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1、安徽医科大学硕士学位论文幽门螺杆菌感染与原发性胆囊癌相关性研究姓名：黄俊申请学位级别：硕士专业：外科学（普外）指导教师：汪宏20100501安徽医科大学硕士学位论文 黄 俊 2幽门螺杆菌感染与原发性胆囊癌相关性研究幽门螺杆菌感染与原发性胆囊癌相关性研究 摘摘 要要 目的目的 探讨人类原发性胆囊癌的发生是否与幽门螺杆菌（Helicobacter pylor，Hp）的感染有关，为原发性胆囊癌的治疗提供新的思路。 方法方法 采用病例对照研究，选取 18 例原发性胆囊癌（primary carcinoma of the gallbladder， PCG） 和 40 例慢性胆囊炎、 胆结石 （chron
2、ic cholecystitis or cholelithiasis，CC）患者的血清标本以及手术后的胆囊粘膜、胆汁标本进行 Hp 检测，选取 20 例排除 PCG 和 CC 的患者作为对照。采用 ELISA 方法检测各组血清中 Hp IgG 的存在；利用微氧培养基从新鲜胆囊粘膜和胆汁标本培养螺杆菌；DNAzol 提取胆囊粘膜和胆汁中的 DNA，采用巢式聚合酶链式反应（polymerase chain reaction，PCR）方法扩增螺杆菌特异的 16S rRNA 基因；对 PCG 组螺杆菌 16S rRNA 基因阳性的样品检测 Hp 特异 26KDa 蛋白基因和 4 个相关功能基因 （ca
3、gA、 vacA、 rps4、 ureA） ，并进行测序分析，确定检出的细菌是否为 Hp；westerblot 检测 Hp 相关功能蛋白UreA、UreB、VacA 和 CagA，初步探讨 Hp 感染导致 PCG 发生的机制。 结果结果 1、术前一天，PCG组中，18份血清标本中有16份显示为Hp IgG阳性，阳性率为88%；CC组中，40份血清标本有25份显示为Hp IgG阳性，阳性率为63%；对照组中，20份血清标本有7份显示为Hp IgG阳性，阳性率为35%；PCG组中Hp IgG阳性率明显高于CC组和对照组。术后，Hp IgG阳性率逐渐下降，术后一年，三组Hp IgG阳性率差异无统计学
4、意义。2、PCG、CC和对照组患者新鲜胆囊粘膜或胆汁标本中均未能培养出螺杆菌。3、PCG组中，36份胆囊粘膜和胆汁标本有29份检出螺杆菌属特异性16S rRNA基因，阳性率为81%；CC组中，80份胆囊粘膜和胆汁标本有40份检出16S rRNA基因，阳性率为50%；对照组中，40份胆囊粘膜和胆汁标本有8份检出16S rRNA基因，阳性率为20%；其中PCG组16S rRNA基因检出率明显高于CC组和对照组，CC组螺杆菌基因检出率显著高于对照组。4、29份螺杆菌16S 安徽医科大学硕士学位论文 黄 俊 3rRNA基因阳性的胆囊粘膜或胆汁标本中，15份cagA基因阳性，阳性率为52%；25份26K
5、Da基因阳性，阳性率为86%；14份ureA基因阳性，阳性率为48%；vacA基因和rps4基因阳性率均为零。27份标本cagA、26KDa、vacA、ureA和rps4基因至少一项阳性，阳性率为93%。5、29份螺杆菌16S rRNA基因阳性的胆囊粘膜或胆汁标本经测序鉴定，与Hp有99以上的同源性。6、PCG组中，UreA、UreB、VacA和CagA蛋白阳性率分别为39%、11%、6%和42%；CC组中，UreA、UreB、VacA和CagA蛋白阳性率分别为24%、11%、8%和29%；对照组中，UreA、UreB、VacA和CagA蛋白阳性率分别为7%、10%、5%和10%；PCG组Ca
6、gA和UreA蛋白的阳性率及平均光密度值明显高于其它两组，UreB和VacA蛋白三者差异不具有统计学意义。 结论结论 PCG 患者中存在 Hp 的高感染率，且明显高于 CC 组和对照组。研究结果初步提示 Hp 感染与 PCG 的发生有关， UreA 蛋白和 CagA 蛋白可能在 PCG 的发生中起到了重要作用。 关键词关键词 幽门螺杆菌 原发性胆囊癌 细胞培养 聚合酶链式反应 蛋白印迹 安徽医科大学硕士学位论文 黄 俊 4Association between Helicobacter pylori infection and primary carcinoma of the gallblad
7、der Abstract Objectives To investigate the relationship betweeen primary carcinoma of the gallbladder (PCG), providing a theoretical basis for the treatment of PCG. Methods The blood serum and the mucosa and bile of gallbladder samples were collected from 18 patients with PCG, 40 patients with chron
8、ic cholecystitis or cholelithiasis (CC), and 20 patients with no PCG and CC (control). The serum IgG against Hp was determined with enzyme linked immunosorbent assay (ELISA). Mucosa and bile fresh samples were submitted to microaerobic culturing. The 16S rDNA-based polymerase chain reaction (PCR) fo
9、llowed by DNA sequence analysis of the obtained PCR fragments was performed. A deveoped searech for Hp was also carried out by PCR, and five genes specific for Hp were amlified. The proteinum UreA, UreB, VacA, and CagA were determined by westerblotting in order to clarity pathogenesis of PCG. Result
10、s 1. One day before operation, 88% of serum IgG against Hp were postive in PCG group, 63% in CC group, and 35% in control group. The positive rate in PCG group were significantly higher than the CC group and the control group. The positive rate decreased in PCG and CC groups after operation. There w
11、ere no statistical difference between the three groups one year after operation. 2. The helicobacters were not successfully cultured in all the three groups. 3. In the PCG group, 81% samples of musca and bile were positive for Helicobacter-specific 16S rRNA gene. In the CC group, 50% samples of musc
12、a and bile were positive for Helicobacter-specific 16S rRNA gene. While in the control group, only 20% samples of musca and bile were positive for Helicobacter-specific 16S rRNA gene. The positive rate in PCG group were 安徽医科大学硕士学位论文 黄 俊 5significantly higher than the CC group and the control group.
13、4. Of these 16S rDNA sequence of Helicobacter pylori positive samples of musca and bile, 52% samples were positive in cagA, 86% samples were positive in 26KDa, 48% samples were positive in ureA separately. The vacA and rps4 genes were never detected in all the samples of musca and bile. At least one
14、 gene were postive in 93% samples of musca and bile. 5. Total of 15 samples of musca and 14 samples of bile were obtained from the PCG patient with Helicobacter-specific 16S rRNA postive. The analysis of these PCR products indicated that they were related most closely to the 16S rDNA sequence of Hp.
15、 6. In the PCG group, 39%, 11%, 6% and 42% samples of musca and bile were positive for the proteinum UreA, UreB, VacA, and CagA separately. In the CC group, 24%, 11%, 8% and 29% samples of musca and bile were positive for the proteinum UreA, UreB, VacA, and CagA separately. In the control group, 7%, 10%, 5% and 10% samples of musca and bile were positive for the proteinum UreA, UreB, VacA, and CagA separately. The positive rate and the mean opitical density of UreA and CagA in PCG group were significantly higher than the CC group and the control group. But there were no statistical differ
