【作者】 李琦；

【导师】 李浩勇；

【作者基本信息】 海南医学院 ， 泌尿外科， 2018， 硕士

【摘要】 目的探讨AID(活化诱导胞嘧啶核苷酸脱氨酶)在人体膀胱尿路上皮细胞癌、癌旁组织中的表达情况以及与疾病的相关性;明确AID对膀胱尿路上皮细胞癌细胞株T24和5637增殖、侵袭、迁移以及凋亡的影响及机制;AID对膀胱尿路上皮细胞癌细胞在裸鼠体内增殖和转移的影响。方法运用Western Bolt以及免疫组织化学染色法检测16例临床膀胱尿路上皮细胞癌组织以及癌旁组织中AID的相对表达量;将携带有抑制AID表达的shRNA序列(shAICDA-T24组)或空载序列(NC-T24组)以慢病毒为载体对T24以及5637细胞进行感染,利用Western Bolt对T24/5637、NC-T24/NC-5637以及shAICDA-T24/shAICDA组进行AID表达量的检测;为了提高抑制率,随后对转染后的细胞进行单克隆细胞筛选并通过Western Bolt结果选取抑制效果最明显的组别用于后续试验;然后运用细胞增值试验(CCK8)、流式细胞术(凋亡)、细胞划痕实验以及细胞小室实验(Transwell)检测抑制AID的表达对T24细胞增殖、凋亡、侵袭以及迁移的影响;建立裸鼠皮下成瘤和尾部静脉注射观察肺部转移灶的动物模型,检测AID在体内实验中对膀胱尿路上皮细胞癌细胞增殖和转移的影响;通过等重同位素多标签相对定量蛋白质组学(iTRAQ)技术对shAICDA-T24组NC-T24组的差异蛋白进行检测,并通过Western Bolt和生物信息学的方法对结果进行验证和分析,筛选最可能位于AID调控下游的因子;最后利用5-氮杂胞苷对shAICDA-T24组细胞进行药物处理并观察对筛选因子以及T24细胞增殖、凋亡、迁移以及侵袭的影响,对AID对膀胱尿路上皮细胞癌细胞的调控机制进行阐述。结果人体膀胱尿路上皮细胞癌组织中存在AID的表达,并且膀胱尿路上皮细胞癌组织中AID的表达显著高于癌旁组织(*P<0.05);通过RNA干扰技术(RNAi)抑制AID的表达可显著降低膀胱尿路上皮细胞癌细胞株T24及5637的增殖、侵袭和迁移能力,并且增加了T24细胞的凋亡率(*P<0.05)。通过等重同位素多标签相对定量蛋白质组学(iTRAQ)分析对T24细胞中6452个蛋白进行检测,富集结果显示有99个蛋白发生上调,142个蛋白发生了下调。最后用生物信息学的方法对实验结果进行分析,最终筛选出MMP14、WLS/GPR177、CXCL12/SDF-1三个在蛋白-蛋白相互作用网络(PPI)中处于中心调控地位并与膀胱尿路上皮细胞癌最具有相关性的蛋白,并通过5-氮胞苷验证了AID通过MMP14促进膀胱尿路上皮细胞癌T24和5637细胞的上皮间-充质转化(EMT)。结论AID在临床膀胱尿路上皮细胞癌患者癌组织中的表达水平显著高于癌旁组织;抑制AID的表达可显著降低T24细胞增殖、侵袭以及迁移能力,并且增加T24细胞的凋亡;AID可促进膀胱尿路上皮细胞癌细胞在裸鼠体内的增殖和转移;AID可能通过脱甲基作用促进MMP14的表达从而促进膀胱尿路上皮细胞癌的进展。AID可能是将来膀胱尿路上皮细胞癌临床治疗的潜在靶点。更多还原

【Abstract】 Objective To investigate the expression of AID(Activation-induced cytosine deaminase)in human bladder cancer and para-cancerous tissues and the relationship between AID and bladder cancer.;clear and definite the effect and mechanism of AID on proliferation,invasion,migration and apoptosis of bladder cancer cell line T24 and 5637;explor the effect of AID on the proliferation and metastasis of bladder cancer cells in nude mice Methods The relative expression of AID in 16 cases of bladder cancer tissues(radical cystectomy)was measured by Western Bolt and immunehistochemical staining;lentivirus-based sh RNA sequence was used to down-regulate the expression of AID in bladder cancer cell line T24 and 5637,nonsense sequence was used as negative control.Western Bolt was used to detected the rate of inhibition.The screening of monoclonal cells was performed,and cells with the lowest expression of AID was selected by Western Bolt for following research;To explor the effect of AID on T24 cells through Transwell assay,Cell Counting Kit 8(CCK8)and Flow cytometry;Observed the proliferation and metastasis of AID-silenced bladder cancer cells in nude mice;i TRAQ-based proteomic analysis was used to identify proteins with significantly different expression,bioinformatics approch was used to screened out the target factors and confirmed by Western Bolt the expression of selected protein was measured by Western Bolt;and 5aza-2’deoxycytidine was used to reveal that AID promote the EMT of T24 and 5637 through MMP14.Result Ectopic expression of AID was found in bladder cancer tissues,and relative expression level is significantly higher than that in paracancerous tissues(*P<0.05).Cell experiments confirmed that the inhibition of AID expression by RANi could significantly reduce the proliferation,migration and invasion ability of T24 cells(*P<0.05).Conclusion The expression of AID can affect the proliferation,migration and invasion of bladder cancer cells in vivo and vitro,and also increased the apoptosis in vitro.and its expression may be related to the occurrence and metastasis of bladder cancer.更多还原