树突状细胞分化发育及功能的表观遗传学调控机制的研究进展

树突状细胞（DCs）是体内功能最强的专职抗原提呈细胞,具有摄取、加工、处理和呈递抗原以及激活初始T淋巴细胞的功能。近年来研究发现,DNA甲基化、组蛋白修饰、染色质重塑、基因组印记以及非编码RNA等表观遗传学修饰能调控相关基因的表达,同时树突状细胞的分化发育及功能也受到表观遗传学调控。研究从DNA甲基化修饰、组蛋白修饰和非编码RNA,了解近年来很有必要树突状细胞的分化发育及功能的表观遗传学调控机制很有意义。     

人附睾蛋白4基因表达下调对卵巢癌细胞生物学行为的影响

目的 探讨人附睾蛋白4(HE4)基因表达下调对卵巢癌细胞增殖、黏附以及侵袭能力的影响.方法 构建含HE4 shDNA的重组质粒,并将其转染卵巢癌细胞系SKOV3细胞,RNA干扰后,通过即时定量聚合酶链反应(qRT-PCR)、Western blot等测定HE4表达下调情况;同时利用平板克隆形成实验、细胞黏附实验、测定稳定转染细胞的侵袭力改变等方法,比较分析HE4相关的生物学效应.结果 Western blot以及qRT-PCR结果均提示HE4干扰重组质粒构建成功,稳定转染SKOV3细胞后能显著抑制HE4基因的表达.体外实验显示,HE4表达下调的细胞侵袭能力明显下降(P＜0.05),黏附率明显下降到27.3％,而增殖能力较转染前无明显变化.结论 所构建的HE4干扰重组表达载体能有效抑制HE4基因表达,而HE4表达下调对卵巢癌的细胞黏附和侵袭能力降低.     

ZAC基因在奥曲肽抑制胃癌细胞体外增殖通路的作用

目的 探讨ZAC基因在生长抑素类似物奥曲肽（OCT）抑制胃癌细胞增殖通路的作用。方法 分别以不同浓度OCT处理胃癌细胞BGC823和 SGC7901不同时间,MTT法筛选其增殖抑制的有效条件。OCT处理胃癌细胞（有效浓度/不同时间,有效时间/不同浓度）,Western blotting检测
OCT对胃癌细胞ZAC基因的效应。设计3条ZAC基因RNA干扰片段,分别插入pSUPER-EGFP-I载体,构建3个ZAC-shRNA表达载体（pSUPER-EGFP-ZAC/1 pSUPER-EGFP-ZAC/2和pSUPER-EGFP-ZAC/3）。经酶切和序列分析鉴定后,分别转染胃癌细胞BGC823和SGC7901。经G418筛选,RT-PCR鉴定,建立
ZAC基因敲低（knock-down）的胃癌细胞系。以有效条件OCT孵育胃癌细胞（对照组）和ZAC基因敲低胃癌细胞（实验组）,MTT法检测OCT对胃癌 细胞的生长抑制效应。结果 OCT抑制胃癌细胞增殖的有效条件为10nmol/L 孵育24h;OCT以时间和浓度依赖的方式诱导胃癌细胞ZAC基因表达。酶切及序列分析鉴定表明ZAC-shRNA表达载体构建成功。转染shRNA-ZAC/2的胃癌细胞,其ZAC mRNA水平明显降低（P<0.05）,为ZAC基因敲低胃癌 细胞。ZAC基因敲低胃癌细胞的增殖明显高于对应的BGC823细胞和SGC7901细胞（P<0.05）。OCT孵育后,BGC823细胞和SGC7901细胞的增殖明显降低（P<0.05）。然而,ZAC基因敲低胃癌细胞的增殖无明显改变（P>0.05）。结论 OCT以时间和浓度依赖的方式诱导胃癌细胞ZAC基因表达;ZAC 基因在OCT抑制胃癌细胞增殖通路中具有重要作用。     

siRNA沉默MIF基因对糖皮质激素抑制脂质炎症介质释放的影响及其机制

 目的： 研究小干扰RNA（siRNA）阻断巨噬细胞移动抑制因子（macrophage migration-inhibitory factor,MIF）基因表达对糖皮质激素抑制脂质炎症介质释放的影响及其细胞内机制。方法：体外培养小鼠巨噬细胞系RAW2647,采用免疫荧光法观测siRNA转染效率,RT-PCR检测MIF mRNA的表达,Western blotting检测MIF蛋白的表达;RAW2647细胞转染MIF siRNA后观察地塞米松（Dex）抗炎作用的变化,用ELISA检测细胞上清中前列腺素E2(PGE2)和白三烯B4(LTB4)的含量,Western blotting检测胞浆膜联蛋白Annexin 1和下游胞浆磷酸酯酶A2α（cPLA2α）的蛋白表达变化。结果：与阴性对照相比,MIF siRNA能有效阻断细胞内源性MIF蛋白的表达,增强RAW2647细胞对Dex作用的敏感性;明显增强Dex抑制PGE2和LTB4产生的效应,增加胞浆蛋白Annexin 1的表达,抑制cPLA2α的磷酸化。结论：MIF siRNA能增强糖皮质激素抑制脂质炎症介质PGE2和LTB4的释放,且可能是通过影响Annexin 1-cPLA2α信号通路实现的。阻断内源性MIF蛋白的表达可显著增强RAW2647细胞对糖皮质激素抗炎作用的敏感性。     

Anti-Soluble Liver Antigen (SLA) Antibodies in Chronic HCV Infection

Hepatitis C infection is associated with autoimmune disorders, such as the production of autoantibodies. Anti-LKM1 and anti-LC1, immunomarkers of type 2 autoimmune hepatitis, have been previously associated with a HCV infection. Anti-Soluble-Liver-Antigen autoantibodies (SLA) are specifically associated with type 1 and type 2 autoimmune hepatitis and more closely related to patients who relapse after steroid therapy. The recent molecular cloning of the soluble liver antigen provides the opportunity to develop more specific tests for the detection of antibodies against it. The aim of this work is to characterize anti-soluble-liver autoantibodies in sera from patients chronically infected by HCV. A recombinant cDNA from activated Jurkat cells coding for the full length tRNP^(Ser)Sec/SLA antigen was obtained. ELISA, Western Blot and immunoprecipitation tests were developed and used to search for linear and conformational epitopes recognized by anti-SLA antibodies in sera from patients chronically infected by HCV. Anti-soluble liver antigen antibodies were found in sera from 10.4% of HCV-infected patients. The prevalence was significantly increased to 27% when anti-LKM1 was also present. Most anti-SLA reactivity was directed against conformational epitopes on the antigen. The means titers by ELISA were lower than those obtained in type 2 AIH. The result of autoantibody isotyping showed a subclass restriction to IgG1 and also IgG4. This study shows the presence of anti-SLA antibodies in approximately 10% of HCV infected patients. The prevalence of SLA autoantibodies in HCV infected patients increases when LKM1 autoantibodies are also present. The relationship between the prevalence of this characteristic autoimmune hepatitis autoantibody and the implication of an autoimmune phenomenon in the liver injury of patients chronically infected by HCV needs further investigation.     

Functional Analysis of a Large set of BRCA2 exon 7 Variants Highlights the Predictive Value of Hexamer Scores in Detecting Alterations of Exonic Splicing Regulatory Elements

Exonic variants can alter pre‐mRNA splicing either by changing splice sites or by modifying splicing regulatory elements. Often these effects are difficult to predict and are only detected by performing RNA analyses. Here, we analyzed, in a minigene assay, 26 variants identified in the exon 7 of BRCA2, a cancer predisposition gene. Our results revealed eight new exon skipping mutations in this exon: one directly altering the 5′ splice site and seven affecting potential regulatory elements. This brings the number of splicing regulatory mutations detected in BRCA2 exon 7 to a total of 11, a remarkably high number considering the total number of variants reported in this exon (n = 36), all tested in our minigene assay. We then exploited this large set of splicing data to test the predictive value of splicing regulator hexamers’ scores recently established by Ke et al. ( 2011 ). Comparisons of hexamer‐based predictions with our experimental data revealed high sensitivity in detecting variants that increased exon skipping, an important feature for prescreening variants before RNA analysis. In conclusion, hexamer scores represent a promising tool for predicting the biological consequences of exonic variants and may have important applications for the interpretation of variants detected by high‐throughput sequencing.