RIPK3 mRNA level acts as a diagnostic biomarker in hepatitis B virus-associated hepatocellular carcinoma

HBV-associated hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related death worldwide, and non-invasive early detection of HBV-associated HCC requires to be improved. To determine the alteration and clinical relevance of necroptosis and its key regulator receptor-interacting protein kinase 3 (RIPK3) in HBV-associated HCC, we detected the mRNA level of RIPK3 in peripheral blood mononuclear cells (PBMCs) and analyzed its correlation with clinical parameters. Here, we demonstrate that the expression of RIPK3 is elevated in patients with HBV-associated HCC compared to patients with chronic hepatitis B (CHB) and patients with HBV-related liver cirrhosis (LC). The mRNA level of RIPK3 is positively correlated with the severity of clinical manifestations and TNM stages. Moreover, the serum levels of RIPK3-asssocited cytokines are altered in consistent with the change of RIPK3 expression. The diagnostic accuracy of RIPK3 mRNA level is comparable to AFP test in discriminating HBV-associated HCC from LC and is better than AFP test in discriminating HBV-associated HCC from CHB. The combination of RIPK3 mRNA level and AFP test significantly improves the diagnosis of HBV-associated HCC. These data suggest that RIPK3 mRNA level is a biomarker in the onset and progression of HBV-associated HCC and may provide novel diagnostic strategies combined with the AFP test.     

Combined detection of insulin-like growth factor-binding protein 7 promoter methylation improves the diagnostic efficacy of AFP in hepatitis B virus-associated hepatocellular carcinoma

This study quantitatively assessed serum insulin-like growth factor-binding protein 7 (IGFBP7) promoter methylation in hepatocellular carcinoma (HCC), and explored its clinical value. A total of 80 patients with hepatitis B virus-associated HCC, 35 patients with chronic hepatitis B (CHB), and 20 healthy controls (HC) were enrolled. MethyLight was used to quantitatively assess the methylation levels of serum IGFBP7 promoter. A logistic regression model was established for the combined evaluation of AFP and serum IGFBP7 promoter methylation. The results showed that mean methylation levels of serum IGFBP7 promoter were significantly higher in HCC (5.33%, interquartile range [IQR] 1.14–15.70%) patients than in individuals with CHB (1.54%, IQR 0.64–2.45%; P < 0.01) and HC (0.63%, IQR 0.22–0.98%; P < 0.01). In HCC subgroups, patients with vascular invasion, tumor size >3 cm and advanced tumor node metastasis (TNM) showed higher methylation levels compared with the remaining groups (P < 0.05). Compared with AFP alone, combined determination based on logistic regression analysis significantly improved the area under the receiver operating characteristic (ROC) curve (AUC) (0.759 vs 0.623, P < 0.05). In addition, the Youden index was increased from 5.71%, 11.25% and 15.18%, when considering AFP alone at cut-off values of 20, 200, and 400 ng/ml, respectively, to 45.71% with IGFBP7 promoter methylation taken into consideration (all P < 0.05). These results suggested that combined quantitative measurement of serum IGFBP7 promoter methylation could enhance the diagnostic ability of AFP in distinguishing hepatitis B virus-associated HCC from CHB.     

Aberrant DNA Methylation of G-protein-coupled Bile Acid Receptor Gpbar1 (TGR5) is a Potential Biomarker for Hepatitis B Virus Associated Hepatocellular Carcinoma

Background: G-protein-coupled bile acid receptor Gpbar1 (TGR5) is a newly identified liver tumor suppressor in carcinogenesis. This present study was therefore to determine the potential value of serum TGR5 promoter methylation in identifying hepatocellular carcinoma (HCC) from chronic hepatitis B (CHB) patients.Methods: The circulating cell-free DNA (cfDNA) was extracted from a retrospective dataset including 160 HCC, 88 CHB and 45 healthy controls (HCs). Methylation status of TGR5 promoter was examined by methylation-specific polymerase chain reaction (MSP).Results: Hypermethylation of the TGR5 promoter occurred significantly more frequent in HCC (77/160, 48.13%) than CHB (12/88, 13.64%; p<0.01) and HCs (2/45, 4.44%; p<0.01). The methylation rate of TGR5 in HCC patients ≥60 years old was significantly higher than those <60 years old (p<0.05). Alpha fetoprotein (AFP) had sensitivity of 58.13%, 30.63% and 24.38% at cut-off points of 20, 200 and 400ng/ml respectively; while TGR5 methylation combined AFP had sensitivity of 81.25%, 68.13% and 65%. AFP had specificity of 47.73%, 92.05% and 98.86% at cut-off points of 20, 200 and 400ng/ml respectively; while TGR5 methylation combined AFP had specificity of 38.64%, 78.41% and 85.23%. AFP had Youden index of 0.06, 0.23 and 0.23 at cut-off points of 20, 200 and 400ng/ml respectively; while TGR5 methylation combined AFP had Youden index of 0.20, 0.47 and 0.50.Conclusions: Our findings strongly suggested the combination of serum TGR5 promoter methylation and AFP enhanced the diagnostic value of AFP alone in discriminating HCC from CHB patients.     

Detection of promoter methylation status of suppressor of cytokine signaling 3 (SOCS3) in tissue and plasma from Chinese patients with different hepatic diseases

SOCS3 as an important negative regulator of IL6/JAK/STAT3 signaling pathway may be early critical determinants of carcinogenesis. This study aimed to explore the aberrant promoter methylation of SOCS3 gene in circulating DNA as a noninvasive biomarker for screening hepatocellular carcinoma (HCC) high-risk individuals and for prognosis of HCC patients after partial hepatectomy. We detected its methylation status in 116 liver tissues and 326 plasma specimens of different hepatic diseases and healthy subjects, and its mRNA and protein expression in tissues. Higher methylation rate was remarkably detected in HCC (47.92%), compared with corresponding non-tumor (25.0%), liver cirrhosis (LC) (10.0%), benign liver diseases (0%) and normal liver tissues (0%) (all P < 0.05). SOCS3 mRNA level was significantly lower in methylated HCC tissues (P < 0.05). The expressions of SOCS3 and pSTAT3 were affected by methylation status. Correlation and consistency of SOCS3 methylation were found between cancer tissue and corresponding plasma (P < 0.001, κ = 0.747). The detection rate of plasma for HCC reached 73.91%, with no false positive error. SOCS3 methylation status both in tissue and plasma was significantly associated with AFP400, tumor size, tumor differentiation, LC, metastasis and recurrence (all P < 0.05). Patients with SOCS3 methylation were followed up a markedly poorer prognosis than those unmethylated for disease-free survival (P < 0.05). These data indicate that methylation status of SOCS3 in plasma cell-free DNA can correctly reflect that in tissue DNA and be used as a noninvasive potential biomarker for chronic liver disease monitoring, predicting the degree of malignancy and poor prognosis of HCC.     

Methylation of serum insulin‐like growth factor‐binding protein 7 promoter in hepatitis B virus‐associated hepatocellular carcinoma

Methylation of gene promoter CpG islands is an important early event in hepatocellular carcinoma (HCC), and detection of cell‐free tumor‐specific DNA methylation is becoming a useful noninvasive method for HCC. This study was aimed at determining the diagnostic value of serum insulin‐like growth factor‐binding protein 7 (IGFBP7) promoter methylation in hepatitis B virus‐associated HCC. A total of 217 subjects, including 136 HCC patients, 46 patients with chronic hepatitis B (CHB), and 35 healthy controls (HCs), were included. The methylation status of the serum IGFBP7 gene promoter was determined using methylation‐specific PCR. The frequency of serum IGFBP7 promoter methylation in HCC patients (89/136, 65%) was significantly higher than that in CHB patients (8/46, 17%; X² = 31.883, P < 0.001) and HCs (5/35, 14%; X² = 29.429, P < 0.001). Moreover, elevated IGFBP7 methylation frequency was also observed in HCC patients with vascular invasion compared with those without vascular invasion (84 versus 60%, X² = 6.633, P = 0.010). The sensitivities of serum IGFBP7 methylation and alpha‐fetoprotein (AFP) in detecting HCC were 65 and 57%, respectively. Of note, the combination of IGFBP7 methylation and AFP showed 85% for sensitivity. These results suggest that methylation of the serum IGFBP7 gene promoter may serve as a useful noninvasive biomarker for HCC diagnosis. © 2013 Wiley Periodicals, Inc.     

Aberrant GSTP1 promoter methylation predicts poor prognosis of acute-on-chronic hepatitis B pre-liver failure

It has been demonstrated that glutathione-S-transferase P1 (GSTP1) could protect cells from DNA damage mediated by oxidizing agents or electrophiles in hepatic inflammatory response. Our study evaluated the methylation status and the predictive value for prognosis of GSTP1 promoter region in patients with acute-on-chronic hepatitis B pre-liver failure (pre-ACHBLF). Methylation status of GSTP1 promoter in peripheral blood mononuclear cells (PBMCs) and plasma was measured in 103 patients with pre-ACHBLF, 80 patients with chronic hepatitis B (CHB) and 30 healthy controls (HCs) by methylation-specific polymerase chain reaction. The mRNA level of GSTP1 was detected by quantitative real-time polymerase chain reaction. The methylation frequency of GSTP1 promoter region in patients with pre-ACHBLF (35/103 in PBMCs and 33/103 in plasma) was significantly higher than CHB (2/80) and HCs (0/30), respectively. The mRNA level of GSTP1 in patients with pre-ACHBLF was significantly lower than CHB and HCs. Additionally, pre-ACHBLF patients with methylated GSTP1 presented strikingly higher incidence of ACHBLF than those without. Of note, GSTP1 methylation presented distinctly better performance than model for end-stage liver disease score [area under the receiver operating characteristic curves (AUCs) 0.825 in PBMCs and 0.798 in plasma VS 0.589; AUC 0.804 in PBMCs and 0.779 in plasma VS 0.622; AUC 0.767 in PBMCs and 0.744 in plasma VS 0.602, respectively] when used to predict the 1-, 2- or 3-month incidence of ACHBLF in patients with pre-ACHBLF. Aberrant methylation of GSTP1 has potential to be a prognostic biomarker for pre-ACHBLF.     

Detection of aberrant p16INK4A methylation in sera of patients with liver cirrhosis and hepatocellular carcinoma

Hepatocellular carcinomas (HCCs) show genomic alterations, including DNA rearrangements associated with HBV DNA integration, loss of heterozygosity, and chromosomal amplification. The genes most frequently involved are those encoding tumor suppressors. The p16INK4A tumor suppressor gene frequently displays genetic alteration in HCC tissues. The present study was performed to examine the incidence of methylated p16INK4A in the sera of liver cirrhosis (LC) and HCC patients, and to evaluate its role as a tumor marker of HCC. The sera of 23 LC patients and 46 HCC patients were examined in this study. The methylation status of p16INK4A was evaluated by methylation-specific PCR of serum samples. Methylated p16INK4A was detected in 17.4% (4/23) of LC patients and in 47.8% (22/46) of HCC patients. No association was demonstrated between p16INK4A methylation and serum AFP level. As the status of p16INK4A methylation was not associated with serum AFP level, it may have a role as a tumor marker of HCC.     

Glutathione S-Transferase P1 Correlated with Oxidative Stress in Hepatocellular Carcinoma

Background: Glutathione-S-transferase P1 (GSTP1) is an important phase II enzyme that can protect cells from oxidative stress in various human cancers. However, few clinical studies were undertaken on the relationship between GSTP1 and oxidative stress in hepatocellular carcinoma (HCC). The present study was therefore aimed to evaluate the potential associations between GSTP1 and oxidative stress in HCC patients.Methods: The GSTP1 expression in peripheral blood mononuclear cells (PBMCs) was determined by flow cytometry from 38 HCC patients and 38 chronic hepatitis B (CHB) patients. The GSTP1 mRNA level in PBMCs was determined by real-time quantitative polymerase chain reaction. Enzyme-linked-immunosorbent-assay (ELISA) was performed to measure the oxidative stress status, including plasma levels of malondialdehyde (MDA), xanthine oxidase (XOD), reduced glutathione hormone (GSH) and glutathione-S-transferases (GST).Results: Significantly decreased GSTP1 protein expression was found in HCC patients than in CHB patients (P<0.05). The GSTP1 mRNA expression of HCC patients was also decreased compared with CHB patients (P<0.05). MDA and XOD levels were significantly higher in HCC patients than in CHB patients, while plasma GSH and GST levels were statistically lower in HCC patients than in CHB patients. GSTP1 expression level was correlated with plasma levels of MDA (P<0.01), XOD (P = 0.01) and GSH (P< 0.01), GST (P< 0.01).Conclusion: We demonstrated that the reduced GSTP1 expression might contribute to oxidative stress in the development of HCC from CHB.     

PreS deletion profiles of hepatitis B virus (HBV) are associated with clinical presentations of chronic HBV infection

BackgroundThe association of hepatitis B virus (HBV) preS1 and preS2 deletions with progressive liver diseases are not fully understood.ObjectiveThe study aimed to investigate characteristics of HBV preS deletion in HBV-infected patients with different illness categories.Study designTotal of 539 HBV-infected patients were enrolled in the study, including 146 with chronic hepatitis B (CHB), 111 with HBV-related liver cirrhosis (LC), 146 with HBV-related acute-on-chronic liver failure (ACLF), and 136 with HBV-related hepatocellular carcinoma (HCC). PreS deletion was determined by sequencing. Replicons containing representative preS1 and preS2 deletion mutants and wild-type were respectively constructed and transfected into HepG2 cells for phenotypic analysis.ResultsThe detection rates of overall preS deletion were 15.8%, 26.1%, 24.0%, and 34.6% in CHB, LC, ACLF, and HCC patients, respectively. PreS1 deletion was most frequently detected in LC patients while preS2 deletion was most frequently detected in HCC patients, both frequencies were significantly higher than that in CHB patients (17.1% vs. 4.8%, P < 0.01; 19.1% vs. 4.8%, P < 0.01). The deletion patterns across preS gene were different among the 4 illness categories. Compared with wild-type strain, the preS1 deletion mutant had defected preS1 expression, significantly decreased viral mRNA level and SP II promoter activity; while preS2 deletion mutant had defected preS2 expression, and significantly decreased viral mRNA level.ConclusionsHBV preS deletion was associated with advancement of liver diseases not only presented in preS deletion incidence, but also in the deletion pattern. Patients with preS2 deletion might have a higher risk to develop HCC.     

Silencing of PCDH10 in hepatocellular carcinoma via de novo DNA methylation independent of HBV infection or HBX expression

PCDH10 is a key tumor suppressive gene for nasopharyngeal, esophageal, and other carcinomas with frequent methylation. In this study, we investigated the potential epigenetic modification of the PCDH10 gene by hepatitis B virus × protein (HBx), a pivotal factor in the progression of HBV replication and potential carcinogenesis. PCDH10 expression was found to be down-regulated in 9/13 (69.2 %) of hepatocellular carcinoma (HCC) cell lines. Decreased PCDH10 expression was correlated with the methylation status of the PCDH10 promoter. Treatment with the DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine (Aza) was sufficient to restore PCDH10 mRNA expression by suppressing PCDH10 promoter methylation in HepG2 cells. Treatment with Trichostatin A alone had no significant effect on PCDH10 expression but enhanced the effect of Aza. PCDH10 methylation was further detected in 76 % (38 of 50) of HCC tissues compared with 40 % (20 of 50) of paired adjacent tissues, with no methylation detected in normal human liver tissues. There were significant correlations between methylation status of PCDH10 and tumor size, serum AFP levels, metastasis or TNM staging (P < 0.05). Moreover, PCDH10 promoter methylation status was not associated with HBV infection in our panel of 50 primary HCC tumors, and transfection with HBX could not alter the status of PCDH10 promoter methylation. Collectively, these observations suggested that the expression of PCDH10 was silenced in HCC via de novo DNA methylation independent of HBV infection or HBX expression, and PCDH10 might form a potentially useful therapeutic target for HCC.     

Promoter hypermethylation of CDH13, DAPK1 and TWIST1 genes in precancerous and cancerous lesions of the uterine cervix

Aberrant DNA methylation is an early event in carcinogenesis and could serve as an additional molecular marker for the early diagnosis. The study was performed to investigate the promoter methylation of DAPK1, CDH13, and TWIST1 genes in uterine cervix lesions in an effort to examine whether this epigenetic event is involved in the process of cervical carcinogenesis, and whether it might be used as a molecular marker of cervical lesions.We conducted a retrospective study of 60 uterine cervix specimens, including 8 normal tissue samples, 10 benign lesions, 28 precancerous lesions (CIN1-3), and 14 squamous cell carcinomas (SCC). DNA hypermethylation was investigated using methylation-specific PCR. Immunohistochemistry was used to find p16^INK4A overexpression.No hypermethylated promoters were detected in normal tissues and benign lesions. However, promoter hypermethylation of CDH13, TWIST1, and DAPK1 increased progressively from CIN1 to cancer, reaching values higher than 50% for cancer. DAPK1 and CDH13 displayed a significantly increased frequency of promoter methylation with progressively more severe cervical neoplasia (p < 0.05). A statistically significant association was observed between p16^INK4A expression and hypermethylation of DAPK1, TWIST1, and CDH13 (p < 0.0001).Hypermethylation of CDH13, DAPK1, and TWIST1 promoters is an early event in the initiation and progression of cervix neoplasia. CDH13, DAPK1, and TWIST1 genes are potential biomarkers of cervical cancer risk.     

Not polymorphism but methylation of class II transactivator gene promoter IV associated with persistent HBV infection.

BACKGROUND: Class II transactivator (CIITA) is the major rate-limiting regulator for expression of class II major histocompability complex (MHC-II). Human CIITA gene expression is controlled by four distinct promoters (pIto pIV). OBJECTIVE: To evaluate the relationship among polymorphism and methylation status of CIITA gene promoters and persistent hepatitis B virus (HBV) infection. METHODS: We recruited 21 patients with hepatocellular carcinoma (HCC), 45 liver cirrhosis (LC), 65 chronic hepatitis B (CHB), 26 acute hepatitis B (AHB) and 95 healthy blood donors. Polymorphism of CIITA gene promoters was assayed by PCR-SSCP-sequencing. Bioinformatics analysis was employed to predict the existence of CpG islands. Methylation-specific PCR (MSP) was used to detect the methylation status of CIITA gene pIV. RESULTS: No sequence differences were observed at CIITA genes pI, III and IV among HCC, LC, CHB, AHB patients and healthy controls. No CpG islands were found in the pI, pII and pIII sequences, but there was a CpG island in pIV. The frequency of methylated POV was not significantly different within persistent HBV infection groups (patients with HCC, LC or CHB). Significance was found between the persistent infection group and acute HBV infection or healthy controls. CONCLUSIONS: CIITA gene promoter sequences are conserved. PIV is highly methylated and associated with host susceptibility to HBV persistent infection.     

Tumor-specific downregulation and methylation of the CDH13 (H-cadherin) and CDH1 (E-cadherin) genes correlate with aggressiveness of human pituitary adenomas.

The gene products of CDH13 and CDH1, H-cadherin and E-cadherin, respectively, play a key role in cell-cell adhesion. Inactivation of the cadherin-mediated cell adhesion system caused by aberrant methylation is a common finding in human cancers, indicating that the CDH13 and CDH1 function as tumor suppressor and invasion suppressor genes. In this study, we analyzed the expression of H-cadherin mRNA and E-cadherin protein in 5 normal pituitary tissues and 69 primary pituitary adenomas including all major types by quantitative real-time RT-PCR (qRT-PCR) and immunohistochemistry, respectively. Reduced expression of H-cadherin was detected in 54% (28/52) of pituitary tumors and was significantly associated with tumor aggressiveness (P<0.05). E-cadherin expression was lost in 30% (21 of 69) and significantly reduced in 32% (22 of 69) of tumors. E-cadherin expression was significantly lower in grade II, III, and IV than in grade I adenomas (P=0.015, P=0.029, and P=0.01, respectively). Using methylation-specific PCR (MSP), promoter hypermethylation of CDH13 and CDH1 was detected in 30 and 36% of 69 adenomas, respectively, but not in 5 normal pituitary tissues. Methylation of CDH13 was observed more frequently in invasive adenomas (42%) than in non-invasive adenomas (19%) (P<0.05) and methylation of CDH1 was more frequent in grade IV adenomas compared with grade I adenomas (P<0.05). Methylation of either CDH13 or CDH1 was identified in 35 cases (51%) and was more frequent in grade IV invasive adenomas than in grade I non-invasive adenomas (P<0.05 and P<0.05, respectively). Downregulation of expression was correlated with promoter hypermethylation in CDH13 and CDH1. In conclusion, the tumor-specific downregulation of expression and methylation of CDH13 and CDH1, alone or in combination, may be involved in the development and invasive growth of pituitary adenomas.     

Humoral immune response to epidermal growth factor receptor in lung cancer

The aim of this study was to explore the potential value of autoantibody to epidermal growth factor receptor (EGFR) in the diagnosis of lung cancer (LC) and its relation with EGFR mutations. Enzyme-linked immunosorbent assay (ELISA) was performed to detect the level of autoantibody to EGFR in sera from 254 LC patients and 222 normal controls (NCs). Besides, the mRNA and protein levels of EGFR were investigated in Gene Expression Profiling Interactive Analysis (GEPIA) and Human Protein Atlas (HPA) database, respectively. The level of autoantibody to EGFR (anti-EGFR) in LC even different types of LC was obviously higher than that in NC (P < 0.05). The area under the curve (AUC) of anti-EGFR was 0.695 (95% CI 0.645–0.742) when comparing LC patients with NC, while the AUC of carcinoembryonic antigen (CEA) was 0.681 (95% CI 0.629–0.730). Moreover, by integrating anti-EGFR with CEA to diagnose LC, the AUC was up to 0.784 (95% CI 0.737–0.826). However, the expression level of autoantibody to EGFR had no difference between LC patients with and without EGFR gene mutation (P > 0.05). EGFR mRNA expression level was obviously upregulated in squamous cell carcinoma (SCC) tissues compared with normal tissues (P < 0.05), but not in adenocarcinoma (ADC) (P > 0.05). The study confirmed that anti-EGFR could be a potential biomarker for LC diagnosis; additionally, it could improve the diagnostic value of CEA in clinical work.

     

慢加急性肝衰竭患者外周血肿瘤坏死因子-α基因启动子甲基化状态的研究

目的 探讨肿瘤坏死因子-α及表观遗传调控在慢加急性肝衰竭发病机制中的可能作用.方法 ELISA法检测外周血TNF-α的表达,并与患者的肝功能进行相关性分析.同时提取外周血单个核细胞DNA,重亚硫酸盐处理后,用针对改变后的DNA序列设计特异性引物并进行聚合酶链反应(PCR).根据修饰后DNA序列的不同,运用在线MethPrimer软件设计引物,甲基化特异性聚合酶链反应(Methylation specific polymerase chain reaction,MSP)检测标本中TNF-α扩增产物,分析TNF-α启动子甲基化与血清TNF-α表达的相关性,并分析甲基化与病情严重程度的相关性.采用SPSS16.0软件进行统计分析.结果 慢加急性肝衰竭患者组外周血TNF-α蛋白水平(44.9260±26.48523)均高于慢性乙型病毒性肝炎患者组(18.92505±9.04461)和健康对照组(11.9172±5.04612),且各组间比较差异均具有统计学意义(P＜0.05).慢加急性肝衰竭患者组外周血清TNF-α水平与血清TBIL及终末期肝脏病模型(MELD)评分之间呈明显的正相关,与PTA呈明显的负相关.慢加急性肝衰竭组、慢性乙型病毒性肝炎患者组、健康对照组TNF-α启动子甲基化状态的差别有统计学意义.慢加急性肝衰竭患者与慢性乙型病毒性肝炎患者及健康对照甲基化组血清TNF-α水平均明显低于非甲基化组(P＜0.05).结论 TNF-α与肝炎活动及肝细胞损害有密切关系,DNA甲基化在慢加急肝衰竭患者中参与调控细胞因子的表达中.     

CDH1基因启动子甲基化在上皮性卵巢癌转移方面的研究

目的探讨CDH1基因启动子甲基化对上皮性卵巢癌转移的影响。方法采用免疫组织化学方法检测38例正常卵巢上皮和80例上皮性卵巢癌组织中E-钙黏附素（E-cadherin）表达；应用甲基化特异的PCR（MSP）检测上述组织中CDH1基因启动子区甲基化；应用5-氮-2＇-脱氧胞苷（5-aza-CdR）使SKOV3细胞去甲基化，观察SKOV3细胞体外侵袭性的改变，并通过RT-PCR检测CDH1基因的改变。结果E-cadherin在正常卵巢组织中表达明显高于上皮性卵巢癌（P〈0．05）。34例CDH1基因启动子区甲基化全部出现在卵巢癌组织中，有淋巴结转移组织中甲基化明显高于无淋巴结转移者（P〈0．05），而CDH1基因启动子区有甲基化的卵巢癌组织中E-cadherin表达明显降低（P〈0．05）。经5-Aza-CdR处理后的SKOV3细胞体外侵袭性降低（P〈0．01），CDH1基因的表达明显上调（P〈0．01）。结论E-cadherin表达降低与上皮性卵巢癌转移关系密切，CDH1基因启动子区甲基化可能是导致E-cadherin蛋白表达减低的重要原因之一，因此启动子区甲基化与上皮性卵巢癌转移有关。     

肝硬化伴肝癌患者血氨、甲胎蛋白、胆碱酯酶、肝纤维化指标与Child-Pugh 分级的相关性

目的:探讨肝硬化伴肝癌(LC-HCC)患者血氨(NH3 )、甲胎蛋白(AFP)、胆碱酯酶(ChE)及肝纤维化指标与Child-Pugh 分级的关系。方法:选取LC 合并HCC 患者58 例(LC-HCC 组)、LC 失代偿期76 例(LC 组)及同期健康体检者60例(HC 组)。采用化学发光法测定AFP,采用化学发光免疫分析法检测透明质酸(HA)、层黏连蛋白(LN)、郁型胶原(-C),采用丁酰基硫代胆碱法测定ChE,连续监测NH3 。比较LC-HCC、LC、HC 三组间,LC-HCC 组不同Child-Pugh 分级间的AFP、ChE、NH3 及肝纤维化指标(HA、LN、-C)水平变化。结果:LC-HCC 组的NH3 、血清AFP、HA、LN、-C 水平显著高于LC 组及HC 组,ChE 水平显著降低,且LC 组与HC 组比较差异均有统计学意义(P<0.05);LC-HCC 组随着Child-pugh 等级上升,NH3 、血清AFP、HA、LN、-C 水平逐渐升高而ChE 逐渐降低,差异有统计学意义(P<0.05);LC-HCC 患者的血清ChE 与Child-pugh分级呈显著负相关(P<0.05),AFP、NH3 、LN、-C 及HA 与Child-pugh 分级呈显著正相关(P<0.05);LC-HCC 患者的ChE 与LN、-C 及HA 呈显著负相关(P<0.05),AFP、NH3 与LN、-C 及HA 呈显著正相关(P<0.05)。结论:LC-HCC 患者存在明显的血清AFP、ChE 和NH3 异常,且与肝纤维化指标及Child-pugh 分级密切相关,联合检测对HCC 的无创性诊断具有重要价值。     

慢加急性肝衰竭中IL-10的表达及其启动子甲基化状态的研究

目的 探讨慢加急性肝衰竭患者血清IL-10的表达及其甲基化状态的意义.方法 慢加急性肝衰竭组25例,慢乙肝组25例,正常对照组10例,ELISA法测定血清IL-10的水平,MSP方法检测IL-10启动子甲基化状态,三组间比较.结果 肝衰竭组、慢乙肝组较正常对照组血清IL-10水平明显升高,有统计学意义(P<0.05);肝衰竭组较慢乙肝组升高,无统计学意义(P>0.05).肝衰竭组IL-10水平与TBIL、MELD明显正相关(分别为r=0.566,r=0.443,P<0.05),与PTA呈明显负相关(r=-0.581,P<0.05),与ALT、HBV-DNA无明显相关性(分别为r=-0.022,r=0.033,P>0.05).肝衰竭组甲基化分布状态较慢乙肝组和正常对照组有统计学差异(P<0.05).结论 肝衰竭、慢乙肝患者IL-10水平明显升高.IL-10水平随肝衰竭的严重程度升高.IL-10启动子区甲基化可能是基因失活的重要机制.     

慢加急性肝衰竭中IL-10的表达及其启动子甲基化状态的研究

目的 探讨慢加急性肝衰竭患者血清IL-10的表达及其甲基化状态的意义.方法 慢加急性肝衰竭组25例,慢乙肝组25例,正常对照组10例,ELISA法测定血清IL-10的水平,MSP方法检测IL-10启动子甲基化状态,三组间比较.结果 肝衰竭组、慢乙肝组较正常对照组血清IL-10水平明显升高,有统计学意义(P<0.05);肝衰竭组较慢乙肝组升高,无统计学意义(P>0.05).肝衰竭组IL-10水平与TBIL、MELD明显正相关(分别为r=0.566,r=0.443,P<0.05),与PTA呈明显负相关(r=-0.581,P<0.05),与ALT、HBV-DNA无明显相关性(分别为r=-0.022,r=0.033,P>0.05).肝衰竭组甲基化分布状态较慢乙肝组和正常对照组有统计学差异(P<0.05).结论 肝衰竭、慢乙肝患者IL-10水平明显升高.IL-10水平随肝衰竭的严重程度升高.IL-10启动子区甲基化可能是基因失活的重要机制.     

乙型肝炎肝硬化患者外周血Toll样受体2和4及CD4+CD25+调节性T细胞的变化

目的 检测乙型肝炎肝硬化患者外周血单个核细胞(peripheral blood mononuelear cells,PBMCs)表面Toll样受体(Toll-like receptor,TLR)2和TLR4以及CD4+CD25+调节性T细胞(regulato-ry T cells,Treg)的表达,并探讨TLR2、TLR4与Treg的相关性.方法 分别应用抗-TLR2-PE、抗-TLR4-APC、抗-CD14-FITC及抗-CD4-PerCP、抗-CD25-FITC、扰-CD127-PE对67例研究对象[乙型肝炎肝硬化患者(HBV related liver cirrhosis,LC)30例、慢性乙型肝炎患者(chronic hepatitis B,CHB)21例、健康对照(normal control,Nc)16例]PBMCs表面分子进行免疫荧光染色,应用流式细胞仪分别对TLR2、TLR4及Treg进行检测.组间比较采用Kruskal-wallis秩和检验,相关性分析采用Spearman秩相关.结果 LC组、CHB组、NC组单核细胞表面TLR2、TLR4的平均荧光强度(MFI)分别为200.3±96.8和32.1±7.2、214.0±72.6和25.2 ±8.3、94.1 ±17.6和17.8 ±3.9.LC组与NC组、CHB组与NC组TLR2 MFI比较差异均有统计学意义(P<0.05),LC组与CHB组TLR2 MFI差异无统计学意义(P>0.05).LC组与NC组、CHB组与NC、LC与NC组TLR4 MFI比较差异均有统计学意义(P<0.05).LC组、CHB组、Nc组Treg占CD4+T淋巴细胞的比例分别为(5.07%±1.43%、5.88%±1.66%、4.21%±1.24%),CHB组与NC组、CHB组与LC组比较差异有统计学意义(P<0.05),而LC组与NC组比较差异无统计学意义(P>0.05).在LC组,TLR4的表达与Treg呈正相关(r=0.469,P=0.032),TLR2与血清病毒载量呈负相关(r=-0.428,P=0.021).结论 LC患者,TLR2、TLR4的表达显著上调,TLR2、TLR4可能与乙型肝炎肝硬化的发生有关.