SLE患者PBMCs雌激素受体mRNA水平与IL-10 mRNA水平的研究

目的：探讨未经刺激外周血单个核细胞（PBMCs）雌激素受体（ER）mRNA水平和白细胞介素10（IL-10）mRNA水平在系统性红斑狼疮发病中的作用。方法：分离25例系统性红斑狼疮（SEE）患者（患者组）和15例正常人（对照组）新鲜外周血单个核细胞，提取总RNA，利用半定量RT-PCR方法逆转录并扩增雌激素受体和IL-10．通过凝胶图像扫描系统对PCR产物的电泳条带进行密度扫描。结果：患者组PBMCsERmRNA水平和IL-10mRNA水平均高于正常对照组（P〈0．01）。患者组和对照组PBMCsERmRNA水平与IL-10mRNA水平均呈正相关（r分别为0．442和0．557，P〈0．05）。结论：SLE患者外周血单个核细胞存在雌激素受体mRNA和IL-10mRNA表达异常．IL-10表达的上调可能与雌激素受体有关．过量的IL-10可能导致了SLE患者B细胞产生大量自身抗体。     

Invariant aphthovirus consensus nucleotide sequence in the transition to error catastrophe

RNA viruses replicate as complex distributions of non-identical but closely related variant genomes termed viral quasispecies. When the error rate during genome replication exceeds a threshold value, the genetic information cannot be maintained and the system enters error catastrophe. This violation of the error threshold results in virus extinction and it is currently being investigated as a new antiviral strategy, based on antiviral activity of some mutagenic agents. Previous studies with the important animal pathogen foot-and-mouth disease virus (FMDV) have shown that FMDV entry into error catastrophe is associated with an increase of complexity (mutation frequency and Shannon entropy) of the mutant spectrum of the quasispecies and that mutated, pre-extinction RNA interferes with the infectivity of standard RNA. Here, we report that despite the increase of complexity, the genomic consensus nucleotide sequence of pre-extinction FMDV RNA remains invariant, and that the fitness of pre-extinction FMDV is at least six-fold lower than the fitness of the parental viral clone, prior to mutagenic treatments. Thus, a low fitness genome ensemble can suppress replication of high fitness virus. Furthermore, the results show that profound genetic modifications associated with fitness decrease of a virus population can take place without any manifestation in the consensus genomic sequence. Thus, increase in mutant spectrum complexity and invariance of the consensus sequence characterizes FMDV extinction through error catastrophe.     

RNA干扰及其技术的应用

RNA干扰(RNAi)是细胞内由双链RNA诱导降解与其配对的特定mRNA的过程。细胞内双链RNA在酶的作用下,形成20-25碱基大小的小干扰RNA(si RNAs),由si RNAs进一步掺入多组分核酸酶并使其激活,从而精确降解与si RNAs序列相同的mRNA,抑制该基因在细胞内的翻译表达。介绍了RNA干扰的分子机制、制备方法、以及RNA干扰技术在功能基因组学、微生物学、基因治疗和信号转导等研究领域里的应用。     

中药热毒清对人巨细胞病毒抑制作用的实验与临床研究(英文)

目的:探讨中药热毒清对人巨细胞病毒(HCMV)体内外的抑制作用及机制。方法:采用定量逆转录聚合酶链反应(RT-PCR)方法检测中药热毒清干预前后感染细胞HCMV晚期mRNA的表达水平,观察感染细胞的病变进展,并在体外实验的基础上将热毒清应用于HCMV活动性感染孕妇的临床治疗,测定治疗前后血清肿瘤坏死因子α(TNF-α)和白细胞介素2(IL-2)的水平。结果:热毒清对     

激光捕获显微切割结合RNA线性扩增技术在膀胱移行细胞癌相关基因研究中的应用

目的当对有间质细胞混杂的膀胱移行上皮细胞与膀胱移行细胞癌进行基因差异表达分析时,激光捕获显微切割技术(lasercap-turemicrodissection,LCM)是不可缺少的,然而从LCM所得的细胞中获取足够RNA量相当困难。本文首次将LCM与RNA线性扩增结合用于膀胱癌相关基因研究,目的是确定一条切实可行的技术路线,将膀胱移行上皮细胞从膀胱粘膜中分离出来,将膀胱癌细胞从肿瘤间质细胞中分离出来,并对其进行RNA体外线性扩增,以备进一步研究。方法采用LCM技术分别从正常膀胱粘膜及膀胱癌组织冰冻切片中获取膀胱移行上皮细胞及膀胱癌细胞,提取RNA,并对微量RNA进行体外线性扩增。然后用RT-PCR验证扩增前、后RNA中β-actin基因表达水平。结果对照实验I证实经LCM后RNA完整性较好;经对照实验Ⅱ初步确定设定条件下LCMshooting次数与可获得RNA量间对应关系。从膀胱粘膜种捕获膀胱移行上皮细胞25万shootings;从膀胱癌组织中获取癌细胞20万shootings。产物RNA扩增后获得片段大小为0.5-2.5kh的aRNA,且β-actin基因表达表达完整。结论LCM结合RNA体外线性扩增技术能成功地获取较为单一的研究目的细胞,扩增后RNA完整性较好,能用于进一步研究中。     

壳聚糖纳米载体提高抗乙肝免疫核糖核酸免疫活性的研究

目的:通过制备抗乙肝免疫核糖核酸(iRNA)的壳聚糖纳米载体,提高其免疫活性.方法:复凝聚方法制备壳聚糖-免疫核糖核酸纳米粒,扫描电镜、原子力显微镜、Zeta电位/粒度分析仪观察测定壳聚糖-免疫核糖核酸纳米粒形态、粒径和表面电位;核糖核酸酶(RNase)保护试验观察壳聚糖对包裹的iRNA的保护作用;白细胞黏附抑制(LAI)试验测定壳聚糖-免疫核糖核酸纳米粒免疫活性.结果:壳聚糖与iRNA形成表面带正电荷的纳米粒,iRNA未被RNase的降解,白细胞黏附抑制率增高.结论:壳聚糖纳米载体提高了抗乙肝免疫核糖核酸的免疫活性.     

Downregulation of Kυ4.2 and Kυ4.3 channel gene expression in right ventricular hypertrophy induced by monocrotaline in rat

INTRODUCTION The calcium-independent transient outward potas-sium current (Ito) is activated by depolarization and playsa key role in controlling the amplitudes and cardiac ac-tion potential duration (APD). Ito contributesto the phase1 repolarization of action potential in myocytes, theprolongation of APD is the result of decrease in Ito den-sity[1]. Ito which is encoded by genes of Kv1.4, Kv4.2,and Kv4.3[2], based on differences in kinetics, as wellas recovery from inactivation…     

RNAi沉默整合素连接激酶基因表达对结肠癌细胞HT-29增殖的影响

目的:利用RNA干扰(RNA interference,RNAi)技术,阻断结肠癌细胞系HT-29中整合素连接激酶(integrin-linked kinase,ILK)基因的表达,研究ILK基因沉默后对HT-29细胞系增殖产生的影响。方法:构建针对ILK基因的真核表达质粒pSUPER-neo-EGFP-ILK(pSNE-ILK),在脂质体介导下稳定转染人结肠癌细胞系HT-29细胞(HT-29/pSNE-ILK组),同时以无关质粒pSUPER-neo-EGFP-C(pSNE-C)转染HT-29细胞(HT-29/pSNE-C组)和未转染细胞(HT-29组)作为对照,分别应用RT-PCR技术及蛋白印迹法检测ILK mRNA及蛋白表达水平,四甲基偶氮唑盐(MTT)比色法检测细胞增殖状况。结果:稳定转染后,HT-29/pSNE-ILK组ILK mRNA及蛋白表达水平分别为0.16±0.11和0.24±0.07,HT-29/pSNE-C组分别为0.53±0.10和0.56±0.08,HT-29组分别为0.64±0.11和0.77±0.02,前组ILK mRNA及蛋白表达水平分别与后两组比较,差异均有统计学意义(P<0.05);稳定转染pSNE-ILK质粒后HT-29细胞中ILK mRNA及蛋白表达抑制率分别为69.8%和57.1%。MTT比色法检测显示,HT-29/pSNE-ILK组细胞增殖率明显下降(P<0.05)。结论:RNA干扰技术能有效抑制靶基因ILK的表达,进而可抑制结肠癌细胞系HT-29的增殖。