蛋白翻译起始因子C_2在原发性肝细胞肝癌中表达的意义

目的研究C_2mRNA及其蛋白在原发性肝细胞肝癌(HCC)中的表达及意义。方法用原位杂交检测C_2mRNA住21例HCC及其癌旁组织中的表达,ABC免疫组化法检测C_2蛋白在60例HCC及其42例癌旁组织中的表达,Western blot法检测C_2蛋白在HCC及其癌旁组织中的表达。结果 21例HCC及其癌旁组织中,C_2mRNA阳性率分别占23.8％(5/21)和85.7％(18/21),60例HCC及42例癌旁组织中,C_2蛋白阳性分别占27.3％(17/60)和83.3％(35/42),27例肝硬变中,C_2蛋白阳性率为77.8％(21/27)。χ~2检验:C_2mRNA及其蛋白在HCC癌旁组织中表达明显高于癌组织(P<0.001);C_2蛋白在肝硬变中的表达明显高于HCC癌组织(P<0.01);C_2的表达与患者年龄、HBsAg及AFP有明显关系(P<0.05);而与癌组织的分化程度、肿瘤大小、淋巴转移无关;Western blot与免疫组化结果一致。结论 C_2基因表达下调可能与HCC的发生、发展及早期诊断有关。     

乙型肝炎病毒cccDNA与HBx蛋白在肝细胞性肝癌中表达的关系及意义

目的:探讨肝细胞性肝癌中乙型肝炎病毒(HBV)occDNA与HBx蛋白表达的关系及其意义.方法:取42例肝细胞性肝癌患者的癌和癌旁组织,采用SABC法检测组织中的HBx蛋白;采用RT-PCR法检测组织中的HBV CCCDNA水平.结果:HBx蛋白在癌及癌旁组织中的阳性例数分别为31例(73.8%)和35例(83.3%),无显著性差异;癌旁组织中cccDNA水平较癌组织中的高,但是统计学检验无显著性差异;癌组织和癌旁组织中HBx蛋白(+)者的cccDNA水平均明显高于HBx蛋白(-)者(P＜0.05).HBx蛋白表达与cccDNA水平呈正相关(r=0.778,P＜0.01).结论:HBx蛋白的表达与cccDNA水平明显相关,他们相互作用、相互影响并在肝癌的发生发展中起重要作用.     

抑癌基因PTEN及p53在肝细胞肝癌中表达的免疫组化研究

为探讨肝细胞肝癌组织中抑癌基因PTEN及p53蛋白的表达情况及临床病理意义。应用免疫组织化学技术检测了41例肝细胞肝癌及其相应的癌旁组织中PTEN和p53蛋白的表达情况。41例癌旁组织PTEN全部阳性表达,肝细胞肝癌组织中PTEN阳性表达率39％,阳性信号显示于胞浆中。p53阳性表达率51％,PTEN蛋白在肝细胞肝癌组织中的阳性表达与组织分化程度明显相关,高分化癌的阳性率为73％,低分化癌阳性率27％。肝细胞肝癌细胞中存在较高比例的PTEN蛋白阴性表达,说明在肝细胞肝癌的发生发展中PTEN基因失活起着重要作用,它的阳性表达可能有一定的预后意义。     

肺癌组织p53基因突变与肿瘤耐药基因表达的相关性及其临床意义

目的探讨p53基因突变与肿瘤耐药基因表达状况及肺癌耐药的关系。方法应用免疫组织化学法检测66例肺癌及其癌旁组织的突变型P53蛋白及耐药相关蛋白的表达水平。其中31例肺癌及其癌旁组织应用聚合酶链测定单链构象多态性(PCRSSCP)银染法及逆转录聚合酶链测定(RTPCR)法检测p53基因第5～8外显子突变情况及各种耐药基因的mRNA表达水平;12例应用三磷酸腺苷肿瘤化疗敏感实验(ATPTCA)检测肺癌细胞对诺维本、卡铂、依托泊苷、表柔比星、5氟尿嘧啶、博莱霉素的敏感性。结果突变型P53蛋白与P糖蛋白(Pgp)、多药耐药相关蛋白(MRP1)、谷胱甘肽S转移酶π(GSTπ)的表达状况存在着相关性(r分别为047、033、044,P均<005);ATPTCA结果显示突变型P53蛋白的表达状况及其与Pgp、MRP的共表达均和诺维本及卡铂的耐药相关(P均<005)。结论肺癌组织耐药相关蛋白的表达与p53基因突变有关,突变型P53蛋白的检出可以预示内源性耐药的存在,恢复野生型P53蛋白的功能可能有助于逆转耐药。     

肝癌组织中HMGA2蛋白、mRNA表达变化及意义

目的观察肝癌组织中高迁移率族蛋白A2(HMGA2)蛋白、mRNA的表达变化,并探讨其临床意义。方法用免疫组织化学SP法检测30例肝癌及其癌旁组织中的HMGA2蛋白,用RT-PCR法检测HMGA2 mRNA。结果 30例肝癌组织中平均HMGA2阳性率35.1%,而癌旁肝组织HMGA2阳性率<1%,两者HMGA2阳性率相比P<0.01。肝癌组织中HMGA2蛋白表达与肝癌Edmondson分级、肝内转移灶和肝门淋巴结转移有关(P均<0.05),与肿瘤直径无关(P>0.05)。癌旁组织和Ⅰ、Ⅱ、Ⅲ、Ⅳ级肝癌组织中HMGA2 mRNA相对表达量分别为0.03±0.02、0.04±0.06、0.10±0.16、0.52±0.87、0.68±0.95。肝癌组织中HMGA2 mRNA相对表达量明显高于癌旁组织(P均<0.05),肝癌组织中HMGA2 mRNA相对表达量与肝癌Edmondson分级有关(P均<0.05)。结论肝癌组织中HMGA2蛋白、mRNA表达升高,其可能参与肝癌的发生发展过程。     

乙型肝炎病毒复制上调肝癌组织TGF-β1和IGF-Ⅱ的表达

目的分析肝癌组织HBV复制与转化生长因子（TGF）-β1和胰岛素样生长因子（IGF）-Ⅱ表达的关系。方法以自身对照法收集人肝细胞癌（HCC）组织及癌旁组织，以生物素标记的HBV—DNA探针检测肝癌组织中HBV—DNA，采用免疫组织化学方法检测组织中TGF-β1和IGF-Ⅱ的表达，分析TGF-β1和IGF-Ⅱ表达与HBV复制的临床病理学关系。结果肝癌组织中TGF-β1和IGF-Ⅱ均呈较高表达，其阳性率均为83．3％，肝癌组明显高于癌旁组（P〈0．01）。癌灶组TGF-β1和IGF-Ⅱ阳性表达与肿瘤分化程度显著相关（P〈0．05）；而与肿瘤直径、数目无关（P〉0．05）；TGF-β1和IGF-Ⅱ表达与HBV复制显著相关，且HBV—DNA阳性组显著高于HBV—DNA阴性组。结论肝癌组织中TG-β1和IGF—II过度表达，且与HBV复制和肝癌的分化程度有关。     

原发性肝细胞癌组织中Smac、XIAP蛋白的表达变化及意义

目的检测原发性肝细胞癌（HCC）组织中Smac、XIAP蛋白的表达,并探讨其意义。方法采用免疫组化法检测50例肝癌组织、25例癌旁组织和15例正常肝组织中的Smac、XIAP蛋白。结果HCC组织、癌旁组织和正常肝组织中Smac蛋白阳性率分别为44．0％、80．0％、100％,XIAP蛋白阳性率分别为86．0％、28．0％、0,三者相比,P均〈0．05;HCC组织中Smac、XIAP蛋白表达均与HCC组织分化程度无关（P均〉0．05）;HCC组织中Smac和XIAP蛋白表达呈负相关（r=-0．270,P〈0．05）。结论HCC组织中Smac蛋白低表达和XIAP高表达,在HCC的发生发展有重要作用。     

抑癌基因PTEN在原发性肝癌中的表达及意义

目的 研究抑癌基因PTEN在原发性肝癌发生过程中的作用及意义。方法 应用免疫组织化学和northern杂交分析60例原发性肝癌及对应癌旁组织中PTEN mRNA及PTEN蛋白表达情况,结合患者的临床病理资料分析PTEN在原发性肝肝癌中的意义。结果 PTEN蛋白明确定位于肝细胞胞浆内。60例HCC的癌组织中,PTEN蛋白阳性率为48.3％(29/60),明显低于癌旁肝组织的阳性率(100％)。PTEN蛋白在肝癌组织内的表达阳性率与肝癌的病理学分级、有无癌栓有关,PTEN蛋白在肝癌组织的Ⅰ～Ⅱ级、Ⅲ级、Ⅳ级的阳性率分别为84.0％、23.8％、21.4％,无癌栓形成组PTEN蛋白表达的阳性率为55.56%,有癌栓形成组PTEN蛋白表达的阳性率为26.7％。Northern杂交显示,PIEN基因在肝癌细胞内存在四个转录子,其大小分别为5.5、4.4、2.4、1.8 kb。患者肝癌组织内PTEN mRNA的表达水平明显低于对应的癌旁肝组织。PTEN 5.5 kb和4.4 kb的转录子表达水平的降低与血清中AFP水平、有无癌栓、有无卫星灶及病理学分级有关;2.4 kb的转录子表达水平降低与患者有无癌栓、有无卫星灶有关;1.8 kb转录子表达水平的降低与临床病理学指标无关。结论 PTEN在肝癌的发生过程中可能起重要作用,其表达水平有可能作为反映肝癌进展和预后的病理学指标。     

子宫内膜癌组织中Mad2和Mtp53蛋白的表达变化及意义

目的 观察子宫内膜癌组织中有丝分裂阻滞缺陷蛋白2(Mad2)和突变型p53( Mtp53)蛋白表达变化.方法 采用免疫组化SP法检测30例正常子宫内膜(A组)、30例复杂型增生子宫内膜(B组)和63例子宫内膜癌组织(C组)中的Mad2和Mtp53蛋白.结果 A、B、C组Mad2蛋白的阳性率分别为23.33％、56.67％、85.71％,Mtp53蛋白的阳性率分别为0、26.67％、69.84％,组间比较P均＜0.05.Mad2蛋白阳性率与子宫内膜癌的分化程度相关,P＜0.05;与临床分期和淋巴结转移无关(P均＞0.05).Mtp53蛋白阳性率与内膜癌临床分期、淋巴结转移及分化程度均相关(P均＜0.05).二者在子宫内膜癌组织中表达呈正相关(r=0.547,P＜0.001).结论 子宫内膜癌组织中Mad2和Mtp53表达升高.检测子宫内膜癌组织中的Mad2和Mtp53有助于估计患者病情和预测预后.     

肝癌P^53基因突变与HBxAg关系的免疫组化研究

本实验对４８例石蜡包埋人肝癌和癌旁组织的Ｐ＾５３蛋白（突变型）和ＨＢｘＡｇ进行了免疫组化（ＡＢＣ法）检测。结果显示肝癌及其癌旁组织Ｐ＾５３蛋白（突变型）和ＨＢｘＡｇ阳性率分别为２９．２％（１４／２８）和９３．８％（４５／１８），Ｐ＾５３蛋白（突变型）与ＨＢｘＡｇ表达不相关（Ｐ〉０．０５），提示ＨＢｘＡｇ在Ｐ＾５３基因突变中的不发挥作用。     

Frequent integration of precore/core mutants of hepatitis B virus in human hepatocellular carcinoma tissues

The development of hepatitis B virus (HBV)-associated hepatocellular carcinoma (HCC) frequently follows persistent HBV infection and may arise in individuals who are hepatitis B e antigen (HBeAg) negative, indicating the possible presence of precore/core mutants. It is unclear whether precore/core mutants are associated with tumour development or are selected for after chromosomal integration of the wild-type viral DNA. We studied the status and sequence variation of the precore/core region of HBV in 56 patients with HBV-associated HCC and in various corresponding non-tumour tissues by Southern blot analysis, polymerase chain reaction and direct sequencing. Southern blot showed that integrated HBV DNA existed in 43 of 56 HCC tissues. Sequence analysis revealed mutations in 65% of the HCC (26/40) and 45% (14/31) of the corresponding non-tumour tissues. The mutation at nucleotide (nt) 1896, known to prevent HBeAg synthesis, was detected in 40% (16/40) of the tumours and in 35.4% (11/31) of the non-tumour tissues. Other mutations were found at nt 1899 (eight of 40 in HCC; three of 31 in non-tumour tissues), nt 1898 (seven of 40 in HCC; two of 31 in non-tumour tissues), nt 1912 (seven of 40 in HCC; none of 31 in non-tumour tissues) and nt 1886 (three of 40 in HCC; none of 31 in non-tumour tissues). To determine whether this finding merely reflected the prevalence of such mutants in this geographical region, HBV DNA from the sera of patients (also in this region) with acute and chronic hepatitis were sequenced. The nt 1896 mutant was found in 5.6% (one of 18) of patients with acute hepatitis B and in 22.8% (nine of 35) of patients with chronic hepatitis B. However, the nt 1898 mutation was not found in any of these sera. The precore/core mutant was observed with increasing frequency from acute hepatitis to chronic hepatitis, non-tumour and HCC, and this difference in frequency was significant between HCC and acute hepatitis B groups (P < 0.01), suggesting that the precore/core mutant or hepatocytes harbouring this mutant may be under immune selection and that such mutations may facilitate integration and subsequent tumour development.     

Microsatellite instability in human hepatocellular carcinoma: relationship to p53 abnormalities

ABSTRACT— Aims/Background: Microsatellite instability was sought in 10 human hepatocellular carcinomas (HCCs) to determine whether defective DNA mismatch repair might be implicated in the multiple genetic alterations observed in the p53 tumor suppressor gene in some of these patients' tumors. Methods: Genomic DNA from HCCs and adjacent non-tumorous livers was subjected to PCR with primers for nine microsatellites, and PCR products were resolved in a denaturing gel. Microsatellite instability was defined as the presence of band shifts or additional bands for at least two microsatellite sequences in an HCC compared to the nontumorous liver tissue from the same patient. Results: Microsatellite instability was detected in four of ten HCCs. Three of these four HCCs did not have p53 exon mutations. However, one HCC had microsatellite instability as well as multiple p53 exon mutations and multiple intron alterations. Four other patients with multiple p53 intron alterations in HCC (compared to their own nontumorous liver), three of whom also had a mutation in the exons, had no microsatellite instability. Conclusions: Defective DNA mismatch repair, as indicated by microsatellite instability, might have played a role in hepatocarcinogenesis in four of the ten patients, but in general it was not associated with p53 alterations. In one of the ten patients, defective DNA mismatch repair might have been the cause of multiple mutations in both the coding and intron sequences of the p53 gene.     

HBsAg-negative hepatitis B virus infections in hepatitis C virus-associated hepatocellular carcinoma

This study was conducted to evaluate reports that hepatitis B virus (HBV) DNA sequences can be found in the serum and/or tumour tissue from some hepatocellular carcinoma (HCC) patients who have no detectable hepatitis B surface antigen (HBsAg) in their sera. Such HBV infections would be highly atypical, because prospective studies have shown a clear succession of specific serologic markers during and after most HBV infections. As most HBsAg-negative HCC patients in Japan have hepatitis C virus (HCV) infections, the present study was conducted to determine whether some of these patients actually have unrecognized HBV infections. Thirty newly diagnosed HCC patients from Kurume, Japan, with antibody to the hepatitis C virus (anti-HCV) were studied. None of the 30 had HBsAg detectable in their serum. Of 22 for whom test results for antibodies to the hepatitis B core antigen (anti-HBc) and antibodies to HBsAg (anti-HBs) were available, 14 (64%) had anti-HBc and anti-HBs, four (18%) had anti-HBc alone, and four (18%) had no HBV markers. Nested polymerase chain reaction was used to detect the HBV surface (S), core (C), polymerase (P) and core promoter gene sequences in the HCC tissues and in the adjacent nontumorous liver tissues. HBV DNA was detected in HCC and/or adjacent nontumorous liver in 22 of 30 (73%) patients [detected in both HCC and nontumorous liver in 19/30 patients (63%)]. Among the 22 patients with detectable HBV DNA, more than one HBV gene was detected in 10 (46%). Among the four patients whose sera were negative for all HBV markers, three had HBV DNA in either HCC and nontumorous liver (two cases) or only in the nontumorous liver (one case); HBV DNA could not be detected in tissues from the fourth patient. In 18 of 21 (86%) patients with detectable HBV core promoter sequences, mutations at both nucleotides 1762 (A-GT) and 1764 (G-A) in the core promoter region were found. No deletions were detected in the core promoter gene region of the type reported to be associated with some cases of HBsAg-negative HBV infection. Thus, HBV DNA was detectable in 22 (73%) HBsAg-negative, anti-HCV-positive HCCs, including three (10%) who were also negative for anti-HBc and anti-HBs. HBV mutations at both nucleotides 1762 (A-GT) and 1764 (G-A) in the core promoter region were found in the majority of cases, mutations that have previously been reported in HBV that is integrated in HCC DNA. In serologic surveys to determine etiologic associations of HCC, patients such as those in this study would have been incorrectly designated as having 'HCV-associated HCC,' whereas the data in this study suggest that HBV could have played a role in the development of their HCCs.     

COOH-terminal deletion of HBx gene is a frequent event in HBV-associated hepatocellular carcinoma

AIM：To investigate the hepatitis B virus （HBV） x gene （HBx） state in the tissues of HBV-related hepatocellular carcinoma （HCC） in Chinese patients and whether there were particular HBx mutations. METHODS： HBx gene was amplified and direct sequencing was used in genomic DNA samples from 20 HCC and corresponding non-cancerous liver tissues from HBsAg-positive patients. HBV DNA integration and HBx deleted mutation were validated in 45 HCC patients at different stages by Southern blot analysis and polymerase chain reaction methods.
RESULTS： The frequencies of HBx point mutations were significantly lower in HCC than their corresponding non- cancerous liver tissues （11/19 vs 18/19, P = 0.019）. In contrast, deletions in HBx gene were significantly higher in HCC than their non-cancerous liver tissues （16/19 vs 4/19, P 〈 0.001）. The deletion of HBx COOH-terminal was detected in 14 HCC tissues. A specific integration of HBx at 17p13 locus was also found in 8 of 16 HCC, and all of them also exhibited full-length HBx deletions. Integrated or integrated coexistence with replicated pattern was obtained in 45.5% （20/45） - 56.8% （25/45） tumors and 40.9% （18/45） - 52.3% （23/45） non-tumor tissues.
CONCLUSION： HBx deletion, especially the COOH- terminal deletion of HBx is a frequent event in HBV-associated HCC tissues in China. HBV integration had also taken place in partial HCC tissues. This supporting the hypothesis that deletion and probably integrated forms of the HBx gene may be implicated in liver carcinogenesis.     

Hepatitis B envelope protein mutants in human hepatocellular carcinoma tissues

Hepatitis B virus (HBV) envelope mutants in the region encoding the highly immunogenic major hydrophilic region (MHR) of surface antigen (HBsAg) have been associated with vaccine failure and chronic infection. To determine if these mutants are associated with the development of human hepatocellular carcinoma (HCC), we measured the frequency and nature of such mutants in 23 HBV-associated HCC and various control tissues by performing Southern blot analysis, the polymerase chain reaction (PCR) and direct sequencing. The HBV genome was present mainly in an integrated form and, in most of the samples, the envelope gene was intact. Amino acid substitutions, involving the MHR region in the HCC tissues, were analysed in 11 (61.1%) of 18 patients with HCC. The mutation Gly145Arg, which has been reported to be associated with immunoevasion, was found in seven of the 18 HCC tissues. A significantly higher frequency of mutations was found in HCC tissues (11 of 18) than in the corresponding non-tumorous tissue of the same patients (one of eight), and in samples from patients with acute (one of 19) or chronic (three of 31) HBV infection ( P < 0.001, Fisher's exact test). The accumulation of these envelope mutants in the HCC tissue suggests that such envelope protein mutations may play a role in the process of oncogenesis and that specific vaccines may need to be developed to prevent the occurrence of mutant HBV-associated HCC. Alternatively, the progressive accumulation of mutants in patients with acute hepatitis, chronic hepatitis and HCC may reflect the increased length of duration of HBV infection in these groups of liver lesions.     

肝细胞癌p53基因249密码子热点突变的研究

目的 研究肝细胞癌(HCC)中p53基因249密码子(p53 E7 cd249)点突变情况。方法 用PCR法及HAEⅢ限制性片段长度多态性分析(HAEⅢ/RFLP)检测河南豫东地区38例HCC石蜡包埋组织及2例肝细胞癌株中p53 E7cd249点突变情况,DNA测序证实。选取广西桂西南地区的10例HCC作对照。结果 来自河南豫东地区的HCC p53 E7 cd249点突变率为10.5％(4/38),对照组广西桂西南地区的HCC p53 E7 cd249点突变为40％(4/10),二者相比具有显著性差异(P<0.05)。2例肝细胞癌株中均未发现HCC p53 E7 cd249点突变。结论 河南豫东地区HCC中p53基因E7 cd249点突变为非高发事件;p53 E7 cd249点突变可能发生在肝细胞癌变的晚期。     

Mutation of p53 gene in regenerative nodules in cirrhotic liver

BACKGROUND/AIMS: Mutations of p53 gene have been detected in precancerous stages of several cancers, and the possible role in multistep carcinogenesis is suggested. The aim of this study was to examine the mutation profile of p53 gene in regenerative nodules in cirrhotic livers.METHODS: Ninety eight tissue specimens of regenerative nodules obtained from 15 cases of cirrhosis were used for analysis. Twenty cases of chronic hepatitis and two cases of fatty liver were used as controls. DNA was extracted from each of manually demarcated regenerative nodules, and nucleotide sequence analysis was performed on p53 gene exon 5.RESULTS: Direct sequencing detected p53 mutations in seven of 98 DNA samples (7.1%) from regenerative nodules in six cases of cirrhosis. Subcloning analysis revealed that mutation sites differed in each subclone and the incidences of the mutation varied from 7.7 to 58.8% depending on individual nodules. The mutation was not detected in any of chronic hepatitis and fatty liver. There were inconsistent p53 sequence with regenerative nodules and accompanied hepatocellular carcinomas in six cases.CONCLUSIONS: Mutations of p53 gene were frequently found in cirrhotic livers compared with livers of patients with chronic hepatitis (P<0.01), suggesting that p53 mutations at the stage of cirrhosis may be a causative factor that may potentially lead to hepatocellular carcinoma.     

HCV核心蛋白、突变p53和Bcl-2在HCC中的表达及相关性

目的探讨HCC组织中的核心蛋白、突变p53、Bcl-2的表达以及其相关性,探讨HCV核心蛋白是否促进p53突变和Bcl-2的表达.方法收集手术切除HCC组织42例,采用免疫组织化学EnVision法检测HCC组织核心蛋白、突变p53、Bcl-2的表达,用统计学分析三者间的关系.结果C蛋白、p53和Bcl-2在HCC癌组织中的表达率分别为40.5%(17/42)、47.62%(20/42)、83.3%(33/42);3组强度等级资料Kruskal-Wallis秩和检验H=16.33,差异性显著,Mann-Whitney U test:C蛋白和p53、Bcl-2蛋白间的P值分别0.43、0.00;C蛋白与突变p53、Bcl-2阳性强度两者间相关性检验P值分别为0.000、0.914,相关系数rs分别为0.67、0.08;突变p53与Bcl-2两者相关性检验P值为0.27,相关系数rp为0.32.结论3种蛋白的表达有相关性;HCV核心蛋白可能促进野生型p53突变和表达;核心蛋白和突变p53都有可能促进Bcb2蛋白的表达.