PCR-RFLP法检测肿瘤坏死因子受体基因多态性

目的：建立聚合酶链反应－限制性片段长度多态性分析（PCR－RFLP）检测肿瘤坏死因子受体Ⅱ（TNF R2）第6外显子基因多态性的方法。方法：用碘化钾法从外周血白细胞中提取基因组DNA，PCR扩增其TNF R2包括编码196位氨基酸残基在内的第6外显子基因，扩增产物用限制性内切酶NIa Ⅲ酶切后干含溴化乙锭的35g/L琼脂糖凝胶中电泳，紫外灯下直接观察TNF R2的基因型。结果：通过观察电泳条带可确定TNF R2的三种基因型，196R／R纯合子、196M／M纯合子、196M／R杂合子分别出现一、二、三条条带；江苏地区健康汉族人TNF R2第6外显子各基因型的频率为：196M／M61．6％，196R／R5．6％，196M／R32．8％；等位基因的频率为196M77．9％，196R22．1％。结论：RCR－RFLP是一种高效敏感、简单快速、准确可靠的分析TNFR2基因多态性的方法，适于普通实验室开展。     

肿瘤坏死因子β基因多态性与脑梗死的相关性研究

目的 探讨脑梗死(CI)患者肿瘤坏死因子β的基因型和等位基因频率分布特点.探讨肿瘤坏死因子β等位基因多态性与CI易感性的关系.方法 采用PCR方法扩增TNFβ第一外显子到第二外显子包含G-A突变在内DNA片段,扩增产物用限制性内切酶Nco I酶切后电泳,分析TNFβ的基因型.对56例脑梗死患者(CI组)和108例健康者(对照组)的肿瘤坏死因子β基因多态性进行分析.结果 CI组TNFβ等位基因分布频率与对照组差异有显著性(P=0.002),其中TNFβ2/2基因型频率(46.4%)明显高于正常人(30.6%),但TNβ*1与TN地Fβ*2等位基因频率差异无显著性(P>0.05).结论 CI患者组TNFβ各基因型频率与健康对照组相比存在显著差异,说明TNFβ*2/2基因型可能与CI发病有关.     

肿瘤坏死因子β基因多态性与江苏子宫内膜异位症患者的相关性研究

目的 探讨江苏子宫内膜异位症(内异症)患者肿瘤坏死因子β的基因型和等位基因频率分布特点.探讨肿瘤坏死因子β等位基因多态性与内异症易感性的关系.方法 采用PCR方法扩增TNFβ第一外显子到第二外显子包含G→A突变在内DNA片段,扩增产物用限制性内切酶Nco I酶切后电泳,分析TNFβ的基因型.对52例子宫内膜异位症患者(内异症组)和43例健康者(对照组)的肿瘤坏死因子β基因多态性进行分析.结果 内异症组TNFβ等住基因分布频率与对照组差异有显著性(P=0.002),其中TNFβ2/2基因型频率(46.4%)明显高于正常人(30.6%),但TNFβ*1与TNFβ*2等位基因频率差异无显著性(P>0.05).结论 内异症患者组TNFβ各基因型频率与健康对照组相比存在显著差异,说明TNFβ*2/2基因型可能与内异症发病有关.     

肿瘤坏死因子β1069位点等位基因多态性与创伤后严重脓毒血症的关系研究

目的 为了探讨江苏汉族人群中肿瘤坏死因子β(TNFβ)等位基因多态性与创伤后严重脓毒血症的关系。方法 采用聚合酶链反应-限制性片段长度多态性(PCR—RFLP)方法对58例创伤后并发严重脓毒血症病人TNFB基因的单碱基突变多态性进行分析。结果 创伤后并发严重脓毒血症病人的TNFβ*2基因频率较正常人明显升高(创伤后严重脓毒血症病人68．9％，正常人55．1％，P=0．015，RR=1．73)。结论 TNFβ*2基因频率与创伤后严重脓毒血症易患性相关。     

肿瘤坏死因子基因多态性与非胰岛素依赖型糖尿病关系的研究

目的　分析中国汉族正常人群和非胰岛素依赖型糖尿病 (NIDDM)患者肿瘤坏死因子 (TNF)基因多态性分布 ,探讨汉族人群TNF基因多态性与NIDDM的关系。方法　应用聚合酶链反应限制性片段长度多态性法 (PCR RFLP)检测 2 15例正常汉族人和 5 5例NIDDM患者TNFα和TNFβ 基因型 ,同时检测血葡萄糖、血脂水平。结果　正常人群和NIDDM人群中TNFβ 基因型均以TNFβ1/ 2型最为多见 ,而TNFα基因型均以TNFα1/ 1型频率最高 ,两组人群均未检测到TNFα2 / 2基因型。 2种多态性基因型分布和等位基因频率在正常和NIDDM人群组间和组内比较均无显著差异 (P >0 .0 5 )。正常对照组和NIDDM组各种临床参数水平在TNFα和TNFβ 基因型间比较均无显著差异 (P >0 .0 5 )。结论　TNF虽然与胰岛素抵抗相关 ,但其基因多态性可能在NIDDM及代谢紊乱的发生发展中不起重要作用     

聚合酶链反应限制性片段长度多态性检测雌激素受体基因型

目的：建立一种简便，实用，准确的人雌激素受体（ER）基因型的检测方法以利于了解基因对骨量变异的调节，方法：采用聚合酶链反应限制性片段长度多态性（PCR－RFLP）来扩增ER基因序列，用限制性内切酶PvuII对223例健康妇女和49例男性的ER基因扩增产物酶切，测定ER基因限制性片段长度多态性（RFLP），结果：汉族妇女ER基因pp,Pp,PP基因型频率分别为0．33，0．47和0．202，男性分别为0．429，0．469和0．102，结论：该方法简便，准确，重复性好，适用于常规实验，ER基因（PvuII)多态性分布有一定的种族差异性。     

重组人肿瘤坏死因子α分泌型真核表达载体的构建及表达

目的构建人肿瘤坏死因子rhTNF—α分泌型真核表达载体,并检测其在中国仓鼠卵巢细胞中的稳定表达。方法以pGEM—T／sig—tumstatin为模板,扩增与TNF带重叠区的Ⅳ型胶原信号肽sig基因片段,以PBV220-TNF为模板,扩增与sig带重叠区的TNF基因片段,以上述2个扩增产物片段为模板,重叠区扩增拼接法（SOEing）扩增得到sig—TNF。sig—TNF片段经酶切后,插入经同样酶切的pIRESneo3质粒,利用克隆PCR、限制性内切酶消化以及序列测定,对获得的sig—TNF基因片段及重组载体进行验证。将重组sig—TNF真核表达载体转染到CHO—K1,并对其表达状况进行检测。结果所获得的sig—TNF片段（558bp）序列与报道的序列完全一致,酶切鉴定结果表明含人肿瘤坏死因子的重组pIRESneo3／sig-TNF表达载体构建成功,转染重组pIRESneo3／sig—TNF的CHO—K1分泌表达了人肿瘤坏死因子rhTNF—α。转染了pIRESneo3／sig—TNF真核表达载体和空载体的CHO—K1和未转染CHO—K1细胞的生长速度没有差别。结论成功构建了重组人肿瘤坏死因子rhTNF—α分泌型真核表达载体,并获得能稳定表达rhTNF—α的CHO—K1,为开展下一步的实验奠定了基础。     

IgA肾病与肿瘤坏死因子β基因多态性的关联性研究

目的：探讨江苏汉族人群肿瘤坏死因子β(tumor necrosis factorβ，TNFβ)1069位点等位基因多态性与IgA肾病的关系。方法：采用聚合酶链反应-限制性片段长度多态性方法对84名健康人和45例IgA肾病患者TNFβ基因的单碱基突变多态性进行了分析。结果：IgA肾病患者的TNFβ*2等位基因频率(67．4％)较正常A．(55．1％)明显升高(P=0．015，RR=1．68)。结论TNFβ*2等位基因频率与IgA肾病发病易患性相关。     

维生素D受体基因型多态性分析方法学探讨

目的探讨聚合酶链反应限制性片段长度多态性(PCR—RFLP)技术检测维生素D受体(vitamin D receptor，VDR)基因多态性的具体方法。方法聚合酶链反应限制性片段长度多态性技术。结果在本院40例骨质疏松患者中检测出了两种VDR基因型，即bb型和Bb型。结论利用PCR—RFLP技术对VDR基因进行分型，操作简单，结果准确，适宜普及。     

哮喘人群中IgE低亲和力受体基因多态性检测

目的：探讨IgE低亲和力受体(FccRⅡ，CD23)基因点多态性与哮喘易感性的关系。方法：聚合酶链反应-限制性片段长度多态性分析(PCR-RFLP)检测外显子9上的G→A碱基突变。结果：本实验未能发现基因外显子9上存在G→A突变，PAGE电泳检测不到限制性片段长度多态性(restriction fragment length polymorphism，RFLP)。结论：所研究人群中FceRⅡ基因不存在(G→A)点多态性。     

基因多态性与2型糖尿病

2型糖尿病(T2D)是一种由遗传和环境因素共同作用的多基因位点疾病。迄今为止,已有多个基因多态性位点被发现与T2D密切相关。最近发现的与T2D密切相关的基因包括胰岛素受体底物1(insulin receptor substrate 1,IRS-1)、蛋白酪氨酸磷酸酶受体类型D(protein tyrosine phosphataes,receptor type,PTPRD)、泛素结合酶E2E2(ubiq-uitin-conjugating enzyme E2E2,UBE2E2)。1 IRS-1IRS-1基因位     

Polymorphism of the ovine calpastatin gene

Calpastatin is a specific inhibitor of calpains and has been implicated in the regulation of beef tenderization. Variation in the ovine calpastatin gene (CAST) was investigated by amplification of a fragment containing the entire exon 6 using polymerase chain reaction (PCR), followed by single-strand conformational polymorphism (PCR-SSCP) analysis and DNA sequencing. Five novel SSCP patterns, representing five different sequences, were identified. Either one or two different sequences were detected in individual sheep and all the sequences identified shared high homology to the published ovine and bovine CAST sequences, suggesting that these sequences represent allelic variants of the ovine CAST gene. Sequence analysis revealed a non-synonymous amino acid variation in exon 6, which would result in a Gln/Leu substitution in Domain L of the mature protein. Considerable variation was detected in an intron region close to the acceptor splice site, with both sequence variation and length variation being observed in this region. Variation detected here might have an impact on both the function and expression of ovine calpastatin.     

Polymorphism of placental phosphoglucomutase in Japanese

Polymorphism of PGM1 and PGM3 types was investigated in placental extracts from 127 unrelated Japanese parturients living in Yamanashi Prefecture. The gene frequencies were 0.787 for PGM1(1), 0.213 for PGM1(2); 0.760 for PGM13, 0.240 for PGM3(2), respectively. There was no significant association between the PGM1 and PGM3 systems. The PGM3(2) frequency in the present sample population was higher than those in Kanto District, but lower than those in Kinki District.     

胱抑素C基因多态性、载脂蛋白E基因多态性与冠心病的相关性探讨

目的 探讨胱抑素C(cys C)基因多态性+148 G/A、载脂蛋白E(apoE) 因多态性与冠心痛的关系.方法 采用聚合酶链反应限制性片段长度多态性(PCR-RFLP)分别检测75例冠心病患者以及40例正常对照组cys C+148 G/A基因型及等位基因频率;等位基因特异性多重聚合酶链反应(PCR-multi-ARMS)检测apoE基因型及等位基因频率,同时结合血脂水平的测定,来探讨它们之间的关系.结果 ①冠心病组apoE4/3基因型及e4等位基因频率明显高于对照组(P<0.05);②E4/3基因型的冠心病患者TC水平明显高于E3/3基因型的冠心病患者(P<0.05); ③cysC+148 G/A基因多态性在冠心病患者反正常对照组的分布差异无统计学意义(P>0.05).结论 裁脂蛋白E,E4/3基因型及e4等位基因频率与冠心病密切相关;cys C+148 G/A基因型及等位基因频率与冠心病无关.     

Polymorphism of theophylline metabolism in man.

To determine whether genetic mechanisms control large interindividual variations in theophylline elimination in normal uninduced human subjects, and, if so, to test the possibility that these genetic factors are transmitted as a simple Mendelian trait, theophylline was administered to 79 unrelated adults, six sets of monozygotic twins, six sets of dizygotic twins, and six two-generation families. Thereafter, in urine collected from each subject at regular intervals for 48 h, concentrations of theophylline and its three principal metabolites were measured and rate constants of formation of these metabolites calculated. The twin study, designed to determine the relative contributions of genetic and environmental factors to large interindividual variation in theophylline elimination, revealed predominantly genetic control. Values for this genetic component, designated heritability (H1(2)), of interindividual variation in rate constants of metabolite formation were 0.61, 0.84, and 0.95 for 3-methylxanthine, 1-methyluric acid, and 1,3-dimethyluric acid, respectively. H1(2) for the overall theophylline elimination rate constant (kel) was lower (0.34). In the 79 unrelated adults, each distribution curve for rate constants of formation of each theophylline metabolite appeared to be trimodal. By contrast, the distribution curve for the overall theophylline elimination rate constant appeared to be either unimodal or bimodal. The extent of interindividual variation was fourfold for theophylline kel and 6-8-fold for the three principal metabolites. High correlations among the three rate constants in individual subjects suggested their regulation by a single shared factor. In six families carefully selected to be under near basal environmental conditions so that hepatic theophylline metabolism of each family member would be neither markedly induced nor inhibited, phenotypes for theophylline metabolite rate constants were assigned. This assignment of phenotype was made by the position of each family member's rate constant on the three distribution curves that were generated from the 79 unrelated subjects. In each family, pedigree analysis of the three phenotypes for each rate constant was consistent with their control by two alleles at a single genetic locus and with autosomal codominant transmission. Frequencies of the two alleles at each genetic locus controlling rate constants of formation of theophylline metabolites were similar (p = 0.49, 0.53, and 0.52). In the three families studied with antipyrine (AP) as well as with theophylline, AP k(el) correlated (r approximately 0.7) with each rate constant of theophylline metabolite formation, as well as with theophylline k(el). While these results are compatible with a common regulatory element in the AP and theophylline polymorphisms, other evidence suggests more than a single genetic polymorphism. This additional evidence includes different gene frequencies for the AP (p approximately 0.1) and theophylline (p approximately 0.5) polymorphisms, different genotype assignments in several families for some theophylline metabolites, different distribution curves for theophylline k(el) from those for the three theophylline metabolites in 79 unrelated subjects, and finally low correlations between AP metabolite rate constants and theophylline metabolite rate constants in the three families receiving both drugs.     

HLA-DRB PCR-单链构象多态性分析

采用聚合酶链技术(PCR)扩增10例HLA-DR血清学定型者的DRB基因,然后作单链构象多态性(SSCP)分析,发现不同基因型的DNA单链区带数及电泳速度不同,提示本方法可用于器官移植时供、受者DRB基因配型及法医犯罪识别。     

PVL阴性MRSA的基因多态性研究

Objective In order to find out the epidemicological spread of MRSA in our hospital,and provide laboratory support for hospital infection control,we investigate the genetic polymorphism of PVL meticillin-resistant Staphylococcus aureus (MRSA) Methods 71 MRSA strains isolated from our hospital from December 2006 to August 2007.The genetic polymorphism a-nalysis of 16 MRSA strains were performed by using pulsed-field gel electrophoresis(PFGE)、staphylococcus cassette chromosome mec (SCCmec) typing.Results PFGE typing:7 genotype( A、B、C、D、G、I、J)were found.They were typeA(1);typeB(1);typeC2(8 subtype);typeD(3);typeG(1);typeI(1)(typeA(1);typeJ(1);SCCmec typing: the main types were SCCmec Ⅱ (50 %);SCCmec Ⅲ (50%).Conclusion By studying the genetic polymorphism of PVL-meticillin-resistant Staphylococcus aureus(MRSA) in our hospital,we get to Know the epidemiological spread of MRSA in our hospital,it provide molecular epidemiological evidence for hospital infection control of MRSA.     

Polymorphism of clinical manifestations of Lyme disease]

The paper describes a clinical picture of a new transmissible disease which got widespread in the Middle zone of Russia. It is caused by a tick-borne organism pertaining to new pathogenic species of Borrelia. Early in its onset Lyme Borrelia (LB) infection presented with tick-borne erythema, followed by neurological and cardiac complications eventuating in locomotor disturbances. Altogether 118 patients at various stages of the disease were examined and treated. Wide-spectrum antibiotics proved an effective therapeutic modality in LB infection management.     

血管紧张素转换酶基因多态性及其与心脑血管病的关系

目的:对近年来关于肾素-血管紧张素系统中血管紧张素转换酶及其多态性的研究现状进行综述。资料来源:应用计算机检索PUBMED1994-01/2006-10期间的相关文章,检索词为“angiotensin I-converting enzymegene”,并限定文章语言种类为English。同时计算机检索万方数据库1994-01/2006-10期间的相关文章,检索词为“血管紧张素转换酶,基因多态性”,并限定文章语言种类为中文。资料选择:对资料进行初审,并查看每篇文献后的引文。纳入标准:文章所述内容应与血管紧张素转换酶基因多态性及同心脑血管病的关系研究相关。排除标准:重复研究或Meta分析类文章。资料提炼:共收集到100篇相关文献,32篇文献符合纳入标准,排除的62篇文献为内容陈旧或重复。符合纳入标准的32篇文献中,3篇涉及肾素-血管紧张素系统的生理作用,10篇涉及血管紧张素转换酶的生物学特征,1篇涉及血管紧张素转换酶基因及其多态性,10篇涉及正常人群血管紧张素转换酶基因I/D多态性,10篇涉及血管紧张素转换酶基因I/D多态性与疾病的相关性。资料综合:肾素-血管紧张素系统是体内重要的体液调节系统,在水盐的代谢平衡和血管张力调节中有重要的作用。血管紧张素转换酶I/D多态性,可能与心脑血管疾病的发生发展相关;且在不同种族不同民族等正常人群中的分布有差异性,说明亦与种族和遗传背景有关;此外,血管紧张素转换酶I/D多态性与心脑血管病相关,在不同种族、不同民族甚至相同种族、相同民族的研究结果是不完全相同的。结论:血管紧张素转换酶是肾素-血管紧张素系统的关键酶,其基因I/D多态性在不同种族、不同民族等正常人群中分布存在差异,与某些心脑血管疾病的易感性,严重性及预后相关。