结直肠癌患者p16基因甲基化的研究

目的检测p16基因启动子区异常甲基化发生情况,探讨p16基因异常甲基化作为结直肠癌临床辅助诊断分子生物学标志物的可能性。方法应用甲基化特异性PCR（MSP）技术,检测结直肠癌患者肿瘤组织中p16基因的异常甲基化情况。结果应用MSP方法,DukesA、B期病人和DLlkes C、D期病人p16启动子区甲基化率分别为23．8%和59．4％。结论分析患者DNA的p16基因异常甲基化有可能成为辅助结直肠癌诊断的有效方法之一。     

粪便HPP1基因甲基化分析在结直肠癌早期诊断中的价值

目的 探讨人粪便中增生性息肉蛋白基因(HPP1)甲基化状态的检测用于结直肠癌早期诊断的可行性.方法 从52例结直肠癌患者、21例结直肠腺瘤患者和24例正常对照者的粪便中分别提取DNA,采用甲基化特异性PCR(MSP)技术分析其HPP1基因甲基化状态.结果 结直肠癌粪便标本HPP1基因甲基化阳性率为71.2% (37/52),腺瘤粪便标本为57.1%(12/21),而正常粪便标本均为阴性.结论 HPP1基因甲基化是结直肠癌疾病进展过程中的早期事件.粪便HPP1基因甲基化检测可望成为结直肠癌早期无创诊断或结直肠癌高风险人群筛查的一个新途径.     

血浆游离DNA中TGR5、RIZ1异常甲基化在肝癌诊断中的价值

目的 研究外周血浆游离DNA中TGR5、RIZ1异常甲基化在肝细胞癌(HCC)诊断中的价值.方法 收集48例HCC和40例健康体检者的血浆,提取血浆游离DNA,运用甲基化特异性PCR(MSP)检测血浆游离DNA中TGR5、RIZ 1甲基化状态,结合其病理资料进行分析.结果 HCC组血浆游离DNATGR5、RIZ1甲基化率分别为68.8％和60.4％;两者联合检测时HCC组甲基化率为72.9％.正常对照组甲基化率分别5.0％和2.5％.TGR5、RIZ1甲基化状态与HCC患者的性别、年龄、肿瘤大小、肿瘤数目、HBeAg阳性及TNM分期等病理参数无关(P＞0.05).结论TGR5、RIA1甲基化水平在早期HCC血浆中升高,通过联合检测患者TGR5、RIZ1甲基化可作为HCC的诊断指标.     

粪便SDC2和TFPI2基因甲基化联合检测对结直肠癌及癌前病变的诊断效能分析

目的 探究粪便硫酸类肝素蛋白多糖（Syndecan 2,SDC2）和组织因子途径抑制物2(Tissue factor pathway inhibitor 2, TFPI2)基因甲基化联合检测对结直肠癌及癌前病变的诊断效能。方法 纳入2020年5月～2021年4月于我院消化内科门诊及住院就诊的45例结直肠病变患者作为研究对象,另纳入同期于我院行体格检查的健康者21例作为对照组,经结直肠镜和术后病理学检查,45例结直肠病变患者中确诊结直肠癌25例,结直肠腺瘤20例。采用甲基化特异性PCR(Methylation-specific PCR,MSP)法检测各组受试者中SDC2和TFPI2基因甲基化情况。应用受试者工作特征（Receiver operating characteristic,ROC）曲线判定SDC2和TFPI2基因甲基化单项检测和联合检测对结直肠癌及癌前病变的诊断效能。结果 结直肠癌组和结直肠腺瘤组SDC2和TFPI2基因甲基化阳性水平均显著高于对照组（P<0.05）。SDC2和TFPI2基因甲基化对结直肠癌诊断的ROC曲线下面积分别为0.744[95%CI(0.624～0...     

DNA甲基化与结直肠癌的预后及治疗进展

DNA甲基化是结直肠癌常见的表观遗传性改变,甲基化的异常可引起肿瘤癌基因的激活以及抑癌基因的失活,因此在结直肠癌的发生发展中有重要的意义。DNA甲基化的改变可导致结直肠癌出现不同的预后。且因为甲基化过程是可逆的,故其有望成为结直肠癌治疗新靶点。现就DNA甲基化在结直肠癌的预后及治疗方面做一简要综述     

ZNF582基因启动子区在结直肠癌组织中的甲基化状态及临床意义

目的 探讨ZNF582基因启动子区在结直肠癌组织中的甲基化状态及临床意义.方法 采用免疫组化法检测89例结直肠癌组织和癌旁正常组织中ZNF582蛋白表达,甲基化特异性PCR检测ZNF582基因启动子区的甲基化状态,分析ZNF582基因启动子区甲基化与结直肠癌患者临床参数及预后的关系.结果 与癌旁正常组织相比,ZNF582蛋白在结直肠癌组织中的表达较低(P＜0.05).结直肠癌组织发生启动子区甲基化比例高于癌旁正常组织(53.9％vs.29.2％)(P＜0.05).ZNF582基因启动子区甲基化与结直肠癌患者TNM分期、淋巴结转移、远处转移和分化程度密切相关(P＜0.05).术后随访3个月～5年,ZNF582基因启动子区甲基化患者的5年生存率低于非甲基化患者(P＜0.01).结论 ZNF582基因启动子区在结直肠癌组织中呈现高甲基化状态,与结直肠癌分化和转移密切相关,有助于结直肠癌患者预后的评估.     

5′-氮杂-2′-脱氧胞苷对 HT-29和 LoVo结直肠癌细胞株中 p16基因甲基化状态、mRNA表达及蛋白表达的影响

目的：探讨甲基化酶抑制剂5′-氮杂-2′-脱氧胞苷（5′-Aza-2′-deoxycytidine,5′-Aza-CdR）对结直肠癌细胞株HT-29和Lo-Vo中p16基因甲基化状态、mRNA及蛋白表达的影响。方法应用TaqMan探针为基础的实时定量PCR法、SYBR Green PCR法及蛋白印迹实验（ Western blot ）检测不同浓度5′-Aza-CdR处理前后HT-29和LoVo细胞中p16基因的甲基化状态、mRNA和蛋白表达。结果 TaqMan 探针为基础的实时定量PCR法检测HT-29和LoVo细胞中p16蛋白在药物作用后异常甲基化得到逆转;实时荧光定量PCR和Western Blot检测到0．5、1．0、1．5μM 5′-Aza-CdR处理后p16基因mRNA和蛋白均重新表达,具有统计学意义（P均＜0．05）。结论结直肠癌细胞株HT-29和LoVo中p16启动子甲基化可能是导致该基因表达下调甚至失活的主要原因。5′-Aza-CdR能够较成功的逆转结直肠癌细胞株HT-29和LoVo中p16基因的甲基化状态,并能恢复mRNA及蛋白重新表达。     

肺癌患者p16基因启动子异常甲基化检测及临床意义

目的评价组织和血浆中p16基因启动子区甲基化对肺癌的诊断价值。方法用甲基化PCR的方法检测106例肺癌患者组织和血浆p16基因启动子区域甲基化,并评价其对肺癌的诊断价值。结果106例肺癌患者血浆及对应的肿瘤组织中,p16基因甲基化的检出率分别为36.8%(39/106)和41.5%(44/106),二者之间差异无统计学意义(P>0.05)。血浆p16基因启动子异常甲基化阳性率在第2轮PCR扩增后显著增高(P<0.05)。结论血浆标本中p16基因甲基化作为一项肿瘤标志物为早期诊断肺癌提供了依据。     

结直肠癌中Kiss-1基因启动子甲基化与Kiss-1基因表达的关系

目的：研究结直肠癌中Kiss-1基因启动子甲基化状态对Kiss-1基因表达的影响。方法：应用甲基化特异性PCR （MSP）方法检测73例结直肠癌、正常结直肠组织和人结直肠癌细胞HCT116、SW480、W1116、LoVo中Kiss-1基因启动子甲基化状态,应用realtime-PCR、Western-blot技术检测相应组织和细胞中Kiss-1基因mRNA和蛋白质（Metastine）的表达量。结果：结直肠癌中Kiss-1基因甲基化阳性率（82.19%）高于正常组织（6.31%）（PSW480〉SW1116〉HCT116,差异有统计学意义（p〈0.05）。结论：结直肠癌中Kiss-1基因启动子甲基化可能引起Kiss-1基因表达下调。     

CDH1基因甲基化状态与结直肠癌临床病理特征及预后的相关性分析

目的 检测原发性结直肠癌患者术中腹腔灌洗液悬浮细胞中的CDH1基因启动子所在5''-CpG岛的异常甲基化,同时对其异常甲基化与临床病情发展、病理变化及术后预后的相关关系进行探讨。 方法 选取该院肿瘤科进行结直肠癌切除术的原发性结直肠癌患者,所有入选患者由专人进行跟踪随访。检测CDH1基因启动子所在5''-CpG岛的异常甲基化,对其异常甲基化与临床病情发展、病理变化及术后预后的相关关系进行探讨。 结果 该研究共纳入患者184例,其中甲基化组86例,非甲基化组98例。对两组患者临床病理结果检查可知甲基化组肿瘤直径大于非甲基化组(P＜0.001),甲基化组肿瘤浸润性所占比例高于非甲基化组(P＜0.001),甲基化组肿瘤分化程度低于非甲基化组(P＜0.001),甲基化组淋巴转移率、远处转移率高于非甲基化组(P＜0.001,P=0.026),甲基化组临床TNM分期更晚(P＜0.001)。根据随访结果显示非甲基化组患者生存率高于甲基化组患者(P＜0.05)。Cox比例风险模型结果显示,患者肿瘤的大体分型、分化程度、侵袭程度和病理分期是影响生存预后的变量,其中腹腔灌洗液悬浮细胞CDH1基因甲基化是影响预后最重要的独立因素,RR=28.514。 结论 原发性结直肠癌患者腹腔灌洗液悬浮细胞CDH1基因甲基化程度提高,恶性程度高,预后差。     

早孕妇女血浆中胎儿游离DNA的检测

目的:建立一种检测早孕妇女血浆中胎儿游离DNA的新方法。方法:从50例妊娠7、8、9周的孕妇外周血血浆中提取胎儿游离DNA,用实时荧光定量聚合酶链反应(FQ-PCR)技术检测其中的Y性别决定区(SRY)基因。结果:38例早孕孕男胎妇女中32例血浆中出现SRY基因,灵敏度84.21%;12例早孕孕女胎的妇女血浆中均未出现SRY基因,特异度100%。结论:用实时荧光定量PCR技术可用来检测早孕妇女血浆中的胎儿游离DNA,这对无创性早期产前基因诊断方法的建立具有重大意义。     

Sensitive quantitation of chromium-DNA adducts by inductively coupled plasma mass spectrometry with a direct injection high-efficiency nebulizer.

A novel method is described for the sensitive detection of chromium-DNA adducts. Chromium-DNA adducts were determined in 1 microgram of DNA from normal human lung fibroblasts exposed to sodium chromate using microscale flow injection analysis with a direct injection high-efficiency nebulizer and inductively coupled plasma mass spectrometry detection. The frequency of Cr-DNA adducts increased in a dose-dependent sigmoidal manner, indicating saturation and toxicity. The low detection limits (on the order of parts per trillion) allows the detection of as few as two Cr adducts per 10,000 bases, which, coupled with the small DNA sample requirement, makes this technique suitable for measuring metal-DNA adducts as biomarkers of exposure to toxic and carcinogenic metals such as Cr, in cultured cells, animals, and humans.     

15例丙戊酸钠玫血浆纤维蛋白原降低分析

目的：研究丙戊酸钠对血浆纤维蛋白原的影响。方法：采用回顾性调查的方法,对符合入选标准的使用丙戊酸钠出现血浆纤维蛋白原降低的15位住院患者的一般情况、丙戊酸钠的用药情况、停药前后纤维蛋白原含量进行调查和分析。结果：患者平均用药时间（18．64±15．46）个月,平均用药剂量（15．49±7．12）mg·kg^-1·d^-1平均纤维蛋白原含量（1．51±0．19）g·L^-1,平均停药时间（7．73±3．76）d后,纤维蛋白原量为（3．52±1．61）g·L^-1。结论：丙戊酸钠可致血浆纤维蛋白原降低。     

HPLC determination of naproxen in plasma

An assay method using isocratic HPLC with fluorometric detection for the determination of naproxen sodium in plasma is presented. A reverse phase Microbondapack column was used with a mobile phase consisting of 42% acetonitrile and 58% water adjusted to pH 3 using phosphoric acid. The fluorometric detector with an excitation wavelength of 270 nm and emission wavelength of 340 nm provided high sensitivity and no interferences from plasma constituents. Plasma samples were injected to HPLC without any extraction. The method was precise and reproducible as was demonstrated by replicate analysis of pooled plasma sample containing 0.5-80 microg/ml naproxen sodium.     

A simple and rapid HPLC method for the determination of atorvastatin in human plasma with UV detection and its application to pharmacokinetic studies

A rapid and sensitive high-performance liquid chromatographic method for the determination of atorvastatin (CAS 134523-00-5) in plasma was developed in this study. Atorvastatin was isolated from plasma using protein precipitation by acetonitrile. Diltiazem (CAS 33286-22-5) was used as internal standard. The chromatographic conditions were as follows: analytical 125 x 4 mm (i.d.) Nucleosil C8 column (5 microm particle size), mobile phase consisting of sodium dihydrogen phosphate buffer-acetonitrile (60:40, v/v) adjusted to pH 5.5 at a flow rate of 1.5 ml/min, UV detection at 245 nm. The detection limit for atorvastatin in plasma was 1 ng ml(-1). The calibration curve was linear over the concentration range 20-800 ng ml(-1). The recovery was complete. The inter-day and intra-day assay coefficients of variation were found to be less than 7%. The present validated method was successfully used for pharmacokinetic studies of atorvastatin in human subjects.     

A quantitative PCR screening method for adeno-associated viral vector 2-mediated gene doping

Gene therapy is currently prohibited in human and equine athletes and novel analytical methods are needed for its detection. Most in vivo products use non-integrating, recombinant viral vectors derived from adeno-associated virus (AAV) to deliver transgenes into cells, where they are transcribed and translated into functional proteins. Although the majority of wild-type AAV (WTAAV) DNA is removed from recombinant AAV (rAAV) vectors, some sequences are conserved. The goal of this study was to develop a quantitative polymerase chain reaction (QPCR) screening test targeting conserved AAV sequences to enable theoretical detection of all rAAV gene therapy products, regardless of encoded transgenes while excluding the presence of WTAAV DNA in horses. Primer sets were developed and validated to target an AAV2 sequence highly conserved across rAAV viral vectors and a sequence only found in wild type AAV2 (WTAAV2). Six horses were administered an intra-articular injection of rAAV. Plasma and synovial fluid were collected on days 0, 1, 2, 4, 7, 14, 28, 56, and 84. Using QPCR, rAAV was detected in plasma for up to 2–4 days in all horses. rAAV DNA was detected for 28 days in synovial fluid from two horses for which synovial fluid samples were available. No WTAAV2 DNA was detected in any sample. This is the first study to develop a QPCR test capable of screening for rAAV vectors that may be used for gene doping in horses.     

Dextran sulfate sodium-induced ulcerative colitis leads to testicular toxicity in mice: Role of inflammation,oxidative stress and DNA damage

Ulcerative colitis is associated with an alteration in gonadal hormones and affects testicular weight in rodents. However, association of ulcerative colitis with testicular damage is not clearly known. Ulcerative colitis was induced using 5% (w/v) dextran sulfate sodium in normal drinking water for 1, 7-day cycle in short-term study and 2.5% (w/v) dextran sulfate sodium in normal drinking water for 4 cycles with 2 weeks remission period between each cycle in long-term study. Ulcerative colitis was associated with a significant increase in inflammation, oxidative stress, DNA damage in testes and sperm DNA damage and a significant decrease in the epididymal sperm count and 3β-HSD expression. No difference was observed in the plasma testosterone levels between control and treatment groups. In the present study, ulcerative colitis was associated with testicular damage, and juvenile mice were found to be more sensitive than adult mice.     

新型抗凝药磺达肝癸钠人血浓度测定方法的建立

目的建立人血浆中磺达肝癸钠浓度的检测方法。方法使用肝素试剂盒拟合标准曲线,测定其检测限、精密度、回收率和稳定性,并检测开胸术后使用磺达肝癸钠进行预防血栓治疗的患者血样156份。结果磺达肝癸钠质量浓度在0．1-1．5mg·L^-1时线性良好（r^2=0．996）,方法回收率99．5％-102％,日内和日间RSD分别为4．2％-8．44％和2．73％-9．51％。结论肝素试剂盒用于检测磺达肝癸钠的血浆浓度简便、快捷,可用于临床药动学研究。     

The effects of sulfur-containing compounds and gemcitabine on the binding of cisplatin to plasma proteins and DNA determined by inductively coupled plasma mass spectrometry and high performance liquid chromatography-inductively coupled plasma mass spectrometry

The aim of this study was to investigate the effects of sodium thiosulfate (STS), glutathione (GSH), acetylcysteine (AC), and gemcitabine on the platinum-protein (Pt-protein) and platinum-DNA (Pt-DNA) binding of cisplatin in whole blood. This was done to obtain more insight into the platinum (Pt) binding in whole blood and the effects of modulators on this process. STS, GSH, AC, and gemcitabine were added before and after the incubation of whole blood with cisplatin. Pt levels in plasma and plasma ultrafiltrate and the Pt that is bound to DNA in peripheral blood mononuclear cells were determined using inductively coupled plasma mass spectrometry. Additionally, information on the major Pt-DNA adducts was obtained by separation of the Pt-DNA adducts by high performance liquid chromatography with off-line inductively coupled plasma mass spectrometry detection. Results showed that the reactive Pt levels in whole blood are reduced by STS, GSH, and AC. This reduction was demonstrated by a reduced Pt-protein and Pt-DNA binding in the presence of sulfur compounds. Furthermore, STS and AC seemed to be able to release Pt from proteins. The compounds could hardly release Pt from the DNA. Gemcitabine slightly inhibited Pt-DNA binding and did not alter Pt-protein binding. The type of Pt-DNA adducts found were not altered in the presence of the modulators. In conclusion, the results of this study illustrate that STS, GSH, and AC affect Pt binding in whole blood, which suggests that these compounds could affect Pt binding in patients. By interfering with Pt-DNA and Pt-protein binding, the compounds could influence side effects and cytotoxicity.     

Regulation of apoptosis in HL-1 cardiomyocytes by phosphorylation of the receptor tyrosine kinase EphA2 and protection by lithocholic acid

Background and Purpose

Heart failure and atrial fibrillation are associated with apoptosis of cardiomyocytes, suggesting common abnormalities in pro-apoptotic cardiac molecules. Activation of the receptor tyrosine kinase EphA2 causes apoptosis in vitro, and dysregulation of EphA2-dependent signalling is implicated in LEOPARD and Noonan syndromes associated with cardiomyopathy. Molecular pathways and regulation of EphA2 signalling in the heart are poorly understood. Here we elucidated the pathways of EphA2-dependent apoptosis and evaluated a therapeutic strategy to prevent EphA2 activation and cardiac cell death.

Experimental Approach

EphA2 signalling was studied in an established model of doxazosin-induced apoptosis in HL-1 cells. Apoptosis was measured with TUNEL assays and as cell viability using a formazan method. Western blotting and siRNA for EphA2 were also used.

Key Results

Apoptosis induced by doxazosin (EC₅₀ = 17.3 μM) was associated with EphA2 activation through enhanced phosphorylation (2.2-fold). Activation of pro-apoptotic downstream factors, phospho-SHP-2 (3.9-fold), phospho-p38 MAPK (2.3-fold) and GADD153 (1.6-fold) resulted in cleavage of caspase 3. Furthermore, two anti-apoptotic enzymes were suppressed (focal adhesion kinase, by 41%; phospho-Akt, by 78%). Inactivation of EphA2 with appropriate siRNA mimicked pro-apoptotic effects of doxazosin. Finally, administration of lithocholic acid (LCA) protected against apoptosis by increasing EphA2 protein levels and decreasing EphA2 phosphorylation.

Conclusions and Implications

EphA2 phosphorylation and activation of SHP-2 are critical steps in apoptosis. Reduction of EphA2 phosphorylation by LCA may represent a novel approach for future anti-apoptotic treatment of heart failure and atrial fibrillation.