脂联素与糖尿病性心脏病

1 脂联素概述 Scherer等通过筛选消减文库的方法在小鼠3T3-L1脂肪细胞中成功克隆出一个新基因，因其编码蛋白的结构与补体因子Clq相似且变性聚丙烯酰胺电泳检测分子量为30kDa而命名为脂肪细胞补体相关蛋白30（adipocyte completment-related protein30，Acrp30）。Acrp30也被称为脂联素（adiponectin，APN）。     

链酶亲和素—生物素ELISA检测人心肌肌钙蛋白T

目的 建立人心肌肌钙蛋白Ｔ（ｃＴｎＴ）链酶亲和素－生物素（ＳＡＢ）双抗体夹心酶联免疫分析（ＥＬＩＳＡ）方法。诊断急性心肌损伤性疾病。方法 以固相化单抗３Ｆ７捕捉样品中ｃＴｎＴ,生物素化３１１１２单抗为检测抗体,加入ＳＡ－ＨＲＰ,显色。根据已知不同浓度标准ｃＴｎＴ显色后Ｄ（λ）值的标准曲线,检测待测样品ｃＴｎＴ含量。结果 ｃＴｎＴ的最低检测值为０．１５ｎｇ／ｍｌ,批内变异系数为３．８％,批间变异系数     

脂联素与心血管疾病的研究现状与进展

脂联素(adiponectin,APN)又称脂肪细胞补体相关蛋白(adipocyte complement related protein of 30 kD,Acrp30)、脂肪组织基因转录最丰富的物质(apM1)、AdipoQ以及凝胶结合蛋白(GBP28).1995年,Scherer等首先从鼠的脂肪细胞分离出一种新基因,将其编码的蛋白质命名Acrp30.1999年Arita等将其命名为脂联素,并建立了可测定人的血浆中apM1产物浓度的方法.人类脂联素由244个氨基酸组成,包含4个功能区:氨基末端的分泌信号序列、胶原样结构域,非同源序列和羧基末端的球型结构域.目前研究发现球形结构域是脂联素发挥功能的主要结构,具有药理学活性.脂联素在血浆中通常以聚合体形式存在,3个单体通过球形结构域连接成同源三聚体,进一步以二硫键形成低分子量六聚体,4～6个三聚体可汇聚成高分子量多聚体.目前,已确认有2种脂联素受体[1]:AdipoR1和AdipoR2.前者在骨骼肌中表达最丰富,是球形Acrp30的高亲和受体及全长型脂联素的低亲和受体;后者在肝脏中表达最丰富,是脂联素和球形Acrp30的中等亲和受体.     

脂联素与糖脂代谢

近年来，人们发现脂肪组织不仅仅是储存和释放能量的场所，还是一个重要的内分泌器官，能够分泌多种活性蛋白，如肿瘤坏死因子（TNF-α）、白介素（IL-6）、瘦素（leptin）、纤溶酶原激活物抑制剂（PAI-1）、脂联素等，它们具有细胞因子的一些结构特点，因此统称为“脂肪因子”，参与能量和代谢的调节。脂联素（adiponectin，又名Acrp30，AdipoQ，apM1，GBp28）是新近发现的由脂肪细胞特异性分泌的一种蛋白质，具有较高的血浆浓度，是所有脂肪细胞因子中唯一负性调节的激素，具有降糖、增加胰岛素敏感性、抗炎、抗动脉硬化等作用。本文就脂联素结构及作用方面的进展作一综述。     

腺病毒载体介导的siRNA对3T3-L1细胞脂联素表达的影响

目的 构建携带小鼠脂联素(Acrp30)siRNA腺病毒载体,并检测其对小鼠脂肪细胞Acrp30表达的影响.方法 首先用KpnⅠ NotⅠ双酶切本课题组前期构建的质粒pSilencer1.0-U6-Acrp30-1和pSilencer1.0-U6-Acrp30-3,得到目的 片段U6-Acrp30-1和U6-Acrp30-3,并将目的 片段亚克隆入穿梭质粒pShuttle,用PmeⅠ线性化穿梭质粒pShuttle-U6-Acrp30-1和pShuttle-U6-Acrp30-3,并与pAdeasy-1在BJ5183内同源重组,筛选、鉴定、测序后,在XL10-Gold中扩增重组腺病毒质粒,最后在293细胞内包装扩增为重组腺病毒Ad-Acrp30-1和Ad-Acrp30-3.用此重组腺病毒感染3T3-L1脂肪细胞,用RT-PCR和ELISA检测其Acrp30 mRNA和蛋白表达.结果 设计并构建了小鼠Acrp30 基因特异性siRNA腺病毒载体,该载体感染脂肪细胞后,能显著抑制其Acrp30 mRNA和蛋白表达.结论 构建的Acrp30 基因特异性siRNA腺病毒载体能有效的抑制脂联素基因在3T3-L1脂肪细胞中的表达.     

脂联素与心房颤动的相关性研究进展

脂联素(又名Acrp30,APN,adiponectin)是迄今为止发现的唯一与体脂呈负相关的脂肪因子,具有抗炎、抗氧化、抗动脉粥样硬化和抗胰岛素抵抗等作用,与肥胖、胰岛素抵抗、糖尿病、血脂异常、冠状动脉粥样硬化性心脏病、高血压等疾病密切相关。近年来国内外研究显示,房颤的发生与脂联素水平存在着相关性。本文就脂联素与房颤的相关性研究进展作一综述。     

层粘连蛋白酶联免疫吸附法的建立及临床应用

层粘连蛋白酶联免疫吸附法的建立及临床应用［高锋，等．中华医学检验杂志，１９９４；１７（１）：３１］以鼠抗层粘连蛋白（ＬＮ）单克隆抗体包被聚苯乙烯微孔板，ＬＮ标准和待测血清夹心，兔抗ＬＮＩｇＧ为桥接一抗，辣根过氧化物酶交联羊抗兔ＩｇＧ为二抗放大显色，建...     

狂犬病抗体免疫金标检测试纸的研制

目的应用酶联免疫原理和胶体金层析技术,研制狂犬病抗体免疫金标检测试纸。方法采用特殊的生产工艺,在玻璃纤维膜包被胶体金标记狂犬病抗原,在硝酸纤维素膜上检测线和对照线处分别包被狂犬病抗原和兔抗狂犬病抗体,制成狂犬病抗体免疫金标检测试纸(人用)。结果当待检样品阳性时,在检测线处形成抗原抗体的免疫复合物而凝聚显色;当待检样品阴性时,检测线处不形成抗原抗体免疫复合物不显色。整个试验过程只需15 m in。试纸与ELISA试剂比较,两者都具有微量、特异、准确的优点,且金标试纸独具操作方便、快速和结果直观、容易判定的优点。结论应用胶体金免疫层析技术建立的"狂犬病抗体免疫金标检测试纸",可以准确地检测出被检样品是否带有狂犬病抗体,同时还可以通过检测线颜色的深浅判定抗体的含量高低。     

非酒精性脂肪肝患者血清胆固醇调节元件结合蛋白一1脂联素变化及其意义

目的探讨非酒精性脂肪肝患者血清胆固醇调节元件结合蛋白-1,脂联素变化及其意义。方法选择30例符合诊断标准脂肪肝患者,并设30倒健康体检者为正常对照粗。应Elisa技术,Real-timePCR技术检测血清胆固醇调节元件结合蛋白-1．脂联素含量及其mRNA变化。结果NAFLD患者脂联素含量减少,其mRNA表达呈下降趋势l而胆固醇调节元件结合蛋白-l含量增多,mRNA表达呈上升趋势。与正常组比较,有统计学意义（P〈0．05）。结论胆固醇调节元件结合蛋白-1和脂联素变化在非酒精性脂肪肝发展占据重要地位,这意味着调控胆固醇调节元件鲒合蛋白-1表达和脂联素分泌对于非酒精性脂肪肝防治是有益的。     

酶联免疫吸附试验一步法检测抗-HBc有关问题探讨

酶联免疫吸附试验(ELISA)是检测抗-HBc的常用方法,根据反应模式可分为一步法和二步法.一步法是将待测样品和酶标记物同时加入到反应孔中进行反应,从而达到简便、快速的目的;二步法是先将样品加入到反应孔中,待反应结束后再加入酶标记物,从而使待测样品的"捕获反应"和酶标记物的"酶联反应"分开进行,因而反应更加稳定,重复性也更好,但反应时间比一步法长.     

自噬与脂联素诱导的人乳腺癌MCF-7细胞凋亡的关系

目的 研究全长脂联素( Acrp30 )在人乳腺癌MCF-7细胞增殖、迁移、自噬、凋亡中的作用,并探讨自噬与凋亡的关系. 方法 体外培养 MCF-7 细胞,分为对照组(未加入Acrp30)、加药组(25、50、100、200 ng/ml Acrp30),四亚基偶氮唑盐( MTT)比色法检测各组细胞增殖情况. 选取100 ng/ml Acrp30浓度组( Acrp组)作用MCF-7 细胞,进行培养,倒置显微镜观察细胞形态、贴壁情况;划痕抑制试验了解细胞迁移能力;Western blot法评价细胞自噬水平;流式细胞仪检测Acrp组及3-甲基腺嘌呤(3-MA) 预处理组不同时间细胞的总凋亡率. 结果 ① MTT结果显示:与对照组相比,50、100 ng/ml组72 h,200 ng/ml 组48、72 h可显著抑制MCF-7细胞增殖( P<0. 05 );② 划痕抑制实验结果显示:与对照组相比, Acrp30 组作用 48、72 h,迁移距离显著减小( P <0. 01);③ Western blot结果显示:与对照组相比,Acrp30 组自噬水平( LC3B-Ⅱ/LC3B-Ⅰ比值) 24、48 h显著提高( P<0. 05,P<0. 01),但随时间延长,比值逐渐下降,作用72 h后LC3B-Ⅱ/LC3B-Ⅰ比值有所升高但差异无统计学意义;④ 流式细胞仪检测凋亡率结果显示:与对照组相比,Acrp30组和干预组48、72 h总凋亡率明显增加(P<0. 05,P<0. 01),与Acrp30组相比干预组72 h凋亡率显著升高( P<0. 05 ). 结论 Acrp30可抑制MCF-7细胞的增殖和迁移,诱导细胞的自噬和凋亡;抑制细胞自噬可促进Acrp30对MCF-7细胞凋亡的诱导.     

脂质体在检测血清子宫内膜抗体中的应用

目的　介绍一种以脂质体作为载体检测血清中抗体的实验方法。方法用磷脂酚胆碱、神经节苷脂、胆固醇制备脂质体,在内部包裹碱性磷酸酶（ＡＫＰ）,表面偶联子宫内膜抗原。取１ｍｌ上述溶液加被检血清和补体（豚鼠血清）作用后,再加入ＡＫＰ作用的底物,于４００ｎｍ处测其ＯＤ值。结果脂质体法检测健康孕妇（ＨＦＷ）和不孕症妇女（ＩＦＷ）血清的子宫内膜抗体阳性率分别为０％（０／３０）和３１．５％（３６／１１４）。与ＥＬＩＳＡ法检测的阳性率３％（１／３０）和２９．８％（３４／１１４）相近,且差别无显著性（Ｐ＞０．０５）。结论脂质体可以作为酶和抗原的载体,检测血清中的抗体。     

ELISA法和免疫印迹法检测抗ENA抗体的比较

目的：将目前国内新开展的两种检测ＥＮＡ技术,即免疫印迹法（ＩＢＴ）和酶免疫法（ＥＬＩＳＡ）进行对比、分析。方法：用 ＩＢＴ法及 ＥＬＩＳＡ法同时检测 １３０例各种结缔组织病、非结缔组织病人及健康人血清中的抗ＥＮＡ抗体。结果：２０例健康人,两法均为阴性;８０例结缔组织病患者,两法检出抗 Ｓｍ抗体的符合率为 ９１．３％,检测抗 ＲＮＰ抗体的符合率为 ８２．５％,以上抗体两法的敏感性无显著差异（Ｐ＞０．０５）。检测抗ＳＳＡ抗体,ＩＢＴ法检出率低。抗ＳＳＢ抗体检出率两法亦无显著差异（Ｐ＞０．０５）,符合率为８７．５０％,在１２例ＳＳ的检测中符合率比较低,为５０％。结论：两种方法在检测抗Ｓｍ、ＲＮＰ、ＳＳＢ抗体时,敏感性无显著差异,ＥＬＩＳＡ法检测抗ＳＳＡ抗体优于ＩＢＴ法。上述两种方法在临床应用于抗ＥＮＡ抗体的检测中于可互相验证和补充。     

129位丝氨酸磷酸化α-突触核蛋白DELFIA检测方法的建立及初步应用

目的 建立解离-增强镧系荧光免疫分析 (dissociation enhanced lanthanide fluorescent immunoassay,DELFIA)检测129位丝氨酸磷酸化α-突触核蛋白的方法及初步应用。方法 使用体外蛋白重组技术纯化得到人源α-syn蛋白单体(α-syn),用Polo 样激酶催化α-syn获得磷酸化α-syn单体(phosphorylated α-syn monomer,p-α-syn),用4-氧代壬烯醛制备α-syn聚集体(aggregates of α-syn,OW)及磷酸化α-syn聚集体(aggregates of p-α-syn,OP)。使用Western blotting法和间接ELISA方法验证本实验室前期制备的识别α-syn蛋白N端的多克隆抗体SN16和识别129位丝氨酸磷酸化α-syn蛋白的单克隆抗体C140S。使用SN16作为捕获抗体,C140S作为检测抗体的双抗夹心DELFIA法建立检测OP的标准曲线,并检测Thy1-α-SYN转基因小鼠血浆内OP的变化。结果 考马斯亮蓝染色和Western blotting法检测结果显示体外纯化的人源α-syn蛋白单体(17 000)纯度高,并通过催化获得了p-α-syn、OW和OP纯蛋白,均可用作建立标准曲线的标准抗原。Western blotting和酶联免疫吸附测定(enzyme-linked immunosorbent assay,ELISA)结果显示SN16 对α-syn、p-α-syn、OW、OP这4种形式的蛋白均可识别,C140S可以识别p-α-syn和OP,说明SN16和C140S可以用作双抗夹心法的配对抗体。间接DELFIA检测结果显示C140S对OP的识别效率更好。双抗夹心DELFIA检测结果显示,SN16-C140S作为配对抗体检测标准抗原的范围为:0.625~20 ng/mL,并建立了检测OP的标准曲线。Wt及Tg组小鼠血浆中OP在12月龄均较6月龄有所增加(P<0.001),Tg组6月龄小鼠血浆中OP显著高于同月龄Wt组(P<0.001),Tg组12月龄小鼠血浆OP较Wt组有增高的趋势,但差异无统计学意义(P>0.05)。结论 本文建立的SN16-C140S配对的双抗夹心DELFIA体系可以检测Thy1-α-SYN转基因小鼠血浆中聚集的磷酸化α-syn变化情况。     

人Aβ42重组抗原的融合表达及Aβ抗体的检测

OBJECTIVE: To clone and express the recombinant amyloid beta-protein (Abeta(42)) antigen and develop a method for detecting Abeta antibody. METHODS: Two partially complementary fragments of Abeta(42) gene were chemically synthesized for constructing the Abeta(42) gene by PCR. The resultant Abeta(42) gene fragment was subcloned into pGEX-2T expression vector for inducing the expression of GST-Abeta(42) fusion protein, which was purified by affinity chromatography. The antigen specificity and reactivity of the purified GST-Abeta(42) fusion protein to Abeta monoclonal antibody were identified with Western blotting. Using either GST-Abeta(42) fusion protein or Abeta(42) peptide as the coating antigen, an indirect enzyme-linked immunosorbent assay (ELISA) was established for detecting Abeta antibodies in the serum of Abeta(42) polypeptide-immunized SD rats. RESULTS: GST-Abeta(42) fusion protein was successfully expressed as an soluble protein, which, after purification, was found to have a relative molecular mass of 31 kD. About 800 mg of GST-Abeta(42) fusion protein were obtained from l L cell culture with a purity over 95%. Western blotting demonstrated specific reaction of this purified GST-Abeta(42) fusion protein with Abeta monoclonal antibody. The sensitivity of the indirect ELISA for detecting Abeta antibody was about 2 ng/ml using GST-Abeta(42) fusion protein as the coating antigen. There was no significant difference in the results of Abeta antibody detection using either GST-Abeta(42) or Abeta(42) as the coating antigen (P>0.05). CONCLUSION: GST-Abeta(42) fusion protein may serve as a substitute for the expensive Abeta(42) peptide for detecting Abeta antibody.     

戊型肝炎IgG抗体检测方法的建立

为建立一种简便、快速诊断戊型肝炎病毒血清抗体的检测方法,以基因工程HEVORF2区重组蛋白抗原和人工会成HEVORF3区合成多肽抗原混合作为包被抗原,辣根过氧化物酶标记的单克隆抗人IgG抗体为第二抗体,建立了间接酶联免疫吸附试验（ELISA）检测血清中HEVIgG抗体技术。检测中国药品生物制品检定所《检测抗HEV抗体国家参考品》全套血清盘：特异性参比品30份,全部为阴性;阳性参比品10份,全部为阳性;精密性参比品,CV（n＝10）＜15％。17例非甲、乙、丙肝炎患者,黄殖出现后4个月内,HEVIgG抗体检出率为100％。普通人群HEVIgG抗体检出率为1％～2％。表明该方法检测HEVIgG抗体,特异性强、敏感性高、操作简便快速,适用于临床对戊型肝炎的诊断和流行病学调查。     

Antibody responses to recombinant and plasma derived hepatitis B vaccines

The antibody response to hepatitis B surface antigen (anti-HBs) induced in 25 recipients of a recombinant hepatitis B vaccine derived from yeast was compared with that induced in 25 recipients of a vaccine prepared from hepatitis B surface antigen (HBsAg) derived from plasma. Anti-HBs affinity and specificity were compared using assays of antibody affinity with two different antigens, a complex of the major polypeptide of HBsAg (p25; molecular weight 25 000 daltons) covalently linked to its glycosylated form (gp30) prepared from native purified HBsAg, and a cyclical synthetic peptide representing amino acid residues 139-147 of the major polypeptide of HBsAg and known to represent a major part of an a determinant. There was no difference in anti-HBs affinity or molar antigen binding sites of the antibody measured with either antigen between the two groups. All subjects in both groups produced antibody that bound to the gp30/p25 complex antigen, whereas 22 of the recipients of the plasma derived vaccine compared with 24 of those receiving the yeast derived vaccine produced antibodies that bound to the cyclical synthetic peptide 139-147. These results support the finding of similar levels of anti-HBs, measured by commercial solid phase radioimmunoassay, in the two vaccine groups after three doses of vaccine. These results show no significant difference in the quantity, quality, or specificity of the anti-HBs response induced by the recombinant hepatitis B vaccine and the plasma derived hepatitis B vaccine.     

Comparison of Dot-ELISA with Sandwich ELISA in detecting circulating antigen in patients with bancroftian filariasis.

Dot-ELISA assay was compared with standard Sandwich ELISA in detecting parasite antigen in sera from patients with bancroftian filariasis. The same monoclonal antibody and the same serum samples were used in both assays. With Dot-ELISA, 67 of 70 serum samples from microfilaremic patients were positive at a dilution of 1:50. End titers ranged from 1:80 to 1:1 280. While with Sandwich ELISA, 64 of the 70 serum samples were positive at a dilution of 1:10. End titers ranged from 1:10 to 1:320. The specificity of both assays was over 91%, but their sensitivity was markedly different. Dot-ELISA could detect as little as 0.055 ng/ml microfilarial antigen added to normal human serum, whereas the lower limit of detection by Sandwich-ELISA was 10 ng/ml parasite antigen. An additional advantage of Dot-ELISA is that it does not require radioactivity or sophisticated equipment and, therefore, can be performed in virtually all filariasis-endemic areas.

     

ELISA法检测可溶性人类白细胞抗原—I及广东人参考值的测定

OBJECTIVE: To explore a method for quantitative detection of soluble human leukocyte antigen class I(HLA-I) antigens, with the assistance of which we attempt to define the reference value of the antigen in the population of Guangdong Province. METHODS: Sandwich enzyme-linked immunosorbent assay (ELISA) was employed with W6/32 monoclonal antibody serving as the solid phase antibody and beta2 microglobulin antibody as the first antibody. HRP-labeled second antibody and the substrate were added for enzyme-catalyzed coloring. On the basis of the standard curve obtained by sHLA-I standard reagent in serial dilution, the concentration of sHLA-I antigens in the samples of 60 normal subjects from the population of Guangdong province was detected. RESULTS: The sensitivity of this assay was 2.84 ng/ml with variation coefficients of 5.80% within the same batch and 9.00% between batches. The recovery rate ranged from 98.57% to 100.15%. The level of sHLA-I antigens in normal individuals was 699.54+/-360.10 ng/ml. CONCLUSION: Sandwich ELISA is sensitive, specific and stable in the detection of sHLA-I.     

乙型肝炎血清标志物四种检测方法的对比分析

目的:应用酶联免疫吸附试验(ELISA)、胶体金免疫层析法(GICA)、时间分辨免疫荧光法(TR-FIA)、化学发光法(CLIA)检测乙型肝炎血清标志物[乙型肝炎表面抗原(HbsAg)、乙型肝炎表面抗体(抗Hbs)、乙型肝炎e抗原(HbeAg)、乙型肝炎e抗体(抗Hbe)、乙型肝炎核心抗体(抗Hbc)],评价不同方法学检测结果之间是否具有一致性。方法:用GICA、ELISA、TRFIA、CLIA分别检测50例乙型肝炎病毒(HBV)感染者和100名正常体检者的乙型肝炎血清标志物。以CLIA作为参考方法,通过Kap-pa检验评价不同方法学检测结果之间的一致程度。结果:4种方法检测乙型肝炎血清标志物的结果具有高度以上一致性,CLIA在检测敏感性上更有优势。结论:目前广泛使用的检测乙型肝炎血清标志物的方法具有较好的一致性,为临床实验室方法学选择提供了依据。