Methylation of CDH13 gene in colorectai cancer and its clinical significance

目的 探讨H-钙黏素(CDH13)基因在结直肠癌(CRC)中的甲基化状态及其临床意义.方法 选取85例CRC患者组织及其相应正常组织,显微切割结合甲基化特异性聚合酶链反应检测CDH13基因启动子区域甲基化.结果 85例CRC组织中,27例(31.8％)检出CDH13基因甲基化,显著多于相应正常组织的8例(9.4％)(P＜0.01).CRC组织中CDH13基因异常甲基化与肿瘤低分化有关(P＜0.05),并且在进展期及淋巴结转移的患者中显示出一定优势.CDH13甲基化与不良总生存(()S)有关(P＜0.01),是其独立预后指标.结论 CDH13基因甲基化有可能成为CRC预后判断的新型分子标记.     

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.     

结直肠癌中CDH13基因甲基化及其临床意义

目的探讨H-钙黏素(CDH13)基因在结直肠癌(CRC)中的甲基化状态及其临床意义。方法选取85例CRC患者组织及其相应正常组织,显微切割结合甲基化特异性聚合酶链反应检测CDH13基因启动子区域甲基化。结果 85例CRC组织中,27例(31.8%)检出CDH13基因甲基化,显著多于相应正常组织的8例(9.4%)(P<0.01)。CRC组织中CDH13基因异常甲基化与肿瘤低分化有关(P<0.05),并且在进展期及淋巴结转移的患者中显示出一定优势。CDH13甲基化与不良总生存(OS)有关(P<0.01),是其独立预后指标。结论 CDH13基因甲基化有可能成为CRC预后判断的新型分子标记。     

肺癌患者CDH13基因启动子甲基化状态研究

目的 应用甲基化特异性PCR(MSP)方法检测非小细胞肺癌(NSCLC) CDH13基因启动子甲基化情况.方法 留取44例非小细胞肺癌患者手术标本及12例非肺癌患者正常肺组织,MSP分析CDH 13基因启动子甲基化情况.结果 44例非小细胞肺癌中CDH 13基因启动子甲基化阳性率为54.5%(24/44),12例正常肺组织中未检测到此基因的甲基化(0/12).CDH13基因甲基化与患者性别及吸烟与否无关;肿瘤分期越晚甲基化的发生率越高.结论 原发性非小细胞肺癌中存在着较高比例的CDH13启动子甲基化,提示CDH13在非小细胞肺癌的发生中起着重要作用.     

分泌型卷曲相关蛋白2启动子区甲基化与β联蛋白异常表达在结直肠癌中的意义

目的研究结直肠癌中Wnt/β-联蛋白(catenin)信号通路调控因子分泌型卷曲相关蛋白(SFRP)2基因启动子区甲基化状态及β-catenin蛋白的表达模式,探讨两者在结直肠癌中的关系及意义。方法用甲基化特异性聚合酶链反应(MSP)法和免疫组织化学法分别检测结直肠癌组、腺瘤组、非腺瘤性息肉组、健康对照组中SFRP2基因启动子区甲基化状态和β-catenin蛋白的表达情况。结果在结直肠癌组、腺瘤组、非腺瘤性息肉组、健康对照组中,SFRP2基因启动子区甲基化率分别为79%、63%、8%、0,差异有统计学意义(P<0.05);β-catenin蛋白表达率分别为74%、48%、4%、0,差异有统计学意义(P<0.05);SFRP2启动子区甲基化与β-catenin异常表达之间存在相关性(P<0.01);SFRP2启动子区甲基化和β-catenin异常表达均与肿瘤分化程度有关(P<0.05)。结论 SFRP2启动子区域甲基化频率及β-catenin异常表达率在结直肠癌中上调,两者可能共同促进了结直肠癌的发生发展。     

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。     

宫颈癌DKK-3基因启动子甲基化检测及其临床意义

目的 检测宫颈癌DKK-3基因启动子甲基化情况并研究其临床意义.方法 采用甲基化特异性PCR检测宫颈癌患者和健康女性宫颈活检组织或术后病理组织DKK-3基因启动子甲基化情况,比较其差异并分析宫颈癌患者DKK-3基因启动子甲基化与临床病理因素的关系.结果 宫颈癌患者DKK-3基因启动子甲基化率显著高于健康女性（P＜0.05）;宫颈癌患者DKK-3基因启动子甲基化率在年龄、吸烟史、酗酒史和病理类型中差异均无统计学意义（P＞0.05）,在HPV感染、分化程度、肿瘤直径、淋巴结转移、远处转移和Figo分期中的差异均有统计学意义（P＜0.05）.结论 宫颈癌患者DKK-3基因启动子甲基化率显著高于健康女性,且与病情及预后密切相关.     

非小细胞肺癌中E-cadherin基因甲基化及蛋白表达的研究

目的 本实验通过探讨非小细胞肺癌癌组织与相应癌旁组织中E-cadherin(E-cad)基因异常甲基化改变及其蛋白表达的特点,以及其与肺癌发生发展的关系,从而为今后肺癌的诊断,治疗及预后提供新思路.方法 应用DNA提取、甲基化特异PCR、蛋白电泳及免疫组化技术对肺癌组织与相应癌旁组织进行相关研究.结果 50例癌组织的E-cadherin蛋白表达阳性率为32％(16/50),相应的癌旁组织阳性率表达为84％(42/50);癌组织中E-cadherin蛋白表达显著低于相应癌旁组织蛋白表达(P＜0.001);同时50例癌组织及其对应癌旁组织的甲基化阳性率分别为74％(37/50)、40％(20/50),癌组织的E-cadherin甲基化阳性率高于相应的癌旁组织(P＜0.001).结论 非小细胞肺癌组织中E-cadhenn蛋白呈低表达,癌组织的E-cadherin基因启动子存在异常甲基化,E-cadherin基因启动子的异常甲基化可能与E-cadherin蛋白表达下调具有相关性,可作为肿瘤恶性程度的良好指标.     

血浆RUNX3基因启动子甲基化检测对宫颈癌早期诊断的价值研究

目的探究血浆人类Runt相关转录因子3(RUNX3)基因启动子甲基化对早期宫颈癌的诊断价值。方法选取80例宫颈癌患者作为A组,选取同期80例宫颈上皮内瘤变(CIN)患者作为B组,另选取80例正常宫颈组织切除者作为C组。对三组研究对象的宫颈癌组织、CIN组织、正常宫颈组织及血浆RUNX3启动子甲基化应用甲基化特异性聚合酶链反应(MCP)进行检测。比较三组组织、血浆RUNX3启动子甲基化异常率,分析宫颈癌病理特征与RUNX3启动子甲基化表达及RUNX3蛋白表达的关系。结果A组组织RUNX3启动子甲基化异常率为86.25%,血浆RUNX3启动子甲基化异常率为73.75%;B组组织RUNX3启动子甲基化异常率为33.75%,血浆RUNX3启动子甲基化异常率为26.25%;C组组织RUNX3启动子甲基化异常率为3.75%,血浆RUNX3启动子甲基化异常率为0;A组组织RUNX3启动子甲基化异常率和血浆RUNX3启动子甲基化异常率均高于B组和C组,差异有统计学意义(P<0.05)。血浆、组织RUNX3启动子甲基化异常与临床分期、肿瘤分化程度、病理类型、淋巴转移存在相关性(P<0.05)。宫颈癌患者RUNX3蛋白异常表达与淋巴转移、肿瘤分化程度、临床分期存在相关性(P<0.05)。结论血浆RUNX3基因启动子甲基化异常可提示宫颈癌的发生发展,为早期诊断宫颈癌提供新方法。     

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     

The adrenergic,cholinergic and NANC nerve-mediated contractions of the female rabbit bladder neck and proximal,medial and distal urethra

1. The nerve-mediated contraction of the female rabbit bladder neck and different portions of the urethra (proximal, medial and distal) was studied in vitro by electrical stimulation (50 V, 30 Hz, 0.05 ms width, trains of 5 s every 5 min) by use of a superfusion system.
2. The amplitude (E_max) and the duration (D_max) of the stimulated contraction were studied in the four tissues. The E_max value was significantly higher in distal urethra (2.07±0.15 g) compared to the bladder neck (1.08±0.10 g), proximal urethra (0.73±0.07 g) and medial urethra (0.87±0.07 g). In contrast, the D_max value appeared slightly but significantly lower (P<0.05) in distal urethra (68.5±2.3 s) than in bladder neck (76.7±6.0 s), proximal urethra (84.5±5.0 s) and medial urethra (81.3±3.5 s).
3. Cocaine (1 μM) significantly increased the basal E_max values in medial and distal urethra and the basal D_max values in the four tissues.
4. Prazosin (1 μM) significantly reduced E_max value in proximal, medial and distal urethra and D_max value in bladder neck and proximal urethra. Atropine (1 μM) also significantly reduced E_max values in bladder neck and proximal urethra and reduced D_max value in bladder neck, but not in other tissues. Yohimbine (0.1 μM) was devoid of effect in the four tissues.
5. The association of prazosin (1 μM) and atropine (1 μM) did not modify the E_max and the D_max values of the electrically-induced contractions, except in proximal urethra and in bladder neck where an additive inhibitory effect (on E_max only) was observed compared to prazosin and atropine alone.
6. The residual contractile response after combined treatment with prazosin and atropine was significantly diminished by tetrodotoxin (TTX; 1 μM) but not completely abolished. These NANC contractions were insensitive to P2X-purinoceptor desensitization by continuous tissue perfusion with α,β-methylene ATP (30 μM).
7. These results demonstrate that bladder neck and proximal urethra are mainly innervated by the parasympathetic nervous system, whereas medial and distal urethras are to a greater extent under the control of the sympathetic innervation. The residual responses, insensitive to prazosin and atropine, may indicate a NANC innervation in the four tissues. However, the nature of the NANC neurotransmitter remains to be identified.

     

The use of biomarkers in occupational health research, practice, and policy

Biomarkers are potentially useful tools for occupational health and safety research, practice, and policy. However, the full realization of this potential has not been achieved. In this paper, the progress made in these three usage areas is reviewed to identify what efforts can be taken to realize the full promise of biomarkers. Biomarker uses are described by a diverse taxonomy that builds on the categories of exposure, effect and susceptibility, and the continuum between exposure and disease prognosis. The most significant uses of biomarkers in occupational health have been in biological monitoring of workers. Other important uses have been in enhancing research and assessing mechanisms of action of occupational toxicants at low exposures. Seven critical areas will influence the extent to which the potential of biomarkers in occupational health and safety is realized. These include: (1) adequate investment in validation; (2) obtaining international agreement on exposure guidelines; (3) exploring the utility of biomarkers in regulation; (4) applying biomarkers to critical occupational safety and health questions; (5) developing the exposome; (6) utilizing biomarkers to address emerging occupational health issues; and (7) continuing to address the ethical and social justice issues related to biomarkers. Overall, if biomarkers are to make a major contribution to occupational health and safety then a more holistic approach to bringing them from the laboratory to practice will be needed.     

The roles of CYP450 epoxygenases and metabolites, epoxyeicosatrienoic acids, in cardiovascular and malignant diseases

Cytochrome P450 (CYP) epoxygenases metabolize arachidonic acid to biologically active eicosanoids. The primary epoxidation products are four regioisomers of cis-epoxyeicosatrienoic acid (EET): 5,6-, 8,9-, 11,12-, and 14,15-EET. CYP2J2, CYP2C8, and CYP2C9 are the predominant epoxygenase isoforms involved in EET formation. CYP2J and CYP2C gene families in humans are abundantly expressed in the endothelium, myocardium, and kidney. The cardiovascular effects of CYP epoxygenases and EETs range from vasodilation, anti-hypertension, pro-angiogenesis, anti-atherosclerosis, and anti-inflammation to anti-injury caused by ischemia-reperfusion. Using transgenic animals for in vivo analyses of CYP epoxygenases revealed comprehensive and marked cardiovascular protective effects. In contrast, CYP epoxygenases and their metabolites, EETs, are upregulated in human tumors and promote tumor progression and metastasis. These biological effects result from the anti-apoptosis, pro-mitogenesis, and anti-migration roles of CYP epoxygenases and EETs at the cellular level. Importantly, soluble epoxide hydrolase (sEH) inhibitors are anti-hypertensive and anti-inflammatory and, therefore, protect the heart from damage, whereas the terfenadine-related, specific inhibitors of CYP2J2 exhibit strong anti-tumor activity in vitro and in vivo. Thus, CYP2J2 and arachidonic acid-derived metabolites likely play important roles in regulating cardiovascular functions and malignancy under physiological and/or pathological conditions. Moreover, although challenges remain to improving the drug-like properties of sEH inhibitors and identifying efficient ways to deliver sEH inhibitors, sEH will likely become an important therapeutic target for cardiovascular diseases. In addition, CYP2J2 may be a therapeutic target for treating human cancers and leukemia.     

The antioxidant activity of chondroitin-4-sulphate, in carbon tetrachloride-induced acute hepatitis in mice, involves NF-kappaB and caspase activation

Background and purpose:

Reactive oxygen species (ROC) are the main causes of carbon tetrachloride (CCl₄)-induced acute liver injury. Chondroitin-4-sulphate (C4S) is known to inhibit lipid peroxidation through antioxidant mechanisms. Activation of nuclear factor (NF)-κB and caspases may strongly intensify inflammation and cell damage, in addition to that directly exerted by ROS. We investigated whether treatment with C4S, besides exerting antioxidant activity, was able to modulate NF-κB and apoptosis activation in CCl₄-induced liver injury in mice.

Experimental approach:

Acute hepatitis was induced in mice by an i.p. injection of CCl₄. Varying doses of C4S were administered i.p. 1 h before, 6 and 12 h after CCl₄ injection. 24 h after CCl₄ injection, the mice were killed for biochemical and histological analysis.

Key results:

CCl₄ injection produced: marked elevation of alanine aminotransferase and aspartate aminotransferase; hepatic membrane lipid peroxidation, assayed by 8-isoprostane levels; and depletion of reduced glutathione and superoxide dismutase. CCl₄ also decreased NF-κB translocation and IkBα, and increased gene expression of mRNA and protein of metalloproteases (MMP)-2 and -9, and of pro- and cleaved forms of caspases-3 and -7. There was also increased liver polymorphonuclear infiltration, evaluated by elastase assay, and hepatic cell disruption.C4S treatment inhibited lipid peroxidation; blocked NF-κB activation and IkBα protein loss; decreased mRNA and proteins for MMPs and caspases; restored endogenous antioxidants; limited hepatic polymorphonuclear accumulation and tissue damage.

Conclusions and implications:

As antioxidants may inhibit NF-κB and caspase activation, we hypothesize that treatment with C4S was able to inhibit NF-κB and apoptosis activation in hepatic injury.     

从热郁痰瘀论治小儿病毒性肺炎的理论研究

在临床研究的基础上,通过对小儿病毒性肺炎病因病机、辨证治疗的分析探讨,提出以热郁痰瘀理论指导小儿病毒性肺炎的辨证论治。这对加强中医药治疗小儿病毒性肺炎的理论认识和临床运用都具有启发作用。     

The toxicity of dimethyl sulphoxide (DMSO) for the dog,pig, rat and rabbit

Dimethyl sulphoxide (DMSO) was tested for oral toxicity in rats and dogs, and dermal toxicity in rabbits and pigs. Oral administration was by gastric intubation as a 50% aqueous solution, 5 days/week at levels equivalent to 9.0, 3.0 or 1.0 ml undiluted DMSO/kg/day. For dermal application 50% and 90% aqueous solutions were used to give levels equivalent to 8.1, 4.5, 2.7 or 1.5 ml DMSO/kg/day, as one daily application for rabbits, and divided into two applications/day for pigs. Dogs were dosed for approximately 2 years and pigs for 1 year, although half the animals of both species were dosed for only 18 weeks. Rats were dosed for 18 months, but some were used for interim sacrifice after a year. Rabbits received applications to normal and abraded skin for 6 months.Minor changes in bodyweight and haematological values were observed, together with a physiological diuretic response to DMSO, but the target organ was the eye, principally the lenticular nucleus. Ocular effects in dogs started after 5–10 weeks dosing at 9 ml/kg and consisted of central (nuclear) lens changes with alteration of the refractive index (myopia); transitory equatorial opacities during the 5th month; central (nuclear) opalescence; and changes in the vitreous humour. Similar changes occurred more slowly at 3 ml/kg, the alterations to the vitreous being first observed after 9–10 months at this level. Progressive nuclear refractive changes occurred after dosing for considerably longer than 6 months at 1 ml/kg, but none of the animals in this group manifested the opalescence. Biochemical investigation of the lenses revealed reduction of soluble protein (mainly α-crystallin), glutathione and water levels, and an increase of insoluble protein. Evidence of recovery was limited mainly to a reduction in the number of dioptres needed to correct nuclear refractive change. Cessation of dosing led to regression of refractive nuclear changes but did not prevent the appearance of opalescence at 3 ml/kg and above.Dogs were the most severely affected of the 4 species, with nuclear effects at 1 ml/kg, extensive changes in the lens, and involvement of the vitreous. Pigs and rabbits were affected by dose levels of 2.7 ml/kg and 1.5 ml/kg respectively. Rats occasionally showed minimal changes at 9 ml/kg.The importance of the findings in dogs is discussed in relation to general toxicology protocols. It is emphasised that reversibility of signs, and adequate duration of administration, must both be considered when ascertaining whether changes occur at levels approximating to those of human intake.     

The Effect of Polyphenols Isolated from Cynanchi wilfordii Radix with Anti-inflammatory,Antioxidant, and Anti-bacterial Activity

Recently, Cynanchi wilfordii Radix has gained wide use in Asian countries as a functional food effective for relieving fatigue, osteoporosis, and constipation, particularly in menopausal disorders. However, its anti-inflammatory and anti-microbial activities have not been explored in detail to date. The anti-inflammatory, antioxidant, and anti-bacterial properties of the Cynanchi wilfordii Radix extracts obtained with water, methanol, ethanol, and acetone were compared. All 4 polyphenol-containing extracts exhibited anti-inflammatory and antioxidant effects. The ethanol extract was found to elicit the most potent reduction of nitric oxide (NO), prostaglandin E₂ (PGE₂), and cytokine (IL-1β, IL-6, IL-10, and TNF-α) levels, as well as inhibit the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in a concentration-dependent manner. The evaluation of antioxidant activity also revealed the ethanol extract to have the highest free radical scavenging activity, measured as 85.3±0.4%, which is equivalent to 99.9% of the activity of α -tocopherol. In the assessment of anti-bacterial activity, only ethanol extract was found to inhibit the growth of the Bacillus species Bacillus cereus and Bacillus anthracis. These results show that polyphenols of Cynanchi wilfordii Radix have anti-inflammatory, antioxidant, and anti-bacterial properties that can be exploited and further improved for use as a supplementary functional food, in cosmetics, and for pharmaceutical purposes.     

The farnesyltransferase inhibitor, LB42708, inhibits growth and induces apoptosis irreversibly in H-ras and K-ras-transformed rat intestinal epithelial cells

LB42708 (LB7) and LB42908 (LB9) are pyrrole-based orally active farnesyltransferase inhibitors (FTIs) that have similar structures. The in vitro potencies of these compounds against FTase and GGTase I are remarkably similar, and yet they display different activity in apoptosis induction and morphological reversion of ras-transformed rat intestinal epithelial (RIE) cells. Both FTIs induced cell death despite K-ras prenylation, implying the participation of Ras-independent mechanism(s). Growth inhibition by these two FTIs was accompanied by G1 and G2/M cell cycle arrests in H-ras and K-ras-transformed RIE cells, respectively. We identified three key markers, p21(CIP1/WAF1), RhoB and EGFR, that can explain the differences in the molecular mechanism of action between two FTIs. Only LB7 induced the upregulation of p21(CIP1/WAF1) and RhoB above the basal level that led to the cell cycle arrest and to distinct morphological alterations of ras-transformed RIE cells. Both FTIs successfully inhibited the ERK and activated JNK in RIE/K-ras cells. While the addition of conditioned medium from RIE/K-ras reversed the growth inhibition of ras-transformed RIE cells by LB9, it failed to overcome the growth inhibitory effect of LB7 in both H-ras- and K-ras-transformed RIE cells. We found that LB7, but not LB9, decreased the expression of EGFRs that confers the cellular unresponsiveness to EGFR ligands. These results suggest that LB7 causes the induction of p21(CIP1/WAF1) and RhoB and downregulation of EGFR that may serve as critical steps in the mechanism by which FTIs trigger irreversible inhibitions on the cell growth and apoptosis in ras-transformed cells.     

The 5-HT2C Receptor Agonist Lorcaserin Reduces Nicotine Self-Administration,Discrimination, and Reinstatement: Relationship to Feeding Behavior and Impulse Control

Lorcaserin ((1R)-8-chloro-1-methyl-2,3,4,5-tetrahydro-1H-3-benzazepine HCl) is a selective 5-HT_2C receptor agonist with clinical efficacy in phase-III obesity trials. Based on evidence that this drug class also affects behaviors motivated by drug reinforcement, we compared the effect of lorcaserin on behavior maintained by food and nicotine reinforcement, as well as the stimulant and discriminative stimulus properties of nicotine in the rat. Acutely administered lorcaserin (0.3–3 mg/kg, subcutaneous (SC)) dose dependently reduced feeding induced by 22-h food deprivation or palatability. Effects up to 1 mg/kg were consistent with a specific effect on feeding motivation. Lorcaserin (0.6–1 mg/kg, SC) reduced operant responding for food on progressive and fixed ratio schedules of reinforcement. In this dose range lorcaserin also reversed the motor stimulant effect of nicotine, reduced intravenous self-administration of nicotine, and attenuated the nicotine cue in rats trained to discriminate nicotine from saline. Lorcaserin also reduced the reinstatement of nicotine-seeking behavior elicited by a compound cue comprising a nicotine prime and conditioned stimulus previously paired with nicotine reinforcement. Lorcaserin did not reinstate nicotine-seeking behavior or substitute for a nicotine cue. Finally, lorcaserin (0.3–1 mg/kg) reduced nicotine-induced increases in anticipatory responding, a measure of impulsive action, in rats performing the five-choice serial reaction time task. Importantly, these results indicate that lorcaserin, and likely other selective 5-HT_2C receptor agonists, similarly affect both food- and nicotine-motivated behaviors, and nicotine-induced impulsivity. Collectively, these findings highlight a therapeutic potential for 5-HT_2C agonists such as lorcaserin beyond obesity into addictive behaviors, such as nicotine dependence.