高分辨熔解技术在疾病关联SNP验证检测中的应用

目的 探讨高分辨熔解(high resolution melting,HRM)技术的常规化检测流程以及实验设计策略和具体操作要点.方法 以疾病关联SNP单位点IL1B-31和双位点IL6-597/-572的检测为例,分别从核酸提取,引物设计、验证和有效性评价以及酶系统和HRM染料的选择与匹配等方面进行最优策略与流程的探索.结果成功建立了单位点IL1B-31和双位点IL6-597/-572的PCR-HRM检测方法并得到验证.柱提基因组DNA模板、特异高效HRM引物和应用热启动酶是PCR-HRM检测方法成功建立的关键,尤其是Beacon designer,uMelt和mfold等工具软件的应用.结论 HRM技术是一种适合疾病关联SNP验证和临床分子诊断的高通量、低成本、易操作、闭管均相检测新方法.     

TNF-α及其受体基因多态性与类风湿性关节炎易感性和血清学指标相关性分析

[摘要] 目的 探讨TNF-α及其受体基因多态性与中国西北地区汉族人群类风湿性关节炎（rheumatoid arthritis, RA）易感性及其血清学指标的相关性。方法 利用高分辨率熔解曲线技术（high resolution melting, HRM)检测452例RA患者和匹配的356例正常对照人群的TNF-α及其受体6个SNP位点的基因多态性（TNFA-857C>T、TNFA -863C>A、TNFA-308G>A、TNFA-238G>A、TNFR1-383A>C、TNFR2-exonT>G)进行病例-对照研究。此外,采用临床常规方法测定RA患者的抗环瓜氨酸肽抗体（anti-CCP）和类风湿因子（RF）,分析其与6个SNP位点的相关性。结果 TNFA-308GA和-863CA基因型在RA病例组和正常对照组中分布频率有统计学差异(P=0.001, P=0.002),TNFA-238GA、TNFA -238AA、TNFR2-exonTG和TNFR2-exonGG基因型在两组间分布频率无统计学差异（P=0.701,P=0.999,P=0.825,P=0.330）。由TNFA三个位点构成的单倍型-238G/-308A/-863C（GAC）、-238G/-308G/-863A（GGA）和-238G/-308G/-863C（GGC）在两组间的分布频率均有统计学差异(P=0.016;P=0.033;P=0.023)。TNF-α及其受体基因多态性与RA血清学指标（RF、anti-CCP)无统计学差异（P>0.05）。结论 TNFA-308GA和TNFA-863CA与中国西北地区汉人群RA易感性相关;TNFA单倍型GAC、GGA和GGC与RA易感性相关;TNF-α及其受体基因多态性与RA患者血清学指标没有相关性。     

深部真菌感染高危因素调查分析

近十余年来,随着广谱抗生素及免疫抑制剂的大量使用,恶性肿瘤的放疗及化疗,各种侵入性医疗措施的增多,医院内深部真菌感染呈上升趋势,对该病的预防和早期诊断显得日益重要。为了解深部真菌感染的高危因素,以便对可控因素进行控制,对兰州大学第二医院2007年1～12月间具有完整临床资料且有微生物依据的120例深部真菌感染病例进行了回顾性分析,现报道如下。     

降钙素基因相关肽、内皮素与2型糖尿病肾病的相关性研究

Objective To investigate the tendency of plasma levels of calcitonin gene-related peptide(CGRP)and endothelin(ET)in type 2 diabetic nephropathy,and explore the relationship between calcitonin gene-related peptide,endothelin and pathogenesis of diabetic nephropathy.Methods A total of 25 healthy controls(healthy control group)and 83 patients with type 2 diabetic mellitus(T2DM group)were enrolled in the investigation.According to level s of urinary albumin excretion rate(UAER),the 83 T2DM patients group were further divided into three subgroups:normal UAER subgroup(n=28),microalbuminuira subgroup(n=29)and clinical proteinuria subgroup(n=26).Plasma contents of calcitonin gene-related peptide and endothelin were measured with radioimmunoassay(RIA).Results The level of ET was significantly higher in T2DM group than that in healthy control group(P＜0.01),positively correlative tO UAER(r=0.65,P＜0.01).The plasma level of CGRP was significantly lower in the patients with diabetic nephropathy than the healthy controls,negatively correlative tO UAER(r=-0.71,P＜0.01).There was negative correlation between plasma levels of ET and CGRP(r=-0.62,P＜0.01).Conclusion The disequilibrium of CGRP and ET in plasma might play an important role in the process of diabetic nephropathy.Therefore,keeping balance of levels of CGRP and ET in plasma contributes to prevention and cure of diabetic nephropathy.     

降钙素基因相关肽、内皮素与2型糖尿病肾病的相关性研究

Objective To investigate the tendency of plasma levels of calcitonin gene-related peptide(CGRP)and endothelin(ET)in type 2 diabetic nephropathy,and explore the relationship between calcitonin gene-related peptide,endothelin and pathogenesis of diabetic nephropathy.Methods A total of 25 healthy controls(healthy control group)and 83 patients with type 2 diabetic mellitus(T2DM group)were enrolled in the investigation.According to level s of urinary albumin excretion rate(UAER),the 83 T2DM patients group were further divided into three subgroups:normal UAER subgroup(n=28),microalbuminuira subgroup(n=29)and clinical proteinuria subgroup(n=26).Plasma contents of calcitonin gene-related peptide and endothelin were measured with radioimmunoassay(RIA).Results The level of ET was significantly higher in T2DM group than that in healthy control group(P＜0.01),positively correlative tO UAER(r=0.65,P＜0.01).The plasma level of CGRP was significantly lower in the patients with diabetic nephropathy than the healthy controls,negatively correlative tO UAER(r=-0.71,P＜0.01).There was negative correlation between plasma levels of ET and CGRP(r=-0.62,P＜0.01).Conclusion The disequilibrium of CGRP and ET in plasma might play an important role in the process of diabetic nephropathy.Therefore,keeping balance of levels of CGRP and ET in plasma contributes to prevention and cure of diabetic nephropathy.     

基质金属蛋白酶-7和组织型金属蛋白酶抑制因子-1在胃癌中的表达及意义

胃癌的发生、发展是一个多因素、多步骤的过程,涉及到癌基因、抑癌基因表达的失调,细胞凋亡、细胞运动和迁移调控的异常等多方面机制。细胞外基质和基底膜降解是恶性肿瘤浸润和转移的关键环节,基质金属蛋白酶是人体内降解细胞外基质（extracel clular matrix,ECM）的主要酶类,在肿瘤发展等众多生理和病理过程中均发挥重要的作用。     

高分辨熔解技术检测类风湿关节炎患者IL-1β和IL-1Ra基因多态性

目的 探讨IL-1β和IL-1 Ra基因多态性与类风湿关节炎(rheumatoid arthritis,RA)易感性及其血清学指标的关系.方法 应用高分辨率熔解(high resolution melting,HRM)技术对452例RA患者和360例对照进行IL-1β和IL-1Ra基因多态性的病例-对照研究,并分析IL-1β和IL-1Ra基因多态性与血清学指标的相关性.等位基因频率比较采用SPSS软件,单倍型构建采用在线SHEsis软件.结果 成功建立了IL-1B-511、-31、+3954和IL-1RN( rs4251961)4个位点的PCR-HRM检测方法.结果显示在女性中,IL-1B+ 3954位点等位基因频率在病例组和对照组之间存在有统计学差异(x2 =5.0,P=0.03),IL-1B-31位点等位基因频率和单倍型IL-1B-511*T/IL-1B-31*C/IL-1B+ 3954*C/IL-IRN*T(TCCT)在抗-MCV阳性组和阴性组之间存在统计学差异(x2 =5.0,P=0.02;x2 =5.3,P=0.02),单倍型IL-1B-511*T/IL-1B-31* C/IL-1B +3954* C/IL-1RN* C(TCCC)在RF、抗CCP和抗MCV 3个指标均为阳性和3指标非全阳性的2组之间存在统计学差异(x2 =5.1,P=0.02).结论 PCR-HRM技术是一种值得推广的常规化基因分型新方法.IL-1B+ 3954*C与女性RA易感性相关,IL-1B-31*C和单倍型TCCT与女性抗MCV阳性相关,单倍型TCCC与女性血清学指标全阳性相关.     

颅内感染患者血清和脑脊液中MMP-9及NSE含量观察

目的观察颅内感染患者血液和脑脊液中基质金属蛋白酶9（MMP-9）、神经元特异性烯醇化酶（NSE）的含量,并探讨其临床意义。方法选择加例颅内感染患者,其中病毒性脑炎患者18例（病脑组）,结核性脑膜炎患者14例（结脑组）,化脓性脑膜炎患者8例（化脑组）,另择健康查体者23例作为对照组。分别应用双抗体夹心ABC-ELISA法和放免法测定各组血清和脑脊液中MMP-9、NSE含量。结果患者组MMP-9和NSE含量均显著高于对照组（P均〈0．05）;MMP-9含量依次为：结脑组〉化脑组〉病脑组,病脑组与结脑组、化脑组比较有统计学差异（P均〈0．01）;NSE含量依次为病脑组〉结脑组〉化脑组（P均〈0．05）。结论血清和脑脊液中MMP-9和NSE含量可作为颅内感染的标志物,其含量高低分别与血脑屏障功能损伤和神经元损害程度有关;二者结合能较全面地反映颅内感染的严重程度。     

抗菌肽MSI-78基因在大肠埃希菌DH5α中的克隆及阳性重组体鉴定

目的 研究抗菌肽MSI-78基因的克隆及鉴定方法 ,为抗菌肽MSI-78基因的表达及抗菌活性研究奠定基础.方法 根据MSI-78氨基酸序列及大肠埃希菌(ECO)偏爱密码子,推出MSI-78编码基因,化学合成获得MSI-78基因,ECOR Ⅰ和Pst Ⅰ消化后与pUC18载体连接并转化大肠埃希菌DH5α,α-互补筛选阳性重组菌,单酶切、双酶切、PCR鉴定阳性重组体.结果 通过ECOR Ⅰ单酶切、ECOR Ⅰ和Pst Ⅰ双酶切抽提的阳性重组质粒,证实阳性重组质粒中含有抗菌肽MSI-78基因,特异性引物PCR扩增也证实阳性重组质粒中含有抗菌肽MSI-78基因,说明MSI-78基因成功在大肠埃希菌DH5α克隆.结论 MSI-78基因的克隆为后期的表达、纯化及生物活性研究奠定了基础.     

降钙素基因相关肽、内皮素与2型糖尿病肾病的相关性研究

Objective To investigate the tendency of plasma levels of calcitonin gene-related peptide(CGRP)and endothelin(ET)in type 2 diabetic nephropathy,and explore the relationship between calcitonin gene-related peptide,endothelin and pathogenesis of diabetic nephropathy.Methods A total of 25 healthy controls(healthy control group)and 83 patients with type 2 diabetic mellitus(T2DM group)were enrolled in the investigation.According to level s of urinary albumin excretion rate(UAER),the 83 T2DM patients group were further divided into three subgroups:normal UAER subgroup(n=28),microalbuminuira subgroup(n=29)and clinical proteinuria subgroup(n=26).Plasma contents of calcitonin gene-related peptide and endothelin were measured with radioimmunoassay(RIA).Results The level of ET was significantly higher in T2DM group than that in healthy control group(P＜0.01),positively correlative tO UAER(r=0.65,P＜0.01).The plasma level of CGRP was significantly lower in the patients with diabetic nephropathy than the healthy controls,negatively correlative tO UAER(r=-0.71,P＜0.01).There was negative correlation between plasma levels of ET and CGRP(r=-0.62,P＜0.01).Conclusion The disequilibrium of CGRP and ET in plasma might play an important role in the process of diabetic nephropathy.Therefore,keeping balance of levels of CGRP and ET in plasma contributes to prevention and cure of diabetic nephropathy.