Prevalence of Helicobacter pylori infection among patients with esophageal carcinoma

BACKGROUND Helicobacter pylori(H.pylori)is a widespread microorganism related to gastric adenocarcinoma(AC).In contrast,it has been reported that an inverse association exists between H.pylori infection and esophageal carcinoma.The mechanisms underlying this supposedly protective effect remain controversial.AIM To determine the prevalence of H.pylori infection in esophageal carcinoma patients,we performed a retrospective observational study of esophageal tumors diagnosed in our hospital.METHODS We retrospectively reviewed the prevalence of H.pylori infection in a cohort of patients diagnosed with esophageal carcinoma.Concomitant or previous proton pump inhibitor(PPI)usage was also recorded.RESULTS A total of 89 patients with esophageal carcinoma(69 males,77.5%),with a mean age of 66 years(range,26-93 years)were included.AC was the most frequent pathological variant(n=47,52.8%),followed by squamous cell carcinoma(n=37,41.6%).Fourteen ACs(29.8%)originated in the gastroesophageal junction and 33(70.2%)in the esophageal body.Overall,54 patients(60.7%)presented at stages III and IV.Previous H.pylori infection occurred only in 4 patients(4.5%),3 with AC(6.3%of all ACs)and 1 with squamous cell carcinoma(2.7%of all squamous cell tumors).All patients with previous H.pylori infection had stage III-IV.Only one patient had received prior H.pylori eradication therapy,whereas 86(96.6%)had received previous or concomitant PPI treatment.CONCLUSION In our cohort of patients,and after histologic evaluation of paraffin-embedded primary tumors,we found a very low prevalence of previous H.pylori infection.We also reviewed the medical history of the patients,concluding that the majority had received or were on PPI treatment.The minimal prevalence of H.pylori infection found in this cohort of patients with esophageal carcinoma suggests a protective role.

Silencing of peroxiredoxin 2 suppresses proliferation and Wnt/β-catenin pathway,and induces senescence in hepatocellular carcinoma

Hepatocellular carcinoma(HCC),a common malignancy worldwide,still lacks effective clinical treatment.The study aimed to investigate the oncogenes that affect the progression of HCC and their possible mechanisms.In our study,we initially confirmed a higher level of PRDX2 in the bile of HCC patients compared to those with choledocholithiasis by 2-DE,LC-MS,and ELISA.Subsequently,we demonstrated the high expression of peroxiredoxin 2(PRDX2)in HCC based on the TCGA database and clinical sample analysis.Furthermore,PRDX2 overexpression enhanced the viability of HCC cells.And PRDX2 silencing induced senescence of HCC cells.In vivo,knockdown of PRDX2 significantly reduced the weight of xenograft tumors.PRDX2 also was found to activate the Wnt/β-catenin pathway by inducingβ-catenin nuclear translocation.Consequently,we proved that silencing PRDX2 could inhibit proliferation and Wnt/β-catenin pathway while promoting senescence in HCC cells.

抗J亚群禽白血病病毒囊膜糖蛋白特异性单克隆抗体的研制及其特性

:J亚群禽白血病病毒 (ALV -J)是一种主要感染肉用型鸡的反转录病毒。本研究用表达ALV -J囊膜蛋白基因产物的Sf9细胞免疫Balb/c小鼠 ,取其脾脏细胞与骨髓瘤细胞NS1进行融合 ,获得了 4株特异性抗ALV -J的单克隆抗体。免疫荧光分析结果表明 ,3株单克隆抗体仅与所试验的ALV -J毒株反应 ,而不能与ALV的A、B、C、D和E亚群的毒株反应。有趣的是 ,有一株单克隆抗体可以与所有试验的外源性ALV毒株反应 ,但不与内源性的E亚群反应。WesternBlot和免疫沉淀试验结果表明 ,单克隆抗体识别的ALV -J囊膜糖蛋白的分子量为 90 - 94kD ,识别未糖基化的囊膜蛋白分子量约为 5 3kD。用这些单克隆抗体能检测出ALV -J病毒感染鸡胚成纤维细胞中的病毒抗原。这些结果提示这些单克隆抗体可用于ALV

小檗碱抗肿瘤作用与Wnt/-βcaten in信号转导关系

目的证明小檗碱抗肿瘤作用机制可能与信号转导过程的调控有关。方法采用细胞增殖抑制和Hoechst 33258染色凋亡实验比较小檗碱和黄连总碱对人结肠癌HCT116和SW480细胞的作用。利用Tcf-4报告基因研究小檗碱对肿瘤细胞的信号转导影响。结果小檗碱在5—40mg·L^-1浓度范围内呈浓度依赖性和时间依赖性抑制人结肠癌HCT116和SW480细胞的增殖；小檗碱（20mg·L^-1）处理72h后的HCT116和SW480细胞出现明显凋亡；相当于小檗碱浓度的黄连总碱有类似于小檗碱的作用。20—40mg·L^-1小檗碱和黄连总碱均能明显抑制β—catenin／Tcf介导的转录活性。结论黄连总碱的抗肿瘤作用可能与其主要成分小檗碱有关；其抗肿瘤作用机制至少与抑制Wnt／β-catenin信号通路有关。

原位PCR和原位杂交检测蛋鸡J亚群禽白血病病毒

根据ALV_J原型株HPRS10 3株gp85基因的内部序列 ,和pol基因的 3′端设计一对引物H5 H7。从发生ML病死蛋用型鸡的肿瘤、骨髓、肝脏、脾脏和输卵管组织中提取总RNA ,反转录为cDNA ,经PCR扩增得到长度为 5 4 5bp的ALV_JcDNA特异性探针。探针定位于 5 2 5 8～ 5 80 2bp。将病鸡的组织石蜡切片置HybaidExpress原位PCR仪平台上 ,以H5 H7为引物进行原位PCR扩增。应用地高辛标记的cDNA探针对原位PCR扩增后切片进行了原位杂交检测。结果在待检组织肿瘤组织、十二指肠、骨髓中出现明显的阳性信号。睾丸、肺、胰腺、大脑、输卵管、肾脏均检出散在的阳性信号。这是国内外首次从分子水平证明蛋鸡J亚群禽白血病。

shRNA表达载体构建方法的优化

目的探讨shRNA表达载体的构建方法 ,以加速RNA干扰研究的进程。方法对shRNA表达载体的构建过程进行分析和监测 ,并加以优化。结果发现shRNA表达载体构建的退火过程容易产生障碍 ,经优化退火缓冲液的NaCl含量后 ,能明显提高退火效率及shRNA表达载体构建的成功率。结论shRNA表达载体构建的退火过程需加以关注 ,退火缓冲液中NaCl含量应提高至 2 0

禽白血病病毒J亚群囊膜蛋白env基因的克隆和表达

禽白血病病毒J亚群 (ALV J)是 90年代鉴定出的ALV的新亚群 ,其囊膜蛋白env基因序别与ALVA E亚群有相当大的差别。为研究ALV Jenv基因及其表达产物的特点 ,用PCR方法扩增出ADOL 4817毒株的env基因 ,并克隆进TA载体 ,经电泳鉴定大小为 1 7kb。将克隆出的env基因与杆状病毒pBlue Bac4表达质粒DNA连接 ,构建成转移性载体 pBac4817env ,通过与Bac N Blue杆状病毒DNA共转染 ,获得了重组病毒rBac4817env 2。该重组杆状病毒感染Sf9细胞 ,能高效表达env基因产物。免疫荧光分析结果证明 ,单克隆抗体G2或多价兔抗envgp37血清能识别Sf9细胞中重组env基因表达的特异性抗原 ;Westernblotting分析结果表明 ,表达的重组基因产物的分子量大小约为 90kD～ 94kD。用这些重组基因产物免疫鸡可以诱导鸡产生出高滴度的抗ALV J特异性抗体。这一结果提示 ,这种杆状病毒表达的重组基因产物有助于ALV

禽白血病病毒J亚群env基因产物的抗原性分析

用PCR扩增方法将ALV Jenv基因不同片段进行了克隆 ,并构建了env基因片段GST融合蛋白载体。用Westernblot实验证明 ,大肠杆菌表达的不同env基因片段的GST融合蛋白能与相应的单克隆抗体产生特异性反应性 ,单克隆抗体JE9和G2识别的抗原位点位于gp85的氨基酸 6 5～ 1 5 5区域 ,而I45识别的抗原表位位于env基因的另一区域 (1 5 6～ 2 3 3位氨基酸 )。ALV J氨基酸多肽而非糖基化位点决定ALV

钙结合蛋白S100A2对Wnt/β-catenin信号途径活性的上调作用

目的研究钙结合蛋白S100A2对Wnt/β-catenin信号途径活性的影响,并探讨其可能的机制。方法原核诱导表达GST-hS100A2,经纯化后加入骨肉瘤细胞株MG63和人结肠癌细胞株HCT116的培养液中,Western blot检测细胞中β-catenin含量的变化;荧光素酶活性分析法检测S100A2对HEK293细胞中β-catenin/TCF4活性的影响;以表达GSK-3β、DVL、Axin的相应质粒分别转染HEK293细胞,GST-Pulldown/Western blot实验检测S100A2与这些蛋白质和β-catenin之间的相互作用。结果S100A2使MG63和HCT116细胞中β-catenin含量增加、β-catenin/TCF4活性增强;S100A2分别与β-catenin和GSK-3β之间存在相互作用,而与DVL和Axin之间则未发现相互作用。结论S100A2可以上调Wnt/β-catenin信号途径的活性,其机制可能涉及S100A2与β-catenin和GSK-3β之间的相互作用。

马立克氏病病毒meq基因功能研究

从马立克氏病病毒(MDV)不同致病型毒株meq基因序列、meq基因产物及其细胞内表达特性和meq蛋白生物学功能的研究探讨马立克氏病病毒致瘤基因meq功能。完成了648A、CVI988/Rispens、814、广西地方毒株G2、N、0093、0095、0297、0304共9个MDV毒株meq基因的序列测定。MDV不同致病型毒株的。meq基因序列相对比较保守,它们相互间核苷酸和氨基酸序列的同源性均很高;与所有7个致瘤的MDV毒株相比,在2个MDV-1弱毒疫苗CVl988/Rispens株和814株发现有二个特征性位点突变;此外,还在其ORF中首次发现含15个氨基酸残基(EELCAQLCSTPPPPI)的2个重复和含6个氨基酸残基(PPICTP)的4个重复,全分布在MEQ蛋白C-端的转录激活域内。MEQ蛋白的表达仅局限于感染细胞的核内,而且随感染时间增加,具有从核质向核仁和核膜转移趋向;Western Blotting和免疫沉淀试验证实重组杆状病毒感染细胞裂解物中有大小约为60 kD的特异带。利用表达的MEQ蛋白产物免疫BALB/c小鼠,获得的杂交瘤细胞被克隆并与MDV感染的鸡胚成纤维细胞(CEF)做免疫荧光试验(FA),获得4株稳定产生抗MEQ蛋白单克隆抗体(McAb)的杂交瘤细胞,其中3G12E6单克隆抗体能够检测到MDV致瘤株感染的CEF及自然MD肿瘤细胞中表达的meq基因产物,而CVl988/Rispens感染的细胞则未检测到。发现细?

RNA干扰抑制结肠癌细胞血管内皮生长因子表达

目的:应用RNA干扰技术抑制结肠癌血管内皮生长因子(VEGF)表达。方法:将VEGF基因作为RNA干扰的靶区,通过E-RNAi网上提供的服务,设计两个特异的RNA干扰序列,将其装入含U6启动子的载体上,构建成抗VEGF基因的小发夹样RNA(shRNA)表达载体,再转染人结肠癌细胞HT29,通过RT-PCR、Northern blotting、免疫荧光和Western blotting,观察VEGF表达受抑的程度。结果:成功构建了两种抗VEGF基因的shRNA表达载体,RT-PCR、Northern blotting、免疫荧光和Western blotting,均发现其能明显抑制HT29细胞VEGF基因的表达,抑制率分别达42%、88%、73%和82%。结论:针对VEGF基因的shRNA表达载体能够明显抑制结肠癌细胞VEGF基因的表达。

凋亡抑制因子survivin基因的克隆及在真核细胞中的表达

目的 :克隆Survivin编码序列并在真核细胞中表达。方法 :RT -PCR扩增Survivin编码序列 ,建立与绿色荧光蛋白(GFP)共表达的融合重组质粒 ,转染哺乳动物细胞 ,利用GFP绿色荧光和WesternBlot检测Survivin蛋白的表达。结果 :RT -PCR扩增出 4 2 1bp的Survivin编码序列 ,构建融合重组质粒pEGFP -C1-Survivin ,利用GFP荧光和WesternBlot证实Sur vivin蛋白的表达。结论 :成功克隆Survivin基因并在真核细胞中表达。

抑制Dicer基因对shRNA功能发挥的影响

本文将Dicer基因的RNA酶III结构域作为靶区,设计并构建了两个抗Dicer基因的小发夹样RNA(shRNA)表达载体,将其转染2215、结肠癌TC细胞和基因组中整合有绿色荧光蛋白基因(GFP)的HepG2A9细胞,通过RT-PCR评价RNA干扰抑制Dicer基因表达的效率;当HepG2A9细胞Dicer基因表达被上述RNA干扰抑制时,再转染抗GFP的shRNA表达载体,通过RT-PCR和荧光显微镜观察GFP表达水平。结果显示,在不同细胞系中,这两个抗Dicer基因shRNA表达载体,均能明显抑制Dicer基因的表达;当Dicer基因受抑时,后续转染抗GFP的shRNA表达载体不能有效抑制GFP的表达。结果表明,抗Dicer基因shRNA表达载体,能够明显抑制Dicer基因的表达;shRNA表达载体的功能发挥需要Dicer酶的直接参与。

Mesenchymal stem cells: Molecular characteristics and clinical applications

Mesenchymal stem cells (MSCs) are non-hematopoietic stem cells with the capacity to differentiate into tissues of both mesenchymal and non-mesenchymal origin. MSCs can differentiate into osteoblastic, chondrogenic, and adipogenic lineages, although recent studies have demonstrated that MSCs are also able to differentiate into other lineages, including neuronal and cardiomyogenic lineages. Since their original isolation from the bone marrow, MSCs have been successfully harvested from many other tissues. Their ease of isolation and ex vivo expansion combined with their immunoprivileged nature has made these cells popular candidates for stem cell therapies. These cells have the potential to alter disease pathophysiology through many modalities including cytokine secretion, capacity to differentiate along various lineages, immune modulation and direct cell-cell interaction with diseased tissue. Here we first review basic features of MSC biology including MSC characteristics in culture, homing mechanisms, differentiation capabilities and immune modulation. We then highlight some in vivo and clinical evidence supporting the therapeutic roles of MSCs and their uses in orthopedic, autoimmune, and ischemic disorders.

Targeting of circulating hepatocellular carcinoma cells to prevent postoperative recurrence and metastasis

Currently,the main treatment for hepatocellular carcinoma(HCC)involves the surgical removal of tumors or liver transplantation.However,these treatments are often not completely curative,as they are associated with a risk for postoperative recurrence and metastasis.Circulating tumor cells(CTCs)are increasingly recognized as the main source for recurrence and metastasis after radical hepatectomies are performed.Many studies have demonstrated the association between the presence of either pre-or postoperative CTCs and an increased risk for HCC recurrence.To improve the therapeutic outcome of HCC,a personalized,comprehensive and multidisciplinary approach should be considered,involving the application of appropriate diagnostic and therapeutic measures targeting HCC CTCs in different stages throughout the course of treatment.This article proposes some HCC CTC-based strategies for the treatment of HCC,including the monitoring of HCC CTCs before,during and after radical hepatectomy,therapeutic targeting of HCC CTCs,prevention of the generation and colonization of CTCs,as well as the use of CTC indexes for the selection of indications,prediction of prognoses,and planning of individualized therapeutic regimens.Innovation and technological development of therapies targeting CTCs,as well as their translation into clinical practice,will help to effectively reduce postoperative recurrence and metastasis,and significantly prolong the survival of HCC patients.

Improved method increases sensitivity for circulating hepatocellular carcinoma cells

AIM:To improve an asialoglycoprotein receptor(ASGPR)-based enrichment method for detection of circulating tumor cells(CTCs)of hepatocellular carcinoma(HCC).METHODS:Peripheral blood samples were collected from healthy subjects,patients with HCC or various other cancers,and patients with hepatic lesions or hepatitis.CTCs were enriched from whole blood by extracting CD45-expressing leukocytes with monoclonal antibody coated-beads following density gradient centrifugation.The remaining cells were cytocentrifuged on polylysine-coated slides.Isolated cells were treated by triple immunofluorescence staining with CD45antibody and a combination of antibodies against ASGPR and carbamoyl phosphate synthetase 1(CPS1),used as liver-specific markers,and costained with DAPI.The cell slide was imaged and stained tumor cells that met preset criteria were counted.Recovery,sensitivity and specificity of the detection methods were determined and compared by spiking experiments with various types of cultured human tumor cell lines.Expression of ASGPR and CPS1 in cultured tumor cells and tumor tissue specimens was analyzed by flow cytometry and triple immunofluorescence staining,respectively.RESULTS:CD45 depletion of leukocytes resulted in a significantly greater recovery of multiple amounts of spiked HCC cells than the ASGPR+selection(P s<0.05).The expression rates of either ASGPR or CPS1were different in various liver cancer cell lines,ranging between 18%and 99%for ASGPR and between 9%and 98%for CPS1.In both human HCC tissues and liver cancer cell lines,there were a few HCC cells that did not stain positive for ASGPR or CPS1.The mixture of monoclonal antibodies against ASGPR and CPS1identified more HCC cells than either antibody alone.However,these antibodies did not detect any tumor cells in blood samples spiked with the human breastcancer cell line MCF-7 and the human renal cancer cell line A498.ASGPR+or/and CPS1+CTCs were detected in 29/32(91%)patients with HCC,but not in patients with any other kind of cancer or any of the other test subjects.Furthermore,the improved method detected a higher CTC count in all patients examined than did the previous method(P=0.001),and consistently achieved 12%-21%higher sensitivity of CTC detection in all seven HCC patients with more than 40 CTCs.CONCLUSION:Negative depletion enrichment combined with identification using a mixture of antibodies against ASGPR and CPS1 improves sensitivity and specificity for detecting circulating HCC cells.

Astragalus polysaccharide enhances immunity and inhibits H9N2 avian influenza virus in vitro and in vivo

This study investigated the humoral immunization of Astragalus polysaccharide （APS） against HgN2 avian influenza virus （H9N2 AIV） infection in chickens. The effects of APS treatment on H9N2 infection was evaluated by an Mqq- [3（4, 5-dimethylthiazol-2-yl）-2, 3-diphenyl tetrazolium bromide] assay and analysis of MFIC and cytokine mRNA expression. The effect on lymphocyte and serum antibody titers in vivo was also investigated. IL-4, IL-6, IL-10, LITAF, IL-12 and antibody titers to H9N2 AIV wet enhanced in the first week after APS treatment. The results indicated that APS treatment reduces H9N2 AIV replication and promotes early humoral immune responses in young chickens.This study investigated the humoral immunization of Astragalus polysaccharide （APS） against HgN2 avian influenza virus （H9N2 AIV） infection in chickens. The effects of APS treatment on HgN2 infection was evaluated by an M]q- [3（4, 5-dimethylthiazol-2-yl）-2, 3-diphenyl tetrazolium bromide] assay and analysis of MHC and cytokine mRNA expression. The effect on lymphocyte and serum antibody titers in vivo was also investigated. IL-4, IL-6, IL-10, LITAF, IL-12 and antibody titers to PIgN2 AIV were enhanced in the first week after APS treatment. The results indicated that APS treatment reduces HgN2 AIV replication and promotes early humoral immune responses in young chickens.

Anti-tumor effect of 5-aza-2'-deoxycytidine by inhibiting telomerase activity in hepatocellular carcinoma cells

AIM:To investigate the effect of the demethylating reagent 5-aza-2'-deoxycitidine(DAC) on telomerase activity in hepatocellular carcinoma(HCC) cell lines,SMMC-7721 and HepG2.METHODS:The related gene expression in cell lines was examined by real-time reverse transcription-polymerase chain reaction and Western blotting analysis.The telomerase activity was examined by telomeric repeat amplification protocol-enzyme-linked immunosorbent assay and DNA methylation was determined by methylation-specific polymerase chain reaction.RESULTS:The telomerase activity was significantly reduced in both cell lines treated with DAC,accompanied by downregulation of telomerase reverse transcriptase(hTERT).We also observed the effect of DAC on the methylation status of hTERT promoter and the expression of regulatory genes,such as c-myc,p15,p16,p21,E2F1,and WT1.The methylation status of hTERT promoter could be reversed in SMMC-7721 by DAC,but not in HepG2 cells.However,p16 expression could be reactivated by demethylation of its promoter,and c-Myc expression was repressed in both cell lines.Moreover,DAC could enhance the sensitivity to the chemotherapeutic agents,such as cisplatin,by induction of apoptosis of HCC cells.CONCLUSION:The DAC exerts its anti-tumor effects in HCC cells by inhibiting the telomerase activity.

Risk factors for intrahepatic cholangiocarcinoma:A case-control study in China

AIM: To carry out a hospital-based case-control study to investigate risk factors for intrahepatic cholangiocarcinoma (ICC) in China. METHODS: A total of 312 ICC cases and 438 matched controls were included in the study. The presence of diabetes mellitus,hypertention,hepatolithiasis,primary sclerosing cholangitis,liver fluke infection (Clonorchis sinensis ),was investigated through clinical records. Blood from all participants was tested for hepatitis B surface antigen (HBsAg) and anti-HCV antibodies. Odds ratios (OR) and 95% confi dence intervals (95% CI) were estimated using conditional logistic regression. RESULTS: Compared with controls,ICC patients had a higher prevalence of HBsAg seropositivity (48.4% vs 9.6%,P < 0.000),and hepatolithiasis (5.4% vs 1.1%,P = 0.001). By multivariate analysis,the signif icant risk factors for development of ICC were HBsAg seropositivity (adjusted OR,8.876,95% CI,5.973-13.192),and hepatolithiasis (adjusted OR,5.765,95% CI,1.972-16.851). The prevalence of anti-HCV seropositivity,diabetes mellitus,hypertention,cigarette smoking,and alcohol consumption were not significantly different between cases and controls. CONCLUSION: These findings suggest that HBV infection and hepatolithiasis are strong risk factors for development of ICC in China.

Growth factor-and cytokine-driven pathways governing liver stemness and differentiation

Liver is unique in its capacity to regenerate in response to injury or tissue loss. Hepatocytes and other liver cells are able to proliferate and repopulate the liver. However, when this response is impaired, the contribution of hepatic progenitors becomes very relevant. Here, we present an update of recent studies on growth factors and cytokine-driven intracellular pathways that govern liver stem/pro-genitor cell expansion and differentiation, and the rel-evance of these signals in liver development, regeneration and carcinogenesis. Tyrosine kinase receptor signaling, in particular, c-Met, epidermal growth factor receptors or fibroblast growth factor receptors, contribute to prolifera-tion, survival and differentiation of liver stem/progenitor cells. Different evidence suggests a dual role for the trans-forming growth factor (TGF)-β signaling pathway in liver stemness and differentiation. On the one hand, TGF-βmediates progression of differentiation from a progenitor stage, but on the other hand, it contributes to the expan-sion of liver stem cells. Hedgehog family ligands are nec-essary to promote hepatoblast proliferation but need to be shut off to permit subsequent hepatoblast differentiation. In the same line, the Wnt family and β-catenin/T-cell fac-tor pathway is clearly involved in the maintenance of liver stemness phenotype, and its repression is necessary for liver differentiation during development. Collectively, data indicate that liver stem/progenitor cells follow their own rules and regulations. The same signals that are essential for their activation, expansion and differentiation are good candidates to contribute, under adequate conditions, to the paradigm of transformation from a pro-regenerative to a pro-tumorigenic role. From a clinical perspective, this is a fundamental issue for liver stem/progenitor cell-based therapies.