弓形虫蛋白激酶C受体1蛋白的表达与抗原性分析

目的构建弓形虫蛋白激酶C受体1基因的pET-30a（＋）-RACK1重组质粒,表达、纯化RACK1蛋白。方法PCR扩增RACK1基因的cDNA序列,用SacI、NcoI限制性内切酶对RACK1基因的PCR产物及pET-30a（＋）质粒进行双酶切,连接,转化大肠杆菌BL21,构建重组质粒。IPTG诱导表达,亲和层析柱纯化表达产物,SDS-PAGE和Western blotting对表达产物进行分析鉴定。结果PCR扩增出 966 bp的RACK1完整基因序列,成功构建RACK1基因的pET-30a（＋）-RACK1重组质粒,表达出约36kDa的RACK1蛋白,蛋白具有抗原性。结论成功构建弓形虫RACK1基因的pET-30a（＋）-RACK1重组质粒,纯化出RACK1蛋白,为进一步进行RACK1蛋白在弓形虫入侵分子机制中的作用研究奠定基础。     

老年原发性高血压内皮功能与高敏C反应蛋白、内皮素1关系的研究

目的研究老年原发性高血压（EH）患者大动脉粥样斑块、内皮功能与血清内皮索1（ET-1）和高敏C反应蛋白（h-CRP）的关系。方法选择69例EH患者，其中男性36例，女性33例，平均年龄（66．8±6．2）岁。应用B超对所有病例的颈动脉、肱动脉进行筛查，检测颈动脉内膜中层厚度（IMT）及粥样斑块，在静息、反应性充血时对肱动脉内径进行检测。用免疫学方法测定血液中ET-1、h-CRP的浓度。结果ET-1、h-CRP与IMT呈正相关，IMT增厚组ET-1[（75．49±6．10）pg／L]、h-CRP[（9．96±1．69）pg／L]分别高于IMT非增厚组ET-1[（52．44±6．52）pg／L]、h-CRP[（4．53±1．52）mg／L]，P〈0．05。加压充血后，肱动脉管径平均扩增率（Flow—MD）与ET-1、h-CRP呈负相关，相关系数分别是-0．607、-0．661，P〈0．01。结论老年原发性高血压患者血液中ET-1—1、h-CRP浓度增高与动脉内皮细胞功能受损、粥样硬化关系密切。     

1型糖尿病患者血浆microRNA-126 变化与内皮功能的相关性

目的 探讨1型糖尿病患者血浆microRNA-126表达水平的变化及其临床意义，并分析microRNA-126与内皮功能的关系。方法 采用实时荧光定量聚合酶链反应检测47例1型糖尿病患者及50例健康对照组人群血浆microRNA-126的表达水平，酶联免疫吸附法检测人内皮型一氧化氮合酶（eNOS）含量，分析血浆microRNA-126表达水平与人内皮型一氧化氮合酶含量的相关性。结果 与健康对照组相比，1型糖尿病组血浆microRNA-126表达水平明显下降（P<0.05），人内皮型一氧化氮合酶含量也明显下降（P<0.05）。相关分析显示血浆microRNA-126表达水平与人内皮型一氧化氮合酶含量呈明显正相关（P<0.05）。结论 1型糖尿病患者血浆microRNA-126水平呈低表达，而糖尿病患者常伴有内皮功能损伤，提示microRNA-126的下调可能与内皮损伤有关。microRNA-126可能通过介导内皮功能的损伤而参与1型糖尿病血管并发症的发生发展。     

TGase 1、ACPase及 APLase A酶活性与皮肤脱屑的关系

收集20例异常脱屑患者[板层鱼鳞病(LI)、寻常银屑病各10例]和10例健康人的皮屑,进行转谷氨酰胺酶1活性 (TGase 1)、酸性磷酸酶活性(ACPase) 和酸性磷脂酶A (APLase A) 活性检测,并对LI患者局部皮肤施以低浓度乙醇酸-甘油,个别重度LI患者进行TGase 1基因测序.结果 :①健康人TGase 1酶活性呈棕色细颗粒,定位于角质形成细胞(KC)胞膜;ACPase酶活性呈黑色细颗粒,主要位于KC的细胞间桥,APLase A酶活性呈蓝色细颗粒,位于KC表面.②LI及银屑病皮屑的以上酶活性皆缺乏或缺如.③多数LI患者治疗1～4周后异常脱屑表型可获得一定改善,但以上酶活性无明显改变.认为TGase 1、ACPase、APLase A活性皆参与正常脱屑机制;低浓度乙醇酸-甘油的疗效可能与其参与KC之间的细胞间桥断裂有关.     

骨肉瘤组织中RASSF1A、Cyclin D1蛋白表达及意义

目的探讨Ras相关区域家族1A（RASSF1A）、细胞周期素（Cyclin）D1蛋白在骨肉瘤发生、发展中的作用。方法采用免疫组化SP法检测RASSF1A、Cyclln D1蛋白在46例骨肉瘤组织（骨肉瘤组）及20例骨软骨瘤组织（对照组）中的表达。结果骨肉瘤组RASSF1A蛋白的阳性率（45．70％）显著低于对照组（95．00％）,P〈0．01.Cyclin D1蛋白的阳性率（63．00％）明显高于对照组（25．00％）,P〈0．01。RASSF1A、Cyclin D1表达呈负相关（r=-0．383,P=0．009）。骨肉瘤组低分化组织RASSF1A的阳性率明显高于高分化组织（P=0．009）。结论RASSF1A低表达与Cyclin D1高表达可促进骨肉瘤的发生发展,二者协同作用;二者联合检测能为骨肉瘤的早期临床诊断和治疗提供有利的生物学信息。     

BRCA1在鼻咽癌中的表达及与预后关系的研究

目的观察乳癌基因1（ BRCA1）在鼻咽癌中的表达水平并分析其表达水平与预后的关系。方法采用免疫组化S-P法检测100例患者鼻咽癌组织中BRCA1蛋白的表达水平,阳性细胞计数≥50％定为BRCA1高表达组;＜50％及不表达者定为BRCA1低表达组。应用χ2检验对BRCA1表达水平与临床资料进行关联性分析。结果100例患者中13例失访,BRCA1高表达者37例（42.5％）,低表达者50例（57.5％）;BRCA1表达与患者的临床分期（ P＝0.383）、原发灶退缩情况（ P＝0.434）、肿瘤发生远处转移（ P＝0.055）无明显关联性,高表达组生存率稍高于低表达组,但差异无统计学意义（ P＞0.05）。结论鼻咽癌组织中BRCA1蛋白的表达与临床分期和原发灶退缩程度无关,但BRCA1低表达者更易出现远处转移的趋势。     

p63蛋白在骨巨细胞瘤穿刺活检及大体标本中的表达及意义

目的探讨p63蛋白在骨巨细胞瘤组织中的表达及意义。方法选择骨巨细胞瘤患者39例（观察组）,非骨巨细胞瘤患者14例（对照组）,采用免疫组化法检测两组穿刺活检及大体标本中p63蛋白表达情况,分析观察组p63蛋白的表达模式。结果观察组及对照组穿刺活检标本中p63蛋白阳性表达率分别为76．9％（30／39）、7．1％（1／14）,P〈0．01;大体标本中阳性表达率分别为92．3％（36／39）、7．1％（1／14）,P〈0．01。观察组36例大体标本阳性者p63呈弥漫着色者17例（47．2％）,其穿刺标本均为阳性;灶片状着色者19例（52．8％）,其穿刺标本中13例为阳性（68．4％）。结论骨巨细胞瘤的穿刺活检和术后大体标本p63表达情况均有助于诊断,由于p63存在灶片状表达模式,故穿刺活检标本的敏感性略低。     

非小细胞肺癌组织中AQP1、nm23-H1的表达变化及意义

目的观察非小细胞肺癌组织中AQP1、nm23-H1蛋白的表达变化,并探讨其意义。方法采用免疫组化SP法检测123例非小细胞肺癌患者（观察组）和12例非肺癌受检者（对照组,包括7例肺炎性假瘤和5例正常受检者）肺组织中的AQP1、nm23-H1蛋白。结果观察组65例nm23-H1表达阳性,对照组1例;观察组77例AQP1阳性率,对照组2例。两组AQP1、nm23-H1阳性表达率相比,P均〈0.05。观察组AQP1表达与临床分期、病理分化程度、淋巴结转移、患者生存期有关（P均〈0.05）;nm23-H1蛋白表达仅与淋巴结转移相关（P〈0.05）。观察组AQP1和nm23-H1的表达呈正相关（P〈0.05）。结论观察非小细胞肺癌组织中AQP1、nm23-H1蛋白表达上调。二者联合检测可作为判断非小细胞肺癌转移潜能的指标     

不稳定型心绞痛患者心外膜脂肪组织厚度及IL-1β表达水平与冠状动脉易损斑块形成相关性分析

目的 通过观察不稳定型心绞痛患者心外膜脂肪组织（EAT）厚度及EAT中白细胞介素1β(IL-1β)表达水平，探讨其与冠状动脉易损斑块（VP）的相关性。方法 选择63例不稳定型心绞痛患者，根据血管内超声检测的斑块性质进一步将不稳定型心绞痛患者分为VP组24例、非VP组39例，另选排除冠状动脉粥样硬化性心脏病需接受换瓣手术的心脏瓣膜病患者45例作为对照组。采用超声心动图测量EAT厚度；收集受试者空腹肘静脉血，ELISA法检测血浆IL-1β；免疫组织化学染色及RT-PCR分别检测EAT中IL-1β蛋白及mRNA表达。结果 EAT厚度VP组>非VP组>对照组（P均<0.05），三组血浆IL-1β表达差异无统计学意义（P>0.05）。免疫组化染色显示，EAT中IL-1β蛋白在VP组表达最多；EAT中IL-1β mRNA表达VP组>非VP组、对照组（P<0.05）。Spearman相关性分析显示，VP组、非VP组EAT厚度及、EAT中IL-1β水平均与冠状动脉VP呈正相关（r分别为0.34、0.67）。多因素Logistic回归分析显示，EAT厚度、EAT中IL...     

非小细胞肺癌组织中AQP1、VEGF的表达变化及临床意义

目的观察非小细胞肺癌（NSCLC）组织中水通道蛋白1（AQP1）和血管内皮生长因子（VEGF）的表达,并探讨其临床意义。方法采用免疫组织化学SP法检测108例手术切除的NSCLC组织（观察组）及20例正常肺组织（对照组）中的AQP1和VEGF,并分析其与患者临床病理参数的关系。结果观察组AQP1和VEGF的阳性表达率分别为46.3%、72.2%,对照组分别为10.0%、25.0%两组相比P均〈0.05。观察组AQP1、VEGF表达与临床分期、病理分化程度、淋巴结转移、患者生存期有关（P均〈0.05）。观察组AQP1和VEGF表达呈正相关（P〈0.05）。结论 NSCLC组织中存在AQP1和VEGF的表达。二者可作为判断NSCLC转移潜能和预后的指标。     

A novel function for transglutaminase 1: Attachment of long-chain ω-hydroxyceramides to involucrin by ester bond formation

Transglutaminases (TGases) are defined as enzymes capable of forming isopeptide bonds by transfer of an amine onto glutaminyl residues of a protein. Here we show that the membrane-bound form of the TGase 1 enzyme can also form ester bonds between specific glutaminyl residues of human involucrin and a synthetic analog of epidermal specific omega-hydroxyceramides. The formation of a approximately 5-nm-thick lipid envelope on the surface of epidermal keratinocytes is an important component of normal barrier function. The lipid envelope consists of omega-hydroxyceramides covalently linked by ester bonds to cornified envelope proteins, most abundantly to involucrin. We synthesized an analog of natural omega-hydroxyceramides N-[16-(16-hydroxyhexadecyl)oxypalmitoyl]sphingosine (lipid Z). When recombinant human TGase 1 and involucrin were reacted on the surface of synthetic lipid vesicles containing lipid Z, lipid Z was attached to involucrin and formed saponifiable protein-lipid adducts. By mass spectroscopy and sequencing of tryptic lipopeptides, the ester linkage formation used involucrin glutamine residues 107, 118, 122, 133, and 496 by converting the gamma-carboxamido groups to lipid esters. Several of these residues have been found previously to be attached to ceramides in vivo. Mass spectrometric analysis after acetonide derivatization also revealed that ester formation involved primarily the omega-hydroxyl group of lipid Z. Our data reveal a dual role for TGase 1 in epidermal barrier formation and provide insights into the pathophysiology of lamellar ichthyosis resulting from defects of TGase 1 enzyme.     

Stimulation of plasminogen activator and inhibitor in the lymphatic endothelium

Chromogenic assays, immunoblotting, and Northern blot hybridization methods were employed to assess the effects of various agonists on the production of tissue plasminogen activator (t-PA) and plasminogen activator inhibitor type 1 (PAI-1) by the lymphatic endothelium (LEC). Fibrin autography showed that plasminogen-dependent fibrinolytic activity occurred at M(r) of 110 kDa, which represents a complex of tPA with PAI-1, and 65- and 55-kDa bands corresponding to tPA and uPA, respectively. The fractionation of lymph collected from ovine lymphatic vessels also produced a prominent lytic band of approximately 110 kDa, suggesting the formation of PA/PAI complexes in lymph. The stimulation of various agonists produced large-scale increases in tPA mRNA, as shown by Northern blot hybridization analyses. The effects of ECGF, histamine, and LPS on the presence of tPA and on enhancing the levels of mRNA reached maximum activity at 4 h and declined to levels below that of controls by 8 h. However, phorbol-treated cells exhibited reduced levels of tPA mRNA at 4 h, but was significantly increased by 8 h. A large-scale increase in PAI-1 mRNA steady-state levels was also stimulated by the agonists used in these studies. Both the 3.4- and 2.4-kb species of PAI-1 mRNA were increased. These observations demonstrated that tPA and PAI-1 are produced and secreted by LEC monolayer cultures and are also present in lymph.     

Antiviral effect of Chinese medicine jiaweisinisan in hepatitis B virus transgenic mice

AIM: To study the antiviral effect of Chinese medicine jiaweisinisan (JWSNS) on hepatitis B virus (HBV) infection in transgenic mice (TGM). METHODS: Twenty two 6-8 wk old HBV TGM in the third generation were divided into TGM control group and TGM treated group randomly. The normal control group included ten normal BC 57L/6 mice at the same age. The mice in treated group were administrated with JWSNS at the concentration of 4 g/mL and the dosage of 50 g/kg per d for 30 d, while the mice in TGM control group and normal control group were administrated with normal saline at the same dosage and the same time. Polymerase chain reaction (PCR) was used to assess the contents of HBV DNA in serum of HBV TGM before and after treatments, whereas blot hybridization was utilized to measure the contents of HBV DNA in the liver of both HBV TGM and normal BC 57L/6 mice. RESULTS: The levels of serum HBV DNA in TGM treated group were remarkably decreased after the treatment of JWSNS (7.662±0.78 vs 5.22±3.14, P<0.05), while there was no obvious change after administration of normal saline in TGM control group (7.125±4.26 vs 8.932±5.12, P>0.05). The OD values of HBV DNA in the livers of the mice in TGM treated group were significantly lower than those of TGM control group (0.274±0.096 vs 0.432±0.119,P<0.01). CONCLUSION: JWSNS exerts suppressive effects on HBV DNA in the serum and liver of TGM.     

恶性疟原虫裂殖子表面抗原MSA1第二区基因在大肠杆菌中的表达

目的 :在大肠杆菌中高效表达恶性疟原虫裂殖子表面抗原 MSA1- R2 ,进一步研究其生物学功能。方法 :将 MSA1- R2基因重组于 p WR4 50 I半乳糖苷酶融合蛋白表达载体中 ,转化大肠杆菌 ,酶切鉴定重组克隆。用 IPTG诱导 MSA1- R2融合蛋白的表达 ,对表达产物进行 SDS- PAGE、β-半乳糖苷酶活性、dot- ELISA和Western- blot鉴定。结果 :表达产物占菌体总蛋白的 35%— 4 0 % ,相对分子量为 70 k Da,与半乳糖苷酶 - MSA1R2融合蛋白的理论分子量相符。IPTG诱导 4 h后 ,β-半乳糖苷酶活性可增高 10倍— 14倍 ,用 dot- ELISA和 Western- blot均证实表达产物具有恶性疟原虫抗原表位。结论 :p WR4 50 -大肠杆菌表达系统可以高效表达具有免疫学活性的疟原虫蛋白。     

Identification of plasma membrane autoantigens in autoimmune hepatitis type 1 using a proteomics tool

Autoimmune hepatitis (AIH) is a liver disease with circulating autoantibodies predominantly directed against widely held cellular components. Because AIH is a liver-specific disease, autoantibodies against plasma membrane antigens may be involved in its pathogenesis and have been reported; however, no definite identification has been described. We thus investigated the fine specificity of anti-hepatocyte plasma membrane autoantibodies in type 1 AIH (AIH-1) using a proteomic tool. A plasma membrane-enriched fraction was validated using enzymatic activity and western blot analysis experiments. Sera from AIH-1 patients (n = 65) and from 90 controls, that is, healthy blood donors (n = 40) and patients with systemic diseases (n = 20) or other liver diseases (n = 30), were studied by immunoblot performed with plasma membrane proteins resolved by either sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) or 2-dimensional (2D) electrophoresis. Proteins contained in the immunoreactive spots were identified by sequences provided by ion-trap mass spectrometry. Hepatocytes probed with sera were also studied using confocal immunofluorescence and immunoelectron microscopy. The more prominent bands stained by patient sera were located at 38 kDa, 48, 50, 52 kDa, 62 kDa, 70 kDa, and a 95-kDa double band. Six proteins with known potential plasma membrane expression were identified: liver arginase (38 kDa), cytokeratins (CK) 8 and 18 (48-52 kDa), heat shock proteins (HSP) of 60, 70, 90 kDa, and valosin-containing protein (VCP) of 92 kDa. The presence of anti-membrane antibodies was confirmed by immunofluorescence and immunoelectron microscopy. CONCLUSION: Overall, our data demonstrate that liver arginase, CK 8/18, HSP 60, HSP 70, HSP 90, and VCP represent potential candidate targets on liver membrane for autoantibodies in AIH-1.     

Isolation, purification, characterization and antigenic evaluation of GPI-anchored membrane proteins from Leishmania (Viannia) braziliensis

GPI-anchored proteins from the plasma membrane of Leishmania (Viannia) braziliensis promastigotes were isolated, characterized and their migration pattern compared with those from other Leishmania species. In all cases the SDS-PAGE migration patterns were obtained under reducing and non-reducing conditions, using DL-dithiothreitol (DTT) as a reducer agent. Our results reveal that under reducing conditions the SDS-PAGE migration pattern is modified as a consequence of the disruption of disulphur-bonds and protein transformation. This is demonstrated when in non-reducing conditions the L. (V.) braziliensis-GPI-anchored proteins pattern showed a group of bands over the 100kDa, and two more bands of 52kDa and 50kDa in four different isolates, whereas under reducing conditions the major GPI-anchored protein fractions were detected as bands of 63kDa, 50kDa and an increase of peptides between 34kDa and 22kDa. Similar modifications were detected in the SDS-PAGE migration patterns of GPI-anchored protein fractions from L. (Leishmania) donovani, L. (L.) mexicana and L. (L.) amazonensis run under the same reducing conditions. Antigenic evaluation carried out by Western blot revealed the presence of two very specific L. (V.) braziliensis-GPI-anchored protein bands of 50kDa and 28kDa. These bands were specifically recognized by anti-L. (V.) braziliensis-GPI-anchored protein serum from experimentally immunized animals. These two peptides were not detected when GPI-anchored protein fractions from L. (L.) donovani, L. (L.) mexicana and L. (L.) amazonensis, were challenged with the same anti-serum. The present results lead us to suggest the use of these two peptides as biochemical markers to identify and differentiate leishmaniasis caused by L. (V.) braziliensis. The lack of immunogenicity observed here with the peptide gp63, a very common protein detected in Leishmania species, is considered.     

Molecular analysis of human T cell lymphotropic virus type 1 and 2 (HTLV-1/2) seroindeterminate blood donors from Northeast Iran: evidence of proviral tax, env, and gag sequences

Human T cell lymphotropic virus type 1 and 2 (HTLV-1/2) Western blot indeterminate results are a problem for blood banks in endemic areas. To determine the prevalence of HTLV-1/2 infection among indeterminate donors, we analyzed 130 cases from Mashhad, an HTLV-1/2 endemic area in Northeast Iran. The most frequent Western blot bands were GD21 alone (37.2%) followed by rgp46-2 alone (32.1%). We further tested 40 available DNA samples of these cases by PCR for viral sequences, tax, gag, and pol, and found five cases (12.5%) to be positive for two or three HTLV-1 genes. There were no significant age, sex, and blood group differences between PCR-positive and PCR-negative cases. Among PCR-positive individuals, the most prevalent Western blot bands were variable combinations of rgp46-1, GD21, and gp21. The mean of the optical density (OD) of the enzyme-linked immunosorbent assay (ELISA) test was significantly higher in PCR-positive individuals. The frequency of the rgp46-1 band was also significantly higher in PCR-positive cases compared to PCR-negative ones. In conclusion, the majority of HTLV-indeterminate donors lack the HTLV provirus and therefore are not considered infected. However, in some cases with higher ODs in the ELISA test and seroreactivity to env proteins, rgp46-1 and GD21 in particular may be indicative of infection and need further evaluation by molecular methods.     

Serine proteinases with gelatinolytic activity in an Aspergillus fumigatus allergenic extract.

The presence of proteolytic activity in Aspergillus fumigatus (Af) extract was analyzed in polyacrylamide gelatine containing gels at different pHs. The gelatinolytic pattern showed three major activities (180, 70, and 34 kDa bands) over a broad pH range (pH 4-8). All bands showed maximum activity at pH 8, and the activity declined at lower pH, showing no activity at pH 2. The susceptibility of the proteolytic activity to protease inhibitors confirmed that the fungal extract contained serine proteinases, and one of them, the 34 kDa band, showed a chymotrypsin-like proteinase. The immunochemical fungal extract properties were studied by western blot, and the samples were run in the presence or absence of denaturing and reducing (DTT) conditions. Two protein bands located at approximately 180 and 84 kDa showed immunoreactivity with allergenic patients' anti-Af serum, when the samples were run under denaturing and reducing conditions. In the native form an additional third immunoreactive band of about 70 kDa was found. There were no immunoreactive bands toward IgE about 34 kDa. Periodate treatment caused the decrease or disappearance of the immunoreactivity in the bands, demonstrating that epitopes recognized by serum IgE specific to Af are partially composed by carbohydrates (180 and 70 kDa bands) while others (84 kDa) are carbohydrates. Although further investigations are needed, these preliminary results suggest alkaline serine proteinases from Aspergillus fumigatus as one of the factors responsible for the fungal allergenicity.     

Notch1与Jagged1蛋白在大肠腺癌组织中的表达及临床病理意义

目的探讨Notch1和Jagged1蛋白在大肠腺癌发生发展的作用。方法采用免疫组织化学PV-9000法和Western blotting法检测78份大肠腺癌组织(大肠癌组),40份大肠腺瘤组织(腺瘤组)及40份正常大肠组织(距大肠腺癌组织远端5㎝,正常组)Notch1与Jagged1蛋白表达情况,分析其与大肠癌临床病理参数的关系。结果大肠癌组Notch1、Jagged1蛋白阳性率明显高于腺瘤组及正常组,P<0.05;Notch1和Jagged1蛋白表达强度与大肠癌组织学分级、Dukes分期和淋巴结转移密切相关,P均<0.05。大肠癌组Notch1与Jagged1蛋白表达关系呈正相关(r=0.407,P<0.01)。结论 Notch1和Jagged1蛋白在大肠腺癌的发生与发展中起重要作用。     

820份蛋白印迹试验HIV-1抗体阳性结果分析

目的 通过研究分析绵阳市1型艾滋病病毒(HIV-1)受试者条带检出情况,提高实验室条带判读能力.方法 对2019年绵阳辖区820份蛋白印迹试验HIV-1抗体检测阳性结果使用描述性分析、卡方检验、Fisher确切概率法和Kappa进行分析.结果 阳性结果中出现全条带的有87.3％(716/820);p51和p66的条带检...