肝癌中细胞凋亡水平和p73基因转录表达的研究

目的　通过观察肝癌组织 ,对应癌旁组织和远癌肝组织中细胞凋亡及p73基因转录表达水平的变化 ,探讨细胞凋亡及p73基因转录表达水平与肝癌发生、发展的关系。方法　采用TUNEL及RT_PCR技术检测 3 2例乙肝相关肝癌癌组织、癌旁组织 ,以及良性肝脏疾病和对应正常肝组织细胞凋亡和p73mRNA的表达水平 ,并结合临床病理学资料进行统计学分析。结果　①肝癌癌组织中的凋亡指数明显高于癌旁组织和远癌组织 (P <0 0 1) ,细胞凋亡水平与肝癌组织学类型和P_TNM分期无明显关系 (P >0 0 5) ;②肝癌组织p73mRNA表达水平和表达率明显高于癌旁肺组织、远癌组织和肝良性病变组织 (P <0 0 1) ;③组织中凋亡指数的升高和p73基因转录表达水平的增加呈显著的正相关 (P <0 0 1)。结论　肝癌组织中存在细胞调亡水平的升高和p73基因的过度转录表达     

STAT3基因在原发性肝癌组织中的表达及意义

目的探讨STAT3基因在肝癌发生发展中的作用及意义。方法应用RT-PCR检测STAT3基因及相关基因c-myc、p53、survivin、VEGF的mRNA水平的表达,应用Western Blot法及免疫组化法检测STAT3基因的蛋白水平及定位表达。结果肝癌组织及癌旁组织中STAT3基因mRNA的表达均明显高于正常肝组织(P<0.05),肝癌及癌旁组织中c-myc、survivin、VEGF基因mRNA的表达上调,p53基因mRNA的表达下调,肝癌组织及癌旁组织中STAT3基因蛋白水平的表达均高于正常肝组织(P<0.05)。结论STAT3基因的持续激活在肝癌的发生发展中起重要作用,可作为早期诊断的指标及治疗的新靶点。     

PeroxiredoxinⅡ基因在肝癌组织中的表达及意义

目的观察peroxiredoxinⅡ（PrxⅡ）mRNA和蛋白在黄曲霉毒素B1（AFB1）诱发树鼩HCC形成过程中的表达变化,并在人肝癌癌旁组织和正常肝组织中进行验证,以探讨PrxⅡ在肝癌形成过程中的作用。方法应用RT-PCR和Western blot方法,检测6例AFB1诱发的树鼩肝癌、癌旁组织和这些动物肝癌发生前的活检肝组织,以及6例对照组动物相应时期的肝活检组织PrxⅡ在mRNA水平和蛋白水平的表达情况,并在18例人肝癌及其相应癌旁组织以及17名正常人肝组织进行验证。结果PrxⅡ在树鼩肝癌组织中的mRNA和蛋白质表达水平均明显高于相应的癌旁和癌前组织,也高于对照组同期活检肝组织（P〈0．05）;人肝癌组织中PrxⅡ的mRNA和蛋白表达改变和树鼩相符,即肝癌中的表达高于癌旁组织和正常肝组织（P〈0．05）。结论PrxⅡ基因可能与肝癌的发生发展有关,并有可能成为防治肝癌的分子靶标。     

Survivin在原发性肝细胞癌中的表达及意义

目的　Survivin是凋亡抑制蛋白中的一种 ,选择性地表达于恶性肿瘤组织。该文研究Sur vivin基因在原发性肝细胞癌中的表达及生物学意义。方法　收集 2株肝细胞癌细胞株 ,4 0例原发性肝癌组织标本及相应的癌旁组织 ,以Westernblotting法检测Survivin蛋白表达 ;半定量RT PCR法检测SurvivinmRNA表达 ;肝癌细胞凋亡指数采用原位末端标记法检测。结果　 2株肝癌细胞株和 85 % (34例 )的肝癌组织表达Survivin蛋白和mRNA ,而癌旁组织内无一例阳性表达。Survivin蛋白表达的阳性率在肝内转移组为 93.5 % ,显著高于肝内无转移组 (5 5 .6 % ,P <0 .0 5 ) ;在门静脉癌栓浸润组为 92 .8% ,显著高于无门静脉癌栓浸润组 (6 6 .7% ,P <0 .0 5 )。RT PCR显示 ,2株肝癌细胞株和 85 .0 % (34例 )的肝癌组织表达SurvivinmRNA ,与Westernblotting的结果一致 ,SurvivinmRNA的表达水平在肝内转移组 (1.10 5± 0 .396 )和门静脉癌栓浸润组 (1.137± 0 .4 0 4 )中 ,显著高于肝内无转移组 (0 .5 72± 0 .0 82 )和无门静脉癌栓浸润组 (0 .6 2 7± 0 .12 2 ,P <0 .0 5 )。所有肝癌组织标本中均可检测到凋亡细胞 ,但Survivin表达阳性组的凋亡指数 (1.15 2 %± 0 .32 6 % )显著低于Survivin表达阴性组 (4.5 0 2 %± 0 .830 % ,P <0 .0 5     

p73基因mRNA表达与老年人肺癌临床病理学特征及预后关系的探讨

目的 探讨p73基因与老年人肺癌发生、发展的关系。方法 采用逆转录多聚酶链反应（RT-PCR）检测65例老年人肺癌组织中p73基因mRNA的表达，并与癌旁组织和正常肺组织对比，结合肺癌的临床病理学特征及预后进行分析；同时采用聚合酶链反应-单链构象多态性分析（PCR-SSCP）技术检测肺癌与癌旁组织中p73基因的突变情况。结果 老年人肺癌组织中p73基因mRNA阳性表达率（78．5％）明显高于癌旁组织（18．5％）和正常肺组织（12．3％）（P〈0．05）；不同年龄组、不同类型和不同分化程度肺癌组织中p73基因mRNA的阳性表达率差异无显著性（P〉0．05）；Ⅰ期和Ⅱ期老年人肺癌组织中p73基因mRNA阳性表达率（90．7％）明显高于Ⅲ期和Ⅳ期（54．5％）（P〈0．05）；生存3年或以上的老年人肺癌组织中p73基因mRNA阳性表达率（91．7％）明显高于生存3年以下者（68．0％）（P〈0．05）。结论 p73基因表达可能参与了调控老年人肺癌发生的过程，但并不能阻止肺癌的进展，可能不是主要的抑癌基因；基因突变不是p73参与肺癌发生发展的主要形式；p73基因表达在各型肺癌发生、发展中可能具有普遍意义，可作为判断老年人肺癌生物学行为和预后的一个参考指标。     

STAT3及相关生长调控基因在胃癌组织中的表达及意义

目的探讨转录信号传导子与激活子3（STAT3）及相关生长调控基因在胃癌发生发展中的作用及意义。方法应用RT—PCR检测STAT3基因及相关基因c—myc，p53，生存素（survivin），血管内皮生长因子（VEGF）的mRNA表达水平，应用Western印迹法及免疫组化法检测STAT3基因的蛋白水平及定位表达。结果胃癌组织及癌旁组织中STAT3 mRNA的表达均明显高于正常胃组织（P〈0．01），胃癌及癌旁组织中c—myc，surovivin，VEGF mRNA的表达上调（P〈0．01），p53 mRNA的表达下调（P〈0．01），胃癌组织及癌旁组织中STAT3蛋白水平的表达均高于正常胃组织（P〈0．01）。结论STAT3基因的持续激活在胃癌的发生发展中起重要作用，可作为早期诊断的指标及治疗的靶点。     

肝癌组织中人宫颈癌癌基因mRNA的表达及意义

目的 观察人宫颈癌癌基因(HCCR)mRNA在肝癌和癌旁正常组织以及肝癌患者和正常人外周血单个核细胞(PBMC)中的表达水平.方法 TRIZOL法提取肝癌组织和癌旁正常肝组织以及肝癌患者和正常人的PBMC的RNA,用RT-PCR技术分析HCCR基因在不同样品中的mRNA表达水平.结果 在肝癌组织中,HCCR的mRNA相对表达强度为0.776±0.101,癌旁正常组织中的相对表达强度0.470±0.154,两者相比,P＜0.05.在PBMC中,肝癌患者HCCR mRNA的表达强度为0.256±0.069,而正常人未检测出,两者相比,P＜0.05. 结论 HCCR基因可能参与了肝癌的发生与发展,与肝癌的病理过程有一定的相关性.     

小干扰RNA沉默巨噬细胞移动抑制因子基因对肝癌细胞p27表达的影响

目的 观察巨噬细胞移动抑制因子(MIF)和细胞周期调控因子p27在肝细胞癌中表达的相互关系,探讨小干扰RNA(siRNA)沉默MIF基因对肝癌细胞p27表达的影响.方法 免疫组织化学法和荧光定量PCR法检测MIF、p27的蛋白和mRNA在肝癌及其癌旁组织中的表达情况.化学合成MIF siRNA和对照siRNA,脂质体法转染肝癌细胞PLC和Hep3B,荧光定量PCR法检测MIF和p27 mRNA在实验组及对照组中的表达情况.根据不同资料分别采用X2检验、Logistic回归分析或单因素方差分析.结果 MIF蛋白及其mRNA在肝癌组织中过表达,在癌旁组织中低表达;p27蛋白及其mRNA在癌组织中低表达,在癌旁组织中高表达.Logistic回归分析提示MIF为肝癌发生的危险因素,p27为保护因素.MIF mRNA在肝癌细胞株中过表达(F=61.036,P＜0.01),p27 mRNA在正常肝细胞L02中高表达(F=529.853,P＜0.01).经MIFsiRNA转染后,MIF mRNA在PLC及Hep3B中的表达水平降低,并且呈剂量依赖关系(F值分别为320.1和201.2,P值均＜0.01);p27 mRNA伴随MIF mRNA的降低而增加(F值分别为419.4和459.9,P值均＜0.01).结论 MIF在肝细胞癌中过表达,MIF siRNA能特异性抑制其在肝癌细胞中的表达;MIF可能参与了p27基因表达的调控.     

GTP连接蛋白4在肝细胞癌中的表达及临床意义

目的分析肝癌组织中GTP连接蛋白(GTPBP) 4的mRNA及蛋白表达水平及其与肝癌患者临床病理特征参数的关系。方法运用实时荧光定量PCR技术检测新鲜肝癌组织中GTPBP4 mRNA的表达水平,采用免疫组织化学方法检测肝癌组织芯片GTPBP4蛋白的表达水平,比较肝癌组织和癌旁组织GTPBP4的表达水平,分析肝癌组织中GTPBP4的表达水平与肝癌患者临床病理参数的关系。结果与癌旁组织相比,新鲜标本中肝癌组织GTPBP4 mRNA的表达水平明显增高(P0. 05)。在肝癌组织芯片中,GTPBP4在肝癌组织中的表达水平显著高于癌旁组织(P0. 05); GTPBP4的表达水平与肝癌患者性别、年龄、肿瘤大小及TNM分期无明显相关性(P0. 05),而与肝细胞癌的病理分级呈正相关,与生存率呈负相关(P0. 05)。结论在肝癌组织中GTPBP4呈高表达状态,其可能在肝细胞癌的发生发展过程中发挥作用。     

膀胱尿路上皮癌中Syk mRNA及蛋白的表达及其意义

目的 检测Syk mRNA及蛋白在膀胱尿路上皮癌组织及其癌旁正常组织中的表达,探讨其在膀胱癌发生发展中的意义.方法 收集52例膀胱尿路上皮癌及其癌旁正常组织标本,采用实时定量PCR(qRT-PCR)、Western印迹方法检测这些标本中Syk mRNA及Syk蛋白的表达情况.结果 Syk mRNA在人膀胱尿路上皮癌中的表达要低于正常膀胱黏膜组织(P＜0.05),Syk蛋白的表达在癌组织中的表达低于其在癌旁正常组织中的表达.结论 Syk mRNA及蛋白在人膀胱黏膜组织及膀胱尿路上皮癌中均有表达,但其表达在膀胱尿路上皮癌降低,并与其临床分期及病理分级相关,可以认为Syk基因的表达下降与膀胱尿路上皮癌发生发展有相关性.     

原发性肝细胞癌及癌旁组织中环氧合酶-2的表达及其意义

目的：研究人原发性肝细胞癌（HCC）组织和癌旁非瘤组织中的环氧合酶－2（COX－2）蛋白及基因表达情况,方法：采用免疫组织化法和原位分子杂交法,研究27对原发性肝细胞癌及癌旁非肿瘤组织,5例正常肝组织中COX－2的蛋白和基因表达,结果：高分化HCC中COX－2蛋白表达显著高于中分化和低分化HCC（P分别＜0．05）以及癌旁组织和正常组织（P分别＜0．01）,癌旁组织的COX－2表达显著高于正常组织（P＜0．05）,癌旁组织,中分化和低分化HCC之间COX－2的表达强度差异无显著性（P＞0．05）,在COX－2蛋白阳性的肝癌细胞和癌旁肝细胞胸胞胞质中可见到COX－2mRNA呈阳性表达,结论：COX－2的过度表达可能参与了高分子HCC的致癌过程。     

Mutation and overexpression of the beta-catenin gene may play an important role in primary hepatocellular carcinoma among Chinese people

AIM: To study the role of beta-catenin gene mutation and expression in hepatocellular carcinogenesis. METHOD: Thirty-four hepatocellular carcinoma (HCC) specimens and adjacent para-cancerous tissues, and four normal liver tissues were analyzed. Subcellular distribution of beta-catenin was examined by immunohistochemistry staining. Mutation and semiquantitative expression of beta-catenin gene exon 3 mRNA were detected by RT-PCR-SSCP and in situ hybridization. RESULT: Immunohistochemistry showed that all normal liver tissues and para-cancerous tissues examined showed membranous-type staining for beta-catenin protein, frequently with weak expression in the cytoplasm, but no beta-catenin accumulation in nuclei was found; while in liver cancer, 21 cases (61.8%) of HCC examined showed accumulated type in cytoplasms or nuclei. On SSCP, 15 cases (44.1%) of HCC altogether displayed three kinds of characteristic mutational mobility shifts. No abnormal shifting bands were found in tissues from normal liver or para-cancerous area. The beta-catenin gene exon 3 mRNA expression index of 34 HCCs was higher than that of para-cancerous tissue and normal liver tissue. Using in situ hybridization, the signal corresponding to beta-catenin gene exon 3 mRNA was particularly strong in cytoplasm of HCC when compared with those of paracancerous tissues and normal liver tissues. CONCLUSION: beta-catenin gene mutation and overexpression may have a critical role in malignant progression of hepatic carcinogenesis among Chinese people.     

Relationship between HBxAg and Fas/FasL in patients with hepatocellularcarcinoma

AIM To assess the relationship between HBV X-gene, X-gene product and Fas/ FasL which mediatehepatocellular apoptosis in patients with hepatocellular carcinoma.METHODS Tissue from 34 patients with hepatocellular carcinoma was tested for the expression of HBxAg.Quantitative ELISA assay was used to detect sFas; and sFasL and PCR were used to detect the HBV X-genein sera from 30 patients with hepatocellular carcinoma, 32 patients with liver cirrhosis and 20 normalcontrols.RESULTS The positive expression of HBxAg, Fas and FasL in carcinoma tissue was 97.06%, 85.29% and100%, respectively. The positive signal was mainly presented in the plasma, and all of these three positivestaining may appear in the same area. Redit analysis showed that there was no significant difference amongthese three positive staining (P >0.05). The mean levels of sFas in sera from hepatocellular carcinoma, livercirrhosis and normal controls were 722.97±321.12, 801.90±419.94 and 224.07±148.23, respectively,showing that sFas levels in patients with hepatocellular carcinoma and liver cirrhosis were significantlyelevated than that in normal controls (P < 0.0l). The mean levels of sFasL in sera from hepatocellularcarcinoma, liver cirrhosis and normal controls were 152.27±7.99, 162.97±12.40 and 154.99 ± 6.96,showing that sFasL level in patients with liver cirrhosis was significantly higher than that in patients withhepatocellular carcinoma and normal controls (P< 0.01). HBV X-gene was found to be positive in sera of30% patients with hepatocellular carcinoma; HBV X-gene was found to be positive in sera of 43.75% ofpatients with liver cirrhosis. There was no significant difference in sFas/sFasL level between HBV X-genepositive patients and HBV X-gene negative patients (P >0.05).CONCLUSION The expression of HBxAg and Fas/FasL in the tissue of hepatocellular carcinoma seemed tobe almost the same, but relation between cause and effect is unclear. The detection of sFas and sFasL inpatient sera may reflect the state of apoptosis mediated by Fas/FasL system. Our data showed that HBV X-gene expression in sera seemed to have no relation to sFas/sFasL level; however, these data also suggestedthat some patients with negative HBsAg in sera might have integrated HBV X-gene in liver tissues, andtherefore X-gene is detectable in those patient sera.     

TRA1 mRNA在肝硬化及肝癌组织中表达的研究

目的探讨肝癌(hepatocellular carcinoma,HCC)组织、肝硬化(liver cirrhosis,LC)组织及正常对照组织中肿瘤排斥抗原1(tumor rejection antigen 1,TRA1)mRNA的表达及关系。方法采用逆转录-聚合酶链反应的方法检测34例肝癌患者肝癌组织、癌旁肝硬化组织、非肝癌的肝硬化患者活检组织、肝血管瘤及肝内胆管结石等对照组织中TRA1 mRNA的表达,用捷达801分析软件对结果进行相对定量分析。结果肝癌组织、肝硬化组织、对照组织TRA1 mRNA表达率分别为95.00%、84.21%、50.00%,肝癌组与对照组间表达率的比较,差异有统计学意义(P0.05),肝硬化组织、对照组织表达率之间差异无明显统计学意义(P0.05);肝癌组织、肝硬化组织、对照组织TRA1 mRNA表达量分别为1.67±0.96、1.49±0.53、0.57±0.27;扩增产物表现出表达量的不同,呈现渐变趋势,肝癌组织、肝硬化组织与对照组之间差异有统计学意义(P0.05),肝癌组织与肝硬化组织比较,差异无统计学意义(P0.05)。结论 TRA1参与了肝硬化、肝癌的发生、发展,TRA1可能是肝硬化和肝癌潜在诊断标志物和治疗靶点。     

HBsAg及AFP在肝细胞癌组织中的表达及其相关性

目的进一步探讨HBV感染在肝细胞癌（HCC）发病中的作用。方法选择40份HCC、癌旁肝组织标本及6份正常肝组织标本,用免疫组化SP法检测其HBsAg与甲胎蛋白（AFP）表达。结果正常肝组织中HBsAg及AFP表达均为阴性;HCC和癌旁肝组织中HBsAg及AFP表达阳性率分别为70%、90%（P〈0.05）,HCC和癌旁肝组织AFP表达阳性率分别为87.5%、82.5%（P〉0.05）;HCC及癌旁肝组织中HBsAg阳性者AFP表达阳性率明显高于HBsAg阴性者（P〈0.05）。结论 HBV感染可能通过诱发肝细胞产生AFP参与HCC发病,具体机制有待进一步研究。     

Expression and clinical significance of TAp73α, p53, PCNA and apoptosis in hepatocellular carcinoma

AIM: To study the prognostic role of TAp73α, p53,proliferating cell nuclear antigen (PCNA) and apoptosis in patients with hepatocellular carcinoma (HCC) after surgical tumor ablation.METHODS: Forty-seven human resected HCC tissues and 42 adjacent non-cancerous tissues were studied with 10 normal liver tissues as control group. TAp73α, p53, and PCNA were detected with Elivision immunohistochemistry.Terminal deoxynucleotidyl transferase (TdT)-mediated d-UTP-biotin nick-end labeling (TUNEL) method was used to detect the apoptosis cells. All clinical and pathological materials were analyzed by SPSS10.0statistical package.RESULTS: TAp73α overexpressed in HCC tissues (36.2%)when compared with adjacent non-cancerous tissues(2.38%, P&lt;0.005) and normal liver tissues (0, P&lt;0.01).Mutant type p53 (rot-p53) overexpressed in HCC tissues(38.3%) when contracted with adjacent non-cancerous tissues (16.7%, P&lt;0.05) and normal liver tissues (0,P&lt;0.01). Proliferation index (PI) level in HCC tissues was significantly higher than that in adjacent non-cancerous tissues (30.34%&#177;4.46% vs27.88%&#177;5.89%, t, P= 0.028).Apoptosis index (AI) level in HCC tissues was higher than that in adjacent non-cancerous tissues (8.62%&#177;2.28%vs7.38%&#177;2.61%, t, P = 0.019). Expression of TAp73α was associated with lymph node metastasis and rot-p53,with r = 0.407 and 0.265, respectively. Expression of rot-p53 was associated with Edmondson‘s stage and AFP,with r = 0.295 and -0.357, respectively. In Kaplan-Meier univariant analysis, TAp73α, AFP, TNM stage, portal vein invasion, liver membrane invasion and HBsAg correlated with prognosis (log rank, P= 0.039, 0.012, 0.002, 0.000,0.014, 0.007, respectively). Multivariant Cox regression analysis showed that TAp73α, AFP, TNM stage, portalve in invasion, liver membrane invasion and age were independent factors of prognosis.CONCLUSION: These results suggest that TAp73α can be used as a prognostic indicator of patients with HCC undergoing surgical tumor ablation. AFP, TNM, portal vein invasion, liver membrane invasion and age also have a potency of predicting the prognosis of HCC.     

Expression and clinical significance of TAp73α, p53, PCNA and apoptosis in hepatocellular carcinoma

AIM: To study the prognostic role of TAp73α, p53, proliferating cell nuclear antigen (PCNA) and apoptosis inpatients with hepatocellular carcinoma (HCC) atter surgical tumor ablation.METHODS: Forty-seven human resected HCC tissues and 42 adjacent non-cancerous tissues were studied with 10 normal liver tissues as control group. TAp73α, p53, and PCNA were detected with Elivision immunohistochemistry.Terminal deoxynucleotidyl transferase (TdT)-mediatedd-UTP-biotin nick-end labeling (TUNEL) method wasused to detect the apoptosis cells. All clinical and pathological materials were analyzed by SPSS10.0statistical package.RESULTS: TAp73α overexpressed in HCC tissues (36.2%) when compared with adjacent non-cancerous tissues (2.38%, P＜0.005) and normal liver tissues (0, P＜0.01).Mutant type p53 (mt-p53) overexpressed in HCC tissues (38.3%) when contracted with adjacent non-cancerous tissues (16.7%, P＜0.05) and normal liver tissues (0, P＜0.01). Proliferation index (PI) level in HCC tissues was significantly higher than that in adjacent non-cancerous tissues (30.34%±4.46% vs 27.88%±5.89%, t, P= 0.028). Apoptosis index (AI) level in HCC tissues was higher than that in adjacent non-cancerous tissues (8.62%±2.28% vs 7.38%±2.61%, t, P = 0.019). Expression of TAp73α was associated with lymph node metastasis and mt-p53, with r = 0.407 and 0.265, respectively. Expression of mtp53 was associated with Edmondson's stage and AFP, with r = 0.295 and -0.357, respectively. In Kaplan-Meier univariant analysis, TAp73α, AFP, TNM stage, portal vein invasion, liver membrane invasion and HBsAg correlated with prognosis (log rank, P = 0.039, 0.012, 0.002, 0.000,0.014, 0.007, respectively). Multivariant Cox regression analysis showed that TAp73α, AFP, TNM stage, portal vein invasion, liver membrane invasion and age were independent factors of prognosis. CONCLUSION: These results suggest that TAp73α can be used as a prognostic indicator of patients with HCC undergoing surgical tumor ablation. AFP, TNM, portal vein invasion, liver membrane invasion and age also have a potency of predicting the prognosis of HCC.     

原发性肝细胞癌中WAF1／CIP1基因的及其与p53基因突变的关系

目的 研究原发性肝细胞癌（ＨＣＣ）组织中ＷＡＦ１基因的表达及其与ｐ５３基因突变和临床病理学指标的关系和意义。方法 应用半定量ＲＴ－ＰＣＲ、免疫组织化学及定量ＤＮＡ图像分析的方法,检测ＷＡＦ１基因在３２例ＨＣＣ及其癌旁肝组织和５例正常肝细胞中的表达,研究其与ｐ５３基因突变及临床病理学指标的关系。结果 肝癌组织中ＷＡＦ１ｍＲＮＡ表达相对值（１．０６±０．３７）Ｕ低于其相应旁旁肝组织（１．３０±０．３７     

人肝癌父系表达基因10的遗传印记状态

目的 研究人肝癌组织及肝癌细胞株中父系表达基因10(PEG10)的遗传印记状态.方法 从40例肝癌及其癌旁组织、15例正常肝组织、5株肝癌细胞(PLC/PRF/5、SMMC 7721、HepG2、Hep3B、SK-HEP-1)、2株正常肝细胞(changliver、HL7702)中提取基因组DNA,针对PEG10基因单核苷酸多态性位点设计引物进行PCR,扩增片段经测序分析基因型;从杂合样本中提取总RNA进行RT-PCR,对扩增产物测序以检测等位基因表达状态,同时进行实时荧光定量RT-PCR检测PEG10表达水平.计量资料以均数±标准差(-x±s)表示,组间比较用t检验与方差分析;两组率的比较用x2检验.结果 40例肝癌及其癌旁组织中16例呈杂合状态,15例正常肝组织中3例呈杂合状态,肝癌细胞HepG2扩增片段测序检测到一杂合突变位点,其余组织及细胞株均为纯合状态.杂合样本中,82.4%(14/17)肝癌样本(包括组织及肝癌细胞株)中PEG10基因呈双等位基因表达,发生印记丢失;17.6%(3/17)肝癌样本呈单等位基因表达,提示印记存在.PEG10在癌旁及正常肝组织中几乎不表达.发生印记丢失的肝癌组织与印记存在的肝癌组织相比,PEG10表达水平的差异无统计学意义(t=1.311,P＞0.05).结论 大多数肝癌组织中存在PEG10印记丢失现象,PEG 10印记状态与其在肝癌组织中的表达水平无明确关系.     

乙型肝炎病毒相关性肝细胞癌中分泌型卷曲相关蛋白基因甲基化分析

目的 研究HBV相关性肝细胞癌(HCC)中分泌型卷曲相关蛋白(SFRP)1、SFRP2基冈甲基化状态及其与肝细胞癌发牛发展的关系.方法 利用甲基化特异性聚合酶链反应(MSP)法检测45例肝细胞癌患者术中取得癌组织、癌旁组织及6例胆囊结石或肝脏血管瘤患者正常肝组织中SFRP1、SFRP2基因的甲基化状态.数据行X2检验、Fisher's 确切概率法统计分析.结果 在45例HCC患者中,癌组织和癌旁组织中SFRP1基因的甲基化率分别占62.2%和35.6%(X2=6.403,P＜0.05);SFRP2基因的甲基化率分别占51.1%和28.9%(X2=4.630,P＜0.05);6例正常肝组织均未检测到甲基化.癌组织中SFRPI与SFRP2基因甲基化在性别、年龄、HBV血清标志物、癌旁组织类型、有无转移和病理分级等因素之间差异均无统计学意义(P＞0.05),癌组织中SFRP1与SFRP2基因异常甲基化具有线性相关性(r=0.381,P=0.01).结论 SFRP1和SFRP2基因甲基化在HBV相关性HCC中是个频发事件,将来有可能作为一种预测HCC形成的分子生物学标志物.