Detection of bcl-2 and bax expression and bcl-2/JH fusion gene in intrahepatic cholangiocarcinoma

AIM:To investigate the relationship between bcl-2 gene and its related protein bax and intrahepatic cholangiocellular carcinoma(CCC). METHODS:Semi-nested in situ PCR(SNISPCR)and imm- unohistochemistry were performed to detect bcl-2/JH fusion gene and bcl-2,bax protein expression in 29 cases of CCC. RESULTS:No bcl-2/JH fusion gene was found in all cases of CCC,72.4% of 29 cases expressed bcl-2 protein.Bcl-2 protein expression was related to histopathological grades (P<0.05).There was no corresponding relationship between bcl-2/JH fusion gene formation and bcl-2 protein expression in CCC(P<0.05).Bax was expressed in 10.3% of 29 cases. The ratio of bcl-2 to bax in normal liver tissues(3.5 to 1)was different from that in tumor tissues(7.0 to 1). CONCLUSION:It is suggested that bcl-2/JH fusion gene formation is not a frequent event and may not play an important role in the pathogenesis of CCC.However,aberrant ratio of bcl-2 to bax protein expression may be involved in the course of tumorigenesis of CCC.Abnormal bcl-2 protein expression may not be solely resulted from bcl-2/JH fusion gene.     

Correlation between expression of gastrin, somatostatin and cell apoptosis regulation gene bcl-2／bax in large intestine carcinoma

AIM: To explore the correlation between expression of somatostatin (SS), gastrin (GAS) and cell apoptosis regulation gene bcl-2/bax in large intestine carcinoma. METHODS: Sixty-two large intestine cancer tissue samples were randomly and retrospectively selected from patients with large intestine carcinoma. Immunohistochemical staining for bcl-2, bax, GAS, SS was performed according to the standard streptavidin-biotin-peroxidase (S-P) method. According to the semi-quantitative integral evaluation, SS and GAS were divided into three groups as follows. Scores 1-3 were defined as the low expression group, 4-8 as the intermediate expression group, 9-16 as the high expression group. Bax and bcl-2 protein expressions in different GAS and SS expression groups of large intestine carcinoma were assessed. RESULTS: The positive expression rate of bax had a prominent difference between SS and GAS high, intermediate and low expression groups (P<0.05, x2SS = 9.246; P<0.05, x2GAs = 6.981). The positive expression rate of bax in SS high (80.0%, 8/10) and intermediate (76.5%, 13/17) expression groups was higher than that in low expression group (40.0%, 14/35) (P<0.05, X2high vslow = 5.242; P<0.05, x2middle vs low = 6.097). The positive expression rate of bax in GAS high expression group (27.3%, 3/8) was lower than that in low expression group (69.4%, 25/36) (P<0.05, x2 = 4.594). However, bax expression in GAS intermediate expression group (46.7%, 7/15) was lower than that in low expression group, but not statistically significant. The positive expression rate of bcl-2 had a prominent difference between SS and GAS high, intermediate and low expression groups (P<0.05, x2ss = 7.178; P<0.05, x2GAS = 13.831). The positive expression rate of bcl-2 in GAS high (90.9%, 10/11) and intermediate (86.7%, 13/15) expression groups was higher than that in low expression group (44.4%, 16/36) (P<0.05, x2high,vslow = 5.600; P<0.05, x2middle vs low = 7.695). However, the positive expression rate of bcl-2 in SS high (40. 0%, 4/10) and intermediate (47.1%, 8/9) expression groups was lower than that in low expression group (77.1%, 27/35) (P<0.05, x2high vs low = 4.710; P<0.05, x2middle vs low = 4.706). There was a significant positive correlation between the integral ratio of GAS to SS and the integral of bcl-2 (P<0.01, r = 0.340). However, there was a negative correlation between the integral ratio of GAS to the SS and bax the integral of (P<0.05, r= -0.299). CONCLUSION: The regulation and control of gastrin, somatostatin in cell apoptosis of large intestine carcinoma may be directly related to the abnormal expression of bcl-2, bax.     

Immunohistochemical expression of mismatch repair genes： A screening tool for predicting mutator phenotype in liver fluke infection-associated intrahepatic cholangiocarcinoma

AIM: To clarify possible contributions of DNA mismatch repair (MMR) system in carcinogenesis of liver fluke infection-associated intrahepatic cholangiocarcinoma (ICC) by using immunohistochemical assay. METHODS: A total of 29 ICC samples, which had been assessed for genomic instability by a PCR-based method, were used for study. They were examined immunohistochemically to demonstrate protein expression of two MMR genes, hMSH2 and hMLH1. Results obtained were compared with their mutator phenotype assessed previously. RESULTS: Either hMSH2 or hMLH1 protein was obviously expressed in 28 of 29 (96.6%) ICC samples. Positive nuclear localization of hMSH2 or hMLH1 protein was observed in 86.2% (25/29) or 93.1% (27/29) ICC cases, respectively, while their negative nuclear reactivity was only detected in 13.8% (4/29) or 6.9% (2/29) ICC cases analyzed, respectively. CONCLUSION: Our study, probably for the first time, showed through immunohistochemical detection of hMSH2 and hMLH1 gene that DNA MMR system does not play a prominent role in liver fluke infection-associated cholangiocarcinogenesis. These results confirm previous findings on mutational status of these genes assessed through a PCR-based method. The immunohistochemical analysis has proven to be an effective and sensitive approach for screening MMR deficiency regardless of somatic inactivation or promoter hypermethylation of hMSH2 and/or hMLH1 gene. Furthermore, immunohistochemistry is more advantageous compared to mutator phenotyping assay in terms of simplicity, less time consuming and cost effectiveness for screening possible involvements of target MMR genes in tumorigenesis.     

姜黄素对实验性肝纤维化大鼠肝脏bax和bcl-2表达的影响

[目的]探讨姜黄素(curcumin,CUR)对大鼠肝纤维化的防治作用及对肝组织bax和bcl-2蛋白及基因表达的影响.[方法]建立CCl4致大鼠实验性肝纤维化模型.用免疫组化法比较正常组、模型组以及CUR组的bcl2、bax蛋白的表达,用RT-PCR法比较各组肝组织bcb-2、bax mRNA的表达.[结果]CUR组肝组织炎症和纤维化程度较模型组显著减轻.bax蛋白主要表达在肝细胞质,而纤维间隔及汇管区呈低表达;beb-2主要表达在纤维间隔及汇管区.bax及bcl-2在模型组及CUR组表达均高于正常组(P<0.01),CUR组均较模型组降低(P<0.01).bax及bcI-2 mRNA在模型组及CUR组表达比正常组增高(P<0.01,<0.05),CUR组均较模型组降低(P<0.05).[结论]CUR可能通过降低肝细胞表达bax、减少肝细胞凋亡而减轻肝脏损伤,通过降低bcl-2表达、诱导肝星状细胞凋亡发挥减轻肝纤维化的作用,防治大鼠肝纤维化.     

Construction of HCV—core gene vector and its expression in cholangiocarcinoma

AIM: To establish an experimental model for exploring the role of hepatitis C virus (HCV) in the development of cholangiocarcinoma. METHODS: Recombinant plasmid of HCV-core gene was constructed with molecular cloning technique and transfected into QBC939 cells with lipofection.After it was selected with G418, resistant colonies were obtained. The colonies were analysed by immunocytochemistry and Western blotting.The morphology was observed under transmission electron microscope(TEM) and microscope. RESULTS: The recombinant plasmid was proved to carry the target gene by PCR and restriction enzymed mapping. Moreover, it could express HCV-C protein efficiently in QBC939 cells. The HCV-like particles were found in the cytoplasm by electron microscope, which were spherical with a diameter of 50 nm-80 nm possessing outer membrane.The transfected cells had lower differentiation and higher malignant degree under microscope. CONCLUSION: Because HCV-core gene could express steadily in cholangiocarcinoma cells,the transfected tumor cells(QBC939-HCVC) could be used to study the effect of HCV in the development of cholangiocarcinoma.     

Identification of osteopontin as the most consistently over-expressed gene in intrahepatic cholangiocarcinoma： Detection by oligonucleotide microarray and real-time PCR analysis

AIM： To investigate the molecular pathways involved in human cholangiocarcinogenesis by gene expression profiling. METHODS： Oligonucleotide arrays （Affymetrix U133A） were used to establish a specific gene expression profile of intrahepatic CCC in comparison to corresponding non- malignant liver tissue. To validate the expression values of the most overexpressed genes, RT-PCR experiments were performed. RESULTS： Five hundred and fifty-two statistically differentially expressed genes/ESTs （221 probes significantly up-regulated, 331 probes down-regulated; P 〈 0.05; fold change 〉 2; ≥ 70%） were identified. Using these data and two-dimensional cluster analysis,a specific gene expression profile was obtained allowing fast and reproducible differentiation of CCC, which was confirmed by supervised neuronal network modelling. The most consistently overexpressed gene （median fold change 33.5, significantly overexpressed in 100%） encoded osteopontin. Furthermore, an association of various genes with the histopathological grading could be demonstrated. CONCLUSION： A highly specific gene expression profile for intrahepatic CCC was identified, allowing for its fast and reproducible discrimination against non- malignant liver tissue and other liver masses. The most overexpressed gene in intrahepatic CCC was the gene encoding osteopontin. These data may lead to a better understanding of human cholangiocarcinogenesis.     

肝内胆管癌细胞9810凋亡和bcl-2基因表达关系研究

应用阿霉素诱导肝内胆管癌细胞 9810凋亡 ,研究其凋亡与bcl- 2基因表达之间的关系。应用流式细胞仪检测肝内胆管癌细胞凋亡的情况 ,并同时检测bcl- 2基因表达。随着阿霉素浓度的增加 ,细胞凋亡率逐步上升 ,同时bcl- 2基因表达下调。bcl- 2基因表达下调是阿霉素诱导肝内胆管癌细胞凋亡机制之一。     

Bcl-2及相关蛋白bax在肝脏良恶性病变中的表达

Ｂｃｌ－２及相关蛋白ｂａｘ在肝脏良恶性病变中的表达郭琳琅１曹长安１王友顺２第一军医大学珠江医院１病理科２普外科广东省广州市５１０２８２Ｓｕｂｊｅｃｔｈｅａｄｉｎｇｓｌｉｖｅｒｃｉｒｈｏｓｉｓ；ｌｉｖｅｒｎｅｏｐｌａｓｍｓ；ｃａｒｃｉｎｏｍａ，ｈ...     

Expression of bcl-2 protein in chronic hepatitis C： Effect of interferon alpha 2b with ribavirin therapy

AIM: Mechanisms responsible for persistence of HCV infection and liver damage in chronic hepatitis C are not clear. Apoptosis is an important form of host immune response against viral infections. Anti-apoptotic protein bcl-2 expression on liver tissue as well as the influence of interferon alpha 2b (IFNa2b) and ribavirin (RBV) were analyzed in patients with chronic hepatitis C. METHODS: In 30 patients with chronic hepatitis C (responders - R and non-responders - NR) treated with IFNα2b+RBV, protein bcl-2 was determined in hepatocytes and in liver associated lymphocytes before and after the treatment. RESULTS: The treatment diminished bcl-2 protein accumulation in liver cells in_patients with hepatitis C (P<0.05). Before and after the therapy, we detected bcl-2 protein in R in 87±15% and 83±20% of hepatocytes and in 28±18% and 26±10% of liver-associated lymphocytes, respectively. In NR, the values before treatment decreased from 94±32% to 88±21% of hepatocytes and 39±29% to 28±12% of lymphocytes with bcl-2 expression. There was no statistical correlation between bcl-2 expression on liver tissue with inflammatory activity, fibrosis and biochemical parameters before and after the treatment. CONCLUSION: IFNα2b+RBV treatment, by bcl-2 protein expression decrease, enables apoptosis of hepatocytes and associated liver lymphocytes, which in turn eliminate hepatitis C viruses.     

Detection of aberrant methylation of RARβ2, GSTP1 and DAPK gene in prostate cancer and its clinical significance

Objective To explore hypermethylation of RARβ2, GSTP1 and DAPK gene in prostate cancer tissues, and to explore its correlation with clinicopathological features of prostate cancer and its diagnostic value. Methods Hypermethylation of RARe2, GSTP1 and DAPK gene was detected by nested methylation-specific PCR (NMSP) in 57 prostate cancer (PCa) tissues and 35 benign prostate hyperplasia (BPH) tissues. The correlation between hypermethylation and clinicopathological features of prostate cancer and its diagnostic value were analyzed. Results The hypermethylation frequencies of RARβ2, GSTP1 and DAPK gene in PCa were significantly higher than those in BPH (RARβ2: 52.6% vs. 0% GSTP1: 61.4% vs. 2.9%;DAPK: 43.9% vs. 8.6%;all P<0.01). The methylation rate of RARβ2 gene was directly correlated with distinct Gleason scores and clinical stage (4～7 score vs. 8～10 score: 34.8% vs. 64.7%;stage B, C vs. stage D: 37.0% vs. 66.7%;x2=4.927 and 5.004, P=0.026 and 0.025). The methylation rate of GSTP1 gene was significantly different in patients with different Gleason scores (4～7 score vs. 8～10 score: 43.5% vs. 73.5%;x2 =11.530, P=0.001), but had no difference in patients with distinct clinical stage (stage B, C vs. stage D: 51.9% vs. 70.0%;x2=1.975, P=0.16) . There was no difference in DAPK gene methylation rate among patients with different Gleason scores and distinct clinical stage (4 ～7 score vs. 8～10 score: 39.1% vs. 50.0%;stage B, C vs. stage D: 33.3% vs. 53.3%;x2= 1.290 and 2.309, both P～0.05). GSTP1 gene showed the highest sensitivity of 61.4% (35/57)with specificity of 97.1%(34/35), while RARβ2 gene had the highest specificity of 100% (35/35) with the sensitivity of 52.6% (30/57). The sensitivity and specificity of DAPK gene were 43.9% and 91.4% (25/57 and 32/35), respeetively. When the hypermethylation of RARβ2, GSTP1 and DAPK gene were detected together, the diagnostic sensitivity was increased, but the specificity was decreased. Conclusions The aberrant methylation of RARβ2, GSTP1 and DAPK gene is correlated with tumorigenesis and progression of prostate cancer, which may be used as an effective diagnostic marker for prostate cancer.     

肝胆管癌bcl-2/JH融合基因检测及bcl-2蛋白表达研究

探讨ｂｃｌ－２基因重排及ｂｃｌ－２蛋白表达与肝胆管癌发生的关系,采用半套式原位聚合酶链反应和免疫组织化学技术对２１例肝胆管癌中ｂｃｌ－２／ＪＨ融合基因和ｂｃｌ－２蛋白表达进行原位观察,结果;２１例肝胆管癌中ｂｃｌ－２蛋白阳性１５例,阳性率为７１．４％,未发现ｂｃｌ－２／ＪＨ融合基因,ｂｃｌ－２蛋白表达与ｂｃｌ－２基因重排之间无相互对应关系,提示肝胆管癌发生可能与ｂｃｌ－２蛋白的异常表达关系密切,而     

5-氟尿嘧啶、阿霉素诱导肝内胆管癌细胞凋亡和bcl-2基因表达研究

目的研究5-氟尿嘧啶(5-FU)及阿霉素诱导肝内胆管癌细胞9810凋亡情况，并对其机制作初步探讨。方法应用流式细胞仪检测肝内胆管癌细胞凋亡的情况，并检测bcl-2基因表达情况。结果随着两种药物浓度的增加，细胞凋亡率逐步上升，bcl-2基因表达下调，两种药物小剂量联合作用效果优于一种药物大剂量应用。结论5-FU及阿霉素能诱导肝内胆管癌细胞凋亡，bcl-2基因表达下调是其作用的一种机制。     

Genome-wide analysis of gene expression in human intrahepatic cholangiocarcinoma

Intrahepatic cholangiocarcinoma is a neoplasm arising in the liver, and its incidence is increasing in Japan as well as in Western countries. Prognosis of patients with this type of tumor remains unsatisfactory because no effective chemotherapeutic drugs are available, we have no sensitive tumor markers to detect this tumor in its early stage, and it is difficult to identify a high-risk group for the disease. To clarify the molecular mechanism of tumorigenesis and identify molecular targets for diagnosis and treatment, we analyzed global gene-expression profiles of 25 intrahepatic cholangiocarcinomas using tumor cell populations purified by laser microbeam microdissection and a cDNA microarray containing 27,648 genes. We identified 52 genes that were commonly upregulated and 421 that were downregulated in intrahepatic cholangiocarcinomas compared with noncancerous biliary epithelial cells. From the 52 upregulated genes, we selected P-cadherin and survivin for further investigation and corroborated enhanced expression of their products in cancer tissues by immunohistochemical staining. Furthermore, comparison between tumors with lymph node metastasis and those without metastasis identified 30 genes that were associated with lymph node involvement. In conclusion, these data should be helpful for a better understanding of the tumorigenesis of intrahepatic cholangiocarcinoma and should contribute to the development of diagnostic and therapeutic strategies for this type of tumor. Supplementary material for this article can be found on the HEPATOLOGY website (http://www.interscience.wiley.com/jpages/0270-9139/suppmat/index.html).     

葡萄糖诱导人血管内皮细胞凋亡及其对bax和bcl-2表达的影响

目的　探讨糖尿病 (DM)状态下葡萄糖 (Glu)对人脐静脉内皮细胞 (HUVEC)凋亡的影响及其分子机制。　方法　细胞凋亡用吖啶橙 (AO)荧光染色、原位末端标记 (TUNEL)法、流式细胞仪分析检测。同时行bcl 2和bax免疫细胞化学染色和定量分析。　结果　高浓度Glu( 2 0mmol/L、4 0mmol/L)能够诱导HUVEC凋亡 ,且呈浓度和时间依赖性。高糖 ( 2 0mmol/L)培养HUVEC 72h后bax表达的MOD×area值由 387± 97升至 136 9± 2 2 5 (P <0 .0 1) ,对照组和高糖组bcl 2染色的MOD×area分别为 15 0± 36和 186± 76 ,两者间差异无显著意义 (P >0 .0 5 )。　结论　DM状态下高血糖能诱导内皮细胞凋亡 ,其机制可能与bax基因表达上调有关。     

自发性高血压大鼠在应激状态下心肌凋亡蛋白表达的特点

目的探讨应激导致的交感神经激活对自发性高血压大鼠心肌细胞凋亡的影响.方法8周雄性自发性高血压大鼠24只(A)组分为应激组A1(n=12)及非应激组A2(n=12),并以8周Wistar24只(B)组作为对照,也分为应激组B1(n=12)及非应激组B1(n=12),每组各取4只测量血压,称重后麻醉、断头处死行称重;余每组8只,应激组采用束缚应激模型各自8周观察至32周,后称体重、麻醉、处死,取心脏称重,Westem blot检测心肌bcl-2及bax蛋白表达,并以bax/bcl-2比值作为心肌细胞凋亡指标.取血测定儿茶酚胺及血管紧张素Ⅱ.结果A1组束缚应激后32周龄鼠血压及心脏重量指数较8周龄明显增加P＜0.01,A2组非应激鼠随着周龄的延长(32周)血压及心脏重量指数显著高于B1、B2组的Wistar鼠及A1组的8周龄鼠.本研究所采用应激模型能够成功激活中枢交感神经系统.A2组非应激组32周龄鼠心肌bcl-2蛋白表达较8周龄时有增高趋势,但无统计学意义;而A1应激组32周龄时bcl-2较8周时显著降低,P＜0.01.心肌bax蛋白随周龄有显著的增高趋势,P＜0.05,束缚应激使该趋势进一步突出.代表凋亡程度的bax/bcl-2比值随周龄而升高,在应激组较非应激组显著增高,P＜0.05.自发性高血压大鼠非应激A2组随周龄的增长,血浆去甲肾上腺素、肾上腺素及血管紧张素Ⅱ均呈上升趋势,但无显著差异.束缚应激A1组,上述血浆活性物质均显著升高P＜0.05.结论束缚应激模型能够有效激活交感神经系统,从而模拟心力衰竭时的神经内分泌状态.自发性高血压大鼠自8周至32周龄较Wistar鼠心肌细胞凋亡增加,应激所致的交感神经激活能够使凋亡过程加剧,因而加速了心力衰竭的进程.     

Influence of stress on the expression of bcl-2/bax protein in spontaneously hypertensive rats

Cardiachypertrophyinhypertensionwasanadaptivereactiontoover－load．Althoughthishyper－trophicreactionmayeffectivelymaintainhemodynamicbalanceinitially，itcanresultinimpairedcardiaccontractility．Themechanismsunderlyingthetransitionfromcardiachypertrophyto