微板式磁化学发光酶免疫分析法对人绒毛膜促性腺激素(HCG)的灵敏快速测定

以磁性酶免疫测量分析为模型 ,建立了微板式磁化学发光酶免疫分析法 .利用 3 (2′ 螺旋金刚烷 ) 4 甲氧基 4 (3″ 磷酰氧基 )苯 1,2 二氧杂环丁烷 (AMPPD) 碱性磷酸酶 (ALP)化学发光体系对人绒毛膜促性腺激素 (HCG)进行测定 ,测量的灵敏度较分光光度法提高了 13倍 .测定的线性范围为 0 15～ 5 0 0mIU/mL ;批内变异系数 (C .V .% )和批间变异系数 (C .V .% )均在 18%之内 ;回收率在 80 %～ 116%之间 ;利用本法对血清样品进行了测定 ,并与其它化学发光免疫分析方法进行了比较 ,其相关系数为 0 965 .首次将酶免疫分析方法用于唾液中人绒毛膜促性腺激素 (HCG)的测定 ,为建立非侵入式样品测定方法打下了基础 .

抗髓过氧化物酶抗体及乳铁蛋白抗体检测在自身免疫性肝炎诊断中的意义

目的探讨抗髓过氧化物酶抗体(AMPA)和乳铁蛋白抗体(ALA)在检测自身免疫性肝炎(AIH)时的临床意义。方法选择2011年4月到2014年4月收治的AIH患者70例(AIH组)和非AIH患者173例(非AIH组),另选择同期行健康体检者60名为对照组。采用酶联免疫吸附试验(ELISA)检测患者AMPA和ALA,采用间接免疫荧光法检测抗核抗体、抗平滑肌抗体、抗中性粒细胞胞质抗体等。通过计算各指标的有效率、特异性、敏感性、阳性似然比、阴性似然比等判断AMPA和ALA检测AIH的效果和准确性。结果70例AIH组患者中AMPA、ALA、ASMA、ANCA阳性率[61.4%(43/70)、20.0%(14/70)、68.6%(48/70)、67.1%(47/70)]明显高于非AIH组[4.0%(7/173)、4.2%(1/173)、7.1%(2/173)、59.3%(15/173)],差异均有统计学意义(P<0.05或0.01)。AIH组患者各检测指标阳性率均高于对照组,差异有统计学意义(P<0.05)。AIH组中AMPA和ALA抗体检测的特异性高达97.1%和99.8%,但ALA的敏感性较低,为20.0%。结论 AMPA、ALA与AIH有密切联系,特别是在检测AIH-Ⅰ型时,AMPA抗体检测有非常明显的优越性;ALA抗体检测结果与ANCA、ASMA并无明显差异性。AMPA和ALA联合其他抗体检测对AIH的诊断具有重要临床意义。

胃癌组织端粒酶活性的表达及其临床意义

目的:研究胃癌组织中端粒酶活性的表达,以探讨胃癌的辅助诊断指标。方法:收集经手术及病理证实的胃癌标本60例,并将胃腺瘤性息肉12例、慢性胃炎20例作对照研究,用TRAP-PCR银染法检测端粒酶活性。结果:60例胃癌标本的端粒酶阳性率为85.0%(51/60),12例胃腺瘤性息肉端粒酶阳性率为16.6%(2/12),20例慢性胃炎端粒酶阳性率为5%(1/20)。胃癌组与胃腺瘤性息肉相比、胃癌组与慢性胃炎相比,端粒酶阳性率均有高显著性差异,胃腺瘤性息肉与慢性胃炎相比端粒酶阳性率亦有显著性差异,而不同组织学分型的胃癌的端粒酶阳性率无显著性差异。结论:大多数胃癌组织端粒酶活性显著升高,而绝大多数胃良性病变无端粒酶活性升高,故端粒酶可作为胃癌辅助诊断的一种肿瘤标记。

以尿激素标记物探讨职业噪声暴露与纺织女工月经功能的关系

目的 通过测定纺织女工尿样中生殖内分泌激素的代谢产物 ,探讨职业噪声暴露与月经功能的关系。方法 以某市纺织厂女工作为暴露组和内对照组 ,社区非噪声暴露女工作为外对照组 ,测量职业噪声暴露水平 ,并通过问卷了解月经情况。用酶免疫学方法测定月经周期尿中的孕酮代谢产物孕二醇 - 3-葡萄糖酸甙 (PdG)和卵泡刺激素 (FSH) β亚单位 ,并以此来判断排卵周期和确定排卵日。用广义线性模型校正重复观测间的相关性 ,并用线性回归模型和Logistic回归模型分析职业噪声暴露对纺织女工月经功能的影响。结果 高噪声暴露〔 >10 0dB(A)〕使女性月经周期长度缩短 ,经统计学分析 ,差异有显著性 (P <0 0 5 )。同时 ,噪声暴露与较短月经周期的发生也有一定的相关关系 (OR =1 6 35 ,95 %CI :0 94 4 ,2 830 ) ,并使较短黄体期的发生率增加 (OR =1 711,95 %CI :1 0 18,2 818)。噪声暴露与长月经周期 (>4 0d)的发生存在明显的负相关 (OR =0 339,95 %CI :0 12 3,0 932 )。噪声暴露对纺织女工的痛经和有无排卵影响并不明显。结论 纺织厂职业噪声暴露可能对纺织女工的月经功能产生影响 ,使月经周期缩短 ,并使短黄体期的发生率增加。用酶免疫学方法测定尿中的PdG、FSH来评价环境和职业暴露对月经功能的影响 ,可应用?

精细化人文关怀应用于ICU危重患者的临床效果

目的探讨精细化人文关怀应用于重症监护室(ICU)危重患者的临床效果。方法选取2018年2~12月在吉林大学第一医院ICU住院的老年危重患者120例,随机按单双数分为对照组和实验组,每组60例;两组均实施对应疾病的临床治疗,护理上对照组实施常规护理,实验组在常规护理基础上实施精细化人文关怀。比较不同护理方式对两组的护理满意度评价;采用焦虑自评量表(SAS)、抑郁自评量表(SDS)和匹兹堡睡眠质量指数(PSQI)评分,比较两组实施前后焦虑、抑郁及睡眠情况;采用酶联免疫吸附试验检测两组血清白细胞介素(IL)-6、IL-10和肿瘤坏死因子(TNF)-α含量,比较两组炎症因子水平,评估患者自身免疫功能状态。结果实验组护理效果明显优于对照组,护理满意度评分高于对照组,差异有统计学意义(P<0.05);护理前实验组SAS、SDS、PSQI评分及血清IL-6、IL-10和TNF-α水平与对照组比较差异无统计学意义(P>0.05),护理后两组各指标均明显低于护理前,且实验组低于对照组,差异有统计学意义(P<0.05)。结论在ICU对危重患者实施精细化人文关怀能降低患者焦虑抑郁情绪、提高睡眠质量及增强机体免疫力。

氯吡格雷联合循证对心血管介入术围术期患者的临床作用

目的评估氯吡格雷联合循证对心血管介入术围术期患者的临床疗效。方法选取心血管介入术患者60例,随机分为对照组和观察组各30例,分别给予氯吡格雷联合常规干预和氯吡格雷联合循证干预。记录两组并发症发生率、患者满意度、评估干预操作和干预模式评分;采用酶联免疫吸附试验试剂盒检测患者血清网膜素-1、血栓素B2、血小板聚集率、组胺及鸢尾素水平,观察氯吡格雷联合不同干预模式在心血管介入术围术期患者中的临床效果。结果观察组并发症发生率明显低于对照组(P<0.05);患者满意度明显高于对照组(P<0.05);干预操作评分、干预模式评分明显高于对照组(P<0.05)。术前两组血清网膜素-1、血栓素B2、血小板聚集率、组胺及鸢尾素水平差异无统计学意义(P>0.05);术后两组比较差异有统计学意义(P<0.05)。结论氯吡格雷联合循证干预能够通过上调心血管介入术患者血清网膜素-1水平,下调血清血栓素B2、血小板聚集率、组胺及鸢尾素水平,从而更有效地促进介入术患者身体康复,明显降低并发症发生率及提高患者满意度,其临床疗效显著。

参芍胶囊对高脂血症大鼠血清SOD及MDA水平的影响

目的:观察参芍胶囊能否影响高脂血症大鼠血清超氧化物歧化酶(SOD)及丙二醛(MDA)水平。方法:将50只大鼠随机分成对照组、模型组、治疗组(低、中、高)剂量,模型组给予高脂饮食,对照组给予普通饮食,治疗组给予高脂饮食的同时给予参芍胶囊治疗(低、中、高)三个治疗剂量;42d后分别测定各组大鼠血清SOD、MDA的含量、治疗组与模型组比较,观察各项指标有无统计学意义。结果:与对照组相比:高脂血症组大鼠血清中SOD水平降低,而MDA水平升高,有统计学意义(P<0.05)。治疗组与模型组相比较:给予参芍胶囊治疗后血清中SOD水平升高,而MDA水平降低,其中中剂量组与模型组相比有显著的统计学意义(P<0.05),低剂量和高剂量组与模型组相比无统计学意义。结论:参芍胶囊可以通过通过影响大鼠血清SOD及MDA水平,从而改善血管内皮细胞功能。

单纯疱疹病毒2型感染与非淋菌性尿道炎的病因关系

目的探讨非淋菌性尿道炎(NGU)患者单纯疱疹病毒2型(HSV2)感染的流行病学状况及实验室诊断方法。方法实验分为NGU患者组和健康对照组。免疫快速测定法(C-C快速法)检测沙眼衣原体(CT),培养法检测解脲支原体(UU),血清酶联免疫吸附法(ELISA法)检测血清HSV2抗体,荧光定量聚合酶链反应(FQ-PCR)检测尿道分泌物HSV2。结果NGU患者组的CT、UU感染率分别为23.27%和30.91%。NGU患者组和健康对照组的HSV2IgGI、gM、HSV2DNA阳性率分别为38.18%/18.03%、10.91%/1.64%、19.63%/4.92%,两组相比差异均有统计学意义(P<0.05)。NGU患者组无CT、UU、阴道毛滴虫、念珠菌感染的HSV2阳性率为33.33%,与对照组相比差异亦有统计学意义(P<0.05)。结论HSV2是NGU的重要病原体之一。FQ-PCR法作为HSV感染的实验室诊断技术具有很好的临床应用价值。

戊二醛聚合对猪、牛和人血红蛋白免疫学特性的影响

猪、牛、人血红蛋白或其相应的戊二醛聚合体分别经皮下或腹腔多次免疫小鼠,ELISA检测抗血清IgG滴度;免疫印迹法分别检测小鼠抗猪、牛、人血红蛋白或其相应的戊二醛聚合体IgG与猪、牛、人血红蛋白或其相应的戊二醛聚合体之间的交叉反应,探讨戊二醛聚合对不同种类血红蛋白免疫学特性的影响。结果表明:猪、牛、人血红蛋白的免疫原性均很弱,其相应的戊二醛聚合体免疫原性明显增强;猪、牛、人血红蛋白具有明显的抗原性差异;戊二醛聚合对这三类血红蛋白免疫学性质的影响完全不同,戊二醛聚合猪血红蛋白没有产生新的抗原决定簇,戊二醛聚合牛血红蛋白某些抗原决定簇被封闭,戊二醛聚合人血红蛋白形成了某些新的抗原决定簇。

ECLIA测定TPOAb与RIA测定TPOAb、TGAb结果的比较

目的:比较电化学发光免疫分析法(ECLIA)检测甲状腺过氧化物酶抗体(TPOAb)和放射免疫分析法(RIA)检测TPOAb、甲状腺球蛋白抗体(TGAb)水平,探讨其临床应用价值。方法:选取215例甲状腺病患者,行甲状腺细针抽吸(FNAB)检查,并采集空腹静脉血,用RIA法分别进行TPOAb、TGAb检测,其中59例自身免疫性甲状腺病(AITD)患者ECLIA法同步行TPOAb检测。结果:1RIA测定TPOAb和TGAb存在正相关。桥本甲状腺炎(HT)患者TPOAb和TGAb阳性率分别为86.5%和56.8%;Graves病(GD)患者为75.6%和56.1%;非AITD患者为16.3%和8.7%。2ECLIA与RIA法检测TPOAb具有相关性。结论:两种方法检测结果的相关性好,RIA法检测TPOAb较TGAb灵敏度高。

Inhibition of high-mobility group box 1 expression by siRNA in rat hepatic stellate cells

AIM:To explore the role of high-mobility group box 1 (HMGB1) protein during liver fibrogenesis and investigate the functional effects of HMGB1 gene silencing in hepatic stellate cells (HSCs) using siRNA.METHODS:Hepatic fibrosis in rats was induced through serial subcutaneous injections of dimethylnitrosamine,and expression of HMGB1 was detected by immunohistochemistry.HMGB1 siRNAs were developed and transiently transfected into HSC-T6 cells using Lipofectamine 2000.HMGB1 expression was evaluated by real-time polymerase chain reaction (PCR) and Western blotting analysis.Expression of α-smooth muscle actin (α-SMA) and collagen typesⅠand Ⅲ was evaluated by real-time PCR.Cell proliferation and the cell cycle were determined using the methyl thiazolyl tetrazolium method.Finally,collagen content in HSC supernatant was evaluated by an enzyme-linked immunosorbent assay.RESULTS:The results showed that HMGB1 was upregulated during liver fibrosis and that its expression was closely correlated with the deposition of collagen.siRNA molecules were successfully transfected into HSCs and induced inhibition of HMGB1 expression in a time-dependent manner.Moreover,HMGB1 siRNA treatment inhibited synthesis of α-SMA and collagen types Ⅰ and Ⅲ in transfected HSCs.CONCLUSION:This study suggests a significant functional role for HMGB1 in the development of liver fibrosis.It also demonstrates that downregulation of HMGB1 expression might be a potential strategy to treat liver fibrosis.

Construction of single chain Fv antibody against transferrin receptor and its protein fusion with alkaline phosphatase

AIM: To construct fusion protein of a single-chain antibody (scFv) against transferrin receptor (TfR) with alkaline phosphatase(AP). METHODS: The VH-linker-VL,namely scFv gene,was prepared by amplifying the VH and VL genes from plasmid pGEM-T-VH and pGEM-T-VL with splicing overlap extension polymerase chain reaction (SOE PCR). After the ScFv gene was modified by 5/71 and Not I,it was subcloned into the secretory expression vector pUC19/119, and then was transformed into E.coli TG1.The positive colonies were screened by colony PCR and their expressions were induced by IPTG.ScFv gene was gained by digesting ScFv expression vector pUC19/119 with 5/71 and NotI restriction enzymes, then subcloned into expression vector pDAP2, followed by transformation in E.coli TG1.The positive colonies were selected by bacterial colony PCR.The expression of fusion protein (scFv-AP) was induced by IPTG.Its activity was detected by enzyme immunoassay. The molecular weights of scFv and scFv-AP were measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). RESULTS: The product of SOE PCR formed a band of 700 bp in agarose gel electrophoresis. SDS-PAGE demonstrated the molecular weight of scFv was 27 ku.Immunofluorescent assay (IFA) demonstrated its reactivity with TfR.The molecular weight of scFv-AP was 75 ku.Enzyme immunoassay showed that scFv-AP could specifically bind to human TfR and play AP activity. CONCLUSION: We have successfully prepared the anti-human TfR scFv and constructed the fusion protein of scFv and AP.It is promising for immunological experiments.

Alteration of chaperonin60 and pancreatic enzyme in pancreatic acinar cell under pathological condition

AIM： To investigate the changes of chaperonin60 （Cpn60） and pancreatic enzymes in pancreatic acinar cells, and to explore their roles in the development of experimental diabetes and acute pancreatitis （AP）. METHODS： Two different pathological models were replicated in Sprague-Dawley rats： streptozotocininduced diabetes and sodium deoxycholate-induced AP. The contents of Cpn60 and pancreatic enzymes in different compartments of the acinar cells were measured by quantitative immunoo/tochemistry. RESULTS： The levels of Cpn60 significanUy increased in diabetes, but decreased in AP, especially in the zymogen granules of the pancreatic acinar cells. The elevation of Cpn60 was accompanied with the increased levels of pancreatic lipase and chymotrypsinogen in diabetes. However, a decreased Cpn60 level was accompanied by high levels of lipase and chymotrypsinogen in AP. The amylase level was markedly reduced in both the pathological conditions. CONCLUSION： The equilibrium between Cpn60 and pancreaUc enzymes in the acinar cells breaks in AP, and Cpn60 content decreases, suggesting an insufficient chaperone capacity. This may promote the aggregation and autoactivation of the premature enzymes in the pancreatic acinar cells and play roles in the development of AR

Osteopontin increases hepatocellular carcinoma cell growth in a CD44 dependant manner

AIM:To investigate the role of osteopontin(OPN) and its splice variants in the proliferation of hepatocellular carcinoma(HCC).METHODS:The expression of OPN variants in HCC cell lines as well as HCC tissue samples and nontumour tissue was studied using polymerase chain reaction.OPN variant cDNAs were cloned into a mammalian expression vector allowing both transient expression and the production of stable OPN expressing cell lines.OPN expression was studied in these cells using Western blotting,immunofluoresnce and enzyme linked immunosorbent assay.A CD44 blocking antibody and siRNA targeting of CD44 were used to examine the role of this receptor in the OPN stimulated cell growth observed in culture.Huh-7 cells stably expressing either OPN-A,-B or-C were injected subcutaneously into the flanks of nude mice to observe in vivo tumour growth.Expression of OPN mRNA and protein in these tumours was examined using reverse transcriptionpolymerase chain reaction and immunohistochemistry.RESULTS:OPN is expressed in HCC in 3 forms,the full length OPN-A and 2 splice variants OPN-B and-C.OPN variant expression was noted in HCC tissue as well as cognate surrounding cirrhotic liver tissue.Expression of these OPN variants in the HCC derived cell line Huh-7 resulted in secretion of OPN into the culture medium.Transfer of OPN conditioned media to na ve Huh-7 and HepG2 cells resulted in significant cell growth suggesting that all OPN variants can modulate cell proliferation in a paracrine manner.Furthermore the OPN mediated increase in cellular proliferation was dependent on CD44 as only CD44 positive cell lines responded to OPN conditioned media while siRNA knockdown of CD44 blocked the proliferative effect.OPN expression also increased the proliferation of Huh-7 cells in a subcutaneous nude mouse tumour model,with Huh-7 cells expressing OPN-A showing the greatest proliferative effect.CONCLUSION:This study demonstrates that OPN plays a significant role in the proliferation of HCC through interaction with the cell surface receptor CD44.Modulation of this interaction could represent a novel strategy for the control of HCC.

Interleukin-2 and Interleukin-6 in Recurrent Genital Herpes Patients

Objective:To study the cellular immunity status of patientswith recurrent genital herpes. Methods: Serum levels of interlukin-2 and its solublereceptor and interlukin-6 were measured by ELISA in 34patients with recurrent genital herpes. Results: Serum levels of IL-2 and IL-6 were significantlydecreased in patients compared to healthy controls (P<0.01),and the level of sIL-2R was significantly increased in patientswith recurrent genital herpes (P<0.01). There were nosignificant differences in all variables amongst patientsregarding relapse stage and remission stage (P>0.05). Conclusion: There was a cellular immune deficiency inpatients with recurrent genital herpes.

Bile-acid-activated farnesoid X receptor regulates hydrogen sulfide production and hepatic microcirculation

AIM: To investigate whether the farnesoid X receptor (FXR) regulates expression of liver cystathionase (CSE), a gene involved in hydrogen sulfi de (H2S) generation. METHODS: The regulation of CSE expression in response to FXR ligands was evaluated in HepG2 cells and in wild-type and FXR null mice treated with 6-ethyl chenodeoxycholic acid (6E-CDCA), a synthetic FXR ligand. The analysis demonstrated an FXR responsive element in the 5'-flanking region of the human CSE gene. The function of this site was investigated by luciferase reporter assays, chromatin immunoprecipitation and electrophoretic mobility shift assays. Livers obtained from rats treated with carbon tetrachloride alone, or in combination with 6-ethyl chenodeoxycholic acid, were studied for hydrogen sulphide generation and portal pressure measurement. RESULTS: Liver expression of CSE is regulated by bile acids by means of an FXR-mediated mechanism. Western blotting, qualitative and quantitative polymerase chain reaction, as well as immunohistochemical analysis, showed that expression of CSE in HepG2 cells and in mice is induced by treatment with an FXR ligand. Administration of 6E-CDCA to carbon tetrachloride treated rats protected against the down-regulation of CSE expression, increased H2S generation, reduced portal pressure and attenuated the endothelial dysfunction of isolated and perfused cirrhotic rat livers. CONCLUSION: These results demonstrate that CSE is an FXR-regulated gene and provide a new molecular explanation for the pathophysiology of portal hypertension.

Hepato-biliary profile of potential candidate liver progenitor cells from healthy rat liver

AIM： To evaluate the presence of progenitor cells in healthy adult rat liver displaying the equivalent ad- vanced hepatogenic profile as that obtained in humans. METHODS： Rat fibroblastic-like liver derived cells （rFLDC） were obtained from collagenase-isolated liver cell suspensions and characterized and their phenotype profile determined using flow cytometry, immunocyto- chemistry, reverse transcription polymerase chain reac- tion and functional assays. RESULTS： rFLDC exhibit fibroblastoid morphology, ex- press mesenchymal （CD73, CD90, vimentin, m-smooth muscle actin）, hepatocyte （UGTIA1, CK8） and biliary （CK19） markers. Moreover, these cells are able to store glycogen, and have glucose 6 phosphatase activity, but not UGTIA1 activity. Under the hepatogenic differentia- tion protocol, rFLDC display an up-regulation of hepatocyte markers expression （albumin, tryptophan 2,3-di- oxygenase, G6Pase） correlated to a down-regulation of the expression of the biliary marker CK19. CONCLUSION： Advanced hepatic features observed in human liver progenitor cells could not be demonstrated in rFLDC. However, we demonstrated the presence of an original rodent hepato-biliary cell type.

Clinicopathological Features of Non-familial Colorectal Cancer with High-frequency Microsatellite Instability

Objective To explore the clinicopathological features of non-familial colorectal cancer with high-frequency microsatellite instability (MSI-H). Methods One hundred and fifty patients with colorectal cancer who had no family history were enrolled in this study from June 2006 to June 2008. Five standard microsatellite loci including BAT25, BAT26, D2S123, D5S346, and D17S250 were amplified with immunofluorescent polymerase chain reaction. The patient information including age, sex, and tumor location was recorded. Pathological features including differentiation, mucinous differentiation, histological heterogeneity, and Crohn's-like reaction were observed under light microscope. The presence of tumor-infiltrating lymphocytes (TLs, CD4+ and CD8+) was detected by means of immunohistochemistry. A regression equation was obtained by stepwise logistic regression analysis to evaluate the relationship between MSI-H phenotype in colorectal cancer ands pathological features. Results MSI-H phenotype occurred in 13.33% of the 150 patients with non-familial colorectal cancer. Poor differentiation, histological heterogeneity, Crohn's-like reaction, and presence of TLs were found to be independent factors to identify MSI-H non-familial colorectal cancer. Logistic regression equation showed an overall sensitivity of 70.0%, specificity of 99.2%, and accuracy of 95.3% in identifying MSI-H non-familial colorectal cancer. Conclusion MSI-H non-familial colorectal cancer manifests specific pathological features, which may be relied upon for effective identification of that disease.