甲基化DNA免疫沉淀-荧光实时定量PCR检测人精浆游离DNA睾丸和附睾特异性甲基化启动子

目的:建立检测精浆游离DNA(cfsDNA)启动子甲基化水平的甲基化DNA免疫沉淀-荧光实时定量PCR(MeDIP-qPCR)方法。方法:提取精液参数正常(6例)和输精管结扎(6例)男性的cfsDNA,将两组不同个体的cfsDNA分别进行混合,超声处理后形成DNA片段,通过MeDIP分离甲基化DNA片段,然后用qPCR方法检测启动子甲基化水平。结果:精液参数正常组的PRAME、PEG10、MORC1、GML、HOXA5、DNMT3L、SNURF、MSH4、DAZ1和CLPB启动子甲基化水平分别为14.93%、2.64%、0.69%、2.66%、17.50%、21.10%、5.98%、2.28%、13.50%和3.86%,明显低于输精管结扎组的121.25%、73.62%、16.25%、42.90%、76.74%、112.40%、59.79%、25.85%、91.90%和64.53%。其中,PRAME、MORC1、GML、HOXA5、DNMT3L、SNURF、MSH4和DAZ1等8个启动子MeDIP-qPCR的检测结果与启动子甲基化芯片结果一致。结论:通过MeDIP-qPCR方法可以定量检测到cfsDNA中的特异性启动子甲基化,为确定cfsDNA中睾丸和附睾特异性甲基化启动子提供了有效途径。     

Effects of antidepressant treatment on mice lacking brain‐derived neurotrophic factor expression through promoter IV

Brain‐derived neurotrophic factor (BDNF) is implicated in the pathophysiology of major depression; mice lacking BDNF expression through promoter IV (BDNF‐KIV) exhibit a depression‐like phenotype. We tested our hypothesis that deficits caused by promoter IV deficiency (depression‐like behavior, decreased levels of BDNF, and neurogenesis in the hippocampus) could be rescued by a 3‐week treatment with different types of antidepressants: fluoxetine, phenelzine, duloxetine, or imipramine. Each antidepressant reduced immobility time in the tail suspension test without affecting locomotor activity in the open field test in both BDNF‐KIV and control wild type mice, except that phenelzine increased locomotor activity in wild type mice and anxiety‐like behavior in BDNF‐KIV mice. The antidepressant treatments were insufficient to reverse decreased BDNF levels caused by promoter IV deficiency. No antidepressant treatment increased the hippocampal progenitors of either genotype, whereas phenelzine decreased the surviving progenitors in both genotypes. The antidepressant treatments differently affected the dendritic extension of hippocampal immature neurons: fluoxetine and imipramine increased extension in both genotypes, duloxetine increased it only in BDNF‐KIV mice, and phenelzine decreased it only in wild type mice. Interestingly, a saline‐only injection increased neurogenesis and dendrite extensions in both genotypes. Our results indicate that the behavioral effects in the tail suspension test by antidepressants do not require promoter IV‐driven BDNF expression and occur without a detectable increase in hippocampal BDNF levels and neurogenesis but may involve increased dendritic reorganisation of immature neurons. In conclusion, the antidepressant treatment demonstrated limited efficacy; it partially reversed the defective phenotypes caused by promoter IV deficiency but not hippocampal BDNF levels.     

多基因启动子甲基化诊断乙型肝炎病毒相关肝癌的研究

目的  探讨血清多基因启动子甲基化在乙型肝炎病毒（HBV）相关肝细胞癌（HCC）中的诊断价值。方法  随机收集98例HCC、75例肝硬化（LC）、90例慢性乙型肝炎（CHB）患者以及80例健康者血清各2ml,以血清BVES、APC、RASSF1A、TIMP3、GSTP1和HOXA9基因作为候选靶标。采用磁珠法提取血清DNA,MethyLight法检测基因启动子甲基化。通过构建受试者工作特征曲线（ROC）评价各检测指标的诊断效能。结果  血清各基因启动子甲基化在HCC患者检测阳性率分别为：RASSF1A（52.04%）、APC（36.73%）、BVES（29.59%）、HOXA9（20.41%）、GSTP1（17.35%）和TIMP3（11.22%）,其中APC甲基化阳性完全与RASSF1A重叠。血清RASSF1A甲基化从慢性HBV感染患者中诊断HCC的效能最高[敏感性=0.520,特异性=0.915,曲线下面积（AUC）=0.718],优于血清AFP（敏感性=0.480,特异性=0.739,AUC=0.609）;血清6个基因启动子甲基化联合使用的敏感性、特异性及诊断效能进一步提高,分别为0.806、0.855和0.845。结论  血清BVES、APC、RASSF1A、TIMP3、GSTP1及HOXA9基因启动甲基化联合检测能显著提高高危人群中HCC的诊断能力。

     

Use of <Emphasis Type="Italic">MET3</Emphasis> promoters for regulated gene expression in <Emphasis Type="Italic">Ashbya gossypii</Emphasis>

A central tool for gene function analysis is the construction mutant strains. This can be done conveniently in A. gossypii using PCR-based tools. The deletion of essential genes can be performed since initial transformants are sheltered in a heterokaryotic mycelium, which contains nuclei with both wild type and mutant alleles. The analysis of mutant phenotypes in A. gossypii is regularly started by germinating spores, which contain only one nucleus. Thus, selection can be used to identify mutant germ cells and germlings. However, such an analysis yields only mutant mycelia if the deleted gene is not essential. We describe the use of the regulatable Saccharomyces cerevisiae and A. gossypii MET3 promoters as novel tools to regulate gene expression in A. gossypii. Conditional expression was tested using GFP and lacZ-reporter genes. Regulation of MET3 promoters was found to be dependent on methionine but not on cysteine and down-regulation to about 1/10 of the initial expression levels was achieved. We used the A. gossypii WAL1 and CYK1 genes as models to demonstrate that MET3 promoters could regulate the expression of these genes and reveal their mutant phenotypes depending on the presence or absence of methionine. Finally, we show that the AgMET3 promoter contains two Cpf1-binding sites and that AgCPF1 can complement the S. cerevisiae cpf1 methionine auxotrophy.     

rs294775 is a cis‐regulatory SNP for human UGT2B10

UGT2B10 is an important metabolism enzyme in human body and its substrates include multiple amine‐containing compounds, especially nicotine, tamoxifen and multiple antidepressants. Multiple common SNPs have been observed in its promoter region, but their role in expression regulation has never been investigated. In this preliminary study, we identified a novel cis‐regulatory SNP, rs294775, for UGT2B10 by plasmid construction, mutagenesis, and luciferase assay, whose mechanism was also investigated. Our work provides a basis for further pharmacogenetics study.     

肿瘤坏死因子-α-238基因多态性及血清学水平与乙型肝炎病毒慢性感染结果的关系

目的 探讨肿瘤坏死因子（TNF-α）基因多态性与乙型肝炎病毒慢性感染结果之间的关系以及乙型肝炎病毒慢性感染患者血清中TNF-α水平在慢性乙肝发展中的临床意义。方法 运用聚合酶链反应-限制性片段长度多态性分析的方法检测TNF-α基因启动子区-238位点单个核苷酸多态性在不同临床类型的慢性HBV感染者及健康对照者中的分布频率;应用ELISA方法检测血清TNF-α浓度水平。结果 TNF-α-238位点G/A和G/G基因型频率以及A等位基因频率分布在实验组和健康对照组的差异无统计学意义。慢性乙型肝炎组、肝硬化组、乙型肝炎肝衰竭组和健康对照组比较,血清TNF-α均有不同程度升高,TNF-α（ng/L）取对数值后经方差分析,差异有统计学意义（P〈0.01）;组间两两比较,差异均有统计学意义（P〈0.01）,血清TNF-α水平乙型肝炎肝衰竭组〉肝硬化组〉慢性乙型肝炎组〉健康对照组。结论 TNF-α-238位点基因型与乙型肝炎易感性无明显相关性。TNF-α与乙型肝炎类型、肝损伤程度有密切关系。