【作者】 姚玉霞；

【导师】 姜慧卿；

【作者基本信息】 河北医科大学 ， 内科学， 2004， 硕士

【摘要】 临床和流行病学资料证实，H.pylori感染是慢性胃炎和消化性溃疡的主要病因，并与胃癌的发生密切相关。目前认为H.pylori感染引起的胃黏膜上皮细胞动力学改变在其致病机制中起重要作用，对活检标本研究显示：H.pylori感染引起胃上皮细胞增殖和凋亡加速，根除H.pylori后增殖和凋亡水平降至正常，H.pylori及其分泌物均对胃上皮细胞生长产生影响。本研究拟用增殖细胞核抗原(PCNA)来评估细胞的增殖状况。PCNA又称周期蛋白，是DNA多聚酶δ的一种辅助因子，静止期细胞含量很少，G1晚期开始增多，S期达到高峰，G2期、M期的含量较低，是评价细胞增殖状态的主要指标。 细胞凋亡是影响细胞生长动力学的重要因素，对维持细胞群体适当的数目及正常功能有重要意义，它是一种不同于坏死的主动死亡形式。在核固缩、碎裂的同时，凋亡细胞裂解为多个有质膜包被的“小体”，可被吞噬细胞吞噬，主要特征是染色质的有控降解，DNA双链在核小体连接部断裂，形成180-200bp的亚单位片段或其数倍的片段。目前常用的检测方法有：用光镜、电镜或荧光显微镜观察其形态学特征识别凋亡细胞；末端转移酶介导的dUTP切口末端标记法(TUNEL)特异识别DNA片段的3′-羟基末端；凝胶电泳检<WP=5>测核小体DNA片段；流式细胞仪或激光扫描细胞形态分析仪对不同细胞周期的凋亡细胞进行定性和定量研究。对活检标本常用一般的形态学检测进行预实验，然后用TUNEL进一步证实。本实验采用HE染色后在光镜下观察，再用TUNEL检测。目前对H.pylori感染引起细胞增殖和凋亡改变的机制仍不清楚，E2F-1在维持组织细胞的增殖和凋亡平衡中起关键作用，能通过诱导多种在G1期起关键作用的基因表达，启动DNA合成，控制细胞周期G1-S期的转换。E2F-1过表达，可引起G1-S期的过渡失调，加速细胞通过G1期，使细胞无限增殖，参与肿瘤的发生。在胃癌、结肠癌、多发性骨髓瘤等恶性细胞中均检测到E2F-1基因的扩增和蛋白表达上调，但亦有报道E2F-1过表达可以通过P53依赖途径及非P53依赖途径诱导细胞凋亡，所以明确H.pylori感染的胃黏膜上皮细胞E2F-1表达及其与胃上皮细胞增殖和凋亡的关系，对研究H.pylori致病机制有重要作用。目前多数学者认为宿主的胃酸分泌状况在一定程度上决定了H.pylori感染的临床结局，胃酸分泌增高的H.pylori感染患者，以胃窦部炎症为主，胃体泌酸黏膜的萎缩较轻，易发生DU；而胃酸分泌无变化或降低的H.pylori感染患者，炎症多累及胃体，往往伴有一定程度的黏膜萎缩，易发展成为萎缩性胃炎，甚至胃癌。国内外最近研究报道有一定比例的DU患者伴有胃癌，与上述观点相悖，本研究同时选取H.pylori 感染的慢性浅表性胃炎(chronic superficial gastritis, CSG)和DU 患者进行对比研究，以更全面的反映H.pylori 感染对胃黏膜上皮细胞的影响及排除患者自身因素的干扰。 <WP=6>第一部分 H.pylori 感染的胃黏膜上皮细胞E2F-1表达与其增殖凋亡的相关性研究目的：研究 H.pylori 感染的胃黏膜上皮细胞E2F-1的表达及与胃黏膜上皮细胞增殖凋亡的关系，为研究H.pylori致病机制提供新的线索。方法：收集快速尿素酶和组织学检测均为H.pylori 阳性的患者46例，包括慢性浅表性胃炎(CSG)患者23例，十二指肠溃疡(DU)患者23例。H.pylori阴性正常对照15例。对DU患者进行根除H.pylori治疗。从胃窦部和胃体部取材，用免疫组织化学方法检测胃黏膜上皮细胞转录因子E2F-1和增殖细胞核抗原(PCNA)的表达，用末端转移酶介导的dUTP切口末端标记法(TUNEL)检测细胞凋亡。结果：H.pylori阳性患者E2F-1阳性率显著高于正常对照组(P<0.05)，DU 组根除H.pylori后E2F-1阳性表达显著减少(P<0.05)。H.pylori感染患者胃上皮细胞增殖和凋亡均显著增强，PCNA与E2F-1表达显著相关(r=0.705 P<0.05)，且表达部位一致；凋亡指数和E2F-1标记指数间也显著相关(r=0.532 P<0.05)，但阳性表达部位不同。结论：H.pylori感染后胃上皮细胞 E2F-1表达增加，并与H.pylori 感染引起的胃上皮细胞增殖密切相关，为探索H.pylori致癌机制提供了一条线索。第二部分H.pylori 感染慢性浅表性胃炎和十二指肠溃疡患者胃黏膜上皮细胞E2F-1表达与其增殖凋亡情况的比较 <WP=7>目的：比较H.pylori 感染慢性浅表性胃炎和十二指肠溃疡患者胃黏膜上皮细胞E2F-1表达与其增殖凋亡情况，以明确H.pylori 感染对胃上皮细胞增殖动力学的作用是否受胃酸分泌状态的影响。方法：收集快速尿素酶和组织学检测均为H.pylori 阳性的患者46例，包括慢性浅表性胃炎(CSG)患者23例，十二指肠溃疡(DU)患者23例。从胃窦部和胃体部取材，用免疫组织化学方法检测胃黏膜上皮细胞转录因子E2F-1和增殖细胞核抗原(PCNA)的表达，用末端转移酶介导的dUTP切口末端标记法(TUNEL)检测细胞凋亡。结果：CSG与DU组胃上皮细胞E2F-1阳性率之间不存在显著统计学差异(73.91% vs 69.56%，P>0.05)；PCNA阳性标记指数（71.89±4.56 vs 63.45±4.96, P>0.05）和细胞凋亡阳性标记指数（13.22±2.58 vs 11.53±0.51, P>0.05）之间差异均无显著性。结论：H.pylori感染后胃上皮细胞 E2F-1表达，及其H.pylori 感染引起的胃上皮细胞增殖凋亡?更多还原

【Abstract】 The evidences from clinical and epidemiological studies show that H.pylori is the major etiological agent of chronic gastritis and peptic ulcer disease and is significantly associated with gastric carcinoma. The alteration of gastric epithelial cell turnover plays an important role in H.ylori associated gastritis. Recent evidences from studies to biopsy specimens have shown that H.pylori induces acceleration of apoptosis and proliferation in gastric epithelial cells; Apoptosis and proliferation decrease to normal level after eradication therapy；H.pylori and its excretion products can accelerate the cell turnover.Our study used proliferation cell nuclear antigen (PCNA) to evaluate the cell proliferation level. PCNA is also named cycle protein, an auxiliary factor of DNA polymerase δ. It’s lowest expressed in Go phase, increases in late G1 phase, highest expressed in S phase, but declines in G2 and M phase. It’s a mainly proliferation cell marker.Apoptosis is an important effect factor to the cell growth kinetics, playing an important role to sustain the cells normal quantity and function. It is initiative style to death different from <WP=9>necrosis, as the nuclear shrunk and broken, the apoptotic cell splits into several ‘apoptosome’ with membrane, can be engulfed by phagocyte. The character of this process is the chromatin resolves in control, DNA strands are broken at the spot of nucleosome joints, forming many 180-200bp fragments. Apoptotic cell can be detected by light microscope, electron microscope, or fluorescence microscope according to its morphology, by terminal deoxynucleotidyl transferase mediated dUTP-biotin nick end labeling(TUNEL) method to recognize 3′-OH bottoms, by gel electrophoresis to detect nucleosome DNA fragments, by flow cytometry and laser-scanning cytometor to do qualitative and quantitative analysis. The biopsy specimens’ apoptosis are commonly used morphology methods to preliminarily test and confirmed by TUNEL. Our study detected hematoxylin-eosin stained tissues by light microscope and confirmed by TUNEL.The mechanism of H.pylori affecting the cell turnover is not clear. E2F-1 transcription factor is pivotal in control of cell proliferation and apoptosis, induces a coordinated expression of genes in G1 phase, initiate DNA synthesis, control the G1 to S transition of cell cycle. Overexpression of E2F-1 often leads to human tumors through deregulation the transition, it’s reported that overexpression of E2F-1 lead to proliferation in the cell line BaF-B03, make the cell line not sensitivity to those cytokines which mediate apoptosis. The gene amplification and increased expression of E2F-1 were detected in human gastric and <WP=10>colorectal carcinoma, osteosarcoma and other cancer tissues. But a variety of experiments also showed that overexpression of E2F-1 mediated apoptosis through p53 dependent pathway or p53 independent pathway. It’s important to investigate the expression of E2F-1 in H.pylori-infected gastric epithelial cell and examine the correlation between such expression and the cell proliferation and apoptosis to study further the etiopathology of H.pylori.It is considered widely that the gastric acid secretion determines the ending of H.pylori associated diseases. The pathogenic mechanism of H.pylori infection inducing duodenal ulcer (DU) distinguishes from gastric carcinoma. H.pylori infection mainly causes the antral inflammation in acid hypersecretion subjects, tends to induces DU. But in acid secretion having no change or decreased subjects, H.pylori infection tends to lead to atrophic gastritis with gastric body inflammation mainly, and furthermore leads to gastric carcinoma. But there were some reports recently of internal and external that duodenal ulcer patients proportion with gastric carcinoma at the same time, this is not consensus with conventional idea that the DU patients would not suffer from gastric carcinoma. Our study selected the chronic superficial gastritis and the DU patients with H.pylori infected, and to reflect the effects of更多还原