谷胱苷肽转移酶抑制剂高通量筛选模型的建立和初步应用

谷胱苷肽 - S-转移酶 -π( GST-π)的活性在某些类型的癌症患者中有显著的升高并且在癌症细胞对抗癌药物抗药性的形成过程中起着重要的作用。抑制过量表达的 GST-π酶的活性将有助于克服肿瘤细胞的耐药性 ,因此 ,GST-π被认为是一个潜在的药物作用的靶位点。为了筛选新的抗癌药物的增敏剂 ,我们建立了一个 GST-π酶抑制剂的高通量筛选模型 ,该模型以 CDNB和 GSH为底物 ,在 96 -孔板上通过对 340 nm处吸光度的变化来检测样品对 GST-π酶的抑制活性。经过初筛和复筛 ,从 4 80 0个微生物发酵提取物中筛选到10个阳性样品 ,阳性率为 0 .2 %。     

A case-control study of cytochrome P450 1A1, glutathione S-transferase M1, cigarette smoking and lung cancer susceptibility (Massachusetts, United States)

Cytochrome P450 1A1 (CYP1A1) and glutathione S-transferase M1 (GSTM1)genetic polymorphisms are involved in the activation and detoxification ofchemical carcinogens found in tobacco smoke; thus they may influence hostsusceptibility to lung cancer. In this study at Massachusetts GeneralHospital (Boston, MA, USA) of 416 cases and 446 controls (mostly White) weevaluated the association between the CYP1A1 MspI and GSTM1 polymorphisms andlung cancer risk, and their interaction with cigarette smoke. The CYP1A1 MspIheterozygous genotype was present in 18 percent of cases and 16 percent ofcontrols, and one percent of cases and controls were CYP1A1 MspI homozygousvariant. The GSTM1 null genotype was detected in 54 percent of cases and 52percent of controls. After adjusting for age, gender, pack-years of smoking,and years since quitting smoking, while neither the CYP1A1 MspI heterozygousgenotype alone nor the GSTM1 null genotype alone were associated with asignificant increas e in lung cancer risk, having both genetic traits wasassociated with a twofold increase in risk (95 percent confidence interval[CI] = 1.0-3.4). Our data did not provide enough evidence for a substantialmodification of the effect of pack-years on lung cancer risk by the CYP1A1MspI and GSTM1 genotypes. However, limitations of our study preclude aconclusion about this potential interaction.     

Polymorphisms in the glutathione S-transferase class mu and theta genes interact and increase susceptibility to lung cancer in minority populations (Texas, United States)

The genes coding for separate isoforms of both the human glutathioneS-transferase class mu and class theta enzymes (GSTM1and GSTT1) arepolymorphic with a variable ethnic distribution. These enzymes detoxifyreactive epoxides, including carcinogens produced by tobacco smoke. Becauseof this, the null polymorphism in the GSTM1 gene (coding for the glutathioneS-transferase class mu enzyme) has been studied widely as a possible sourceof inherited susceptibility to smoking-related lung cancer. The more recentlydescribed null polymorphism in the GSTT1 gene also could contribute to anincreased risk of smoking-related lung cancer. As the incidence of lungcancer is known to differ by ethnicity, we have conducted a case-controlstudy in the United States of 108 African-Americans (Blacks) and 60Mexican-Americans (Hispanics) with lung cancer and 132 African-American(Black) and 146 Mexican-American (Hispanic) controls to investigate theassociation of the GSTT1 and GSTM1 polymorphi sms with lung cancer inminority populations. In the unadjusted data, there was a borderlinesignificant association of the GSTM1 null polymorphism with lung cancer inMexican-Americans (odds ratio [OR] = 1.8, 95 percent confidence interval [CI]= 1.0-3.3 ) that was not observed in African-Americans. The GSTT1 nullpolymorphism also had a higher prevalence in cases than controls in bothracial/ethnic groups, but this increase was not statistically significant.When the data were analyzed using logistic regression controlling for age,gender, race, and smoking, no significant association of either trait withlung cancer was observed, with ORs for both traits of approximately 1.3.However, when the prevalence of individuals who were null for bothpolymorphisms was compared by case status, a significant interaction wasobserved. Logistic regression models showed the OR for the association oflung cancer and the presence of both null polymorphisms compared with one(either GSTT1 or GSTM1) or no null genotype to be 2.9 (P < 0.04). Theseresults suggest that there may be carcinogenic intermediates in cigarettesmoke that are substrates for both the GSTT1 and GSTM1 enzymes, and that lungcancer risk is increased more than additively for individuals who have bothGSTT1 and GSTM1 null polymorphisms.     

肺癌核素显像与Pgp、PCNA、GST-π关系的初步研究

目的：探讨肺癌核素显像＾９９ｍＴｃ－ＭＩＢＩ滞留分数与Ｐ糖蛋白（Ｐｇｐ）、增殖细胞核抗原（ＰＣＮＡ）和胎盘型谷胱甘肽－Ｓ－转移酶（ＧＳＴ－π）的关系。方法：１２例肺癌病人术前分别作肺部早期和延迟＾９９ｍＴｃ－ＭＩＢＩ断层显像,计算肿瘤部位＾９９ｍＴｃ－ＭＩＢＩ滞留分数,与手术后肿瘤标本测定的Ｐｇｐ、ＰＣＮＡ、ＧＳＴ－π水平作相关分析。结果：＾９９ｍＴｃ－ＭＩＢＩ滞留分数与Ｐｇｐ明显负相关（ｒ＝－０     

注射用还原型谷胱甘肽质量标准分析方法--HPLC法的建立与验证

目的建立注射用还原型谷胱甘肽含量及有关物质氧化型谷胱甘肽(GSSG)的质量标准分析方法--HPLC法,并对其进行验证.方法采用Waters Nova-Pak(R) C1g色谱柱(3.9 mm×150 mm,4 μm),0.05 mol·L-1磷酸二氢钾的溶液(含0.01 mol·L-1庚烷磺酸钠,用磷酸调节pH至3.0)-甲醇(973,V/V)为流动相,检测波长210 nm,柱温30℃.结果GSH在0.01～1 g·L-1浓度范围内呈良好的线性关系,r=0.999 9,n=6;平均回收率为99.72%,RSD=0.43%,n=9;日内精密度RSD为0.41%(n=6),日间精密度RSD为0.49%(n=5).GSSG在0.5～50 mg·L-1浓度范围内呈良好的线性关系,r=1.000 0,n=7;最低检测限为1 ng(rSN=3),定量限为5ng(rSN=10);平均加样回收率为99.0%,RSD=0.61%,n=9.结论本法简便、专属性好、准确可靠,可作为注射用还原型谷胱甘肽的质量标准分析方法.     

中国汉族196人红细胞谷胱甘肽硫转移酶的活性分布

目的采用分光光度法测定人RBC谷胱甘肽硫转移酶(GSTs)活性,研究196名(男101名,女95名)健康汉族人的GSTs活性分布。方法制备人RBC裂解后,参照Habdous等的方法测定GSTs活性。反应体系如下:0.1 mol.L-1磷酸钠缓冲液(pH 6.5)2.6 mL,RBC裂解液0.1 mL,30 mmol.L-1谷胱甘肽0.1 mL,10 mmol.L-11-氯-2,4-二硝基苯(CDNB)0.1 mL。结果方法的日内和日间精密度均<10%。RBC GSTs活性呈正偏态分布,196例健康汉族人的平均GSTs活性为(3.89±0.96)U.(gHb)-1。不同年龄组平均GSTs活性无差异,而女性的平均GSTs活性比男性的平均值高,但并不显著。结论确定了可供临床参考的健康汉族人GSTs活性分布范围。     

人谷胱甘肽硫转移酶A1原核表达系统的构建及高效表达

目的　对人谷胱甘肽硫转移酶A1(GSTA1)原核表达系统的构建及高效表达。方法　采用RT -PCR方法将肝组织中提取总RNA扩增人GSTA1基因的cDNA序列 ,将其插入到原核表达载体pET30a多克隆位点中 ,构建重组表达质粒 ,并用PCR扩增、酶切分析及序列测定等方法对重组质粒进行鉴定 ,并进行了高效表达。结果　人GSTA1克隆到原核表达载体pET30a多克隆位点中 ,测序结果同GenbankGSTA1(GI:2 2 0 914 5 3)cDNA序列相比较 ,在 5 12位点T→C ,氨基酸由Met→Thr,同源性为 99% ,基因登陆号为AY5 32 92 8。通过IPTG诱导表达 ,在 34.4KDa处 1、2、3、4小时的表达量分别为 4 1.8%、6 8%、6 8.3%、83%。结论　经Westernblot分析 ,证实GSTA1基因在原核表达系统中有蛋白质表达     

低碘对大鼠脑组织抗氧化能力的影响

目的 :研究低碘对大鼠脑组织抗氧化能力的影响。方法 :20只清洁级Wistar大鼠随机分为低碘组 (LI)、适碘组 (NI) ,6个月后测定24h尿碘含量、甲状腺质量、脑组织匀浆中超氧化物歧化酶 (SOD)、谷胱甘肽过氧化物酶 (GSH -Px)活性及丙二醛 (MDA)含量。结果 :LI组甲状腺质量、24h尿碘含量明显低于NI组 (P<0.01) ;与NI组相比LI组GSH -Px降低 ,而MDA升高 (P<0.01) ,2组间SOD差别无统计学意义。结论 :低碘6个月的大鼠脑组织抗氧化能力降低 ,无法保护脑组织免受自由基的损伤     

妊娠高血压疾病患者氧化应激状态的改变

目的探讨妊娠高血压疾病患者氧化应激状态的改变情况。方法选取妊娠高血压疾病患者60例,其中妊娠高血压患者15例,轻度子痫前期患者15例,重度子痫前期患者30例。另取同期住院分娩的正常孕妇30例作为对照组。所有研究对象均测血清同型半胱氨酸、丙二醛、超氧化物歧化酶、谷胱甘肽过氧化物酶水平。结果妊娠高血压疾病组MDA水平为7.02±3.05 nmol/L,显著高于对照组5.16±2.79 nmol/L(P<0.01),妊娠高血压疾病组GSH-PX水平105.41±32.95 U/0.1 ml,明显低于对照组127.52±40.64 U/0.1 ml(P<0.05),两组SOD水平无显著差别。妊娠高血压疾病组Hcy为11.8±3.4μmol/L,显著高于对照组9.7±3.0μmol/L(P<0.01)。并且妊娠高血压疾病组Hcy与MDA呈正相关。结论妊娠高血压疾病患者由于氧化物质增加,抗氧化物质减少,氧化还原系统平衡失调,呈现氧化应激状态。     

大豆黄酮抗热应激作用

目的研究大豆黄酮对热应激小鼠谷胱甘肽过氧化物酶(GSH-Px)活力及丙二醛(MDA)含量的影响。方法将30只小鼠随机分为正常对照组、热应激组和大豆黄酮热应激组,以(45±1)℃持续20 min的热应激模式,观察热应激后小鼠肝、脾、肾及血清中GSH-Px活力及MDA含量变化。结果与正常对照组相比,热应激后小鼠肝脏、脾脏及肾脏GSH-Px活力升高,其中肝脏酶活力显著升高(P<0.05);大豆黄酮热应激组酶活力显著高于正常对照组(P<0.05或P<0.01),肝脏、脾脏酶活力显著高于应激组(P<0.05)。热应激后2组MDA含量均比正常对照组升高,其中肝脏和血清上升显著(P<0.05);大豆黄酮热应激组比应激组有所下降,但差异无统计学意义(P>0.05)。结论大豆黄酮可增强应激机体的抗氧化能力。