小鼠子宫内胚胎电转技术方法的改良

目的 优化小鼠子宫内胚胎电转技术,用于检测胎鼠大脑新生神经元的迁移和分化特征。 方法 通过选择异氟烷气体麻醉方式,改进毛细玻璃针的制备方法,减少手术中胚胎的暴露时间,优化电转参数等,对小鼠子宫内胚胎电转技术进行一系列优化。采用优化的子宫内胚胎电转技术给6只怀孕16.5 d的孕鼠进行了手术,共电转胚胎42只。 结果 电转后孕鼠和胚胎的存活率达到100%,其中大脑表达外源基因的胎鼠占85%±3%。 结论 优化的小鼠子宫内胚胎电转技术可以获得令人满意的胎鼠存活率和基因电转效率。     

免疫性肝纤维化大鼠血浆氨基酸浓度与肝纤维化程度的相关性研究

目的探讨血浆氨基酸浓度与肝纤维化程度的相关性。方法通过注射猪血清构建不同分期大鼠肝纤维化模型。采用Agilent 1100,通过高效液相色谱法(high performance liquid chromatograph,HPLC)测定大鼠血浆中的氨基酸浓度。利用统计学方法分析血浆氨基酸浓度与肝纤维化程度之间的相关性。结果根据柱前衍生高效液相色谱法测定的大鼠血浆中16种游离氨基酸含量及Spearman等级相关分析和方差分析发现:血浆组氨酸(histidine,His)、苯丙氨酸(phenylalanine,Phe)、苏氨酸(threonine,Thr)、酪氨酸(tyrosine,Tyr)、缬氨酸(valine,Val)等与肝纤维化程度具有相关性(His:rs=0.512,P=0.015;Phe:rs=0.713,P<0.01;Thr:rs=0.567,P=0.006;Tyr:rs=0.753,P<0.01;Val:rs=0.508,P=0.016);His、Phe和Tyr在不同肝纤维化病理分期中的浓度均与正常组有统计学差异,但在不同病理分期间无统计学差异。结论血浆中His、Phe和Tyr浓度与肝纤维化程度正相关,但对肝纤维化病理分期无判别意义。     

葛根素对支气管上皮细胞和中性粒细胞共培养体系中黏附分子表达的影响

目的 探讨葛根素抑制支气管上皮细胞(BEAS-2B)与中性粒细胞(NEU)的相互作用进而调控气道炎症反应的机制.方法 实验分为BEAS-2B单独培养组、NEU单独培养组、BEAS-2B+NEU共培养组、BEAS-2B+NEU+50μg/ml葛根素共培养组、BEAS-2B+NEU+100μg/ml葛根素共培养组、BEAS-2B+NEU+200μg/ml葛根素共培养组,应用流式细胞术检测BEAS-2B和NEU两种细胞中胞间黏附分子ICAM-1、ICAM-3蛋白的表达,Real-time PCR检测两种细胞中ICAM-1、ICAM-3 mRNA的表达.结果 与单独培养组相比,BEAS-2B+NEU共培养组中BEAS-2B细胞ICAM-1、ICAM-3及NEU细胞ICAM-1蛋白和mRNA表达均明显增强(P＜0.05,P＜0.01),而NEU细胞ICAM-3蛋白和mRNA表达无明显改变(P＞0.05).在BEAS-2B+NEU共培养体系中分别加入50、100、200μg/ml葛根素干预后,流式细胞术检测显示仅葛根素浓度为200μg/ml时BEAS-2B细胞ICAM-1蛋白表达显著低于BEAS-2B+NEU共培养组(P＜0.05),余与BEAS-2B+NEU共培养组比较差异均无统计学意义,而Real-time PCR检测显示,除NEU细胞的ICAM-3 mRNA与BEAS-2B+NEU共培养组比较差异无统计学意义外,余均显著低于BEAS-2B+NEU共培养组(P＜0.05,P＜0.01).结论 葛根素可有效抑制BEAS-2B与NEU共同培养诱导的BEAS-2B及NEU细胞ICAM-1、ICAM-3基因和蛋白的表达.     

逆转录—聚合酶链反应—酶联夹心杂交法定量检测IL—6mRNA

目的：建立一支高灵敏度、高特异性、精确、简便的逆转录－聚合酶链反应－微孔板酶联夹心杂交技术定量检测人IL－6mRNA的方法。方法：从人外周静脉血单个核细胞提取总RNA，进行RT－PCR扩增，PCR产物经热变性后与检测探针一起加入已包被捕获探针的微孔板内进行夹心杂交，用链亲和素－辣根过氧化物酶（SAV－HRP）及底物系统检测杂交信号。结果：该方法检测IL－6mRNA的灵敏度明显高于逆转录－聚合酶链反应－琼脂糖凝胶电泳；特异性高，RT－PCR和酶联夹心杂交均无非特异性阳性信号出现；重复性良好。结论：本方法操作简单、灵敏度高、特异性强、结果数据化，适合于细胞因子mRNA的定量检测。     

应用多重连接依赖式探针扩增技术快速高通量诊断胎儿染色体非整倍体异常

目的 评价多重连接依赖式探针扩增技术(multiplex ligation-dependent probe amplification,MLPA)在染色体非整倍体诊断中的应用价值,为我国羊水染色体诊断提供一种快速、特异、高通量的分子诊断手段.方法 应用MLPA技术检测了500份羊水标本,所有标本均进行荧光原位杂交(fluorescence in situ hybridization,FISH)技术检测和常规染色体核型分析,应用RH-MLPA-v511数据分析软件获得MLPA结果,比较MLPA技术与FISH和染色体核型分析结果的准确性,总结MLPA技术临床应用过程中的关键要点.结果 在500份羊水标本中,MLPA检测成功率97%.3个工作日完成结果的为92%,需重复检测的为5%,失败为3%.对染色体非整倍体异常检测敏感性和准确性100%.证实38例非整倍体病例探针信号比值＞正常二倍体4s,2例疑似三体结果＞2s.分析了21号染色体8条探针的杂交效率,21三体患者8条探针中平均4条探针比值＞1.3.结论 MLPA技术具有快速、特异、敏感、高通量、成本低等特点,可用于产前染色体非整倍体数目的快速检测,是传统染色体培养方法的补充,临床应用价值较高.

Abstract：

Objective To assess the diagnostic value of multiplex ligation-dependent probe amplification (MLPA) for detection of common chromosome aneuploidy in amniotic fluid (AF) cells in order to obtain an accurate, rapid, cost-effective and high-throughput method in routine prenatal clinical practice.Methods The MLPA test was performed on 500 AF samples by using kit P095 and the results were obtained by using analysis software RH-MLPA-v511. The results were compared with that from fluorescence in situ hybridization (FISH) and traditional karyotyping (TK). The technical critical issues were analyzed in routine diagnostic application. Results The absolute specificity and sensitivity of the MLPA test to detect the aneuploidy were 100%. For the 500 AF samples, the success rate of the MLPA tests was 97%. Among them 92% were finished within three working days and 5% required more days for repeating. The test failure rate was 3%. The results confirmed that for the 38 detectable aneuploid samples,the probe reliability weighted mean ratio values were more than 4SD compared to normal diploids and the 2 suspected trisomy samples were more than 2SD. In this study, authors analyzed hybridization efficiencies of 8 probes for chromosome 21, and the presence of a trisomy was considered if at least 4 of the 8 probes gave probe ratio of ＞1.3. Conclusion The data suggested that MLPA is a rapid, simple and reliable method for large scale testing for aneuploidy of chromosomes 13, 18, 21, X, or Y in AF. The MLPA technology is complementary to AF culture and valuable for prenatal diagnosis.     

细胞间黏附分子-1基因K469E多态性与类风湿性关节炎的关系

目的 探讨细胞间黏附分子-1(intercelluhr adhesion molecul-1,ICAM-1)基因K469E多态性与类风湿性关节炎(rheumatoid arthritis,RA)的关系.方法 对275例类风湿性关节炎患者和254名体检健康者作为对照组进行研究.采用聚合酶链反应-限制性片段长度多态性方法分析ICAM-1基因K469E的多态性.结果 RA组K469E位点KK、KE和EE基因型频率为0.535、0.411和0.054;健康对照组K469E位点KK、KE和EE基因型频率为0.512、0.437和0.051.RA组K469E基因型频率与健康对照组相比差异无统计学意义(x2=0.371,P=0.831).RA组K等位基因频率(0.74)与健康对照组(0.73)相比差异无统计学意义(x2=0.127,P=0.721,OR=1.051,95%CI为0.800～1.381),在RA组中KK+KE基因型频率与对照组相比,差异无统计学意义(P=0.863,OR=0.935,95%CI为0.436～2.005).结论 ICAM-1基因K469E多态性分布与RA的易感性无明显相关性.

Abstract：

Objective To investigate the association of the intercellular adhesion molecule-1 gene (ICAM-1)K469E polymorphism and rheumatoid arthritis (RA). Methods Two hundred and seventy five patients with RA and 254 healthy individuals were collected and enrolled in the study. The K469E polymorphism of ICAM-1 gene was analyzed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Results The genotype frequencies of KK, KE and EE of K469E polymorphism were 0. 535,0.411 and 0. 054 respectively in the RA patients, and 0. 512,0. 437 and 0. 051 respectively in the healthy individuals, and there was no significant difference between the two groups (x2 =0. 371,P=0. 831). The frequencies of the K469 allele were 0. 74 and 0. 73 in the RA patients and the controls respectively (x2 = 0. 127, P = 0. 721, OR = 1.051,95 % CI: 0. 800-1. 381 ). No significant difference was observed in KK+KE genotype frequencies between the two groups (P=0. 863), with an odds ratio of 0. 935 (95% CI: 0. 436-2.005). Conclusion The K469E polymorphism of the ICAM-1 gene was not associated with the susceptibility of rheumatoid arthritis.     

度洛西汀与艾司西酞普兰治疗精神分裂症后抑郁的对照研究

目的探讨度洛西汀与艾司西酞普兰治疗精神分裂症后抑郁的疗效及安全性。方法将68例精神分裂症后抑郁患者随机分为度洛西汀组(34例)和艾司西酞普兰组(34例),治疗6周。分别于治疗前及治疗1、2、4、6周末用汉密尔顿抑郁量表(HAMD)、简明精神病量表(BPRS)、临床疗效总评量表(CGI-SI)及治疗中出现的症状量表(TESS)评定疗效及不良反应。于基线及6周末用健康状况问卷(SF-36)评估生活质量。结果 6周末,度洛西汀组和艾司西酞普兰组治疗有效率分别为78.13%和81.82%,差异无统计学意义(χ2=0.138,P0.05)。两组治疗后各时点HAMD、CGI-SI、BPRS评分与基线相比差异均有统计学意义(P均0.01);而各评定指标评分组间同期比较差异无统计学意义。6周末,度洛西汀组和艾司西酞普兰组SF-36总分与基线相比差异均具有统计学意义(χ2=3.167,3.203;P均0.01)。两组不良反应发生率低,程度均较轻。结论度洛西汀和艾司西酞普兰对精神分裂症后抑郁患者同样安全有效,且有助于提高患者的生活质量。     

树突状细胞来源的趋化因子的免疫调节作用研究进展

树突状细胞是一类重要的免疫调控细胞，它所产生的趋化因子有重要的免疫调节作用；趋化吸引免疫细胞参与Th1或Th2类免疫反应，促进免疫细胞的成熟；介导其他细胞与树突状细胞的交互反应以及优化炎症反应，使其向有利于机体的方向发展等。树突状细胞可能还有更多的免疫学作用由趋化因子实现，有待进行深入的研究。     

线粒体基因组np16181～16193区基因变异与2型糖尿病的关联研究

目的 探讨线粒体基因组np16181～16193区基因变异与2型糖尿病(type 2 diabetes mellitus,T2DM)之间的关联.方法 随机收集199例浙江地区2型糖尿病患者和205名正常对照,采用聚合酶链反应、基因直接测序检测该区域基因,同时分析变异与2型糖尿病及主要临床指标间的关联.结果 发现np16181～16193区域为高度变异区,存在多种变异形式,考虑为基因多态性位点;在该区域发现有4种变异碱基排列形式仅存在于T2DM中;在np16181～16193区域中变异组餐后1 h血糖明显高于未变异组(P<0.05),而其余各生化指标差异无统计学意义(P>0.05).结论 线粒体基因组np16181～16193区域引起的变异尚不能视为2型糖尿病的易感因素.     

基于创新型医学检验人才培养的医学遗传学教学改革探索

培养创新型医学检验人才是为了适应21世纪社会对医学人才的需求。笔者在医学遗传学的教学中融入对学生科学精神、创新性思维品质和创新能力等方面的培养,取得了显著的成绩,提高了创新型医学检验人才的培养质量。