)吉西他濱的敏感性。
[Abstract]:Part 1 the effect of autophagy on the chemotherapeutic drug resistance of bladder cancer. Bladder cancer is the most common malignant tumor of the urinary system. In our country the incidence is at the top of the urological tumor. About 25% of the initial patients with bladder cancer are musculocutaneous invasive bladder cancer, although the other 75% of the bladder cancer patients are non muscularis infiltrating bladder. Cancer, but most of the recurrence or invasion of the muscle layer. Therefore, early surgery, postoperative routine bladder perfusion chemotherapy is the key to the treatment of bladder cancer, which can effectively reduce the recurrence rate after bladder cancer surgery, improve the prognosis of patients and improve their survival rate. However, there are still 2/3 patients with recurrence, and 15% to 20% of the recurrent patients. There will be a higher pathological stage and / or higher grade. Most of the bladder cancer related deaths are due to local infiltration or distant metastasis caused by myometrium infiltrating bladder cancer. Higher mortality is urgent for Department of Urology physicians to find new and more effective treatments for bladder cancer. In recent years, although gemcitabine (Gemcitabine) The combination of Cisplatin and cisplatin (cisplatin) (cisplatin), a new combination of GC, is effective as a chemotherapy for advanced bladder cancer, but the occurrence of drug resistance greatly restricts the long-term effect of chemotherapeutic drugs. Doctors are aware of the dangers of gemcitabine resistance. Although the initial chemotherapy effect of bladder cancer patients is significant, 60 to 70% effective patients have a recurrence within 5 years, with an average survival of only 12~14 months. This is due to the phenomenon of drug resistance in the course of treatment. Many studies have shown that chemotherapeutic drugs can induce apoptosis of cancer cells. However, chemotherapy can also induce tumor cells to produce autophagy, which is induced by autophagy as a protective mechanism to inhibit cell apoptosis, and this protective mechanism can also make tumor cells against various chemotherapeutic drugs, thus making the tumor cells resistant to cancer and is not conducive to the treatment of tumors. Therapy. It has been shown that tumor cells can increase autophagy death by using RNAi technology to interfere with autophagy related genes after chemotherapy. In addition, some autophagic inhibitors, such as 3- methyl adenine, chloroquine, and other antitumor drugs, have been combined with anti tumor chemotherapeutic agents. Therefore, we assume that in tumor chemotherapy, we can activate tumor related apoptosis by inhibiting the resistance to autophagy in tumor cell chemotherapy, which may increase the process of apoptosis and increase the effect of chemotherapeutic drugs. For example, the inhibition of autophagy by tumor cells in some way can be achieved. In order to improve the effect of cell autophagy on the chemotherapeutic resistance of gemcitabine in bladder cancer, the effect of cell autophagy (cell autophagy) on the drug resistance of gemcitabine in bladder cancer. 112-GR) and detection of cell resistance by MTT cytotoxicity test. RT-PCR and Western boltting were used to study the difference in the level of mRNA and protein expression of autophagic marker protein (LC-3) in RT112 and RT112-GR two groups, and the difference in the level of autophagic corpuscles in the cell lines of RT112 and RT112-GR two groups was detected by electron microscopy. MTT cell viability test and CCK8 cytotoxicity test were used to detect the change of RT112-GR cell line resistance to gemcitabine after chloroquine (CQ) inhibition of RT112-GR autophagy. Results MTT experimental results showed that the median inhibitory concentration (IC50) of gemcitabine in RT112-GR cell line was 4.2 umol/L, which was 350 times of its parent cell RT1 12 (RT112 IC50 value was 12). Ol/L), and there was no cross resistance to doxorubicin and cisplatin resistant RT112-GR cells resistant to gemcitabine. When the drug resistant cell line was treated with autophagy inhibitor CQ, the MTT test showed that the IC50 value was 10.9 nmol/L, compared with common bladder cancer cells, there was no statistically significant difference (NS).45 nM for RT112 and RT112-GR. Two groups of cell lines, RT-PCR results showed that the mRNA expression level of LC-3 in RT112-GR group was significantly higher than that of normal RT112 cell group (P=0.023).Western boltting results showed that the expression of LC3 in the drug resistant cell line of the bladder cancer cell line was significantly higher than that of the common RT112 cell group (P0.05). The 45 nM gemcitabine treated two groups of cell lines. The autophagic corpuscle in 12-GR cytoplasm increased significantly (P0.05).MTT cytotoxicity test, and the toxicity of gemcitabine was restored to the drug resistant group after CQ. Conclusion we successfully constructed the cystine resistant gemcitabine resistant strain (RT112-GR). The equivalent treatment amount of 45 nM of gemcitabine treatment RT112 and RT112-GR two cell lines, RT112-GR self The level of phagocytosis was significantly higher than that of the parent RT112 cells, indicating the production of protective autophagy, and the decrease of autophagy and the lack of drug resistance when adding the autophagic inhibitor CQ to the above - mentioned gemcitabine resistant cells. Second part of the gene transfection cell construction and related functional study of the tumor suppressor columnoma gene (Cylindr Omatosis, CYLD) encodes a ubiquitin, CYLD gene mutation or deletion that leads to a cylindric tumor, and is closely associated with the occurrence of a variety of tumors, closely related to the ubiquitin chain of K63 connected by.CYLD through its own ubiquitonasase activity to remove specific substrates, and to regulate multiple signaling pathways, including NF- kappa B, JNK, and AKT, in the tumor and in the tumor. There has been no study at home and abroad. It is suggested that CYLD may affect the autophagy of lysine48 and lysine6 in the postsynaptic synapses of adult rats, and autophagy has been clearly related to the chemotherapeutic resistance of various tumors. Whether CYLD affects the growth of bladder cancer, apoptosis and chemotherapy sensitivity of bladder cancer by regulating autophagy. The perceptual ability needs to be further studied. Our previous study showed that the reduction of CYLD expression level in rat vascular smooth muscle cells by adenovirus infection could improve the activation level of NF- kappa B, JNK and Akt conduction pathway. The activation of the above three pathways is the key signal pathway for autophagy activation. The study confirmed that the chemotherapeutic drugs are activating JNK, phosphorylating the downstream substrate c-Jun, up regulating the expression of Beclinl gene, mediating the tumor cell Hep3B, and the autophagic death of CNE2, while Gao Junling et al. Research points out that blocking JNK conduction pathway can effectively inhibit autophagy of neurons after traumatic brain injury and have cellular protective effect.Wan G W and others have confirmed that the regulation of NF-k, B can affect the protective effect of autophagy on cells. In addition to this, our recent study shows that the expression of CYLD can be significantly reduced after the use of adenovirus infection to increase the expression of CYLD in the vascular smooth muscle cells of rats; and the expression of the autophagic protein LC3 can be significantly reduced. The expression level of CYLD can significantly increase the expression of autophagin LC3, which indicates that the level of CYLD expression is closely related to autophagy. To sum up, previous studies have confirmed that autophagy may affect tumor growth, apoptosis and chemotherapeutic resistance. The autophagy may be regulated by CYLD, and its modulation mechanism is likely to be activated by the NF- kappa B, JN, Akt conduction pathway In our previous study, we put forward the hypothesis that the expression of CYLD in bladder cancer cells was up-regulated, and that the activation level of NF- kappa B, JNK, Akt pathway was down regulated, and autophagy inhibited, thus inducing autophagic cell death or apoptosis and reversing chemosensitivity of bladder cancer. The relationship between the CYLD and the autophagy of bladder cancer. Methods 55 patients with bladder cancer in our hospital case library were collected by immunohistochemical technique. The tumor tissues were removed by operation and 10 cases of benign bladder tissue were used as control to construct the tissue microarray. Immunohistochemical staining was used to stain the tissue of the microarray. LC-3 staining was applied to the microarray tissue by immunohistochemical staining. The correlation between LC-3 and CYLD expression was studied by Pearson correlation analysis; the bladder cancer cells that knocked out CYLD were constructed, and Western Blot and RT-PCR technology were used to detect the experimental group (knockout CYLD, Ad-Cyld shRNA silent adenovirus). CYLD expression of adenovirus), empty virus transfection group and blank control group; Western Blot and RT-PCR technique were used to detect bladder cancer cells that knocked out CYLD, overexpressed group, empty virus transfection group and common cell LC-3 expression. Results immunohistochemical staining results showed that the CYLD table in bladder cancer tissue was compared with the control group (normal bladder tissue). The difference was significantly decreased (P0.05). The difference was statistically significant in the expression of.LC-3 in the bladder cancer tissue and the expression of CYLD, and the Pearson correlation coefficient was -0.511 (P0.05). The results of real-time quantitative PCR showed that the LC-3 gene water in the experimental group (RTl12) and the unloaded virus transfected group was in the experimental group (the CYLD RT112). The expression of CYLD increased significantly, while the expression of CYLD was significantly down, and the difference was statistically significant (P0.05). Conclusion the common bladder cancer cells that knocked out CYLD were successfully constructed, and the role of CYLD in bladder cancer may be related to autophagy. The recognized tumor suppressor gene, when the mutation or deletion of CYLD gene, may affect the occurrence and development of tumor. Microtubule related light chain protein -3 (Microtubule associated protein light chain3, LC-3) is the homologous gene.LC-3 of the yeast cell autophagy related gene ATG8, which presents abnormal expression in a variety of tumor diseases. The tumor invasion depth, lymph node metastasis, lymph node invasion and bad prognosis are negatively correlated. Compared with the low expression of LC-3, the overall survival time of the tumor patients with high expression of LC-3 is better, and the LC-3 protein is a better autophagy correlation marker. B, JNK, Akt conduction pathways are associated with activation levels. In the course of chemotherapy for bladder cancer, the interaction between CYLD and LC-3 requires us to further study the regulatory role of.CYLD on autophagy of bladder cancer and its mechanism, to understand the precise molecular mechanism of autophagy, and to design novel and reasonable targeting It is of great significance to further improve the level of diagnosis and treatment of bladder cancer in China. Objective to study the relationship between the regulation of autophagy and the drug resistance of gemcitabine in bladder cancer. Methods using real-time quantitative PCR technique to detect the gene of CYLD in the experimental group (knockout CYLD:RT112-CYLD shRNA), drug resistance group (RT112-GR) and common cell group (RT112). The difference between the experimental group (knockout CYLD) and the two groups of IC50 was detected by CCK8; the use of autophagy inhibitor CQ in the experimental group (knockout CYLD) was used to detect the difference between the IC50 and the first three groups. After the same treatment, gemcitabine was used to detect the mRNA expression of LC-3 in the three groups of cells by real-time quantitative PCR and the application of CCK8 experiment. The test group was tested for cytotoxicity in each group, the experimental group was added with CQ, and the mRNA level of LC-3 was detected by RT-PCR technique and the cytotoxicity of CQ was detected by CCK8 test. The results of real-time quantitative PCR showed that the CYLD of the drug resistance group of bladder cancer was similar to that of the experimental group, and the difference between the group and the common cell group was statistically significant. The LC-3 expression in the CYLD test group was similar to the high expression of the drug resistance group in the bladder cancer group; the knockout CYLD experimental group showed a similar drug resistance in the drug resistant group, and the gemcitabine IC50 was 4.2 u mol/L in the drug resistant group, and the IC50 in the knockout CYLD experimental group was about 4.12 u mol/L, and there was no significant difference between the IC50 and the drug resistant group, that is, there was no difference between the IC50 and the drug resistant group. Statistical significance (P0.05); after adding autophagy inhibitor CQ, there was no statistical difference in drug resistance reversal or IC50 reduction compared with normal cell group.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R737.14

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