经典Wnt信号途径相关基因异常甲基化与肺癌发生关系的探讨＊

[摘要] 目的 检测肺癌中经典Wnt信号途径相关基因(WIF-1,sFRP-1,DKK-3)启动子的甲基化状态及β-连环蛋白(β-catenin)的表达情况,探讨其在肺癌发病中的作用。方法 应用巢式甲基化特异性聚合酶链反应(nMSP),检测66例肺癌组织、25例相应的癌旁组织和10例正常肺组织以及肺腺癌A549细胞株中WIF-1,sFRP-1和DKK-3基因启动子区域的甲基化;免疫组化SP法检测β-catenin的表达情况。结果 正常肺组织均未检测到所选基因的甲基化,肿瘤组织和癌旁组织中WIF-1、sFRP-1、DKK-3基因启动子甲基化的发生率分别为：50.0% vs 24.0% (P＜0.05)、36.4% vs 12.0% (P＜0.05)和36.4% vs 8.0% (P＜0.01);重度吸烟患者肿瘤组织3个基因的甲基化发生率明显增高(P＜0.05)。肺腺癌A549细胞株中WIF-1和DKK-3基因呈甲基化状态,而sFRP-1基因呈未甲基化状态。免疫组织化学显示肺癌组织中β-catenin的阳性表达率为81.2%,异常表达率为64.4%,且与组织学类型无关(P＞0.05)。结论 经典Wnt信号途径相关基因启动子甲基化可能与吸烟引起的肺癌有关,甲基化的WIF-1、sFRP-1和DKK-3基因可能成为肺癌表观遗传干预治疗的新靶点。 [关键词] 肺肿瘤 经典Wnt信号途径相关基因 β-连环蛋白     

经典Wnt信号途径相关基因异常甲基化与肺癌发生的关系

目的检测肺癌中经典Wnt信号途径相关基因(WIF-1,sFRP-1,DKK-3)启动子的甲基化状态及β-连环蛋白(β-catenin)的表达情况,探讨其在肺癌发病中的作用。方法应用巢式甲基化特异性聚合酶链反应(nMSP),检测66例肺癌组织、25例相应的癌旁组织和10例正常肺组织以及肺腺癌A549细胞株中WIF-1,sFRP-1和DKK-3基因启动子区域的甲基化;免疫组化SP法检测β-catenin的表达情况。结果正常肺组织均未检测到所选基因的甲基化,肿瘤组织和癌旁组织中WIF-1,sFRP-1,DKK-3基因启动子甲基化的发生率分别为:50·0%vs24·0%(P<0·05),36·4%vs12·0%(P<0·05)和36·4%vs8·0%(P<0·01);重度吸烟患者肿瘤组织3个基因的甲基化发生率明显增高(P<0·05)。肺腺癌A549细胞株中WIF-1和DKK-3基因呈甲基化状态,而sFRP-1基因呈未甲基化状态。免疫组织化学显示肺癌组织中β-catenin的阳性表达率为81·2%,异常表达率为64·4%,且与组织学类型无关(P>0·05)。结论经典Wnt信号途径相关基因启动子甲基化可能与吸烟引起的肺癌有关,甲基化的WIF-1,sFRP-1和DKK-3基因可能成为肺癌表观遗传干预治疗的新靶点。     

血浆WIF-1基因启动子甲基化检测在肺癌早期诊断中的临床意义

目的检测肺癌患者血浆Wnt抑制因子-1(WIF-1)基因启动子的甲基化情况并分析其在肺癌早期诊断中的意义。方法应用巢式甲基化特异性PCR(n MSP)法,首先检测肺癌患者血清癌胚抗原(CEA)水平,再分别检测78例肺癌患者、40例肺良性病变患者和40例健康志愿者血浆中WIF-1基因启动子区甲基化状态。结果肺癌患者血清癌胚抗原阳性率为35.90%(28/78),大致接近血浆标本中WIF-1基因启动子区甲基化率34.62%(27/78)。肺良性病变患者血浆中WIF-1基因启动子区甲基化率为2.50%(1/40),健康志愿者血浆中WIF-1基因启动子区未检测出甲基化。肺癌患者血浆WIF-1基因启动子甲基化和血清癌胚抗原二项联合检测的灵敏度为(62.82%),特异度为(90.00%)。不吸烟者WIF-1基因启动子甲基化率低于吸烟者。结论外周血浆中WIF-1基因启动子甲基化有成为肺癌的新的检测手段的可能性,并且对肺癌的初步筛查具有一定的临床意义。     

巢式甲基化特异性PCR检测不同类型组织标本中WIF-1基因启动子异常甲基化

目的:采用一种高灵敏度的DNA甲基化分析方法,即巢式甲基化特异性PCR法(nested-MSP,nMSP),检测外科手术切除新鲜组织、石蜡包埋组织及纤维支气管镜活检组织中WIF-1基因启动子的异常甲基化状态。方法:将基因组DNA变性成为单链,用亚硫酸氢盐修饰单链DNA,所有未甲基化的胞嘧啶被转变为尿嘧啶,而甲基化的胞嘧啶则不变。设计针对甲基化和非甲基化等位基因的特异引物,进行巢式PCR扩增,最后经凝胶电泳检测目的片段。结果:在3种类型的原发性非小细胞肺癌标本中都检测出了WIF-1基因启动子的异常甲基化。结论:巢式甲基化特异性PCR是一种灵敏度高、特异性强的甲基化检测方法,可广泛应用于不同类型标本基因启动子甲基化的分析。     

siRNAi沉默DNMT1基因对肺癌细胞WIF-1启动子甲基化的影响

目的 探讨DNA甲基转移酶(DNMT1)siRNAi对人肺癌A549细胞内抑癌基因wnt抑制因子-1(WIF-1)基因启动子高甲基化的影响.方法 利用pSilencer 4.1-CMV neo载体,通过siRNAi表达载体法制备DNMT1 siRNAi.将DNMT1 siRNAi转染肺癌A549细胞,采用RT-PCR和Western blot检测观察转染前后肺癌A549细胞DNMT1内DNMT1基因的表达改变,并利用甲基化PCR,直接测序法检测WIF-1基因启动子甲基化水平.结果 构建的DNMT1 siRNAi转染肺癌A549细胞后,明显抑制DNMT1基因的表达,mRNA表达下降75%,蛋白表达下调82%;同时,WIF-1基因启动子序列较转染前甲基化水平明显降低.结论 靶向DNMT1的siRNAi可明显下调靶基因DNMT1的表达,并逆转WIF-1基因启动子高甲基化水平.     

WIF-1、DKK基因启动子甲基化对COLO 320、

目的 探讨Wnt抑制性基因甲基化对结直肠癌细胞株Wnt/β-catenin信号通路的影响。 方法 使用甲基特异性PCR和逆转录实时定量PCR方法,分别检测结直肠癌细胞株COLO 320、HT-29及正常细胞株CCD-18Co中WIF-1、DKK-1、DKK-3的启动子CpG岛甲基化、mRNA水平,和测定β-catenin蛋白磷酸化情况,并观察5-氮杂-2′-脱氧胞苷（5-aza-dC）去甲基化对各指标的影响。 结果 COLO 320细胞的DKK-1及DKK-3 mRNA水平明显降低、甲基化程度高;HT-29细胞的DKK-3、WIF-1 mRNA水平低、甲基化程度高;两个肿瘤细胞株的β-catenin蛋白总量均明显增高,且主要为非磷酸化的状态。5-aza-dC可减少这些指标的改变。 结论 抑制性基因甲基化调节的Wnt/β-catenin通路的异常激活,可能在结直肠癌的形成中有重要作用。在不同的肿瘤细胞株之间、不同Wnt抑制基因的甲基化程度存在差异;该领域的深入研究有助于开发出新的治疗药物。     

肺腺癌组织与血清中半胱氨酸双加氧酶1基因启动子区甲基化在早期肺癌诊断中的意义

目的观察肺腺癌患者肿瘤组织和血清中半胱氨酸双加氧酶1(CDO1)启动子区甲基化状态,探索其与肺腺癌发生进展的关联,及CDO1基因甲基化作为肺腺癌血清学标记物的可能性。方法收集早期肺腺癌患者和肺部良性肿瘤组织及血清样本,采用基于磁珠的DNA提取和亚硫酸氢钠修饰技术,结合实时荧光定量PCR技术检测CDO1基因启动子区甲基化状态。结果肺腺癌组织中CDO1基因甲基化检测率为71.4%(25/35),对应的血清检出率为51.4%(18/35)。对照组中CDO1基因甲基化率为10.0%(2/20),对应的血清检出率为5.0%(1/20),肿瘤组与对照组之间比较差异有统计学意义(P0.05)。从肺腺癌患者血清检测CDO1启动子区甲基化与从肿瘤组织中直接检测其结果具有很高的一致性(r=0.732,P0.05)。结论肺腺癌组织和血清DNA中存在CDO1基因启动子区的异常甲基化状态,且具有极高的特异性。血清游离DNA的甲基化检测可能为肺腺癌的早期诊断、复发监测和预后判断提供全新的临床思路。     

甲状腺及乳房多原发癌病人甲状腺癌组织WIF-1基因mRNA表达及其启动子甲基化状态分析

目的探讨甲状腺、乳房多原发癌病人甲状腺癌组织WIF-1基因mRNA表达及其启动子的甲基化状态。方法采用RT-PCR及甲基化特异性PCR(MSP)方法检测33例甲状腺、乳房多原发癌病人甲状腺癌组织WIF-1基因mRNA表达及其启动子甲基化状态,以30例单纯甲状腺乳头状癌和20例甲状腺良性病变组织作为对照。结果多原发癌病人甲状腺癌组织、单纯甲状腺乳头状癌组织和甲状腺良性病变组织WIF-1基因的表达缺失率分别为51.5%(17/33)、36.7%(11/30)、15.0%(3/20),3组比较差异有统计学意义(χ2=7.105,P<0.05)。多原发癌病人甲状腺癌组织、单纯甲状腺乳头状癌组织WIF-1基因的甲基化率分别为45%(15/33)、43%(13/30),高于甲状腺良性病变组织的10%(2/20),差异有显著性(χ2=6.349、7.185,P<0.01),但多原发癌病人甲状腺癌组织与单纯甲状腺乳头状癌组织相比差异无显著性(P>0.05)。结论 WIF-1基因mRNA表达缺失在甲状腺、乳房多原发癌的发生过程中起关键作用;WIF-1基因启动子区的高甲基化状态可能是其mRNA低表达的机制之一。     

ASC和p16基因启动子甲基化与非小细胞肺癌发生发展的关系

目的 探讨ASC和p16基因启动子甲基化与非小细胞肺癌发生发展的关系.方法 应用甲基化特异性PCR（MSP）检测非小细胞肺癌中ASC和p16基因启动子的甲基化发生率.结果 48例非小细胞肺癌中ASC和p16基因启动子甲基化的发生率分别为47.9%和37.5%,显著高于癌旁正常组织（P＜0.001）;重度吸烟患者的肿瘤组织中ASC和p16基因启动子甲基化的发生率明显增高（P＜0.05）;ASC基因启动子甲基化在较小的肿瘤组织中的有较高的发生率（P＜0.05）.而且,重度吸烟患者的正常肺组织中亦可检测到ASC基因启动子的甲基化.结论 ASC和p16基因启动子的甲基化在非小细胞肺癌中有较高的发生率.ASC基因启动子的频繁甲基化可能与吸烟引起的非小细胞肺癌有关,并且ASC基因甲基化有可能是非小细胞肺癌发展过程中的一个早期事件。     

人肺癌组织细胞中HIN-1基因甲基化分析

目的:HIN-1(High in normal-1)是新近鉴定的一个抑癌基因,在乳腺癌细胞中,HIN-1启动子区的高甲基化导致其表达沉默。本研究拟探讨肺癌组织和6种肺癌细胞株中抑癌基因HIN-1启动子区内的甲基化修饰水平。方法:甲基化特异性PCR(MSP)和Biosulfite(亚硫酸氢钠)测序法鉴定样品中HIN-1启动子内CpG甲基化状态,RT-PCR法检测5-aza-dc处理前后HIN-1在细胞内的表达水平。结果:在60例肺癌样本中,HIN-1启动子甲基化阳性样本为35例(阳性率58.3%),而5例正常肺组织样品均为阴性反应。采用RT-PCR法分析了肺癌细胞株H157,H358,H1299,A549,H727,DMS53内HIN-1基因的表达水平,发现前4种细胞株内HIN-1表达水平极低,也是在这4种肺癌细胞中,HIN-1基因启动子呈高甲基化状态。用去甲基化试剂5-aza-dc处理上述4种细胞96h后,所有这4种细胞内均出现HIN-1基因的重新活化。结论:抑癌基因HIN-1启动子高甲基化导致的HIN-1表达抑制可能是肺癌发生发展的重要因素。     

Value of D-Dimers in patients with acute aortic dissection

Objective: To evaluatel the value of D-dimers in patients with acute aortic dissection (AAD). Methods: This study consisted of 16 patients with AAD and 27 non-AAD patients. Serum D-dimets were measured by Sta-Liatest D-DI immunoturbidimetric assay. Results: D-dimer level was higher (P ＜ 0.001) in patients with AAD(7.91 ± 5.52 μg/ml) than that in non- AAD group(1.57±1.24 μg/ml). D-dimer was positive (＞0.4 μg/ml) in all patients with AAD and in 10 control group patients (37%). Among patients with acute AAD, D-dimers tended to be higher in Stanford A than in Stanford B (8.67 ± 4.31 μg/ml vs. 3.24±1.27 μg/ml, P ＜0.01). D-dimer values tended to be higher in more extended disease(3.84 ± 1.65 μg/ml, 8.57 ± 3.58 μg/ml and 11.87 ± 5.69 μg/ml in thoracic aorta, thoracic and abdominal aorta, thoracic and abdominal aorta and iliacal arteries, respectively, P ＜ 0.05 for both 8.57 ± 3.58 and 11.87 ± 5.69 vs. 3.84 ± 1.65 ). Including the control group into the analysis, we found a sensitivity of 100%, a negative predictive value of 100%, and a specificity of 66% and a positive predictive value of 64% for D-dimer in diagnosis of AAD in our patients with suspected AAD. Conclusion: D-dimer was elevated in patients with AAD. A negative D-dimer test result could be useful in excluding AAD.     

Research of combined liver-kidney transplantation model in rats

Objective： To set up a simple and reliable rat model of combined liver-kidney transplantation. Methods： SD rats served as both donors and recipients. 4℃ sodium lactate Ringer＇s was infused from portal veins to donated livers,and from abdominal aorta to donated kidneys, respectively. Anastomosis of the portal vein and the inferior vena cava （IVC） inferior to the right kidney between the graft and the recipient was performed by a double cuff method, then the superior hepatic vena cava with suture. A patch of donated renal artery was anastomosed to the recipient abdominal aorta. The urethra and bile duct were reconstructed with a simple inside bracket. Results： Among 65 cases of combined liver-kidney transplantation, the success rate in the late 40 cases was 77.5%. The function of the grafted liver and kidney remained normal. Conclusion： This rat model of combined liver-kidney transplantation can be established in common laboratory conditions with high success rate and meet the needs of renal transplantation experiment.     

BLOOD PRESSURE CHANGE WITH AGE IN SALT-SENSITIVE TEENAGERS

Objective To observe blood pressure change with age in salt-sensitive teenagers whose salt sensitivity were determined by repeated testing.Methods Salt sensitivity was determined through intravenous infusion of normal saline combined with volume-depletion by oral diuretic furosemide in 55 teenagers. After five years, salt sensitivity was re-examined and subject blood pressure was followed up. Blood pressure changes in salt-sensitive teenagers were compared to that of non-salt sensitive teenagers over five years.Results After 5 years, the repetition rate of salt sensitivity determined by intravenous saline loading is 92.7%. In teenagers with salt sensitivity on the baseline, both the systolic blood pressure increments and increment rates were much higher than non-salt sensitive teenagers (12.7±12.1 mmHg vs. 2.8±5.2 mmHg, P＜ 0.01; 12.2%± 12.0% vs. 2.5% ±4.4%, P＜ 0.001,respectively). There was a similar trend for diastolic blood pressure (8.4 ± 6.4 mmHg vs. 3.7 ± 6.4 mmHg, P = 0.052; 13.2% ±10.6 % vs. 6.8%± 10.1%, P = 0.053, respectively).Conclusions Salt sensitivity determined by intravenous saline loading showed good reproducibility. Blood pressure increments with age were much higher in salt-sensitive teenagers than non-salt sensitive teenagers, especially in terms of systolic blood pressure.     

安心颗粒对急诊经皮冠状动脉介入术后生活质量影响的研究

目的：评价使用安心颗粒对急诊经皮冠状动脉介入术（PPCI）术后生活质量的影响.方法：将160例接受PPCI的急性ST段抬高型心肌梗死患者随机分为安心颗粒组（术前顿服安心颗粒8.8g,术后安心颗粒4.4 g/次,每日2次）和对照组（仅接受基础药物治疗）.所有患者均服用阿司匹林、氯吡格雷和阿托伐他汀.分别在入院时、出院前1d、出院后180 d时,应用心肌梗死多维度量表（MIDAS）、中文版SF-36评价量表对患者生活质量评分.并观察术后30 d以内的出血并发症、血小板减少症发生情况.结果：入院时和出院前1d,两组患者的心肌梗死MIDAS、SF-36量表评分比较无差异（P＞0.05）;出院后180 d时,与对照组比较,安心颗粒组MIDAS、SF-36评分明显减低（P＜0.05）;组内与入院时比较,两组出院前1d、出院后180 d时,MIDAS、SF-36评分均降低（P＜0.05）.两组患者在随访期间均无大量出血、少量出血、重度和极重度血小板减少症发生,安心颗粒组有4例、对照组有7例发生不明显出血（P＞0.05）.两组发生轻度血小板减少症的患者数比较无差异（P＞0.05）.结论：PPCI使用安心颗粒,能改善急性ST段抬高型心肌梗死患者的生活质量,且不增加出血风险.     

The comparative studies of the influences of Urapidil and Nicardipine on sino-atrial node function, atrio-ventricular node function and hemodynamics

Objective:To investigate the influences of urapidil and nicardipine on rabbit sinus function,atrio-ventricular node function and hemodynamics.Methods:Thirty-two Angora's rabbits were selected and randomly divided into four groups.U1 group:urapidil 0.25 mg/kg;U2 group:urapidil 0.5 mg/kg;N1 group:nicardipine 10 μg/kg;N2 group:nicardipine 20 μg/kg.All these medicine were administrated within 30 seconds.Measurements were taken before and after the administration of urapidil or nicardipine for the following data:mean blood pressure(MAP),heart rate(HR),sino-atrial conduction time(SACT),maximal sinoatrial recovery time(SNRTmax)corrected sinus node recovery time(CSNRT),index of sinus node recovery time(SNRTI),Wenckebach A-V conduction frequency (WB),and P-R interval.Results:Significant MAP and HR changes were identified in all of the four groups before and after administration of both urapidil and nicardipine.No significant changes could be found in the rest of the parameters.Intergroup analysis showed that SACT and CSNRT of N1 and N2 groups were shorter than those of the U2 group(P＜0.01);the MAP decreased(P＜0.01)and the HR increased drastically(P＜0.01).Conclusions:Neither urapidil(0.25 mg/kg,0.5 mg/kg)nor nicardipine(10μg/kg,20μg/kg)has any significant influence on rabbit sinus function or rabbit atrio-ventricular node function.Nicardipine could be a better choice than urapidil for parafunctional sinus node patients.     

Expression of OPGmRNA and ODFmRNA in the patients of hip fracture due to osteoporosis

Objective：To investigate the gene expression of osteoprotegerin（OPG） and osteoclast differentiation factor（ODF） in the bone tissue of patients with hip fracture due to osteoporosis. Methods：OPGmRNA and ODFmRNA in the bone tissue in 50 cases of osteoporosis sufferers（over 50 years old） with hip fracture（Observer Group） and 30 cases of hip facture sufferers with no osteoporosis（Control group） were analyzed with the Semi-Quantitative RT-PCR method. Results：The mRNA expressed of ODF, OPG were both high in the patients with hip fracture. In the control group, the expression of OPG mRNA was observed, while the expression of ODF mRNA was very slight. Conclusion：Aged patients contained all signals including OPG, ODF that are essential for inducing osteoclastogenesis and promoting bone resorption.     

The clinicopathological and immuohistochemical analysis of solid-pseudopapillary tumor of the pancreas： report of 9 cases

Objective：To investigate the clinical features, pathological characteristics and immunophenotype of solid-pseudopapillary tumor of the pancreas（SPTP）. Methods：Nine surgically treated cases of SPTP were retrospectively reviewed. Hematoxylin and Eosin（HE） staining and immunohistochemical staining were used to analyze all cases, and the general clinical data was collected. Results：Six patients were asymptomatic except for a palpable mass. Two patients complained of vague-epigastric pain. One patient appeared jaundice. The tumor was encapsulated and solid tissues alternately with cystic tissues. Histologically, the histological structure of solid portion was pseudopapillary with a fibrovascular core. Tumor cells were uniform and medium-sized which were arranged in sheets ets or nests or pseudopapillary patterns. Immunohistochemical studies demonstrated that SPTP proved positive in vimentin（9/9 cases）, AAT（9/9 cases）, NSE（9/9 cases）, ACT（7/9 cases）, CK20（2/9 cases）, CgA（1/9 cases）, S-100（3/gcases）, PR（4/gcases）, Syn（3/9 cases） and CD56（5/9cases）, negative in CEA and ER. Conclusion：SPTP is a tumor predominantly occurring in young women frequently without special symptoms. This tumor has various characteristical histological patterns with different immunophenotype.     

Dynamic Changes in Serum Estradiol and Progesterone levels in Patients of Premenstrual Syndrome with Adverse Flow of Liver-qi

Objective:To probe into the influence of changes of ovarian hormones on the pathogenesis of the specific sub-type premenstrual syndrome(PMS)and reveal partial microcosmic mechanisms of adverse flow of liver-qi.Methods:Estradiol(E2)and progesterone(P)levels in serum were determined at different phases of menstrual cycle by radioimmunoassay.Results:In the group of PMS with adverse flow of liver-qi.the secretive peak value Of E2 and P at the follicular phase significantly decreased,and the secretive peak value at the luteal phase did not come into being.Conclusions:Low E2 and P secretive peak at the follicular phase and absence of secretive peak at the luteal phase is one of the microcosmic mechanisms of PMS with adverse flow of liver-qi.One of the pathophysiologic mechanisms of specific sub-type PMS is probably the continuous low level of E2and P.     

Real-time Three-dimensional Echocardiography in Assessment of Left Ventricular and Right Ventricular Volumes

Real-time three-dimensional echocardiography (RT3DE)is a new ultrasound technique that enables dynamic threedimensional visualization and quantification of the heart in real time. Investigation of feasibility and methodology of RT3DE in determining left ventricular (LV) and right ventricular (RV) volumes, RT3DE was performed in 35 normal adults using Philips SONOS 7500 system with a 2-4 MHz matrix array transducer. The 60°×60° "pyramid" volume database was obtained and analyzed on a TomTec echo workstation. Both LV and RV volumes were calculated with four 3DE methods (i.e. apical 2, 4, 8, and 16-plane) through manually tracing ventricular endocardial borders in end diastole and end systole. Stroke volumes were then calculated. LV volume was also measured by 2DE Simpson's rule using GE VIVID 7 ultrasound machine.     

SCREENING FOR A 21-CHROMOSOME ABNORMALITY IN PREIMPLANTED EMBRYOS OF ELDERLY WOMEN

Increasing maternal age is the only etiological factor unequivocally linked to Down's syndrome in humans. The occurrence rate of newborns with Down's syndrome is about 1/220 in women over 35 years old. However, the occurrence rate in embryos fertilized in vitro, of the elder woman is unclear. Using FISH we screened the number of chromosome 21 in preimplanted embryos of 5 elderly women (average age, 38.4 years) to study the feasibility and necessity of screening trisomy 21 in embryos in patients over 35 years old at the in vitro fertilization (IVF) center.