乙型肝炎病毒C基因启动子双变异患者中医证型特点研究

目的：探讨HBVC基因启动子（BCP）双变异慢性乙型肝炎患者的中医证型特点。方法：选择HBsAg阳性慢性乙型肝炎患者168例进行观察。中医证型分为湿热中阻、肝郁脾虚、肝肾阴虚、瘀血阻络、脾肾阳虚5型；BCP双变异检测，采用微板核酸杂交法。结果：BCP双变异的总检出率为36．31％（61／168），其中，湿热中阻型BCP双变异检出率最高（54．24％），与其他各型比较差异均有显著性意义（P〈0．052）；肝郁脾虚型BCP双变异检出率（36．36％）虽低于湿热中阻型（P〈0．025），但却明显高于肝肾阴虚型（13．04％）及瘀血阻络型（10．53％），且差异有显著性意义（P〈0．05）；肝肾阴虚与瘀血阻络两型BCP双变异检出率均较低，两者比较差异有显著性意义（P〉0．05）。结论：HBV BCP双变异的慢性乙型肝炎患者中医证型特点以湿热中阻为主，肝郁脾虚居次，临床治疗要重视清热利湿，舒肝健脾治法或方药的选用。     

新基因TAHCCP1编码蛋白对NS3TP6基因启动子转录活性的调节

目的探讨丙型肝炎病毒（HCV）核心蛋白反式激活基因TAHCCP1编码蛋白对NS3TP6启动子转录活性的调节。方法以我室前期克隆的TAHCCP1基因表达谱芯片结果为基础,利用生物信息学方法确定NS3TP6基因的启动子区域（NS3TP6-p）,聚合酶链反应（PCR）扩增并克隆至真核报告载体pCAT3-basic中,构建重组报告质粒pCAT3-NS3TP6-p;以该质粒单独或与pcDNA3．1（-）-TAHCCP1共转染肝癌细胞系HepG2细胞,用酶联免疫吸附法（ELISA）检测氯霉素乙酰转移酶（CAT）的表达活性;观察细胞中表达的TAHCCP1蛋白对NS3TP6启动子转录活性的调节。结果构建的重组表达载体pCAT3-NS3TP6-P在HepG2细胞中能够启动报告基因CAT表达,表明克隆的NS3TP6启动子有指导下游基因转录表达的活性;共转染实验中pCAT3-NS3TP6-p与pcDNA3．1（-）-TAHCCP1组CAT的表达活性是对照组的3．1倍,说明TAHCCP1蛋白能够在转录水平反式激活NS3TP6基因启动子活性。结论本实验验证了基因芯片的实验结果准确性,进一步完善了新基因TAHCCP1的生物学功能,为深入理解HCV核心蛋白的反式激活调节机制提供了新的依据。     

Hepatitis B virus subgenotype C2- and B2-associated mutation patterns may be responsible for liver cirrhosis and hepatocellular carcinoma,respectively

The objective of this study was to examine hepatitis B virus (HBV) subgenotypes and mutations in enhancer II, basal core promoter, and precore regions of HBV in relation to risks of liver cirrhosis (LC) and hepatocellular carcinoma (HCC) in Southeast China. A case-control study was performed, including chronic hepatitis B (CHB; n=125), LC (n=120), and HCC (n=136). HBV was genotyped by multiplex polymerase chain reaction and subgenotyped by restriction fragment length polymorphism. HBV mutations were measured by DNA sequencing. HBV genotype C (68.2%) predominated and genotype B (30.2%) was the second most common. Of these, C2 (67.5%) was the most prevalent subgenotype, and B2 (30.2%) ranked second. Thirteen mutations with a frequency >5% were detected. Seven mutation patterns (C1653T, G1719T, G1730C, T1753C, A1762T, G1764A, and G1799C) were associated with C2, and four patterns (C1810T, A1846T, G1862T, and G1896A) were associated with B2. Six patterns (C1653T, G1730C, T1753C, A1762T, G1764A, and G1799C) were obviously associated with LC, and 10 patterns (C1653T, G1730C, T1753C, A1762T, G1764A, G1799C, C1810T, A1846T, G1862T, and G1896A) were significantly associated with HCC compared with CHB. Four patterns (C1810T, A1846T, G1862T, and G1896A) were significantly associated with HCC compared with LC. Multivariate regression analyses showed that HBV subgenotype C2 and C2-associated mutation patterns (C1653T, T1753C, A1762T, and G1764A) were independent risk factors for LC when CHB was the control, and that B2-associated mutation patterns (C1810T, A1846T, G1862T, and G1896A) were independent risk factors for HCC when LC was the control.     

The conserved lymphokine element 0 is a powerful activator and target for corticosteroid inhibition in human interleukin-5 transcription

The role of eosinophilia in allergic disorders indicates hIL-5 as a potential target for therapy. The conservation of hIL-5 gene proximal elements suggests they are important in controlling expression. Corticosteroids are important in the treatment of allergy, and are powerful inhibitors of IL-5 expression. This study aimed at understanding the role of hIL-5 conserved proximal elements, and elucidating the target of corticosteroid activity, in hIL-5 gene expression. Methods used include transient transfection of PBMC and PER117 cells with hIL-5 deletion constructs, EMSA, Western Blotting, and RT-PCR.

The conserved proximal CLE0/TATA elements driving a reporter gene gave similar or higher expression than a 500 bp promoter in primary human T cells and a T-cell line. Two and three copies of IL-5 CLE0 upstream of the silent IL-4 minimal promoter gave 30–45 fold increases in expression in forward orientation, but little activity in reverse orientation. Consequently, CLE0 is a powerful activator but not a classical enhancer. Deletion analysis identified CLE0 as the key element in the inhibition of IL-5 reporter constructs by dexamethasone, and RT-PCR analysis indicated that GILZ expression correlated with dexamethasone-induced inhibition of IL-5. Ectopic expression of GILZ, confirmed by western blotting, gave a 90% inhibition of promoter constructs in absence of dexamethasone. CLE0 is a powerful activator sufficient for the inducible expression of IL-5, and functions when moved upstream in a heterologous promoter. CLE0 is also the main target for IL-5 inhibition by dexamethasone, and we present evidence consistent with a role of GILZ in this.     

Promoter Methylation of LEP and LEPR before and after Bariatric Surgery: A Cross-Sectional Study

IntroductionDNA methylation constitutes one important epigenetic mechanism that regulates gene expression in human cells. With regard to obesity, bariatric surgery-induced weight loss has been associated with promoter methylation changes in several genes. Hyperleptinemia is a characteristic feature of obesity. The underlying regulating mechanisms have not yet been completely elucidated.MethodsWe investigated the methylation of the promoters of the leptin gene (LEP) and the leptin receptor gene (LEPR) as well as leptin expression in pre- and postbariatric surgery patients using a comparative cross-sectional design.ResultsOur results revealed significantly higher LEP promoter methylation patterns in prebariatric surgery patients compared to postoperatively. DNA methylation of the LEPR promoter was significantly higher in the postoperative group. Moreover, we found significantly higher leptin serum levels in patients before the bariatric surgery than afterwards.DiscussionThese findings strengthen the suggestion that there is an association between LEP expression and LEP methylation in obesity. We suggest that the epigenetic profile of LEP might be influenced by leptin serum levels in the form of a regulating feedback mechanism.     

MGMT启动子与垂体腺瘤侵略性行为及ADC值的相关性研究

目的探讨垂体腺瘤O-6-甲基鸟嘌呤DNA甲基转移酶(MGMT)启动子甲基化状态与肿瘤侵略性行为及ADC值的相关性。方法采用前瞻性研究,选取我院2019年01月至2020年08月收治的垂体腺瘤的患者,所有患者术前接受MRI检查(平扫+增强+解剖弥散),术后对肿瘤组织标本进行苏木精-伊红(HE)染色、免疫组化检查,通过焦磷酸测序检测肿瘤MGMT启动子甲基化状态。对比MGMT启动子甲基化状态与垂体腺瘤侵袭性分组,功能分型,增值性分组以及ADC值之间的相关性。结果17例增值组患者中有2例患者伴有MGMT突变,MGMT无突变患者15例,MGMT启动子甲基化突变状态与肿瘤的增值性存在明显相关(P<0.05)。30例影像学侵袭组患者中,8例患者有MGMT突变,MGMT无突变患者22例,两组差异无统计学意义(P>0.05)。21功能型垂体腺瘤患者中,有7例患者有MGMT突变,MGMT无突变患者14例,两组间差异无统计学意义(P>0.05)。此外,18例MGMT甲基化突变组的垂体腺瘤ADC平均值为844.67,37例MGMT无突变组ADC平均值为791.14,差异无统计学意义(P>0.05)。结论垂体腺瘤MGMT启动子甲基化状态与Ki-67表达密切相关,有望成为预判垂体腺瘤侵略性行为的重要指标。     

DNA Methylation Profiling Revealed Promoter Hypermethylation-induced Silencing of p16, DDAH2 and DUSP1 in Primary Oral Squamous Cell Carcinoma

Background: Hypermethylation in promoter regions of genes might lead to altered gene functions and result in malignant cellular transformation. Thus, biomarker identification for hypermethylated genes would be very useful for early diagnosis, prognosis, and therapeutic treatment of oral squamous cell carcinoma (OSCC). The objectives of this study were to screen and validate differentially hypermethylated genes in OSCC and correlate the hypermethylation-induced genes with demographic, clinocopathological characteristics and survival rate of OSCC.Methods: DNA methylation profiling was utilized to screen the differentially hypermethylated genes in OSCC. Three selected differentially-hypermethylated genes of p16, DDAH2 and DUSP1 were further validated for methylation status and protein expression. The correlation between demographic, clinicopathological characteristics, and survival rate of OSCC patients with hypermethylation of p16, DDAH2 and DUSP1 genes were analysed in the study.Results: Methylation profiling demonstrated 33 promoter hypermethylated genes in OSCC. The differentially-hypermethylated genes of p16, DDAH2 and DUSP1 revealed positivity of 78%, 80% and 88% in methylation-specific polymerase chain reaction and 24% and 22% of immunoreactivity in DDAH2 and DUSP1 genes, respectively. Promoter hypermethylation of p16 gene was found significantly associated with tumour site of buccal, gum, tongue and lip (P=0.001). In addition, DDAH2 methylation level was correlated significantly with patients'' age (P=0.050). In this study, overall five-year survival rate was 38.1% for OSCC patients and was influenced by sex difference.Conclusions: The study has identified 33 promoter hypermethylated genes that were significantly silenced in OSCC, which might be involved in an important mechanism in oral carcinogenesis. Our approaches revealed signature candidates of differentially hypermethylated genes of DDAH2 and DUSP1 which can be further developed as potential biomarkers for OSCC as diagnostic, prognostic and therapeutic targets in the future.     

CD14 promoter polymorphism in Chinese alcoholic patients with cirrhosis of liver and acute pancreatitis

AIM: To investigate the relationship between genetic polymorphism of the CD14 promoter and the occurrence of alcoholic cirrhosis and alcoholic pancreatitis, and to challenge the conclusion made earlier that the patients with acute alcoholic pancreatitis and patients with alcoholic cirrhosis of liver are two different subpopulations. METHODS: Using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method, we determined the polymorphism of CD14 gene and aldehyde dehydrogenase gene 2 (ALDH 2) in 335 alcoholic patients with different organ complications i.e., cirrhosis of liver (n = 100), acute pancreatitis (n = 100), esophageal cancer (n = 82) and avascular necrosis of hip joint (AVN) (n = 53) and 194 non-alcoholic controls in a Chinese group. RESULTS: The results showed that the carriage of T allele was not different among alcoholic patients with cirrhosis of liver, alcoholic patients with other complication and non-alcoholic controls. On the other hand, the carriage of the C allele was significantly more prevalent for alcoholic pancreatitis than for esophageal cancer (0.79 vs 0.60, P<0.001), alcoholic AVN (0.79 vs 0.65, P<0.025) and nonalcoholic controls (0.79 vs 0.68,P<0.025). Furthermore, when only subjects with ALDH2 1-1 genotype were examined, the C allele frequency was significantly more prevalent for alcoholic pancreatitis than for alcoholic liver cirrhosis (0.82 vs 0.69,P<0.025), esophageal cancer (0.82 vs 0.61,P><0.01), alcoholic AVN (0.82 vs 0.64, P<0.01) and non-alcoholic controls (0.82 vs 0.69, P<0.05). CONCLUSION: The C allele may be associated with some mechanism, which is important in the pathogenesis of alcoholic pancreatitis, and that alcoholic patients with acute pancreatitis and cirrhosis of liver are probably two different subpopulations.