针对PAI基因的小干扰RNA特异性调节PAI及tPA表达的实验研究

目的 设计针对纤溶酶原激活物抑制物(plasminogen activator inhibitor,PAI)基因的小干扰RNA(PAI siRNA),研究其对PAI和组织型纤溶酶原激活物(tPA)表达的影响.方法体外化学合成PAI siRNA,电转染法转染人主动脉平滑肌细胞(HASMC),采用RT-PCR及Western印迹法分别从mRNA和蛋白水平上检测转染细胞PAI及tPA蛋白的表达情况.结果转染细胞PAI mRNA和蛋白表达水平均较对照组明显下降(P＜0.01),tPA蛋白表达较对照组明显升高(P＜0.01).结论 PAI siRNA可快速特异性封闭PAI基因表达,并调节tPA蛋白表达,使其升高,为进一步研究PAI在血栓性疾病中作用机制以及探讨PAI与tPA之间关系提供了实验基础.     

PAI siRNA表达载体的构建及鉴定

目的 构建和鉴定针对PAI 的发卡样小干扰RNA（siRNA）真核表达载体PAI siRNA.方法 人工合成一对互补并编码相应短发夹状PAI siRNA 的寡核苷酸链, 将其插入到pSilencerTM 2.1-U6 neo 载体中,经测序鉴定所构建的重组载体是否正确;采用电转染法将构建的重组载体导入人肝癌细胞HpG2中, 采用逆转录聚合酶链反应 （RT-PCR） 、West ern印迹法分别检测转染细胞PAI mRNA 和蛋白的表达.结果 经测序证明PAI siRNA 序列正确;转染PAI siRNA 载体后,HpG2细胞PAI mRNA 和蛋白表达均较对照组明显下降（P ＜0.01）.结论 测序结果表明发卡样PAI siRNA 真核表达载体构建成功,转染Hp G2细胞后获得稳定表达,并可特异性封闭PAI的表达, 为进一步研究PAI siRNA 载体提供了实验基础.     

血浆纤溶酶原激活物抑制因子基因启动子多态性与2型糖尿病伴发冠心病的关系

目的从分子遗传水平进行基因多态性研究，以探讨２型糖尿病易伴发冠心病的内在原因。方法对８１例２型糖尿病患者、９３例非糖尿病（非ＤＭ）患者进行口服糖耐量试验、血脂分析、血浆纤溶酶原激活物抑制因子（ＰＡＩ１）基因多态性分析。结果２型糖尿病和非ＤＭ相比ＰＡＩ１基因启动子４Ｇ频率明显增加（４２％和３１％，Ｐ＜０００１）；２型糖尿病伴冠心病与不伴冠心病相比，ＰＡＩ１基因启动子４Ｇ频率及４Ｇ／４Ｇ基因型频率存在明显差异（５２％和３０％，３２％和１４％；Ｐ分别＜０００１、００５）；非２型糖尿病伴冠心病与不伴冠心病相比，ＰＡＩ１基因启动子４Ｇ频率及４Ｇ／４Ｇ基因型频率则无明显差异（３１％和３１％，８％和１６％，均为Ｐ＞００５）。结论ＰＡＩ１基因４Ｇ等位基因可能是糖尿病合并冠心病的内在危险因素     

MTHFR C677TT, PAI1 4G-4G, V Leiden Q506, and prothrombin G20210A in hepatocellular carcinoma with and without portal vein thrombosis

We studied thrombophilic genetic factors (TGFs) MTHFR C677TT, PAI1 4G-4G, V Leiden Q506, prothrombin G20210A as risk factors in 94 patients with HCC with and without portal vein thrombosis (PVT), compared with 214 patients with liver cirrhosis (LC) with and without PVT and 94 healthy controls (HC). The OR (95% CI) for MTHFR C677TT with HCC was 3.85 (1.55–7.39) vs. HC. The OR for PAI1 4G-4G in HCC, was 2.87 (1.27–6.55) vs. HC. Also prothrombin G20210A was significantly more frequent among HCC, mainly in patients with PVT, while V Leiden factor was equally distributed among HCC and HC. Differences were more significant in patients with associated PVT. These findings suggest that frequently TGFs are needed for patients to be at risk of HCC and PVT. We conclude that in all patients with chronic liver disease TGF screening should be performed to individuate patients at risk of HCC and PVT.     

中老年冠心病患者血浆t-PA、PAI活性变化的探讨

本文对55例各临床类型冠心病患者血浆t-PA、PAI活性进行了初步探索,并与正常对照组对比。结果表明:各组患者血浆t-PA活性均明显低于正常对照组,其中心绞痛和急性心肌梗塞(AMI)组降低更为显著、但此二组的血浆PAI活性明显高于正常对照组;各组患者血浆t-PA/PAI比值均明显低于正常对照组。与心绞痛 AMI组相比:心律失常、心衰、陈旧性心梗组的血浆t-PA活性及t-PA/PAI均明显增高,但血浆PAI活性均明显减低。心绞痛 AMI组的血浆t-PA活性及t-PA/PAI与年龄有明显负相关,提示血浆t-PA、PAI活性异常在冠心病、心绞痛和AMI的发生、发展中可能有重要的病理意义。     

t-PA, PAI, and protein C before and after vascular occlusion of the upper limb in patients with Raynaud's phenomenon

Tissue plasminogen activator (t-PA) and its inhibitor (PAI) were assessed in venous blood drawn before and after venous occlusion (bvo, avo) for 33 patients with Raynaud's phenomenon (RP), 14 with primary RP (PRP), 9 with suspected secondary RP (SSRP), and 10 with definite collagen disease and secondary RP (SRP). There were significant differences in PAI values avo between PRP (and controls), SSRP, and SRP. PAI activity decreased significantly avo only in controls and in PRP, and there was significant t-PA antigen elevation avo in the same groups. In addition, since PAI is neutralized by activated protein C (PC), both PC antigen and PC activity were assessed avo and bvo. PC Ag remained unchanged in all groups, with PC activity significantly lower than controls in SRP and SSRP. Finally the authors looked for interference of anticardiolipin antibodies (ACA) and lupus-like anticoagulant (LAC) with the PC system in collagen disease-associated RP. Specific IgG ACA were found in only 1 patient with SRP. In conclusion, there is an endothelial derangement, involving t-PA release and PAI, in SSRP and SRP patients. The reduced PC activity in these latter groups appears to be due to increased PAI influence rather than to ACA/LAC.     

下调PAI对HpG2细胞组织型纤溶酶原激活物和尿激酶纤溶酶原激活物的影响

目的探讨纤溶酶原激活物抑制剂(PAI)的发卡样小干扰RNA(siRNA)真核表达载体对人肝癌HpG2细胞组织型纤溶酶原激活物(tPA)和尿激酶PA(uPA)表达的影响。方法设计短发夹状PAI siRNA的寡核苷酸链,将其插入到真核细胞表达载体pSilencerTM 2.1-U6 neo载体中构建重组载体后,采用脂质体转染方法将构建的重组载体导入人肝癌细胞HpG2中,采用逆转录聚合酶链反应(RT-PCR)、Western印迹法分别检测转染细胞PAI mRNA和蛋白对tPA和uPA表达的影响。结果转染后细胞中PAI含量降低,tPA和uPA的表达减少(均P<0.01)。结论测序结果表明发卡样的PAI siRNA真核表达载体转染HpG2细胞后获得稳定表达,并可特异性封闭PAI的表达,PAI siRNA能有效阻断PAI的蛋白合成,同时抑制细胞中tPA和uPA的表达。     

蛛网膜下腔出血后知浆t—PA、PAI—1变化的临床意义

对28例蛛网膜下腔出血（SAH）后患者的血浆组织型纤溶酶原激活物（t-PA）、纤溶酶原激活物抑制物（PAI-1）变化进行了观察,并与30例健康者进行对照。结果显示,与对照组比较,SAH后1～14天患者血浆纤溶性明显增高;其中伴发脑血管痉挛（CVS）者出血第7天、14天血浆PAI-1活性均明显高于无CVS者;出血量多者血浆t-PA、PAI-1活性较高。提示血浆t-PA、PAI-1活性在SAH后呈动态变化,二者可能均参与SAH的发生、发展、其活性测定可望成为监测SAH、CVS及出血量的有效指标。     

Common polymorphisms of the PAI1 gene do not play a major role in the development of diabetic nephropathy in Type 1 diabetes.

AIM: Plasminogen activator inhibitor 1 (PAI1) plays a key role in the regulation of extracellular matrix (ECM) degradation. ThePAI1 gene is therefore an excellent candidate gene for diabetic nephropathy. The aim of this study was to employ gene resequencing to identify common DNA polymorphisms in thePAI1gene, and subsequently assess haplotype tagged single nucleotide polymorphisms(htSNPs) using a case control design. METHODS All nine exons, exon-intron boundaries, introns 1, 4 and 7 and approximately 3 kb upstream and 5 kb downstream of thePAI1 gene were screened for DNA polymorphisms in 15 case and 15 control subjects using WAVE denaturing high-performance liquid chromatography technology and confirmed by DNA sequencing. Polymorphisms were genotyped in 86 healthy individuals using direct sequencing and haplotype tagged single nucleotide polymorphisms (htSNPs) identified. Genotyping of the htSNPs was performed in 583 Type 1 diabetic patients (222 with nephropathy, 361 without nephropathy)using Pyrosequencing. RESULTS: Twenty-one polymorphisms with a minor allele frequency (MAF)>1%were identified; 14 had a MAF> or =10%. Five htSNPs [c.-1968_69insG, c.43 G-->A (Ala15Thr), c.1092-105 A-->G, c.*1737 G-->A, c.*3711 C-->T] were identified.Haplotype frequencies were similar in case and control groups (likelihood ratio chi2 test,P=0.66). CONCLUSION: It is unlikely that common polymorphisms of thePAI1 gene strongly influence susceptibility to diabetic nephropathy in the White Type 1 diabetic population.     

Association of the metabolic syndrome with PAI 1act and clot lysis time over a 10-year follow up in an African population

Background and aimsThe association between the metabolic syndrome (MetS) and plasminogen activator inhibitor-1 (PAI-1) has been well established in cross-sectional studies. It is less clear whether this translates into decreased clot lysis rates and very little information is available on non-European populations. Little is known regarding prospective associations and whether clot lysis progressively worsens in MetS individuals over time. We determined the prospective association of MetS with PAI-1 activity (PAI-1_act) and clot lysis time (CLT) over a 10-year period.Methods and resultsAs many as 2010 African men and women aged ≥30 years were stratified according to MetS status and number of MetS criteria (0–5). We also determined the contribution of the PAI-1 4G/5G polymorphism to these associations and identified which MetS criteria had the strongest associations with PAI-1_act and CLT. Both PAI-1_act and CLT remained consistently elevated in individuals with MetS throughout the 10-year period. PAI-1_act and CLT did not increase more over time in MetS individuals than in controls. The 4G/5G genotype did not influence the association of PAI-1_act or clot lysis with MetS. Increased waist circumference, increased triglycerides and decreased HDL-C were the main predictors of PAI-1_act and CLT.ConclusionsBlack South Africans with MetS had increased PAI-1_act and longer CLTs than individuals without MetS. The inhibited clot lysis in MetS did, however, not deteriorate over time compared to controls. Of the MetS criteria, obesity and altered lipids were the main predictors of PAI-1_act and CLT and are thus potential targets for prevention strategies to decrease thrombotic risk.     

心力衰竭大鼠肾素-血管紧张素系统、纤溶酶原激活物抑制物激活及依那普利干预的影响

目的:检测心力衰竭(HF)大鼠循环肾素-血管紧张素系统(RAS)、血浆纤溶酶原激活物抑制物(PAI)活性变化以及依那普利干预的影响,以证明HF时RAS对内源性纤溶系统的作用并讨论其意义.方法:建立大鼠动静脉瘘HF模型,将其分成单纯HF组及依那普利治疗组,另将大鼠作假手术作为对照组(每组各10只).分别于手术前3 d、术后9 d及30 d检测血浆肾素活性(PRA)、血管紧张素Ⅱ(AngⅡ)及血浆纤溶酶原激活物抑制物活性(PAI-Ⅰ).结果:①PRA及AngⅡ浓度:各组间于手术前及假手术组手术前后均无显著差异(均P＞0.05).手术后9 d时HF组及依那普利组均较对照组明显升高,也较手术前明显升高(均P＜0.05),尤以HF组更明显,手术后30 d时均下降,HF组仍明显高于对照组(P＜0.05),而依那普利组已回到手术前水平.②PAI-Ⅰ活性:手术前各组间及对照组手术前后均无明显差异(均P＞0.05).术后依那普利略高于对照组,但差别不显著(P＞0.05),手术后HF组较另两组明显升高(P＜0.05),但亦呈下降趋势.结论:HF时RAS激活与体内纤溶功能间有密切联系,RAS激活对导致机体纤溶系统功能失衡有重要作用,可用以解释严重心功不全患者血液呈高凝状态,并且发生血栓栓塞疾病危险增高的原因,通过抑制HF时RAS过度激活,血管紧张素转换酶抑制剂治疗对降低严重HF患者血栓栓塞性疾病的发生可能有重要意义.     

Personalized Activity Intelligence (PAI) for Prevention of Cardiovascular Disease and Promotion of Physical Activity

Purpose

To derive and validate a single metric of activity tracking that associates with lower risk of cardiovascular disease mortality.

Characterization of MTHFR,GSTM1, GSTT1, GSTP1, and CYP1A1 genotypes in childhood acute leukemia

The role of methylenetetrahydrofolate reductase (MTHFR C677T), glutathione S-transferases (GSTM1 and GSTT1 null, GSTP1 Ile105Val), and cytochromes p450 (CYP1A1*2A) genotypes in the etiology of childhood leukemia was simultaneously investigated. 144 Turkish children with acute lymphoblastic leukemia (ALL) and 33 with acute nonlymphoblastic leukemia (ANLL) were studied and compared with 185 healthy pediatric controls. The frequency of MTHFR genotype was insignificantly higher in ALL (7.7%) and ANLL (6.3%) than in controls (4.4%). Equal distribution of the GSTM1 null genotype was detected between ALL patients and controls (55%), while its incidence was slightly higher in ANLL patients (61.3%). Although GSTT1 null genotype was insignificantly lower in ALL patients (20.9%) than controls (22.7%), it was significantly underrepresented in ANLL patients (6.5%) (P = 0.05, OR 0.24, 95% CI 0.05-1.03). The homozygous frequency of GSTP1 genotype did not differ significantly between groups of ALL (3.7%), ANLL patients (9.1%) and controls (4.9%). Homozygous CYP1A1*2A genotype was underrepresented in ALL patients (1%) as compared to control (4.8%) but the differences did not reach to statistical significance (OR 0.21; 95% CI 0.03-1.72). Homozygosity for this genotype was not detected in ANLL patients. No particular association was noted between different combinations of combined genotypes and risk of development of childhood ALL and ANLL. These results suggested that there are no significant associations between the studied genotypes and the risk of developing either form of acute leukemia except GSTT1 null and homozygosity for CYP1A1 genotypes that may play protective roles in the development of ANLL in Turkish children.     

CYP1A1, CYP2E1, GSTM1, GSTT1, EPHX1 exons 3 and 4, and NAT2 polymorphisms, smoking, consumption of alcohol and fruit and vegetables and risk of head and neck cancer

Purpose As risk-modifiers of alcohol and tobacco effects, metabolic genes polymorphisms were investigated as susceptibility candidates for squamous cell carcinoma of the head and neck (SCCHN). Methods A total of 210 cases and 245 hospital controls, age and gender matched, were genotyped for CYP1A1, CYP2E1, GSTM1, GSTT1, EPHX1 exons 3 and 4, and NAT2 polymorphisms. A measurement of the biological interaction among two risk factors was estimated by the attributable proportion (AP) due to interaction and its 95% confidence interval (CI). Results SCCHN risk was associated with high-levels of alcohol intake [OR = 3.50 (95%CI: 1.93–6.35) and OR = 6.47 (95%CI: 2.92–14.35) for 19–30 g/day and >30 g/day, respectively], cigarette smoking [OR = 3.47 (95%CI: 1.88–6.41) and OR = 7.65 (95%CI: 4.20–13.90) for 1–25 and >25 pack-years of smoking, respectively] and low-fruit and vegetables consumption (OR = 2.45; 95%CI: 1.53–3.92). No differences were observed for the genotypes or haplotypes distributions among cases and controls, and no biological interaction emerged from gene–gene and gene–environment interaction analyses. An attributable proportion (AP) due to biological interaction of 0.65 (95%CI: 0.40–0.90) was detected for heavy drinkers with a low intake of fruit and vegetables, and an AP of 0.40 (95%CI: 0.10–0.72) resulted forever smokers with low fruit and vegetables consumption. Conclusions Even in presence of high alcohol consumption or cigarette smoking, a high intake of fruit and vegetables might prevent the development of around one quarter of SCCHN cases. The lack of interaction between the studied polymorphisms and the environmental exposures suggests that chronic consumption of tobacco and alcohol overwhelm enzyme defences, irrespective of genotype.     

CYP1A1 , GSTM1 , GSTT1 and NQO1 polymorphisms and colorectal adenomas in Japanese men

AIM: To investigate the role of functional genetic poly-morphisms of metabolic enzymes of tobacco carcinogens in the development of colorectal adenomas. METHODS: The study subjects were 455 patients with colorectal adenomas and 1052 controls with no polyps who underwent total colonoscopy in a preretirement health examination at two Self Defense Forces hospitals. The genetic polymorphisms studied wereCYP1A1*2A (rs 4646903), CYP1A1*2C (rs 1048943), GSTM1 (null or non-null genotype), GSTT1 (null or non-null genotype) and NQO1 C609T (rs 1800566). Genotypes were determined by the polymerase chain reaction (PCR)-restriction fragment length polymorphism or PCR method using genomic DNA extracted from the buffy coat. Cigarette smoking and other life-style factors were ascertained by a self-administered questionnaire. The associations of the polymorphisms with colorectal adenomas were examined by means of OR and 95%CI, which were derived from logistic regression analysis. Statistical adjustment was made for smoking, alcohol use, body mass index and other factors. The gene-gene interaction and effect modification of smoking were evaluated by the likelihood ratio test. RESULTS: None of the five polymorphisms showed a significant association with colorectal adenomas, nor was the combination of GSTM1 and GSTT1 . A borderline significant interaction was observed for the combination of CYP1A1*2C and NQO1 (P = 0.051). The OR associated with CYP1A1*2C was significantly lower than unity among individuals with the NQO1 609CC genotype. The adjusted OR for the combination of the CYP1A1*2C allele and NQO1 609CC genotype was 0.61 (95%CI: 0.42-0.91). Although the interaction was not statistically significant (P = 0.24), the OR for individuals carrying the CYP1A1*2C allele and GSTT1 null genotype decreased significantly compared with those who had neither CYP1A1*2C allele nor GSTT1 null genotype (adjusted OR: 0.69, 95%CI: 0.49-0.97). Smoking did not modify the associations of the individual polymorphisms with colorectal adenomas. There w     

Glutathione S-transferase M1, T1, and P1 genotypes and rheumatoid arthritis

OBJECTIVE: To determine the effects of genetic polymorphisms of glutathione S-transferase (GST) M1, GSTT1, and GSTP on risk and severity of rheumatoid arthritis (RA) in a Korean population. METHODS: A total of 258 patients with RA and 400 disease-free controls were enrolled. GST genotypes were determined by RFLP-PCR. HLA-DRB 1 typing and further subtyping of all alleles was performed using sequence-specific oligonucleotide probe hybridization after PCR. Severity of RA among cases was assessed by Steinbrocker anatomical stage. Risk was assessed by calculating the age and sex adjusted odds ratio (OR) and 95% confidence intervals (CI). RESULTS: The OR for risk of RA with the GSTM1-null genotype was 1.40 (95% CI 1.02- 1.92, p = 0.04), and 1.86 (95% CI 1.12- 3.09, p = 0.005) among individuals without the shared epitope (SE). Among patients with RA, the OR for risk of severe RA for the GSTM1-null genotype was 2.45 (95% CI 1.04- 5.77, p = 0.02). No association was observed between the GSTT1 or GSTP1 genotypes and either risk or severity of RA. CONCLUSION: These results suggest that the deletion polymorphism of GSTM1 is associated with increased susceptibility for RA, particularly among individuals who are not carriers of the HLA-DRB 1 SE.