摘要
胰腺癌是恶性程度最高的肿瘤之一,严重威胁人类的健康。大多数病人在发现时已有局部浸润或远处转移,丧失了治疗最佳时期,预后较差。除年龄和吸烟是胰腺癌一个比较确切的危险因素外,有相当一部分胰腺癌患者的病因尚不十分明确,因此,有关胰腺癌病因的探讨仍是该研究领域的热点问题之一。近年来研究发现胰腺癌患者的血清H.pylori感染率显著高于对照组,提示H.pylori感染可能与胰腺癌的发生发展相关。有学者用PCR方法在5/6的胰腺癌组织中发现了螺杆菌菌属特异性16SrDNA,提示螺杆菌菌属感染可能在胰腺癌的发生发展中发挥作用。Fox等在慢性胰腺炎患者的胆汁和胆囊组织中发现有H.pylori的存在,故推测H.pylori可能是特发性胰腺炎的病因之一。意大利一研究小组,通过PCR技术对40例酒精性慢性胰腺炎患者的胰液进行H.pylori尿素酶-C基因序列检测,没有发现其序列的存在,而在40例阳性对照患者的胃粘膜活检标本中均检测到。虽然未发现H.pylori可以在胰腺直接定植,但定植在胃粘膜的H.pylori有可能引起上消化道病理和生理学的改变,并建立一个有利于胰腺炎症甚至肿瘤发生的环境。所以进行H.pylori感染与胰腺癌的相关性研究以及探讨对胰腺炎症的影响有意义重大。
本研究对214例胰腺癌患者H.pylori感染率进行了调查,同时对部分胰腺癌患者的胰腺组织进行了螺杆菌菌属特异性16SrRNA及H.pylori不同致病因子检测。通过H.pylori感染慢性胰腺大鼠模型的建立,探讨了H.pylori感染对胰腺炎症的作用。主要研究结果如下:
1、采用ELISA方法和Western-Blotting方法检测胰腺癌患者血清抗-H.pylori-IgG抗体,抗-H.pylori-CagA抗体和抗-H.pylori-VacA抗体检测,按照病例对照原则比较胰腺癌患者与对照组之间的差异。结果显示胰腺癌患者的H.pylori感染率高于对照组,胰腺癌患者H.pylori CagA+ VacA+毒力亚型感染率高于其他毒力亚型;发生于不同部位的胰腺癌患者之间H.pylori感染率无明显差异,但合并H.pylori感染者以胰腺导管细胞腺癌多见。由此得出:H.pylori感染,尤其是H.pylori CagA+VacA+毒力亚型菌株感染可能是胰腺癌发生的危险因素;H.pylori感染与胰腺癌的发生部位不相关;H.pylori感染可能与胰腺导管细胞腺癌发生更为密切。
2、采用PCR技术对胰腺癌组织进行螺杆菌菌属特异性16SrRNA及H.pylori CagA、VacA和glmM基因检测,并分离培养螺杆菌或H.pylori。结果发现螺杆菌16SrRNA在胰腺癌组织检出率为高于对照组,合并H.pylori感染螺杆菌菌属16SrRNA扩增的DNA产物序列分析,与肝螺杆菌基因序列的同源性为97.8%,与H.pullorum同源性为98.3%,而未分离培养出螺杆菌或H.pylori。由此得出:在胰腺癌患者的胰腺组织中有螺杆菌菌属特异性16SrRNA存在;螺杆菌感染,尤其是致病性较强的螺杆菌感染与胰腺癌的发生相关;螺杆菌(包括H.pylori)在胰腺癌发生发展中的作用并非直接在胰腺定植所致。
3、采用PCR技术对H.pylori感染慢性胰腺炎大鼠胰腺组织进行螺杆菌菌属特异性16SrRNA及H.pylori CagA、VacA和glmM基因检测,并分离培养螺杆菌或H.pylori。与对照组比较H.pylori感染慢性胰腺炎Wistar大鼠的体重、血淀粉酶和脂肪酶下降,血糖的升高以及较为严重的胰腺的炎症改变;在H.pylori感染慢性胰腺炎大鼠的胰腺组织中可找到螺杆菌菌属特异性16SrRNA基因片断,而未分离培养出螺杆菌或H.pylori。
由此得出: H.pylori可加重0.5%-乙硫氨酸诱导慢性胰腺炎症,纤维化明显,血糖升高,血淀粉酶和脂肪酶显著降低;H.pylori诱导的胰腺炎症并非通过H.pylori CagA、VacA和glmM因子直接发挥致病作用。
PART I The prevalence of H. pylori infection in patients with pancreatic cancer and the association between pancreastic cancer and different type of
H.pylori
[Objective] To investigate the prevalence of H.pylori infection in pancreatic cancer patients and the association between pancreatic cancer and different virulence subtype of H.pylori.
[ Methods] H.pylori IgG antibodies were assessed by ELISA in 214 patientswith pancreatic cancer and 232 with chronic gastric and 236controls. Cytotoxin associated protein A (CagA) and VacA proteins were tested by Western-blotting assay in the patients with H. pylori seropositivity.
[Results] Prevalence of H. pylori IgG was higher in pancreatic cancer patients than in controls (63.1% vs 47.0%, P<0.05), the H. pylori seropositivity was associated with pancreatic cancer. The association was more seen (OR=1.71,95%CI 1.12～2.66), excluding age, gender, body mass index, inhabited environmen, case history and living habit. Pancreatic cancer patients also had a higher pre-valence of CagA and VacA positive H. pylori strains(39.3% vs 11.2%,P<0.05), and adjusted odds ratio was 2.12(95%CI 1.12-2.66).This relationships were similar for tumors located in different part of pancreas, but the most patients of H. pylori infection were pancreatic ductal cell cancer.
[Conclusion] H. pylori seropositivity is an risk factor for pancreatic cancer, infection with CagA and VacA positive strains appear to be more related to the disease.The most patients of H. pylori infection were pancreatic ductal cell cancer.
PART II The study of helicobacter genus specific 16SrRNA and H.pylori specific CagA,VacA and glmM DNA in the tissues of pancreatic and clinic significance
[Objective] To determin whether there is helicobacter genus specific 16SrRNA and the causative agent of H.pylori and and their clinic significance.
[Methods] Not only helicobacter genus specific 16SrRNA, but also CagA, VacA and glmM DNA of H.pylori was detected and analyzed in the tissue of 28 cases of pancreatic cancer by PCR.
[Results] Detected 53.6% prevalence of Helicobacter genus specific 16SrRNA in the tissue of pancreatic cancer,it was higher than controls.The helicobacter genus specific 16SrRNA in 10/13 cases with H.pylori was 97.8% similer to the helicobacter species liver and clustered to a phylogenetic group that includes H.pylori, others was 98.3% similer to the H.pullorum and clustered to a phylogenetic group that includes H.bilis
[Conclusion] There was helicobacter genus specific 16SrRNA in the tissue of pancreatic cancer, along with higher prevalence of H.pylori carried Cag A+ VacA+ infection. Helicobacter genus including H.pylori may play a indirect role in pancreatic cancer.
PART III
The effect of H.pylori in the development of chronic pancreatitis by induced H.pylori animal model
[Objective] To approach the chronic pancreatitis rats model with H.pylori infection established and H.pylori pathopoiesis.
[Methods] The weight , biochemstry and histopathological changes of the chronic pancreatitis in the rats model with H.pylori infection were observed. Not only the helicobacter genus specific 16SrRNA , but also CagA, VacA and glmM DNA of H.pylori was detected in pancreatic tissue of the chronic pancreatitis rats by PCR. Moreover, the relation to H.pylori and pancreatic inflammation was analyzed
[Results] The weight of the chronic pancreatitis rats, amylase and lipase in serum were decreasing, nevertheless glucose was increasing。The histopathological changes of the chronic pancreatitis in the Wistar rats with H.pylori infection were more serious than the control groups,.The helicobacter genus specific 16SrRNA was found and not foud CagA,Vac,Aand,glmM DNA of H.pylori in the pancreatic tissue of the Wistar rats with H.pylori infection, but not isolated and cultured helicobacter or H.pylori species.
[Conclusion] H.pylori infection influenced on weight and lipase in the serum of chronic pancreatitis rats and was able to aggravate chronic pancreatic inflammation,which maynot be induced by CagA,Vac and glmM DNA of H.pylori.
引文
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