【作者】 李伟；

【导师】 吕农华；

【作者基本信息】 南昌大学 ， 内科学， 2014， 硕士

【摘要】 研究背景和目的：幽门螺杆菌（Helicobacter pylori, Hp）是一种常见的、定植于胃黏膜的革兰阴性杆菌，与许多胃肠道疾病发病密切相关，目前普遍认为在慢性胃炎—肠上皮化—异型增生—胃癌这一演变模型中，Hp感染起着先导作用，因此，1994年国际抗癌联盟（UICC）将Hp列为I类致癌因子。最近国外有研究报道，Hp可诱导胃黏膜上皮细胞发生DNA双链断裂（Double-strand breaks，DSBs）；DSBs是DNA损伤最严重的一种形式，其修复机制较其他DNA损伤类型亦更为复杂。DSBs发生后可触发机体的DNA损伤反应（DNAdamage response，DDR），DSBs对于细胞是严重的威胁，如果不能修复或错误修复将导致突变或染色质重排，最终可能导致肿瘤发生或细胞死亡。在哺乳动物细胞，DSBs主要通过非同源末端连接(non-homologous DNAending joining, NHEJ)和同源连接(homologous recombination, HR)两种途径修复。NHEJ不需要序列同源而能够直接端对端地重连接断裂的DNA末端，在整个细胞周期都发挥作用，但主要是在G0和G1期对DSBs进行修复，对保持基因组的完整性十分重要。NHEJ修复通路涉及一系列的信号通路和功能蛋白，主要包括DSBs反应感受器DNA-PK复合物（包括DNA-PKcs、Ku70/80二聚体）的激活，转导蛋白介导信号级联反应（Artemis、XRCC4）以及效应器发挥损伤修复功能（DNA连接酶IV、XLF）等。NHEJ通路通常被认为是参与调控细胞致死性损伤的主要途径。然而，目前关于Hp是否对胃黏膜细胞损伤后NHEJ修复机制产生影响尚无研究，因此，探讨Hp感染正常胃粘膜是否打破细胞正常NHEJ信号通路平衡，从而导致增值、凋亡紊乱参与疾病发生，可进一步阐明Hp的致病机制，为Hp感染相关性疾病防治策略的制定提供新的理论和依据。研究方法：1、收集Hp阳性和阴性的正常胃黏膜（共34例，其中Hp阳性18例和阴性16例）、胃癌病理标本（共146例，其中Hp阳性57例和阴性89例），免疫组织化学法检测DNA-PKcs和Ku70/80蛋白的表达。2、按MOI=100使用ATCC43504Hp标准菌株分别作用于GES-1和AGS细胞，同时以同体积细胞培养液作用的GES-1和AGS细胞作为对照组，1、3、6、12和24h后提取总蛋白，Western blot检测NHEJ信号通路相关蛋白DNA-PKcs和Ku70/80的表达。3、采用ATCC43504Hp标准菌株悬液灌胃蒙古沙鼠，第6、12个月处死动物，收集胃组织，免疫组化法检测胃黏膜组织DNA-PKcs和Ku70/80蛋白表达。研究结果：1、人胃癌组织中NHEJ信号通路相关蛋白DNA-PKcs和Ku70/80的变化及其与Hp感染的关系（1）DNA-PKcs蛋白在胃癌组织中的表达水平显著高于正常胃黏膜组织(p<0.05)。实际上，DNA-PKcs在正常胃黏膜组织中几乎不表达，在正常组的阳性表达率为0%(0/34)，在胃癌组其阳性表达率为93.8%(137/146)，两组间差异具有统计学意义(p<0.05)。此外，胃癌组DNA-PKcs蛋白表达的平均免疫组化得分显著高于正常组(p<0.05)。将正常组和胃癌组标本按Hp阳性和阴性分两组，比较各组中Hp阳性和阴性组间DNA-PKcs蛋白的表达差异，结果发现DNA-PKcs蛋白表达在各组的Hp阳性和阴性组间均无显著性差异（p>0.05）。（2）Ku70/80在正常组的阳性表达率为79.4%(27/34)，在胃癌组的阳性率为96.6%(141/146)，Ku70/80蛋白在胃癌组织中的表达水平显著高于正常胃黏膜组织(p<0.05)。将正常组和胃癌组标本按Hp阳性和阴性分两组，比较发现Ku70/80蛋白表达在胃癌组织的Hp阳性表达显著高于Hp阴性组（p<0.05）。（3）采用Spearman相关性分析发现，在胃癌患者胃黏膜组织中，DNA-PKcs蛋白表达水平与胃癌组织分化程度呈负相关(p<0.05)，其中从高分化(G1)、中等分化(G2)到低分化(G1)表达逐渐降低，低分化(G1)和中等分化(G2)期之间表达差异具有统计学意义(p<0.05)，低分化(G1)和高分化(G3)期之间表达差异具有统计学意义(p<0.05)，而中等分化(G2)和高分化(G3)期之间表达差异无统计学意义(p＞0.05)。在胃癌患者胃黏膜组织中，Ku70/80蛋白表达水平与Hp感染呈负相关(p<0.05)，此外，Ku70/80蛋白表达水平与临床分期呈负相关(p<0.05)。在胃癌组织中，DNA-PKcs表达水平与Ku70/80表达水平之间不具有相关性(p＞0.05)。2、Hp感染对GES-1和AGS细胞NHEJ信号通路相关蛋白DNA-PKcs和Ku70/80表达的影响按感染复数MOI=100将Hp活菌悬液作用GES-1细胞1h后，DNA-PKcs蛋白相对表达量分别为1.16±0.08，高于对照组的1.04±0.32，差异具有统计学意义（P<0.05）。Hp作用GES-1细胞1、3、6、12和24h后，Ku70/80蛋白的相对表达量分别为1.58±0.33、1.84±0.41、1.97±0.34、3.72±1.43和3.74±1.55，均高于对照组的1.24±0.32，差异均具有统计学意义（P<0.05）；Hp作用AGS细胞1、3、6、12和24h后，Ku70/80蛋白的相对表达量分别为4.69±0.86、3.67±0.66、2.41±0.25、1.35±0.36和1.32±0.11，均比对照组的相对表达量4.84±0.77低，差异均具有统计学意义（P<0.05）。3、Hp灌胃感染蒙古沙鼠胃黏膜NHEJ信号通路相关蛋白DNA-PKcs和Ku70/80表达的变化（1）Hp灌胃后第6和12个月，DNA-PKcs蛋白在感染组沙鼠胃黏膜中的阳性率分别为96.6%(29/30)和100.0%(24/24)，感染组6个月和12个月之间差异无统计学意义(P＞0.05)。在Hp感染第6个月和12个月，DNA-PKcs蛋白在正常对照组中均不表达(0/25)，而在感染组中的总阳性率为98.1%(53/54)，两组间差异具有统计学意义(P<0.01)。此外，灌胃感染后第6个月和12个月，实验组DNA-PKcs蛋白免疫组化总体得分为5.39±1.72，高于对照组的0.92±0.87，差异具有统计学意义（P<0.01）。（2）在Hp感染后第6个月和12个月，感染组的Ku70/80蛋白阳性率分别为60.0%(18/30)和79.2%(19/24)，感染组6个月和12个月之间差异无统计学意义(P＞0.05)。正常对照组的Ku70/80蛋白总阳性率为92.0%(23/25)，而感染组的Ku70/80蛋白总阳性率为68.5%(37/54)，正常组和感染组之间总阳性率差异具有统计学意义(P<0.05)。灌胃第6个月和12个月Ku70/80蛋白免疫组化总体得分为4.17±2.46高于对照组的总体得分6.96±2.23，差异具有统计学意义（P<0.01）。研究结论：1、体内实验提示，DNA-PKcs和Ku70/80蛋白的高表达与胃癌关系密切。2、体外实验表明，Hp感染主要通过调节Ku70/80的表达来影响NHEJ活动，从而参与胃黏膜细胞损伤的异常修复活动。3、本研究通过成功建立Hp感染的蒙古沙鼠模型证明，体内感染Hp情况下，DNA-PKcs和Ku70/80表达的不一致可能导致了NHEJ启动异常的发生。4、Hp感染可能通过影响DNA-PKcs和Ku70/80的表达，从而导致异常的NHEJ活动，参与胃黏膜病变的发生。更多还原

【Abstract】 Backgroud andAim:Gastric carcinoma is a worldwide malignant disease with high incidence andmortality, and it is characterized by genome instability through severe DNA damagecaused by various factors, including Helicobacter pylori (H.pylori) infection, heredityand living habits. It is well-established that H.pylori infection is a definite etiologicalfactor in gastric carcinogenesis. However, the mechanism through which H.pyloriinfection contributes to the development of gastric cancer has not been fullyelucidated. The latest study showed an increased rate of DNA double strand breaks(DSBs) in H.pylori infected gastric mucosa cells. The catalytic subunit of theDNA-dependent protein kinase (DNA-PKcs) and the Ku70/Ku80heterodimer(Ku70/80)are crucial promoters of the non-homologous end joining (NHEJ) pathway.Accumulating evidence has shown that dysregulation of DNA-PKcs and Ku70/80isassociated with pathological processes in various tumors. The expression changes andbiological function of DNA-PKcs and Ku70/80in gastric cancer are still unclear. Wedetermined a possible pathological role of DNA-PKcs and Ku70/80-mediated DNArepair pathways in H.pylori related human gastric pathology processes.Materials and Methods：1. A total of146gastric biopsies were obtained from gastric carcinoma patients[H.pylori-negative:89and H.pylori-positive:57] and34from normal subjects[H.pylori-negative:16and H.pylori-positive:18] via surgery and endoscopic detection.The expression of DNA-PKcs and the Ku70/80protein was detected byimmunohistochemistry.2. GES-1and AGS cells were co-cultured with H.pylori (ATCC43504, Cag A+Vac A+) at a cell/bacteria ratio of1:100from1h,3h,6h,12h to24h, the whole celllysated were harvested and subjected to western blot using specific antibodies,western blot for β-actin served as loading control.3. The Mongolian gerbils were divided into two groups:(1) control (n=30)housed normally according to the feeding standard, and randomly sacrificed fifteen gerbils at6,12months of experiment;(2) H.pylori infected group (n=60) receivingintragastric administration of living H.pylori levitation liquid in concentration of109CFU/ml five times in each other day,0.5ml/per time and randomly sacrificed thirtyof them at6,12months of the experiment for analysis.Immunohistochemistry assaywere used to estimate DNA-PKcs and Ku70/80protein expression in gerbils.Results:1. Expression of DNA-PKcs and Ku70/80in human gastric mucosa tissues andthe correlation between the expression of DNA-PKcs and Ku70/80and differentclinical parameters in gastric carcinoma patientsOverall, the positive rates of both DNA-PKcs and Ku70/80were significantlyincreased in gastric cancer (χ2=133.04, P <0.001for DNA-PKcs and χ2=13.06, P <0.01for Ku) compared with normal gastric mucosa (NGM). There was hardly anydetectable expression of DNA-PKcs in NGM, and the positive rate of DNA-PKcsprotein expression in patients with NGM was0%(0/34), whereas the rate in gastriccancer (GC) was93.8%(137/146). The difference between the two groups wasstatistically significant. Additionally, the positive rate of Ku70/80was79.4%(27/34)in NGM and96.6%(141/146) in GC. The DNA-PKcs protein level was significantlyincreased in GC (Mann-Whitney U=39.00, P <0.001), compared with NGM. Inaddition, there was a significant difference in the expression of Ku70/80(Mann-Whitney U=1117.00, P <0.001) between GC and NGM. There was also asignificant difference in Ku70/80protein expression between GC patients with andwithout H.pylori infection (P <0.05). Spearman analysis showed a negativecorrelation between tumor differentiation and DNA-PKcs expression (r=-0.447, P <0.05). Moreover, Ku70/80expression was negatively correlated with both clinicalstage (r=-0.189, P <0.05) and H.pylori colonization (r=-0.168, P <0.05).2.The effect of H.pylori on the expression of DNA-PKcs and Ku70/80in gastriccell linesAfter incubation with Hp at a MOI of100, the expression of DNA-PKcs was1.16±0.08in GES-1cells co-cultured with H.pylori for1hour, while it’s1.04±0.32inthe control, the difference was statistically significant (t1h=4.67，P<0.05). With theextended response time, the expression of Ku70/80were1.58±0.33,1.84±0.41, 1.97±0.34,3.72±1.43and3.74±1.55in GES-1cells co-cultured with H.pylori for1,3,6,12and24hours respectively, and it’s1.24±0.42in GES-1cells without H.pylori,the difference were statistically significant (t=3.57,4.20,5.03,8.11and8.14,P<0.05). Ku70/80protein expression in AGS cells were4.69±0.86,3.67±0.66,2.41±0.25,1.35±0.36and1.32±0.11after co-cultured with H.pylori for1,3,6,12and24hours respectively, all decreased significantly than that in AGS cells withoutH.pylori, which was4.84±0.77(t=34.13,27.68,19.81,4.47and5.68，P<0.05).3. Effects of H.pylori on the expression of DNA-PKcs and Ku70/80inMongolian gerbilsAt six and twelve months post-challenge, the total positive rate of DNA-PKcs ininfected gerbils was98.1%(53/54), while it’s all negative in the controls (0/25), thedifference between the two groups was statistically significant (χ2=74.55,P<0.01).However, the total positive rate of Ku70/80heterodimer was68.5%(37/54) and92%(23/25) in infected gerbils and control gerbils respectively, the differencebetween the two groups was statistically significant (χ2=5.16,P<0.05).Conclusions:1. Enhanced DNA-PKcs and Ku70/80expression may be closely associated withgastric carcinoma.2. In Vitro, H.pylori could increase the expression of Ku70/80in GES-1cells, aswell as inhibit Ku70/80heterodimer expression inAGS cells.3. In vivo, H.pylori infection could promote the expression of DNA-PKcs anddown-regulate Ku70/80expression in gerbil gastric mucosa, suggested that theimbalanced expression of DNA-PKcs and Ku70/80exacerbated H.pylori inducedDNAdamage in gastric mucosa cells.4. The abnormal DNA repairing activities mediated by DNA-PKcs and Ku70/80heterodimer may be associated closely with pathological process in gastric mucosacaused by H.pylori.更多还原