CagA+幽门螺杆菌与胃黏膜上皮细胞凋亡的分子研究

目的：观察CagA^ Hp对胃黏膜上皮细胞凋亡的影响，进而探讨CagA^ Hp增加胃癌发生危险性的机制。方法：以TUNEL染色法研究30例CagA^ Hp阳性患者Hp根除前后胃黏膜上皮细胞凋亡的情况；通过免疫组织化学法和RT－PCR检测凋亡相关基因bcl-2和bax的表达。结果：CagA^ Hp阳性患者胃黏膜上皮细胞凋亡指数为13．42％，Hp根除后，胃黏膜上皮细胞凋亡指数降为4．8％，较治疗前明显减少（P＜0．01），而CagA^ Hp仍为阳性患者胃黏膜上皮细胞凋亡指数无明显减少（P＞0．05）；bcl-2蛋白表达阳性的CagA^ Hp阳性患者Hp根除后，胃黏膜上皮细胞bcl-2蛋白表达阳性明显增多（P＜0．01），且胃黏膜上皮细胞bcl-2的mRNA表达明显增强（P＜0．01），而CagA^ Hp仍为阳性患者胃黏膜上皮细胞bcl-2蛋白表达和mRNA表达无明显增强（P＞0．05）；bax蛋白表达阳性的CagA^ Hp阳性患者Hp根除后，胃黏膜上皮细胞bax蛋白表达阳性明显减少（P＜0．01），且胃黏膜上皮细胞bax的mRNA表达明显减少（P＜0．01）；而CagA^ Hp仍为阳性患者胃黏膜上皮细胞bax蛋白表达和mRNA表达无明显减少（P＞0．05）。结论：诱导胃黏膜上皮细胞凋亡是CagA^ Hp参与胃癌发生的机制之一，CagA^ Hp通过下调bcl-2的表达、促进bax的表达诱导胃黏膜上皮细胞凋亡。     

重组人p53腺病毒提高胃癌细胞对顺铂敏感性的实验研究

目的：研究重组人p53腺病毒增加胃癌细胞对顺铂敏感性的作用。方法：重组人p53腺病毒感染胃癌细胞BGC-823，Western blot法检测p53蛋白在胃癌细胞中高表达；MTT法测定重组人p53腺病毒单独及联合顺铂用药的不同浓度处理细胞的生长抑制率，流式细胞仪检测细胞周期分布和凋亡率。结果：重组人p53腺病毒感染BGC-823 48h，p53蛋白在BGC-823中高表达，并产生G2／M期阻滞和细胞凋亡。重组人p53腺病毒联合顺铂用药增加顺铂的敏感性，有剂量时间的依赖性。结论：腺病毒介导p53基因感染BGC-823细胞诱导凋亡并增加胃癌细胞对顺铂的敏感性，为p53基因治疗与胃癌化疗临床结合提供了可靠的实验依据。     

Hp感染与胃炎性病变,不典型增生及胃癌关系的研究

目的　通过检测 392例不同胃部疾病 :慢性胃炎 (2 13例 )、消化性溃疡 (77例 )、不典型增生 (5 3例 )及胃癌(4 9例 )中幽门螺杆菌 (Hp)感染的不同阳性率 ,探讨幽门螺杆菌感染与胃部疾病的关系。方法　应用快速尿素酶法 ,Giemsa染色法及C1 4呼气实验等 3种不同方法检测胃粘膜Hp感染。应用免疫组织化学ABC法检测人胃癌组织中caspase 3和bcl xl的表达情况。并对比不同胃粘膜病变中Hp阳性率及caspase 3和bcl xl表达的差异。结果　Hp感染与胃炎性病变及不典型增生的严重程度密切相关 ,但在胃癌中的检出率较前二者低 (P <0 0 5 )。胃炎性病变、不典型增生及胃癌中caspase 3的表达依次降低 (P <0 0 5 ) ,bcl xl的表达依次升高 (P <0 0 5 )。Hp阳性组较Hp阴性组caspase 3表达降低 (P <0 0 5 ) ,而bcl xl表达升高 (P <0 0 5 )。结论　Hp感染与胃粘膜炎性病变及不典型增生程度正相关 ,Hp在胃癌的发生发展过程中可能是早期事件 ,可能与改变细胞凋亡有关     

消化性溃疡患者CagA-Hp感染与血清IL-8、IL-10的相关性研究

目的：探讨消化性溃疡（PU）患者伴有CagA-Hp感染与其血清IL-8、IL-10之间的关系。方法：取H.pylori阳性PU患者100例，其中CagA-Hp抗体阳性患者50例，CagA-Hp抗体阴性患者50例，采用ELISA法分别测定其血清IL-8和IL-10水平。结果：消化性溃疡患者中CagA-Hp抗体阳性组血清IL-8和IL-10显著高于CagA-Hp抗体阴性组。CagA-HpIgG抗体阳性患者中，胃溃疡与十二指肠球溃疡患者血清中IL-8和IL-10水平与溃疡发生的部位无关。     

Recombinant, refolded tetrameric p53 and gonadotropin-releasing hormone-p53 slow proliferation and induce apoptosis in p53-deficient cancer cells

The p53 tumor suppressor is mutated in over 50% of human cancers. Mutations resulting in amino acid changes within p53 result in a loss of activity and consequent changes in expression of genes that regulate DNA repair and cell cycle progression. Replacement of p53 using protein therapy would restore p53 function in p53-deficient tumor cells, with a consequence of tumor cell death and tumor regression. p53 functions in a tetrameric form in vivo. Here, we refolded a wild-type, full-length p53 from inclusion bodies expressed in Escherichia coli as a stable tetramer. The tetrameric p53 binds to p53-specific DNA and, when transformed into a p53-deficient cancer cell line, induced apoptosis of the transformed cells. Next, using the same expression and refolding technology, we produced a stable tetramer of recombinant gonadotropin-releasing hormone-p53 fusion protein (GnRH-p53), which traverses the plasma membrane, slows proliferation, and induces apoptosis in p53-deficient, GnRH-receptor-expressing cancer cell lines. In addition, we showed a time-dependent binding and internalization of GnRH-p53 to a receptor-expressing cell line. We conclude that the GnRH-p53 fusion strategy may provide a basis for constructing an effective cancer therapeutic for patients with tumors in GnRH-receptor-positive tissue types.     

幽门螺杆菌细胞毒素相关蛋白与胃癌的相关性

目的 探讨幽门螺杆菌细胞毒素相关蛋白与胃癌的相关性。方法 采用免疫印迹法检测218例慢性浅表性胃炎(CSG)、慢性萎缩性胃炎(CAG)和胃癌(GC)患者血清细胞毒素相关蛋白抗体(CagA)。结果 CagA抗体在218例患者中总检出率为69．27％。在慢性浅表性胃炎、慢性萎缩性胃炎、胃癌患者中CagA抗体阳性率分别为55．43，74．11和90．24％，经x^2检验x^2分割法显示：三组胃疾病阳性率总体间差异有显著性；慢性浅表性胃炎与慢性萎缩性胃炎阳性率比较，差异有显著性；慢性浅表性胃炎与胃癌阳性率比较，差异有非常显著性；慢性萎缩性胃炎与胃癌阳性率比较，差异无显著性。结论 慢性萎缩性胃炎和胃癌的发生与CagA阳性Hp感染有关，定期随访CagA抗体阳性Hp感染特别是已进入慢性萎缩性胃炎阶段的患者，可能有利于胃癌的早期诊断。     

CagA^＋幽门螺杆菌菌株培养滤液对胃黏膜上皮细胞生长及DNA的影响

目的研究CagA^＋Hp菌株培养滤液对胃黏膜上皮细胞（GES-1）生长的作用。方法分为CagA^＋组、CagA^-组、布氏肉汤组（空白对照组）,Hp菌株制备培养滤液处理GES-1细胞,连续培养1个月。MTT法观察GES-1细胞生长,单细胞微凝胶电泳检测GES-1细胞DNA的损伤,并采用电镜观察GES-1细胞的形态变化。结果 CagA^＋培养滤液可使GES-1细胞增生活跃,GES-1细胞出现彗星现象显著高于CagA^-组（41.2%vs.12.5%）（P〈0.05）。经CagA^＋组处理的GES-1细胞,细胞核增大、畸形、核染色质变粗、核仁肥大、核分裂。结论 CagA在Hp培养滤液促使GES-1细胞呈肿瘤细胞的形态学改变及生长特征中起重要作用。DNA损伤可能是CagA诱导GES-1生长特征改变的机制之一。     

Interaction of CagA with Crk plays an important role in Helicobacter pylori-induced loss of gastric epithelial cell adhesion

CagA protein is a major virulence factor of Helicobacter pylori, which is delivered into gastric epithelial cells and elicits growth factor-like responses. Once within the cells, CagA is tyrosine phosphorylated by Src family kinases and targets host proteins required to induce the cell responses. We show that the phosphorylated CagA binds Crk adaptor proteins (Crk-II, Crk-I, and Crk-L) and that the interaction is important for the CagA-mediated host responses during H. pylori infection. H. pylori-induced scattering of gastric epithelial cells in culture was blocked by overexpression of dominant-negative Crk and by RNA interference-mediated knockdown of endogenous Crk. H. pylori infection of the gastric epithelium induced disruption of E-cadherin/catenin-containing adherens junctions, which was also dependent on CagA/Crk signaling. Furthermore, inhibition of the SoS1/H-Ras/Raf1, C3G/Rap1/B-Raf, or Dock180/Rac1/Wiskott-Aldrich syndrome protein family verprolin homologous protein pathway, all of which are involved downstream of Crk adaptors, greatly diminished the CagA-associated host responses. Thus, CagA targeting of Crk plays a central role in inducing the pleiotropic cell responses to H. pylori infection that cause several gastric diseases, including gastric cancer.     

The effect of triptolide on apoptosis of glioblastoma multiforme (GBM) cells

Triptolide, derived from the traditional Chinese herb, Tripterygium wilfordii, sensitizes cancer cells to apoptosis. Glioblastoma multiforme (GBM), which accounts for most cases of central nervous malignancy, has a very poor prognosis and lacks effective therapeutic inventions. We, therefore, investigated the effects of different concentrations of, and different periods of exposure to, triptolide on cell proliferation and apoptosis in the glioma cell lines, U251MG and U87MG, and in normal human fetal astrocytes. Cell proliferation was investigated by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) assay and growth curve analysis, and apoptosis was assessed from genomic DNA fragmentation. Triptolide showed dose-dependent inhibition of cell proliferation and induction of apoptosis in glioma cells. It also increased the ratio of the pro-apoptotic protein, Bax, to the anti-apoptotic protein, Bcl-2. Since U87MG has the wild-type p53 gene whereas U251MG harbours a mutated p53 gene, our results indicate that triptolide induces apoptosis in GBM cells via a p53-independent pathway. The dose-dependent inhibition of cell proliferation and induction of apoptosis by triptolide may involve upregulation of Bax and downregulation of Bcl-2.     

吴茱萸碱抑制胃癌SGC-7901细胞生长及诱导凋亡作用的研究

目的观察吴茱萸碱对胃癌SGC-7901细胞凋亡的生长抑制作用,并探讨可能的分子机制。方法体外培养人胃癌SGC-7901细胞,分别用0.5、1.0、1.5μmol/L吴茱萸碱及吴茱萸碱＋20μmol/L胱天蛋白酶抑制剂（Z-VAD-FMK）作用于SGC-7901细胞12、24和36 h。四唑盐（MTT）比色法观察吴茱萸碱对SGC-7901细胞增殖活性的影响。采用膜联蛋白V-异硫氰酸荧光素/碘化丙啶（Annexin V-FITC/PI）双染细胞,流式细胞仪检测SGC-7901细胞凋亡的情况。结果吴茱萸碱能抑制SGC-7901细胞增殖,MTT结果显示具有时间和剂量的依赖性;流式细胞仪结果显示吴茱萸碱可诱导SGC-7901细胞凋亡,且随剂量和时间的增加作用增强;Z-VAD-FMK可部分抑制吴茱萸碱诱导该细胞的凋亡作用。结论吴茱萸碱能抑制人胃癌SGC-7901细胞增殖并诱导其凋亡,且在加入Z-VAD-FMK后吴茱萸碱仍可诱导其凋亡,但作用减弱。该研究结果提示吴茱萸碱诱导胃癌SGC-7901细胞凋亡除胱天蛋白酶途径外,还存在其他的诱导凋亡途径。     

Adenovirus-mediated p53 treatment enhances photodynamic antitumor response

Photodynamic therapy (PDT) has been reported to be effective for treating various tumors and to induce apoptosis in many tumor cells. In this study, we evaluated the ability of PDT combined with a tumor suppressor factor, recombinant adenovirus p53 (AdCMVp53), to induce apoptosis as well as cell growth inhibition in CaSki human cervical cancer cells and in nude mice with implanted CaSki cells. To examine levels of apoptosis, CaSki cells were treated with PDT and/or AdCMVp53, and an annexin V-staining assay was then conducted. In addition, Western blot analysis was done to identify p53 induction at the cellular and tumor tissue levels. PDT+AdCMVp53 cotreatment caused remarkable inhibition of CaSki cell proliferation, as compared with the individual treatments. In parallel with the inhibition of cell proliferation, the cotreatment caused a significantly greater increase in the annexin V-stained cell population compared with the individual treatments, as determined by fluorescence-activated cell-sorting analysis. The Western blotting assay also showed significantly more cellular p53 expressed after PDT+AdCMVp53 cotreatment than after each separate treatment. This was consistent with observations of tumor tissue in the mouse system. However, apoptosis- related protein, p21, was significantly suppressed by PDT+AdCMVp53 cotreatment, contrary to treatment with AdCMVp53 alone. Taken together, these findings suggest that PDT plus AdCMVp53 gene therapy exerts more potent antitumor effects on human cervical cancer cells, with induction of apoptosis at least through activation in p53 protein at the cellular and tumor tissue levels.     

幽门螺杆菌根除前后胃癌组织中MUC5AC表达的变化

目的调查幽门螺杆菌（Hp）根除前后的胃癌组织中MUC5AC表达的变化。方法采用Western Blot 和 PCR RT-PCR方法检测根除Hp前后的胃癌组织中MUC5AC蛋白及mRNA的相对含量。以Hp阴性的胃癌组织作为对照组。结果胃癌组织中MUC5AC蛋白及mRNA的表达在根除Hp后较根除前明显增高（P〈0.05）,但仍然明显低于对照组。Hp阳性胃癌组织中MUC5AC的表达明显低于Hp阴性胃癌组织的表达;转移淋巴结数目大于5的Hp阳性胃癌组织中的MUC5AC相对含量明显低于Hp阴性胃癌组织。结论胃癌组织中Hp感染与MUC5AC的降低相关,但是它可能不是胃癌组织中MUC5AC减少的唯一因素,MUC5AC的减少可能只是Hp引起胃癌的一个中间环节,但可能是胃癌发生和进展的一个重要因素。     

幽门螺杆菌感染与慢性特发性血小板减少性紫癜相关性的临床初步探讨

目的 初步探讨幽门螺杆菌(Hp)感染与慢性特发性血小板减少性紫癜(ITP)的关系.方法 以查体人群(118例)和ITP患者(38例)作为研究对象.经血清Hp检查、13C呼气试验确定有无Hp感染,Hp阳性者测定抗CagA抗体滴度.Hp阳性ITP患者行Hp根除治疗并随访12周观察血小板是否增加.结果 入选查体人群118例,Hp阳性者79例,Hp阴性者39例.其外周血PIJT、WBC、RBC差异无统计学意义(P均＞0.05).ITP患者38例,Hp阳性22例,其中Caga阳性18例;Hp阴性16例.查体人群中,血清CagA阳性者29例.ITP患者中Hp阳性率为57.9%,查体人群中Hp阳性率为66.9%;二者阳性率相比,差异无统计学意义(Pearson Chi-square 1.032;P=0.310).而在Hp阳性者中,ITP患者CagA阳性率(81.8%)明显高于查体人群(36.7%),差异有统计学意义(Pearson Chi-square 14.074;P=0.001).ITP患者根治Hp后疗效观察:无效:12例,2例男性,4例cagA阴性.有效+显效10例,100%为女性,100%cagA阳性.治疗有效率45.5%.结论 正常人群感染Hp后,外周血白细胞、Hb、血小板无显著性改变.ITP患者与查体人群,二者Hp阳性率相比,差异无统计学意义.而在Hp阳性者中,ITP患者CagA阳性率明显高于查体人群.ITP患者,经抗Hp治疗后,部分治疗有效.随着随访时间的延长,经根治Hp治疗后,部分患者PLT水平逐渐回升,平均5周可恢复至正常范围.     

Sepsis from Pseudomonas aeruginosa pneumonia decreases intestinal proliferation and induces gut epithelial cell cycle arrest

OBJECTIVES: To evaluate whether the up-regulation in sepsis-induced gut epithelial apoptosis is balanced by an increase in intestinal proliferation and to assess mechanisms affecting the gut's regenerative response to overwhelming infection. DESIGN: Prospective, randomized, controlled study. SETTING: Animal laboratory in a university medical center. INTERVENTIONS: Mice were subjected to intratracheal injection of Pseudomonas aeruginosa and killed between 1.5 and 24 hrs after induction of pneumonia-induced sepsis to assess for gut epithelial proliferation and cell division and for apoptosis. Animals were compared with sham-operation controls, septic transgenic mice that overexpress Bcl-2 throughout their small intestinal epithelium, and septic p53-/- mice. MEASUREMENTS AND MAIN RESULTS: Proliferation and cell division were assessed by measuring S-phase and M-phase cells in intestinal crypts. The number of S-phase cells showed a progressive decline at all time points measured, with a 5-fold decrease in proliferation between control animals and septic mice 24 hrs after intratracheal injection of pathogenic bacteria (p <.0001). In contrast, cells in M-phase remained constant for the first 12 hrs after the onset of sepsis, but increased nearly 50% at 24 hrs after instillation of P. aeruginosa (p <.005). Both the decrease in S-phase cells and the increase in M-phase cells were partially suppressible in Bcl-2 overexpressors, but cellular proliferation and division were similar between septic p53-/- and p53+/+ mice. Crypt apoptosis was increased at all time points, with maximal death occurring between 12 and 24 hrs. CONCLUSIONS: Sepsis from P. aeruginosa pneumonia induces a p53-independent decrease in gut epithelial proliferation. Despite an increase in sepsis-induced intestinal apoptosis, there is no compensatory increase in intestinal epithelial proliferation, and there is evidence of a cell cycle block with an accumulation of cells in M-phase. Decreasing gut apoptosis by overexpression of Bcl-2 is associated with a partial reversal of the effect of sepsis on the cell cycle.     

Green Tea Polyphenols Reduce Gastric Epithelial Cell Proliferation and Apoptosis Stimulated by Helicobacter pylori Infection

Recently the finding of gastric cancer in Helicobacter pylori (H. pylori)-infected mouse models was reported. Studies of humans and animal models have shown that H. pylori infection stimulates gastric epithelial cell proliferation and apoptosis. Polyphenols contained in green tea and related compounds were reported to have a variety anti-tumor effects and bactericidal properties. We studied the effect of green tea polyphenols on gastric cell proliferation and apoptosis in an H. pylori-infected mouse model. This model was prepared by inoculating Balb/c mice with 10(8) cfu of H. pylori (NCTC 11637 strain) by gavage. Beginning 18 weeks after inoculation, 0.5% polyphenols were given in drinking water every day for 2 weeks. Mice were sacrificed 1 h after bromodeoxyuridine (BrdU) was given i.p. for preparation of paraffin-embedded specimens. Cell proliferation and apoptosis were examined by the avidin-biotin complex method using anti-BrdU antibody and the TUNEL method, respectively. H. pylori infection resulted in increased BrdU-labeled cells in both the antrum and the bodies. Administration of polyphenols suppressed this increased proliferation. H. pylori infection increased apoptotic cells in both the antrum and the corpus in comparison with controls. This increase was not seen in H. pylori-infected mice given polyphenols. We conclude the administration with polyphenols might suppress gastric carcinogenesis that is in part related to H. pylori infection.     

慢性胃炎病人血清CagA抗体和胃黏膜炎症活动性及Hp密度检测

目的探讨细胞毒素相关基因A（CagA）对慢性胃炎胃黏膜组织炎症活动性程度、幽门螺杆菌（Hp）密度的影响。方法对慢性胃炎病人80例进行血清CagA抗体检测及胃黏膜组织病理学检查,分析CagA抗体与慢性胃炎病人胃黏膜病理变化之间的关系,以及CagA抗体对慢性胃炎病人Hp密度的影响。结果慢性胃炎病人血清CagA抗体阳性52例（65.0%）,阴性28例（35.0%）。同性别CagA抗体阳性组与阴性组炎症活动性程度比较,差异有显著性（χ2=12.161、6.111,P〈0.05）;肠上皮化生、萎缩、Hp密度差异均无显著性（P〉0.05）。不同性别间各指标差异无显著性（P〉0.05）。结论慢性胃炎CagA抗体阳性病人的胃黏膜中性粒细胞浸润更明显,人体不能有效清除Hp在胃黏膜中的定植。     

Replication and cytolysis of an E1B-attenuated adenovirus in drug-resistant ovarian tumour cells is associated with reduced apoptosis

Therapeutic approaches which are effective in tumour cells resistant to conventional chemotherapy would be of value. An E1B 55 kDa-deleted adenovirus (ONYX-015) induces lysis in cells with mutant p53, although the specificity of these observations for different cell types is unclear. We have used a matched set of drug-resistant human ovarian tumour cell lines to examine the potential of ONYX-015 for preferential replication and lysis of drug-resistant ovarian tumour cells with documented alterations in p53 function. Marked preferential replication of ONYX-015 is observed after infection of mutant p53 transfectant and cisplatin-resistant derivatives, compared to the wild-type p53 expressing parental A2780 line. Infection causes increased cytopathic effects in vitro and inhibition of tumour growth in vivo of the drug-resistant derivatives, but not the parental line. In apparent contrast, increased apoptosis and reduced clonogenic survival is induced by ONYX-015 infection of the chemosensitive parental cell line. ONYX-015 induces increased pro-apoptotic BAX and reduced anti-apoptotic BCLX(L) in parental cells, but not in the resistant derivative A2780/cp70. We propose that induction of apoptosis is one factor which prevents ONYX-015 spread and cytolysis after infection of chemosensitive cells, while it is the failure to engage apoptosis in drug-resistant cells that allows preferential viral replication, spread and cytolysis.     

慢性胃炎镜下病理特点与幽门螺杆菌血清抗体类型的相关性研究

目的探讨不同菌型幽门螺杆菌(Hp)血清抗体类型与慢性胃炎发生率及其病理特点的关系。方法选取该院2014年11月-2016年11月收治疗的上腹部不适的患者650例作为研究对象,所有患者均行胃镜检查,所有患者均行Hp呼气实验检查Hp,并采用酶联免疫吸附法检查患者血清中Hp抗体类型,采用Logistic分析Hp感染与慢性胃炎发生及其病理特点的关系。结果 650例患者中慢性胃炎93例,以Hp(-)为对照组,Hp(+)组患者慢性胃炎风险是Hp(-)患者的1.6倍,Hp(+)Cag A-Ig G+患者慢性胃炎是Hp(-)患者的2.3倍,Hp(+)Cag A-Ig G-患者慢性胃炎是Hp(-)患者的1.8倍。慢性萎缩性胃炎(CAG)患者27例中,以Hp(-)为参照,Hp(+)组患者CAG是Hp(-)患者的2.1倍,Hp(+)Cag A-Ig G-患者CAG是Hp(-)患者的1.9倍,Hp(+)Cag A-Ig G+患者CAG是Hp(-)患者的2.9倍;CAG伴不典型增生(Dys)患者13例中,以Hp(-)为参照,Hp(+)组患者是Hp(-)患者的2.4倍,Hp(+)Cag A-Ig GCAG伴Dys是Hp(-)患者的3.5倍,Cag A-Ig G+的慢性胃炎患者炎症反应程度明显比Hp(-)及Cag A-Ig G-的患者高,且炎症活动度明显更高。结论 Hp(+)Cag A-Ig G+感染加重慢性胃炎患者胃黏膜患者的炎症反应,同时促进慢性胃炎向CAG伴Dys发展。