应用富含GC区SNP分型的序列特异性PCR新方法检测子宫内膜癌特定位点C729T多态性

目的建立适合富含GC区SNP分型的序列特异性PCR方法,分析子宫内膜癌特定ERαExon3位点C729T多态性基因分型。方法选择22例子宫内膜癌患者与42例良性病患者为研究对象。建立富含GC区序列特异性PCR(SS-PCR)。将两对引物分别进行PCR扩增,所设计的两条特异性引物即内引物分别与DNA双链的两条单链相同,其3′端正好与SNP位点重合,由引物的3′端控制着引物的延伸反应,根据延伸产物的条带确定SNP类型;并通过在引物的3′端区域倒数第3位引入一个人为不匹配碱基来提高延伸反应的特异性。应用该方法鉴别Exon3 SNP位点C729T基因型,并通过直接测序法验证。结果建立的序列特异性PCR方法适合富含GC区SNP分型,能够准确分析子宫内膜癌的特定ERαExon3位点C729T多态性基因分型。结论富含GC区SNP分型的序列特异性PCR方法,可以用于子宫内膜癌的特定ERαExon3位点C729T多态性基因分型。     

宁夏回族高脂血症患者CYP2C9*3、MSA2756G基因多态性的研究

目的 研究高脂血症治疗的药物相关基因CYP2C9*3、甲硫氨酸合成酶(MSA2756G)在宁夏回族高脂血症患者中的分布及其与高脂血症的关系.方法 通过扩增引进限制性酶切位点(ACRS)和应用聚合酶链式反应-限制性片段长度多态性(PCR-RFLP)技术对高脂血症患者进行基因型分析.结果 180名宁夏回族高脂血症患者中,CYP2C9*3等位基因频率为3.33%,突变频率男性(3.05%)高于女性(0.28%),差异有显著性(P<0.01),而与健康对照组(3.00%)比较,差异无显著性(P>0.05);MSA2756G等位基因频率为15.83%,显著高于健康对照组(10.25%)(P<0.05),未发现性别差异.结论 宁夏回族人群携带CYP2C9*3基因型的男性患高脂血症风险更大;MSA2756G可能是高脂血症的危险因子.     

宁夏回族高血压患者MSA2756G、CYP2C9*3多态性的研究

目的观察高血压患者致病基因MSA2756G和药物代谢酶相关基因CYP2C9*3多态性位点在宁夏回族高血压患者中的分布及其与高血压的关系。方法通过扩增引进限制性酶切位点(ACRS)和聚合酶链式反应-限制性片段长度多态性(PCR-RFLP)技术对高血压患者进行基因型分析,利用χ2和t检验分析以上两个多态性位点的各基因型与宁夏回族高血压的相关性。结果①宁夏回族人群MSA2756G位点等位基因G在对照组中的频率为10.25%,而在高血压组中的频率为14.04%,两组等位基因G的频率分布差异无统计学意义(P>0.05);在男性中,等位基因G在高血压组(8.79%)与对照组(11.50%)中的分布差异无统计学意义(P>0.05);在女性中,等位基因G在高血压组(19.54%)与对照组(9.00%)中的分布差异有统计学意义(P<0.05);在高血压组,等位基因G在男性、女性中的频率各为8.79%、19.54%,差异有统计学意义(P<0.05)。②宁夏回族人群CYP2C9*3位点等位基因C在对照组中的频率为3.00%,而在高血压组中的频率为3.37%,两组等位基因C的频率分布差异无统计学意义(P>0.05);在男性中,等位基因C在高血压组(4.40%)与对照组(3.50%)中的分布差异无统计学意义(P>0.05);在女性中,等位基因C在高血压组(2.30%)与对照组(2.50%)中的分布差异无统计学意义(P>0.05)。结论 MSA2756G等位基因G是宁夏回族女性患高血压的危险因子,而与男性无关。     

内脂素基因多态性与2型糖尿病的关系

目的探讨内脂素(visfatin)rs11977021、rs12537455、rs2110385位点单核苷酸多态性与2型糖尿病易感性的关系。方法收集陕西西安地区汉族人群192例受试对象,其中106例单纯2型糖尿病(T2DM)患者和86例健康对照者,采用标准化的聚合酶链反应-限制性片段长度多态性(PCR-RLFP)方法,分别检测内脂素(visfatin)rs11977021/DdeⅠ、rs12537455/AluⅠ和rs2110385/AluⅠ基因型并进行比较。结果 rs2110385位点的T等位基因频率和GT基因型频率在T2DM组均高于健康对照组(分别为28.3、14.0和14.2、7.0,P均<0.05);两组的rs11977021位点和rs12537455位点C、T等位基因及基因型频率分布均无统计学差异(P>0.05);单倍型分析显示T-C-T和T-T-T单倍型在T2DM组均显著高于健康对照组(分别为0.066、0.026和0.029、0.012,P均<0.05)。结论 Visfatin基因rs2110385位点在陕西西安汉族地区人群中存在遗传变异,其rs2110385多态性位点的T等位基因可能与T2DM的发病风险有一定的关系。     

陕西人群环氧化酶-2(COX-2)启动子区基因多态性及与结直肠癌易感性的关联研究

目的探讨陕西人群环氧化酶-2(COX-2)基因启动子区的rs20417G/C及rs2745557G/A 2个位点的基因多态性并分析其与结直肠癌(CRC)发病风险及病理参数的相关性。方法采用病例-对照研究,利用聚合酶链式反应和限制性片段长度多态性(PCR-RFLP)技术,对198例CRC患者和200例健康人(入组者均长期居住在陕西省西安市及周边县、市)的COX-2基因的2个位点的多态性进行检测,并运用SPSS 19.0软件统计分析各位点的基因型分布和等位基因频率。分析其与CRC发病风险及病理参数的相关性。结果在陕西人群中,COX-2rs20417G/C位点多态性的GC或CC基因型频率与对照组的差异无统计学意义,而GC+CC基因型频率在病例组的频率显著高于对照组(OR=1.61;95%CI:1.02².56)。病例组含有C等位基因的GC或CC基因型的淋巴结转移以及肿瘤分期与GG基因型比较,差异有统计学意义(P<0.01及P<0.01);两个等位基因频率分布均与发病部位无显著相关性。而COX-2rs2745557G/A位点多态性结果显示:GA基因型在病例组的频率较对照组显著增高(OR=1.98,95%CI:1.152.56)。病例组含有C等位基因的GC或CC基因型的淋巴结转移以及肿瘤分期与GG基因型比较,差异有统计学意义(P<0.01及P<0.01);两个等位基因频率分布均与发病部位无显著相关性。而COX-2rs2745557G/A位点多态性结果显示:GA基因型在病例组的频率较对照组显著增高(OR=1.98,95%CI:1.15³.40),AA基因型在病例组中的频率亦较对照组高(OR=1.87,95%CI:1.093.40),AA基因型在病例组中的频率亦较对照组高(OR=1.87,95%CI:1.09³.19),A等位基因携带者在病例组中的频率高于对照组(OR=1.92,95%CI:1.183.19),A等位基因携带者在病例组中的频率高于对照组(OR=1.92,95%CI:1.18³.13)。含有A等位基因的GA或AA基因型相对于GG基因型,淋巴结转移以及肿瘤分期差异均有统计学意义(P均<0.01);两个等位基因频率分布均与发病部位无显著相关性。结论相同环境条件下,陕西人群中携带COX-2rs20417C等位基因型的个体和rs2745557A等位基因型的个体患CRC的风险增加,rs20417C等位基因频率、rs2745557的A等位基因频率与CRC发生发展及淋巴结转移密切相关。两个基因位点的各等位基因频率与CRC的发生部位均无明显相关性。     

PPARγ2基因Pro12Ala多态性与2型糖尿病一级亲属的相关性

目的探讨PPARγ2基因Pro12Ala多态性与2型糖尿病(T2DM)一级亲属的相关性。方法将研究对象按WHO的诊断与分型标准分为:①T2DM组,79人,为T2DM患者且其家族中有血缘关系的T2DM患者≥2人;②正常糖耐量一级亲属(NFDR)组,84人,为T2DM的一级亲属中糖耐量正常者;③无家族史的正常糖耐量(NC)组,104人,为与T2DM无血缘关系,且经口服糖耐量试验排除T2DM和糖耐量异常者。每组按体重指数(BMI)再分为2个小组,即BMI≥25kg/m2小组和BMI<25kg/m2小组。应用聚合酶链反应—限制性内切酶片段长度多态性(PCR-RFLP)方法检测PPARγ2基因Pro12Ala多态性,比较各组基因型频率及等位基因频率。结果T2DM组、NFDR组、NC组基因型频率、等位基因频率之间无显著性差异(P>0.05)。T2DM组、NFDR组中BMI≥25kg/m2小组和BMI<25kg/m2小组基因型频率、等位基因频率之间亦无显著性差异(P>0.05);NC组中BMI≥25kg/m2小组和BMI<25kg/m2小组基因型频率无显著性差异(P>0.05),而等位基因频率有显著性差异(P<0.05)。结论PPARγ2基因Pro12Ala多态性与T2DM一级亲属无明显关联而与汉族无T2DM家族史的正常人群肥胖有关。     

GSTM1基因多态性及启动子甲基化在结肠癌细胞系中的研究

目的研究参与内毒物代谢的重要酶谷胱甘肽-S-转移酶M1(glutathione-s-transferase M1,GSTM1)在结肠癌细胞系中的基因表达状况与其遗传基因型及启动子甲基化的关系。方法采用多重PCR方法检测GSTM1基因型,甲基化特异PCR(MSP)方法检测GSTM1基因启动子甲基化状态;用5-aza-dC处理结肠癌细胞系,RT-PCR方法检测GSTM1基因转录水平。结果结肠癌细胞系HT29、SW480、SW1116为GSTM1非空白基因型,而HCT116、Lovo、Colo结肠癌细胞系为GSTM1空白基因型。MSP检测发现SW1116细胞中GSTM1基因主要呈非甲基化状态,其他5个细胞系中呈甲基化状态,而且基因表达缺失。经5-aza-dC处理后,HT29和SW480细胞的GSTM1基因表达复活,而HCT116、Lovo、Colo细胞中GSTM1仍无扩增。结论 GSTM1基因转录受到遗传基因型和表观遗传启动子甲基化双重调控。     

β2肾上腺素能受体基因多态性与维吾尔族肥胖的关系

目的探讨β2肾上腺素能受体(β2-adrenergic receptor,β2-AR or ADRB2)基因R16G、Q27E多态性与新疆维吾尔族人群肥胖发生的相关性。方法采用Taqman技术对新疆754例维吾尔族受试者进行β2-AR基因R16G、Q27E位点基因分型,并分析各基因型和等位基因频率在不同体重指数(BMI)水平的分布及其与超重、肥胖的关系。结果维吾尔族人群中超重组与肥胖组的血压、甘油三酯及胆固醇水平显著高于正常体重组(P<0.05)。R16G、Q27E两个多态位点的基因型频率与等位基因频率在正常体重组、超重组与肥胖组之间的分布均无统计学差异(P>0.05);按性别分层后基因型与等位基因频率的分布在3组间亦未见统计学差异。R16G、Q27E多态构成的4种单倍型在正常体重组、超重组与肥胖组的分布无统计学差异(P>0.05)。维吾尔族与汉族人群间比较,Q27E基因型与等位基因频率分布的差异有统计学意义(P<0.001)。结论β2-AR基因R16G、Q27E多态性与新疆维吾尔族人群肥胖发生无关,但Q27E基因型与等位基因频率分布在汉族与维吾尔族人群间不同。     

脂联素基因启动子区多态性与肥胖、2型糖尿病的相关性

目的探讨中国人群内脂联素基因启动子区-11391G/A、-11377C/G基因多态性以及突变与肥胖、2型糖尿病(T2DM)的相关性。方法选取研究对象共305名,利用聚合酶链反应-限制性内切酶片段长度多态性(PCR-RFLP)方法鉴定-11391G/A、-11377C/G基因型。结果①在所选群体中未发现-11391G/A突变;②以基因型分组:-11377 GG基因型体重指数(BMI)值明显高于CC,CG基因型,且具有统计学意义(P<0.05);GG基因型舒张压、收缩压明显高于CC,CG基因型,且与CG基因型差别有统计学意义(P<0.05);CC基因型高密度脂蛋白(HDL)最高且与CG基因型差别有统计学意义(P<0.05)。结论在西安人群内未发现-11391G/A突变;-11377C/G基因多态性与BMI的相关性具有统计学意义;未发现11377C/G基因多态性与2型糖尿病的相关性。     

PPARγ_2基因Pro12Ala多态性与2型糖尿病及其一级亲属血脂的相关性研究

目的探讨过氧化物酶体增殖物激活受体(PPAR)γ2基因Pro12Ala多态性与2型糖尿病(T2DM)及其一级亲属血脂的相关性。方法将研究对象按WHO 1999年T2DM的诊断与分型标准分为T2DM组(156例,为T2DM患者且其家族中有血缘关系的T2DM患者≥2例)、T2DM一级亲属中正常糖耐量组即NFDR组(168例,为T2DM的一级亲属中糖耐量正常者)、无糖尿病家族史的正常糖耐量组即NC组(150例,与T2DM无血缘关系,且经口服糖耐量检测排除T2DM和糖耐量异常IGT者)。每组按体重指数(BMI)再分为肥胖组(BMI≥25kg/m2)和非肥胖组(BMI<25kg/m2)。应用聚合酶链反应-限制性内切酶片段长度多态性(PCR-RFLP)方法检测PPARγ2基因Pro12Ala多态性,比较不同基因型患者的血脂水平。结果 T2DM组患者BMI显著高于NC组(P<0.05);NC组、NFDR组、T2DM组甘油三酯(TG)依次增高,3者之间差异有统计学意义(P<0.05);胆固醇(TC)依次增高,但3者之间差异无统计学意义(P>0.05);T2DM组高密度脂蛋白胆固醇(HLD)显著低于NC组,差异有统计学意义(P<0.05);3组的低密度脂蛋白胆固醇(LDL)差异无统计学意义(P>0.05)。T2DM组PA/AA基因型患者较PP基因型患者的TG、TC、LDL高(P<0.05),其中肥胖组PA/AA基因型较PP基因型患者的TG、TC、LDL高,差异有统计学意义(P<0.05),而在非肥胖组中的差异均无统计学意义(P>0.05)。NFDR组PA/AA基因型较PP基因型中TG、TC、LDL有增高趋势,但差异无统计学意义(P>0.05),其中肥胖组中PA/AA基因型较PP基因型中TG、TC、LDL高,差异有统计学意义(P<0.05),而在非肥胖组中的差异均无统计学意义(P>0.05)。NC组PA/AA基因型与PP基因型患者的TC、TG、HDL、LDL水平差异无统计学意义(P>0.05),在NC肥胖组及NC非肥胖组中两种基因型患者的各血脂水平差异无统计学意义(P>0.05)。结论 PPARγ2基因Pro12Ala多态性与西北地区汉族T2DM及其一级亲属血脂相关,以肥胖者明显,但可能对血脂的影响甚微。     

Simultaneous determination of berberine, matrine and oxymatrine in traditional Chinese medicines by using nonaqueous capillary electrophoresis

A rapid method for the simultaneous determination of berberine (BBR), matrine (MT) and oxymatrine (OMT) by nonaqueous capillary electrophoresis (NACE) was developed. Optimum separation of the analytes was obtained on a 50cm×50μm i.d. fused-silica capillary using a non-aqueous buffer system of 70mM ammonium acetate, 7.0% acetic acid and 10% acetonitrile at 25kV and 20℃. The relative standard deviations (R.S.D.) of the migration times and peak areas of the three active components were 0.06%-0.20% and 0.12%-3.41% for berberine, 0.11%-0.60% and 0.74%-1.63% for matrine, 0.15% and 0.45% for oxymatrine, respectively. Detection limits of berberine, matrine and oxymtrine were 0.18μg/mL, 4.08μg/mL and 4.16μg/mL, respectively. In the tested concentration range, good linear relationships (0.9992 for berberine, 0.9988 for matrine and 0.9988 for oxymatrine) were observed. The linear calibration ranges were 0.45-360.0μg/mL for berberine, 8.16-408.0μg/mL for matrine and 20.8-416.0μg/mL for oxymatrine. This method has been successfully applied to the phytochemical analysis of alkaloids extracts from two commonly used traditional Chinese herbal drugs: Sophora flavescens Ait. (Kushen) and Cortex phellodendri chinensis (Huangbai) and their medicinal preparations.     

Electrogenerated chemiluminescence sensor for the determination of metoclopramide using ordered mesoporous carbon for immobilizing tris(2, 2'-bipyridyl) ruthenium

A novel electrogenerated chemiluminescence (ECL) sensor for the determination of metoclopramide was developed by employing ruthenium complex as an ECL signal producer and an ordered mesoporous carbon (OMC) material as modified material. The ECL sensor was fabricated by adsorption ruthenium complex into a mixture of OMC and Nafion, which showed good electrochemical and ECL behaviors. It was found that the ECL intensity of the sensor fabricated was greatly enhanced in the presence of metoclopramide. Based on this finding, a highly sensitive and reproducible ECL method was developed for the determination of metoclopramide. The result showed that the ECL intensity was linear with the concentration of metoclopramide in the range from 1.0×10-10 to 5.0×10-7M and the detection limit was 3×10-11M. The ECL sensor exhibited a long-term stability and a fine reproducibility with relative standard deviation of 1.0 % for 1.0×10-10M metoclopramide in 18 continuous determinations. The developed method has been applied to the determination of metoclopramide in tablet samples with satisfactory results.     

Ghrelin基因Arg51Gln多态性与西安汉族2型糖尿病、高血压病的相关性

目的 研究ghrelin基因Arg51Gln多态性在中国西安汉族人群的分布情况,并探讨该多态性与2型糖尿病、高血压病发病的相关性.方法 应用PCR-限制性片段长度多态性法分析中国西安地区汉族人305例(正常对照组80例;2型糖尿病组125例;高血压病组48例;2型糖尿病合并高血压病组52例)Ghrelin基因Arg51Gln多态性的分布及其与临床指标间的相关性.结果 2型糖尿病组、高血压病组、2型糖尿病合并高血压病组的基因型频率分别与正常对照组相比,均无统计学意义(P值分别为:0.652、0.201、0.212);等位基因分布分别与正常对照比较无统计学意义(P值分别为:1.002、0.199、0.124);2型糖尿病组Gln51等位基因携带者的BMI、FINS、HOMA-IR均高于野生型(P值分别为:0.045、0.003、0.041).结论 该基因多态性与2型糖尿病胰岛素抵抗相关,Ghrelin基因Arg51Gln多态性与高血压病无相关性.     

A rapid method for the determination of dopamine in porcine muscle by pre-column derivatization and HPLC with fluorescence detection

A rapid method has been developed based on the sample preparation procedure named as QuEChERS （Quick, Easy, Cheap, Effective, Rugged and Safe）, combined with reversed-phase high performance liquid chromatography with fluorescence detector and C18 column after precolumn derivatization using o-phthalaldehyde and 2-mercaptoethanol to determine dopamine in porcine muscle. Methanol and deionized water （0.1% acetic acid, v/v） with a ratio of 60：40 was used as mobile phase. The flow rate was 0.8 mL/min and dopamine was eluted within 15 min. The linearity range was 0.003-8 μg/mL with r=0.9992. The detection limit for dopamine was 4 μg/kg and the quantification limit was 9 μg/kg. Recovery studies were carried out at 0.1, 0.5 and 1.0 mg/kg fortification levels and the average recoveries obtained ranged from 90.4% to 98.2% with relative standard deviations between 3.5% and 8.1%. The method was found to be suitable for detection of dopamine in animal product tissues at the maximum residue level.     

中国西北地区汉族人群5-羟色胺2A受体启动区多态性与心境障碍发病、性别、症状、自杀的相关性

目的探讨中国西北地区汉族人群5-羟色胺2A受体(-1438A/G)基因多态性与心境障碍的发病、性别、亚型以及自杀相关是否关联。方法应用聚合酶链反应(PCR)扩增技术测定160例患者(包括单相抑郁症和双相障碍-抑郁相)和160例正常对照的5-HTR2A的基因型和等位基因,分别验证各种基因型与心境障碍的性别、亚型、自杀的相关性。结果病例组的A/G、G/G基因型和G等位基因频数均高于正常对照组(47.5%vs.40.6%;38.7%vs.34.4%;62.5%vs.54.7%;均P<0.05),两组性别分层比较,女性组与男性病例组相比差异无统计学意义(P>0.05)。单相抑郁症与双相障碍-抑郁相两组间进行比较差异无统计学意义(P>0.05)。病例组有无自杀相关分层比较差异无统计学意义(P>0.05)。自杀相关性别分层比较差异无统计学意义(P>0.05)。结论中国西北地区汉族人群5-HTR2A(-1438A/G)基因多态性与心境障碍的发病相关,主要是与单相抑郁症相关;A/G、G/G基因型可能是心境障碍的易感基因型,G等位基因可能是心境障碍的易感基因。     

Development and validation of a RP-HPLC method for the simultaneous determination of Embelin, Rottlerin and EUagic acid in Vidangadi churna

Vidangadi churna is a popular Ayurvedic formulation described in the chapter Krimicikitsa of the Ayurvedic literature Cakradatta for the treatment of Krimiroga. The preparation is a composite mixture o...     

RP-HPLC method for the simultaneous determination of daidzein,genistein and formonetin in Solanum Lyratum Thunb

A rapid method for the simultaneous determination of daidzein, genistein and formonetin in solanum Lyratum Thunb by high performance liquid chromatography (HPLC) was developed. Separation was achieved on a Diamonsil C18 column (250 mm×4.6 mm, 5 μm) with isocratic elution, using a mobile phase of methanol-tetrahydrofuran-water (44∶3∶53, v/v). The wavelength was set at 260 nm and column was maintained at 35 ℃. The linear ranges of daidzein, genistein and formonetin were 1.0-40.0, 0.1-4.0 and 0.1-4.0 μg/mL, respectively. The average recoveries were between 98.4% and 101.3%. This method could be used for the quality control of Solanum lyratum Thunb due to its simplification, reliability, rapidity and excellent precision.     

Identification and determination of the major constituents in traditional Chinese medicine Longdan Xiegan Pill by HPLC-D

A novel and sensitive HPLC-UV method has been developed for the simultaneous determination of twelve major compounds in Longdan Xiegan Pill.The chemical profile of the twelve compounds,including geniposidic acid（1）,geniposide（2）,gentiopicroside（3）,liquiri     

中国新疆哈萨克族人群CSF1PO、TH01和TPOX三个STR位点的遗传多态性

目的　获得CSF1P0、TPOX和TH0 1三个短串联重复序列 (shorttandemrepeat ,STR)在新疆哈萨克族人群中等位基因频率、基因型频率及相关法医学数据。方法　应用PCR技术、4 0 g·L-1(4% )变性聚丙烯酰胺凝胶电泳及银染技术对上述三个STR位点分型。结果　新疆哈萨克族人群CSF1P0位点有 8个等位片段 ,TPOX位点有 8个等位片段 ,TH0 1位点有 7个等位片段 ;3个位点的基因型分布均符合Hardy Weinberg平衡 ;各位点杂合度分别为 0 .875 3、0 .8777、0 .932 1,多态信息量分别为 0 .74 0 1、0 .75 6 8、0 .75 0 9。结论　得到的上述三个位点的基因频率数据可为新疆哈萨克人群遗传学研究和法医学应用提供依据     

LC-ESI-MS/MS, a modified method for simultaneous quantification of isoflavonoids, flavonoids, alkaloids and saponins in a Chinese herbal preparation Gegen-Qinlian decoction

A sensitive and reliable liquid chromatography-electrospray ionisation-tandem mass spectrometry (LC-ESI-MS/MS) method was established to simultaneously quantitate four categories of compounds (isoflavonoids, flavonoids, alkaloids and saponins) in Gegen-Qinlian decoction (GQD). These compounds were separated by a Shiseido CAPCELL PAK C18 column with a linear gradient consisting of 0.1% (v/v) formic acid in water (A) and 0.1% (v/v) formic acid in acetonitrile (B), and delivered at a flow rate of 0.3 mL/min. All the analytes were determined by electrospray positive ionization tandem mass spectrometry in a multiple reaction monitoring (MRM) mode. Linearity, accuracy, precision, recovery and stability of the method were evaluated with the validation over the range of 4.0-538 5 ng/mL. The proposed method was applied to the analysis of a Chinese herbal preparation GQD successfully.