大肠癌患者外周血CD4^＋CD25^＋CD127^lo/-调节性T细胞的含量及临床意义

目的探讨大肠癌患者外周血CD4^＋、CD25^＋、CD127^lo/-调节性T细胞（Treg）的变化及意义。方法流式细胞术检测36例大肠癌患者及30例健康人外周血中CD4^＋、CD25^＋、CD127^lo/-Treg的变化；ELISA检测同一标本血清中转化生长因子β（TGF—β）的表达水平。结果36例大肠癌患者外周血中CD4^＋、CD25^＋、CD127^lo/-Treg占CD4^＋、淋巴细胞的比例为（6．92±1．31）％，与正常对照组（4．91±1，24）％相比差异有显著性（P〈0．05）。20例腺癌、7例黏液癌、9例未分化癌患者外周血中CD4^＋、CD25^＋、CD127^lo/-Treg比例分别为（6．81±1．52）％、（6.99±1．21）％、（7．08±1．17）％，各组间比较差异无显著性（P〉0．05），均高于对照组的（4．91±1．24）％，差异有显著性（P〈0，05）。IV期大肠癌患者外周血CD4^＋、CD25^＋、CD127^lo/-Treg高于Ⅱ期大肠癌患者（P〈0．05）。大肠癌组外周血清中TGF—β水平显著高于正常对照组，随着TGF—β水平增加，CD4^＋、CD25^＋、CD127^lo/-Treg比例逐渐增高，两者呈正相关。结论大肠癌患者外周血中CD4^＋、CD25^＋、CD127^lo/-Treg表达增高，与临床分期相关，但与组织学分类无关，导致机体免疫抑制，可能参与大肠癌发生。     

Fibroblast-derived exosomal miRNA-133 promotes cardiomyocyte-like differentiation

ObjectiveTo investigate the role of exosomal miRNA-133 secreted by cardiac fibroblasts (CFs) in promoting cardiomyocyte differentiation.MethodsNeonatal rat CFs were cultured in vitro, and the cultured CFs were divided into three groups as follows: induction, miRNA-133 high expression, and miRNA-133 inhibition. miRNA-133 was transfected into CFs with lentivirus as a vector. CFs were transfected with the miRNA-133 inhibitor, and the markers of cardiomyocyte were detected through immunofluorescence staining, Western blotting, and real-time quantitative polymerase chain reaction (qRT-PCR) at 3, 8, and 14 days, respectively. The expression levels of cardiac troponin T (cTnT) and cardiac actin (α-actin) were determined, and qRT-PCR was used to detect the expression of miRNA-133 in the fibroblast exosomes.ResultsCFs subjected to immunofluorescence staining expressed vimentin and discoid domain receptor 2. The exosomes secreted by CFs were observed as small vesicles of 30–100 nm via transmission electron microscopy, and Western blotting was used to detect exosome-specific protein CD63 and CD9 expression. The expression levels of cTnT, α-actin, and exosomal miRNA-133 secreted into the supernatant of the miRNA-133 high-expression group increased gradually at different time points and reached the highest level at 14 days. The expression levels of cTnT, α-actin, and exosome miRNA-133 in the miRNA-133 inhibition group were the lowest.ConclusionThe exosomal miRNA-133, which is derived from CFs, can promote the differentiation of fibroblasts into cardiomyocyte-like cells.     

血清miRNA-182检测在结直肠癌中的诊断价值研究

目的探讨血清miRNA-182作为结直肠癌(CRC)潜在标志物的诊断价值。方法运用荧光定量PCR法检测53例CRC患者血清miRNA-182相对表达量,并与肠良性病变患者及健康体检者进行比较分析。结果 CRC患者血清miRNA-182相对表达量明显高于肠良性病变组及对照组,差异均有统计学意义(P0.05)。ROC曲线显示血清miRNA-182诊断CRC的曲线下面积为0.846,95%置信区间为0.768~0.925;诊断灵敏度为77.1%,特异度为81.1%。血清miRNA-182相对表达量与CRC患者TNM分期(P=0.011)及远距离转移(P=0.002)有关;而与其他临床特征无关。CRC患者术后血清miRNA-182相对表达量明显低于术前水平(P0.01);而术后出现复发/转移的患者血清miRNA-182相对表达量又明显升高(P=0.019)。结论 CRC患者血清miRNA-182相对表达量明显升高,并与病情及恶性进展密切相关,血清miRNA-182可作为CRC潜在标志物用于临床诊断及病情评估。     

pH-responsive nanoparticles releasing tenofovir intended for the prevention of HIV transmission

This study is designed to test the hypothesis that tenofovir (TNF) or tenofovir disoproxil fumarate (TDF) loaded nanoparticles (NPs) prepared with a blend of poly(lactic-co-glycolic acid) (PLGA) and methacrylic acid copolymer (Eudragit® S-100, or S-100) are noncytotoxic and exhibit significant pH-responsive release of anti-HIV microbicides in the presence of human semen fluid simulant (SFS). After NPs preparation by emulsification diffusion, their size, encapsulation efficiency (EE%), drug release profile, morphology, and cytotoxicity are characterized by dynamic light scattering, spectrophotometry, transmission electron microscopy, and cellular viability assay/transepithelial electrical resistance measurement, respectively. Cellular uptake was elucidated by fluorescence spectroscopy and confocal microscopy. The NPs have an average size of 250 nm, maximal EE% of 16.1% and 37.2% for TNF and TDF, respectively. There is a 4-fold increase in the drug release rate from the 75% S-100 blend in the presence of SFS over 72 h. At a concentration up to 10 mg/ml, the PLGA/S-100 NPs are noncytotoxic for 48 h to vaginal endocervical/epithelial cells and Lactobacillus crispatus. The particle uptake (∼50% in 24 h) by these vaginal cell lines mostly occurred through caveolin-mediated pathway. These data suggest the promise of using PLGA/S-100 NPs as an alternative controlled drug delivery system in intravaginal delivery of an anti-HIV/AIDS microbicide.     

CD4(+)CD25(high)CD127(low) regulatory T cells in patients with stable angina and their dynamics after intracoronary sirolimus-eluting stent implantation

Rapamycin contributes to the expansion of regulatory T cells (Tregs) in vitro. We investigated CD4(+)CD25(high)CD127(low) Treg level dynamics as well as the major parameters of cell immunity and sCD25 and highly sensitive C-reactive protein (hsCRP) concentrations in the blood of patients after coronary stenting (CS) with sirolimus (rapamycin)-eluting stents (SES; n = 43). The relation between initial Treg values and the severity of coronary atherosclerosis was observed. Treg and sCD25 levels were increased 1 month after CS versus baseline values and versus data in the control group (coronary angiography [CA], n = 20). A positive correlation between Treg and sCD25 levels was reported, whereas no relation was observed with the length of SES implanted. HsCRP level was increased during the first 7 days and returned to baseline values 1 month after CS/CA. Treg content is lower in patients with multivessel CAD. Elevated levels of Tregs and sCD25 after SES implantation might occur because of the immunomodulating effect of rapamycin.     

HIV-1感染者CD4+CD25nt/hiCD127lo调节性T细胞PD-1表达水平与疾病进展的关系

目的:阐明HIV-1感染者外周血中具有CD4+CD25nt/hiCD127lo特征的调节性T细胞(Treg)表面PD-1的表达水平与疾病进展的关系.方法:选取108名未经治疗的不同进展期的HIV-1感染者和27名健康人对照, 采集静脉血, 用Ficoll-Hypaque密度梯度离心法分离获得PBMC, 加入PerCP-CD4抗体、 FITC-CD25抗体、 PE-CD127抗体和APC-PD-1抗体, 经细胞表面四色染色、流式细胞术(FCM)分析Treg表面PD-1的表达;另将50 L全血加入Trucount绝对计数管, 采用Multitest CD3/CD8/CD45/CD4试剂盒检测CD4+T细胞绝对数;分离静脉血血浆, NucliSens EasyQ测定血浆HIV-1病毒载量;实验数据采用SPSS14.0 统计学软件分析处理.结果:HIV-1感染者Treg表面PD-1表达水平显著高于健康人(5.33%±2.24% vs 1.72%±0.65%, P<0.01);AIDS期(7.87%±2.23%)明显高于进展期(5.21%±1.72%, P<0.05)和新近感染者(3.22%±1.01%, P<0.05);HIV-1感染者Treg表面PD-1表达水平与血浆中的HIV-1病毒载量和CD4+T细胞绝对数密切相关.结论:首次证实HIV-1感染者外周血中Treg表面PD-1表达增加, 且表达水平与病程进展相关.该结果为进一步揭示HIV-1感染中Treg的效应机制、探索新的免疫治疗方案提供了理论及实验依据.     

类风湿关节炎患者外周血CD4+T细胞CD25及CD127的表达

目的 探讨类风湿关节炎(RA)患者外周血调节性T细胞(Tregs)比例的变化情况,了解Tregs变化在RA发病及炎症活动中的意义.方法 采用流式细胞仪三色荧光抗体标记法分别对25例RA患者和31名健康志愿者(HV)的外周血淋巴细胞进行CD4、CD25及CD127标记,分析比较RA患者组与HV组间淋巴细胞中CD4+CD25+、CD4+CD25high、CD4+CD25+CD127-及CD4+CD25highCD127-细胞比例的差异情况,并将RA组相应细胞比例与RA疾病活动性评分(DAS28-4)、压痛关节数、肿胀关节数、晨僵时间、医生和患者对疾病活动性的整体评价(VAS)、血红细胞沉降率、C-反应蛋白行相关分析.结果 RA组外周血淋巴细胞中,CD4+CD25+CD127-和CD4+CD25highCD127-细胞占CD4+T淋巴细胞的百分率分别为(2.53±0.85)%和(0.91±0.32)%,而HV组则分别为(3.22±0.97)%和(1.25±0.41)%.CD4+CD25+CD127+和CD4+CD25highCD127-细胞比例在RA组均明显低于HV组,其组间差异均有统计学意义(均P<0.05);另外,RA患者CD4+CD25+CD127-和CD4+CD25highCD127-T细胞比例均与DAS28-4及压痛关节数存在负相关(P<0.05),CD4+CD25highCD127-T细胞还与肿胀关节数及患者VAS存在负相关(P<0.05).结论 RA患者外周血淋巴细胞存在Tregs比例的降低,Tregs数量的异常可能是RA发病及炎症活动的重要影响因素.     

CD4+CD25+/highCD127low在传染性单核细胞增多症患儿中的"永生细胞"抑制作用

目的 探讨CD4+CD25CD25+/highCD127low 在传染性单核细胞增多症(IM)EB病毒(EBV)转化的"永生细胞"抑制途径的作用.方法 流式细胞仪测定23例急性期IM患儿和其中10例恢复期随访EBV感染IM患儿CD3+、CD3+CD4+、CD3+CD8+、CD4+CD25+/highCD127low和CD19+、CD19+CD23+表达情况,并与20例正常对照组比较.结果 IM患儿CD3+、CD3+CD8+与正常对照组比较有明显升高,而CD3+CIM+、CD4+/CD8+比值、CIM+CD25+/highCD127low、CD19+、CD19+CD23+明显低于正常对照组.CD3+、CD3+CD8+急性期明显高于恢复期,CD3+CD4+、CD4+/CD8+比值、CD+CD25+/highCD127low、CD19+、CD19+CD23+恢复期都明显下降.在疾病恢复期,CD3+CD8+、CD4+/CD8+比值、CD19+、CD19+CD23+仍有显著性差异,其他指标与正常对照组之间差异无统计学意义.在急性期CD3+、CD3+CD8+与CD19+、CD19+CD23+细胞均呈负相关,而CD4+CD25+/highCD127low与CD19+、CD19+CD23+细胞均呈正相关.结论 在EBV感染儿童IM发病过程中,CD4+CD25+/highCD127+的降低可能对EBV转化的"永生细胞"在体内的有效抑制起着主要的作用.     

HeLa细胞中miRNA-214靶基因Mek3的确定

目的 探索miRNA-214在HeLa细胞中的与其靶基因Mek3相互作用.方法 通过miRNA靶基因预测网站寻找可能与miRNA-214相互作用的靶基因,合成miRNA-214和对照序列,将miRNA-214、对照序列、Mek3的3'非翻译区(3' UTR)以及突变的Mek3 3'UTR分别克隆到表达载体上,转染HeLa细胞,转染48 h后提取蛋白,检测绿色荧光蛋白的表达水平; HeLa细胞转染miRNA-214后,Trizol抽提RNA,通过荧光定量PCR检测Mek3 mRNA的表达水平;Western印迹检测Mek3的蛋白表达水平.经过以上实验从mRNA和蛋白水平上验证了在HeLa细胞中miRNA-214对靶基因Mek3的作用效应.结果 生物信息学方法显示miRNA-214和Mek3存在可能的结合位点.经过实验验证了miRNA-214可以下调Mek3的mRNA和蛋白水平.结论 miRNA-214可以负调节靶基因Mek3的表达.