乙型肝炎肝硬化患者外周血单核细胞基因表达谱研究

目的：探索基因表达谱技术在肝硬化形成的分子生物学机制研究及其诊断方法研究的应用。方法：应用含8192个人体cDNA的微阵列芯片和来自外周血单核细胞的标记cDNA,分析慢性乙型肝炎、肝炎肝硬化各15例基因表达谱。通过CenePix4000B扫描芯片仪和ImaGene3．0软件分析Cy3、Cy5两种荧光信号的强度和比值。结果：在8192个基因中,2组中筛选出有差异的基因60个,占0．73％,其中主要是炎症、凋亡基因、细胞外基质蛋白基因、细胞生长调节基因,占71．6％。结论：基因表达谱技术可为乙型肝炎肝硬化形成的分子生物学机制及其诊断研究提供大量有益的生物学信息。     

绝经对血液单核细胞基因表达谱的影响:基因芯片初步研究

绝经是女性一生中很重要的生理现象之一,它能增加一系列复杂免疫、神经退化、新陈代谢和心血管方面的疾病。血液单核细胞能分化成各种各样的细胞,这些细胞在组织形态发生和免疫应答方面起着很重要的作用。本研究中采用了包含大约14,500个基因探针的Affymetrix Human U133A基因芯片来研究健康的绝经前和绝经后女性外周血液单核细胞中的基因表达谱。样本之间的对比分析表明有20个基因上调,20个基因下调。其中的28个基因根据它们的生物过程如细胞繁殖、免疫应答、细胞代谢等等被分成了6个主要的GO类别;剩下的12个基因其生物学功能还没有被鉴定。研究结果支持了我们的假设:血液单核细胞的功能状态确实受到绝经的影响,而且由此带来的改变可能是由全基因组范围的基因表达谱而决定的。本研究中鉴定的一些差异表达基因有可能作为以后研究与绝经相关的系统免疫、神经退化和心血管疾病的候选基因研究。此工作是这个研究方向的第一次尝试,为将来的进一步研究奠定了基础。     

哮喘患者外周血单核细胞血红素氧合酶-1表达水平的研究

目的探讨血红素氧合酶-1(HO-1)在哮喘患者外周血单核细胞(PBMC)的表达及与肺通气功能的关系.方法应用免疫组织化学和逆转录聚合酶链反应技术分析18例哮喘患者PBMC的 HO-1蛋白及mRNA水平的表达,测定全血一氧化碳血红蛋白(COHb)的百分比含量、血清总IgE含量、肺通气功能,并与18名健康正常者的结果进行比较.结果哮喘组PBMC中HO-1表达阳性的细胞百分比41.7%±7.44%与正常对照组10.5%±4.36%比较,差异有显著性(P<0.01),哮喘组PBMC HO-1 mRNA表达的平均吸光度值(26.05±4.14)与正常对照组(10.82±4.26)比较,差异亦有显著性(P<0.01),HO-1染色阳性细胞的百分比与FEV1占预计值%、PEFR和MEFR50%均呈显著负相关(r值分别为-0.89,-0.56,-0.51,均P<0.01),与全血COHb的百分比含量及血清总IgE含量呈显著正相关(r值分别为0.80, 0.48, 均P<0.05).HO-1 mRNA的表达水平与1s用力呼气容积(FEV1)占预计值%、顶峰呼气流速(PEFR)和50%肺活量时的最大呼气流速(MEFR50%)均呈显著负相关(r 值分别为-0.89,-0.65,-0.67, 均P<0.01),与全血COHb的百分比含量和血清总IgE含量呈显著正相关(r分别为 0.85和0.62, 均P＜0.01).结论哮喘患者PBMC 的HO-1表达水平显著增加,提示HO-1可能参与了哮喘的发病过程,HO-1表达的变化与哮喘患者的病情程度有一定关系.     

聚肌胞免疫刺激下仔猪外周血单核细胞的基因表达分析

聚肌胞(Poly I:C)是一种天然双链RNA(Double strand RNA, dsRNA)的拟似物,能够模拟病毒感染后所形成的dsRNA及刺激机体产生抗病毒免疫反应。文章以抗病力存在差异的大蒲莲和长白仔猪为研究对象,分离外周血单核细胞(Peripheral blood mononuclear cell, PBMC),在20 μg/mL的Poly I:C的免疫刺激下体外培养24 h,对影响免疫应答过程中的7个细胞因子(IRF3、IL6、IL8、IL10、TNFα、IFNγ和IFNα)和3个模式识别受体(TLR3、TLR4和RIG1)利用实时荧光定量PCR检测Poly I:C免疫刺激组相对于对照组的基因表达变化倍数。结果表明：检测的大部分细胞因子和受体(6个)表达量变化倍数很大,其中3种白细胞介素IL6、IL8和IL10免疫刺激变化倍数最大,平均变化倍数分别为20.71、10.87和5.18倍。对不同个体和品种间的比较发现,不仅大蒲莲和长白两品种间(大蒲莲猪的变化倍数平均高于长白猪)而且同品种的3头全同胞仔猪间对Poly I:C免疫刺激的应答也存在较大的变化。文章利用Poly I:C体外模拟dsRNA对PBMC的感染,为下一步筛选仔猪对Poly I:C刺激的免疫应答基因及鉴定大蒲莲猪特殊的抗性基因奠定了基础。     

乙型肝炎肝硬化患者血脂变化的临床意义

目的探讨乙型肝炎肝硬化患者血清总胆固醇(TC)、甘油三酯(TG)、高密度脂蛋白(HDL-C)、低密度脂蛋白(LDL-C)及极低密度脂蛋白(VLDL-C)的临床意义。方法回顾性分析浙江大学医学院附属第一医院120例乙型肝炎肝硬化患者临床资料,同时选用我院30例健康体检者临床资料为正常对照。结果与正常体检者相比,乙型肝硬化患者血脂水平明显降低(P0.05),TC、TG、HDL-C、LDL-C水平与白蛋白、胆碱酯酶水平正相关(P0.01),TC、TG、LDL-C水平与凝血酶原时间、MELD分级负相关(P0.01)。结论乙型肝硬化患者血脂水平明显降低,血脂水平与乙肝肝硬化患者肝功能损害明显相关。     

基因芯片技术与基因表达谱研究

基因芯片技术是近年来出现的分子生物学与微电子技术相结合的最新DNA分析检测技术,该技术将成为信息科学与生命科学之间的联系纽带,为后基因组时代基因功能的分析提供一种最重要的技术手段,目前基因芯片技术已在基因表达谱等研究中得到广泛应用。     

IgA 肾病患者与膜性肾病患者外周血单核细胞mRNA分析

为了寻找诊断、鉴别IgA肾病(IgAN)和膜性肾病(MN)的血液特异性标记物,利用公共数据库中的IgAN和MN患者的外周血单核细胞(PBMCs)的转录组表达谱数据集识别特异性生物标记物,为诊断和鉴别提供简便、可靠的依据补充。从公共基因表达数据库(GEO)下载IgAN患者组(n=15)和MN患者组(n=8)芯片数据集,筛选前250个差异表达基因(DEGs)。通过分析筛选关键基因和途径,进行基因本体(GO)富集分析、京都基因与基因组百科全书(KEGG)通路分析和蛋白质与蛋白质相互作用关系(PPI)分析等进一步了解DEGs。通过分析共发现75个显著DEGs,其中73个上调基因,2个下调基因。GO富集分析的生物学过程(BP)主要包括蛋白质转运、内溶酶体到溶酶体转运、趋化因子介导的信号通路作用等。显著富集差异表达基因KEGG通路分析包括Endocytosis和Hepatitis B的相关信号通路。PPI筛选出EPS15、STAT4、CCL2、SUN2、SEC24C、SEC31A、GOLGB1、F2R,RAB12和PTK2B等关键基因。成功筛选出核心差异表达基因,为IgAN和MN的诊断和鉴别提供简便、可靠的依据补充,甚至提供治疗的新靶点。     

人外周血单核细胞的分离和电穿孔法转染

目的:建立分离纯化人外周血单核细胞及转染的有效方法。方法:应用基于HISTOPAQUE的密度梯度离心及抗体标记纯化的方法分离单核细胞,并以电穿孔技术转染绿色荧光蛋白(GFP)表达质粒、肿瘤坏死因子受体相关因子6(TRAF6)小干扰RNA。结果:基于外周血单核细胞的表面标志分子CD14的流式细胞分析表明得到了高纯度(89.95%)的外周血单核细胞;GFP荧光照片显示GFP表达质粒的转染效率为60%～70%;免疫印迹显示TRAF6的表达抑制(90%)下调了脂多糖(LPS)对JNK的激活,说明通过电穿孔技术有效递送了TRAF6小干扰RNA,表明单核细胞若缺少TRAF6将抑制LPS-TLR4信号通路的激活。结论:建立了一种高效的从人外周血中分离原代单核细胞的方法,且应用基于NucleoFectorⅡ的电穿孔技术实现了对此原代单核细胞的高效转染,为进一步外周血单核细胞的相关功能研究奠定了方法学基础。     

应用cDNA微阵列研究肿瘤基因表达谱

随着研究水平的深入 ,对基因在发育过程中、生理反应中和疾病发展中表达上的时空差别 ,基因蛋白质产物的亚细胞定位和细胞分子间的相互作用等研究领域的开拓 ,所涉及到的基因的广泛性用传统方法研究已很难达到要求 ,因此一些新的研究方法应运而生 ,基因表达谱的研究即为其中之一。基因表达谱 ( ｇｅｎｅｅｘｐｒｅｓｓｉｏｎｐｒｏｆｉｌｅ)是指细胞中所有基因表达的格局 ,具体说来就是指细胞中产生所有ｍＲＮＡ的总和。对于所有的ｍＲＮＡ来说 ,通常采用的研究方法是将其逆转录成ｃＤＮＡ ,通过构建ｃＤＮＡ文库 ,再进行分析研究。原先采用…     

泌乳期奶牛乳腺基因表达谱研究

从青春期到泌乳期以至干乳期,奶牛乳腺经历复杂的生物学功能和代谢水平的变化.通过基因芯片分析奶牛乳腺的基因表达谱,通过泌乳旺盛的泌乳期与不泌乳的青春期和干乳期相比较,共筛选出122个差异表达的基因,其中包括79个泌乳期上调基因和43个泌乳期下调基因.GO分析表明,在泌乳期奶牛乳腺中上调的基因主要与物质转运、生物合成、信号转导、催化活性、免疫防御、细胞凋亡以及促进发育相关.这些数据提示了奶牛乳腺泌乳期所发生的分子事件.     

Specific gene expression patterns in liver cirrhosis

Liver cirrhosis (LC) is a complex disease that can develop into hepatocellular carcinoma (HCC). In an effort to investigate genetic differences between LC and HCC, we used cDNA microarray analysis to characterize the gene expression profiles in LC and HCC tissues. Consistent differences were observed among the expression patterns in LC, HCC, and normal liver tissues. Interestingly, the expression patterns of LC without tumor association (LCT) were also readily distinguished from those of LC tissues near hepatic tumor tissues (near-tumor tissue, NTT). Moreover, 25 cirrhosis-specific genes could be used to divide the NTT samples into two groups: inflammatory active cirrhosis (NTTa) and inflammatory inactive cirrhosis (NTTi). We found that NTTa samples showed gene expression patterns similar to those of the LCT and HCC groups, whereas the expression patterns of the NTTi group were significantly different from those of the LCT, NTTa, and HCC groups. Finally, we selected two of the 25 LC-specific genes and showed that these markers could be used to successfully discriminate among the different LC subtypes. Collectively, these novel results allow the identification of new genetic subgroups of LC and provide new candidate genes for use as early markers for active cirrhosis and HCC.     

Peripheral blood monocytes from the decompensated liver cirrhosis could migrate into nude mouse liver with human hepatocyte-markers expression

Adult stem cells provide a promising alternative for the treatment of decompensated liver cirrhosis. Our previous study showed that peripheral blood monocytes (PBMCs) from decompensated liver cirrhosis could differentiate into hepatocyte-like cells in vitro. We here aimed to investigate the differential potential of the PBMCs under liver environment. PBMCs were isolated from one cirrhotic patient, who was mobilized by the recombinant human granulocyte colony stimulating factor for consecutive 3 days, and then PBMCs were transplanted into nude mice by tail vein after labeled with PKH26-GL. The location of the transplanted PBMCs was identified by PKH26-GL staining and PRINS for human SRY gene. The expressions of human hepatocyte-markers were detected by immunohistochemistry, RT-PCR, and Western blot analysis. Our results demonstrated that PBMCs from decompensated liver cirrhosis could migrate into the liver of nude mice with human hepatocyte-markers expression, indicating that autologous PBMCs transplantation might be one alternative therapy for the decompensated liver cirrhosis.     

肺癌相关基因芯片的制作

探讨肺癌相关基因芯片的制作过程及其在基因表达谱方面的应用,同时对一些传统的技术环节进行了改进。首先有目的搜集了60个肺癌相关基因,用修饰后的引物将其分别克隆,经纯化、变性后,用Cartesian PinSys5500 cDNA Microarray点样仪以微阵列的形式将其点布于醛基化的玻璃片上;然后将人支气管上皮恶性转化细胞模型BEP2D细胞的原代、20代、35代的总RNA逆转录后再经LD PCR标记成荧光探讨与基因芯片进行杂交。ScanArray 3000荧光扫描仪扫描后显示,图象背景均匀、信号清晰,证实该芯片制作成功,并能取得良好的杂交效果。     

肝硬化合并结核病患者的临床特征

目的 分析肝硬化合并结核病患者的临床特征,为该类患者的治疗提供参考。 方法 回顾性分析2014年9月至2017年7月于浙江大学医学院附属第一医院住院的肝硬化合并结核病患者的相关资料,总结患者的临床特征。 结果 共收治68例肝硬化合并结核病患者,其中男性50例(73.5%),女性18例(26.5%),平均年龄(59.9±13.7)岁。在所有患者中,肝硬化最常见的原因是HBV感染(46例,67.7%)和酒精(9例,13.2%)。肺结核(43例)、结核性腹膜炎(13例)、结核性胸膜炎(12例)是最常见的结核类型,此外骨关节结核、结核性脑膜炎、结核性心包炎、泌尿系结核、肠结核、淋巴结结核均可见。该类患者常见临床表现为乏力、腹胀,出现发热的患者不足半数,有盗汗表现的更为罕见。 结论 肝硬化患者并发结核后可使病情复杂化,增加治疗难度。此外部分患者临床表现不典型,需提高警惕,避免漏诊。     

用cDNA微阵列研究辐射损伤小鼠髓细胞基因的表达

为了探讨辐射引起小鼠髓细胞损伤后基因表达的变化,运用常规分子生物学和cDNA微阵列杂交技术,观察小鼠肥大剂量射线全身一次性照射后,骨髓细胞中588种已知功能基因的表达情况,初步得到辐射损伤后骨髓细胞某些功能基因的差异表达变化谱,结果表明,辐射可诱导小鼠骨髓细胞中一系列基因表达变化,这些基因可能参与了辐射所致骨髓细胞损伤的过程。     

Restoration of mycobacterial antigen-induced proliferation and interferon-gamma responses in peripheral blood mononuclear cells of tuberculosis patients upon effective chemotherapy

Peripheral blood mononuclear cells (PBMC) were obtained from culture-proven tuberculosis (TB) patients before and after 2 and 6 months of chemotherapy with a multi-drug regimen. PBMC were tested for cellular responses in antigen-induced proliferation and interferon-gamma (IFN-gamma) assays in response to complex mycobacterial antigens (whole cell Mycobacterium bovis BCG and M. tuberculosis, cell walls and short-term culture filtrate [ST-CF] of M. tuberculosis), fractionated ST-CF antigens (fractions F1-F10) and ESAT-6. The responses in TB patients before anti-TB treatment were low (median stimulation index (SI)=1-7, median delta IFN-gamma=0-12 U ml(-1), and percent responders=13-67%) to all the antigenic preparations. Following the administration of anti-TB chemotherapy for 2 months, there were significant (P<0.05) improvements in the cellular responses (median SI=9-76, median delta IFN-gamma=3-70 U ml(-1), and percent responders=33-100%) to most of the antigenic preparations tested. However, concanavalin A-induced proliferation responses of PBMC from the same patients before and after 2 months of chemotherapy were high and comparable (median SI=101 and 114, respectively, P>0.05, 100% responders). A further increase in IFN-gamma responses (median delta IFN-gamma=14-250 U ml(-1) and percent responders=43-100%) to mycobacterial antigens was observed in patients receiving chemotherapy for 6 months. Among the ST-CF fractions, F1 and F2 containing low molecular mass proteins resulted in the highest responses, whereas ESAT-6 showed responses comparable to these fractions only in a minority of the patients. HLA-DR typing of these patients showed heterogeneity in the expression of molecules encoded by HLA-DRB genes. These results show that effective chemotherapy restores cellular responses of TB patients to a large number of M. tuberculosis antigens, which could be useful in monitoring the efficacy of anti-TB treatment.     

Comparative analysis of amplified and nonamplified RNA for hybridization in cDNA microarray

Limiting amounts of RNA is a major issue in cDNA microarray, especially when one is dealing with fresh tissue samples. Here we describe a protocol based on template switch and T7 amplification that led to efficient and linear amplification of 1300x. Using a glass-array containing 368 genes printed in three or six replicas covering a wide range of expression levels and ratios, we determined quality and reproducibility of the data obtained from one nonamplified and two independently amplified RNAs (aRNA) derived from normal and tumor samples using replicas with dye exchange (dye-swap measurements). Overall, signal-to-noise ratio improved when we used aRNA (1.45-fold for channel 1 and 2.02-fold for channel 2), increasing by 6% the number of spots with meaningful data. Measurements arising from independent aRNA samples showed strong correlation among themselves (r(2)=0.962) and with those from the nonamplified sample (r(2)=0.975), indicating the reproducibility and fidelity of the amplification procedure. Measurement differences, i.e, spots with poor correlation between amplified and nonamplified measurements, did not show association with gene sequence, expression intensity, or expression ratio and can, therefore, be compensated with replication. In conclusion, aRNA can be used routinely in cDNA microarray analysis, leading to improved quality of data with high fidelity and reproducibility.     

应用基因芯片技术检测肝组织的基因表达

为检测正常肝组织中的基因表达状况,采用cDNA microarray技术对正常肝组织中表达的1500个基因进行定量。结果为97 个基因较高表达,1010个基因中度表达,380个基因低表达。结果是cDNA microarray技术是大规模检测基因表达的有效方法。