苯对小鼠淋巴细胞端粒酶活性的影响及硒的调节作用
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摘要
苯系化合物为一类芳香族化合物,多为无色至浅黄色透明、易挥发的油状液体,具有强烈芳香气味,主要包括苯(Benzene)、甲苯(Toluene)、二甲苯(Xylene)等。苯系化合物多引起骨髓抑制及免疫系统抑制,主要产生血液毒性,可导致细胞及基因毒性。它可产生多种生物学效应,引起造血细胞凋亡增多、肝细胞及祖细胞复制增殖。抑制细胞因子分泌,影响T淋巴细胞的功能及外周血中亚群的改变。引起外周血血象改变,淋巴细胞及红细胞减少,引起微核。导致细胞凋亡、突变、细胞复制,引起DNA断裂,致淋巴细胞结构及其它方面染色体畸变,染色体损伤,非整倍体改变、染色体缺失、染色体易位等。苯进入人体,首先输入肝,在相关酶的作用下,活化代谢,其代谢产物及中间产物随即分布至血液、肾脏、脾脏、胸腺、骨髓等造血器官,作用于淋巴细胞和骨髓细胞,经过对染色体损害,代谢产物可引起血液疾病及癌样变。它可引起再障贫血,引起急性髓性白血病、血管瘤、骨髓异常增生综合症、腺淋巴瘤、慢性淋巴瘤、慢性白血病等造血器官肿瘤,苯亦可导致多器官肿瘤。苯及其代谢物选择性作用于某些非致死性染色体,端粒是苯的敏感作用位点及检测的选择点。针对苯的遗传毒性、染色体损伤、及肿瘤建模的研究已经多见。苯中毒的研究已从染色体改变深入到基因水平研究。
端粒酶是逆转录酶,由RNA和蛋白质组成的核糖蛋白酶,它主要有三个组成成分:端粒酶RNA组分(telomerase RNA component, TERC)、端粒酶相关蛋白(telomerase-associated protein, TELPI)和端粒酶逆转录酶催化蛋白亚单位(telomerase reverse transcriptase, TERT)。端粒酶能合成新的端粒DNA,添加到染色体末端,来补偿端粒的丢失、粘连、重组阻止端粒的缩短,使细胞逃脱程序性死亡。在大多数体细胞和正常组织中,端粒酶活性不表达或低水平表达,生殖细胞、胚胎细胞、造血干细胞、骨髓细胞、肿瘤、癌组织中高表达。它使端粒,染色体末端重复DNA系列呈帽子结构维持染色体稳定性,与细胞衰老、分化、肿瘤形成有关,维持长度并跨过生物钟,进行分裂,细胞衰老、凋亡、永生化过程中激活端粒酶高表达,引起肿瘤和癌症,并且表达水平同肿瘤恶化程度正相关。端粒酶激活是肿瘤形成的必经途径,它是肿瘤具特异性和普遍性的效应标志物和早期分子生物学标志物。
硒具有营养作用,在体内发挥消除自由基、抗氧化等作用。营养剂量硒具有激活端粒酶活性、延长端粒、延长细胞寿命的功能。它在肿瘤的发生发展过程中起预防作用,它可以抗衰老、抑制肿瘤、癌症。
苯对细胞产生毒性,我们采用动物实验构建模型对其进行毒性评价。选用健康昆明小鼠,随机分成几组,分别设不同剂量组用苯对其进行染毒,并设不同剂量苯联合硒组进行处理,同时设阴性对照和试剂对照。染毒后观察变化,收获外周血淋巴细胞,采用端粒重复扩增分析法(telomere repeat amplification protocol assay, TRAP)法检测其端粒酶活性。分析苯及硒对淋巴细胞端粒酶活性的影响,观察两者的相关性。鉴于此,我们研究了苯对小鼠淋巴细胞端粒酶活性的毒性影响及硒的调节作用,进一步了解苯致肿瘤及癌症的机制,旨在从端粒系统获得新的苯暴露早期效应生物学标志物和易感性标志物。
第一部分苯对小鼠淋巴细胞端粒酶活性的影响及硒的调节作用
目的:本部分通过研究苯对小鼠淋巴细胞端粒酶活性的影响及硒的调节作用,评价端粒酶作为生物标志物对苯致癌症的分子机制的影响,旨在获得新的苯暴露早期效应生物学标志物和易感性标志物。方法:选择6~8周龄的健康雄性昆明小鼠,25-30g左右,40只随机分成8组,每组5只。分别设100.0 mg/kg苯、200.0 mg/kg苯及400.0 mg/kg苯染毒组,200.0 mg/kg苯+ 0.75 mg/kg亚硒酸钠、200.0 mg/kg苯+ 1.5 mg/kg亚硒酸钠及200.0 mg/kg苯+ 3.0 mg/kg亚硒酸钠处理组,阴性对照组及试剂对照组。对小鼠采用腹腔注射染毒,连续5天,末次染毒48h后,眼球摘除取外周血,分离淋巴细胞。用TRAP-ELISA法定量分析淋巴细胞端粒酶活性,在前引物P1-TS的作用下,端粒酶先以自身为模板合成端粒DNA,然后在前引物P1-TS和后引物P2作用下对TTAGGG结构进行PCR特异性扩增。取5μl PCR产物进行变性,再与地高辛标记的探针进行杂交,加入到生物素标记的微孔板中,最后加入TMB底物,在450nm和690nm波长处读取溶液的OD值,A450-A690的值即我们的端粒酶活性值。比较不同组间的端粒酶活性差异,分析苯对端粒酶的毒性作用和硒的调节作用,利用统计软件分析实验数据。结果:阴性对照和试剂对照呈现微弱的端粒酶活性表达,活性值均在正常范围内。各剂量苯处理组与阴性对照和试剂对照相比,端粒酶活性阳性,均值范围均升高,其中100.0 mg/kg苯组有统计学差异(P<0.01),说明苯刺激了端粒酶的表达。与阴性对照和试剂对照相比,各剂量苯联合硒作用组端粒酶活性均增高,其中200.0 mg/kg苯+ 0.75 mg/kg亚硒酸钠组有显著性差异(P<0.001),三组活性值均为阳性。同时,与相应的200.0 mg/kg苯组相比,端粒酶活性均增加,说明硒激活了端粒酶活性。结论:本实验中,活体不同浓度苯作用均增高端粒酶活性表明小鼠淋巴细胞得到激活和增殖,端粒酶可能是苯刺激淋巴细胞的一种敏感的早期效应标志物。硒可以上调小鼠淋巴细胞端粒酶活性。说明端粒酶是潜在的苯暴露致肿瘤的早期效应生物标志物。
第二部分荧光定量RT-PCR快速检测重要肠道病毒的研究
目的:利用荧光定量RT-PCR技术建立一种快速检测重要肠道病毒的方法。方法:从GeneBank下载相应肠道病毒的基因系列,利用TaqMan技术,根据肠道病毒基因的相对保守序列参考国外文献分别设计一对共引物及相应的TaqMan探针。首先检测荧光定量RT-PCR的特异性,以PolioⅠ、Ⅱ、Ⅲ、EV71、CA16和CA24标准株作为阳性标准,轮状病毒、诺沃克病毒为阴性标准,并设阴性对照。对反应体系及反应条件进行优化。其次建立荧光定量RT-PCR的标准曲线,以EV71为标准株,用通用引物构建质粒标准品,测定OD值定量后,十倍倍比稀释标准品(108-104拷贝/μl)并用建立的方法构建定量标准曲线。同时,以EV71为标准株进行病毒效价滴定后作为参考株,根据TCID50十倍倍比稀释至8个浓度,并设置阴性对照用建立的方法构建灵敏度标准曲线。最后,收集2006年深圳市疑似肠道病毒感染的病人粪便标本共69份,用通用引物进行传统的RT-PCR检验,再用荧光定量RT-PCR进行检验,验证其应用价值。结果:所有对照经我们建立的体系检验,该方法具有高度的特异性,PolioⅠ、PolioⅡ、PolioⅢ、EV71、CA16和CA24阳性标准均观察到荧光信号增强,轮状病毒、诺沃克病毒和阴性对照均未观察到荧光信号增强,说明无假阳性结果。阳性标准和阴性标准间无交叉反应。我们用EV71标准株构建的质粒标准品,建立的定量标准曲线最低检测限为105拷贝/μl,曲线斜率为-3.474,在Y轴截距为53.67,R2=0.999,PCR扩增效率为94%。用EV71标准株进行病毒效价滴定后的TCID50,建立了灵敏度标准曲线,本体系检测到的最低浓度值为5.0 TCID50,曲线斜率为-3.230,在Y轴截距为16.36,R2=0.991,PCR扩增效率为104.0%。说明我们建立的标准曲线各个浓度的相关系数非常好,PCR反应对模板的扩增很完全。临床标本验证中,89份肠道病毒疑似病例的粪便标本用建立的荧光RT-PCR和传统的RT-PCR进行检测,普通RT-PCR检出56份手足口病阳性,检出率为62.92%,我们建立的荧光RT-PCR检出72份阳性结果,检出率为80.9%,荧光RT-PCR检出率显著高于普通RT-PCR,两者符合率为79.78%。说明荧光RT-PCR可以用于日常疑似肠道病毒感染的临床标本的筛检工作。结论:我们成功建立了快速检测重要肠道病毒共引物的荧光定量RT-PCR方法,该方法具有敏感性高、特异性好、效率高、稳定性好、准确、简单等优点,筛检标本检出率及符合性好,可以作为肠道病毒的快速检测方法,用于疑似肠道病毒感染的日常临床标本筛检工作。
Benzene Hydrocarbon is a kind of aromatic hydrocarbon, mainly achromatous or pale yellow transparent and evaporable oleaginous liquid, rich in aromatic odor, they including Benzene, Toluene, Xylene and so on. Benzene Hydrocarbon can lead to inhibiting of bone marrow and immune system, mainly cause hematotoxicity, can result in cytotoxicity and genotoxicity. They can give rise to many kinds of biological effects, cause hematopoietic cells apoptosis and influence hepatocytes and progenitor cells replication. They inhibit cytokine secretion, influence on T lymphocytes function and variation of its subpopulation in peripheral blood. They give rise to peripheral blood hemogram variation, cause lymphocytes and erythrocytes reduction, lead to micronuclei. They induce cells apoptosis, mutation and replication, resulting in DNA breakage, leading to lymphocytes aberration in structural and other aspects, cause chromosome damage, including aneuploidy, chromosome deletion, chromosome translocation, and so on. After benzene was absorbed in human bodies, it was transported into liver, after activated and metabolized under the mediation by cytochrome P-450 monooxygenase, their metabolin hydroquinone, Phenol, intermediates were distributed to blood, Kidney, Spleen, Thymus Gland and Bone Marrow Hematopoiesis system. They targeted at lymphocytes and myeloid cells, injured chromosome, they lead to Hematologic disease and carcinogenesis. It can cause Aplastic Anemi, Acute Myeloid Leukaemia (AML), Hemangioma, Myelodysplastic Syndrome (MDS), Thymic Lymphoma, Chronic Lymphadenoma, Chronic Myelogenous Leukemia (CML) and other Hematogenic Neoplasma. Benzene is showed to be a carcinogen cause multi-organ neoplasma. Benzene and its metabolite effect preferential on specific non-lethal chromosome, elimination of telomere has been reported to be a selective chromosome, it is a sensitive chromosome preferential effect on and damage in the chromosome will be detected more selectively. Researches on benzene ranged from its genotoxicity, chromosome aberration and carcinogenesis mechanism. Recent work on benzene toxicity has provided insight into its gene level from chromosome damage.
The telomerase, is a ribonucleoprotein comprised by RNA and protein that functions as a reverse transcriptase, it consists of three components, telomerase RNA component (TERC), telomerase-associated protein (TELPI) and catalytic subunit that possesses reverse transcriptase activity (TERT). Telomerase uses its RNA component as a template for the addition of G-rich repeats to the 3’end of telomeric DNA, thereby compensating for the telomere loss, fusion, and preventing homologous recombination to avoid shortening associated with cell division. This escape cells from apoptosis. Telomerase activity is undetectable or express low level in most somatic cells and normal tissues, but activated and expressed high level in germ cell, embryonic cell, hemopoietic stem cell, myeloid cell and most carcinoma such as neoplasma and hematologic malignancies. Telomeres, the protective caps of eukaryotic chromosomes are the physical ends of eucaryotic chromosomes, prevent the chromosome from end-to-end fusion, recombination, and degradation, which lead to chromosome instability and cell death. The gradual loss of telomere repeats in cells appears to be a major component of cell senescence and tissue ageing, limit cellular proliferation and develop to tumor. Telomerase function as elongating telomeric RNA and bypass crisis, allowing indefinite replicating to immortal cells. Cells senescence, apoptosis and immortalization resulting in critical telomere shortening, telomerase is activated and then triggering the carcinogenesis and neoplasma, telomerase activity is strong expressed in 80~90% of human cancers, telomerase activity rise with the acuteness and aggressiveness of tumor and carcinoma. It is well accepted that the re-activation of telomerase is the common final pathway of malignant tumor occurrence and telomerase plays an important role in tumorigenesis. Telomerase is expressed to detect tumors as a specific and popular effect biomarker and early biomarker. Since regulation of telomerase activity and telomere length are critical in maintaining stability of chromosome and benzene is known for its tumorigenesis in human cells, alterations in telomerase activity and telomere may be associated with the chromosomal damage exerted by benzene.
Selenium, a trace element, is nourished to cells, it functions nutrition by antioxidant effects and clearing free radical on human bodies. Low dose selenium can activate telomerase activity, enlongate telomere length and increase cells life span. It has protection role on preventing tumor initiation, development, meanwhile, it has prevention of ageing, specific cancers and antitumorigenic effects in postinitiation phases of cancer.
Benzene is toxic to cells after metabolized by enzymes, therefore, we select animals to construct models to observe its toxicity and appreciate influence performance. Healthy Kunming mice were selected to perform on, they are randomly divided into several groups. We designed different benzene treated groups with different doses to exert on mice, defined different doses of benzene combined with selenium to treat on mice, meanwhile, negative control and reagent control was established. After treated with different groups, we observed the indications, obtained peripheral blood then isolated lymphocytes, and then TRAP (telomere repeat amplification protocol assay) was used to detect telomerase activities in lymphocytes. According to the telomerase activity expression in different treated groups, we observed their different influence on telomerase activity, analyzed benzene and selenium treated groups varied on influence, we found out their correlation. In the present paper, we shed light on the telomerase activity changes by benzene toxic influence on mice lymphocyte and the regulation function of selenium in vivo, and to further understand the molecular mechanism of benzene induced neoplasma and tumorigenesis, aim to find out a new biomarker of the early effect and biomarker of susceptibility for benzene exposure from telomerase system.
PartⅠEffects of benzene on mice lymphocytes telomerase activity and regulations of selenium in vivo
Objective: We researched on the telomerase activity changes by benzene toxic influence on mice lymphocytes and the regulation function of selenium in vivo, and to further understand the molecular mechanism of benzene induced neoplasma and tumorigenesis, aim to find out a new biomarker of the early effect and biomarker of susceptibility for benzene exposure from telomerase system. Methods: 40 male healthy Kunming mice aged from 6~8 weeks, weighted from 25-30g were randomly divided into 8 groups, with 5 animals in each group. Three benzene treated groups were designed with different doses, separately they were 100.0 mg/kg benzene, 200.0 mg/kg benzene and 400.0 mg/kg benzene; three benzene combined with selenium treated groups were established with different doses, they were 200.0 mg/kg benzene + 0.75 mg/kg selenium, 200.0 mg/kg benzene + 1.5 mg/kg selenium and 200.0 mg/kg benzene + 3.0 mg/kg selenium individually; two control groups were also set up, they were negative control and reagent control. Abdominal injection was adopted to treat mice, injection lasted for 5 days in succession, after last injection and 48h later, 1 ml heparinized PBMC was obtained from eyeball removal, lymphocytes were then isolated by kit. To quantitative detect telomerase activity, TRAP-ELISA Kit was used to assay. In the first step, telomerase adds telomeric repeats (TTAGGG) to the 3’end of the biotinlabeled synthetic P1-TS-primer. In the second step, primers P1-TS and P2 were used by PCR to amplify elongation products with telomerase 6 nucleotide increments. Then, after PCR, 5μl PCR product was denatured and hybridized to a digoxigenin-(DIG)-labeled probe, the mixture was immobilized to a streptavidin-coated microplate, finally, the probe was visualized by virtue of peroxidase metabolizing TMB to form a colored reaction product. The absorbance of the samples at 450 nm was measured with a reference wavelength of 690 nm. The telomerase activity was valued by A450nm– A690nm units. Telomerase activity differences were compared between different groups, effects of benzene on telomerase and regulations of selenium were analyzed. Data was analyzed with one-way ANOVA by SPSS13.0. Results: The telomerase activity of lymphocytes from negative control group and reagent control group located in normal range which was judged as negative. It indicated that normal mononuclear cells do have demonstrated a low but marginal level telomerase activity. Among lymphocytes from mice treated with benzene at different doses, the telomerase activity was positive in all groups. The mean values were increased and higher than negative control and reagent control groups, at the dose of 100.0 mg/kg it was significantly increased compared to control groups with a P<0.01. It indicating that benzene activated lymphocytes telomerase expression. For cases of benzene combined with selenium at different concentrations, the telomerase activity was positive in all groups, the mean values were higher than negative control and reagent control groups. Compared with 200 mg/kg benzene, they were much increased, at the dose of 200 mg/kg benzene + 0.75 mg/kg selenium it was significantly enhanced with a P<0.05. Compared with negative control and reagent control groups, they were remarkably increased in all groups, at the dose of 200 mg/kg benzene + 0.75 mg/kg selenium it has a significance of P<0.01. It demonstrated that selenium activated telomerase activity. Conclusions: It is presented in our paper that, telomerase activity is increased after treated by benzene at different concentrations demonstrated that mice lymphocytes are activated and proliferated in vivo, telomerase activity might be a sensitive early biomarker of lymphocytes proliferation by benzene. Selenium can up-regulate mice lymphocytes telomerase activity. It is suggested that telomerase can be used as a biomarker of the early effect for benzene exposure to induce neoplasma and tumorigenesis.
PartⅡRapid detection of Enterovirus by Real Time Quantitative RT-PCR
Objective: To develop a method of rapid detection of Enterovirus by using Real Time Quantitative RT-PCR. Methods: Enterovirus gene sequences were downloaded from Genebank, using TaqMan technique, we designed a set of universal primers and TaqMan probe according to the highly reserved sequence of enterovirus gene and foreign literature. Firstly, the specificity of real time RT-PCR was detected. PolioⅠ, PolioⅡ, PolioⅢ, EV71, CA16 and CA24 standard virus were used as positive standards, Rotavirus and Norwalk virus were used as controls, a set of negative control was established to examine specificity. Then primers, probe and mixture concentrations and reactive condition were optimized. Secondly, standard curves of real time RT-PCR were established. Use EV71 as standard, we constructed plasmid standard by common primers. After quantified by detected absorbance at 260nm and 280nm, standard plasmid was serially diluted by ten-fold (108-104 copies/μl) and detected by real time RT-PCR we developed to construct quantitative standard curve. Meanwhile, a titrated viral culture from the EV71 strain was used as standard to detect sensitivity curve, according to the TCID50 the cultured virus stock was serially diluted ten-fold from 10 to 107 times in sterile water, real time RT-PCR we developed was used to construct sensitivity curve by setting a negative control. Lastly, to test the clinical specimens, we collected 2006 suspected enterovirus infection patients’feces samples in Shenzhen city, a total of 89. The specimens were tested by conventional RT-PCR using common primers, then we used real time RT-PCR to detect the samples. Compared with the two results, we testified the real time RT-PCR application in clinical diagnoses. Results: The real time RT-PCR we developed has high specificity, PolioⅠ、PolioⅡ、PolioⅢ、EV71、CA16 and CA24 positive standards had fluorescence signal increased, Rotavirus, Norwalk virus and negative control didn’t observed fluorescence value, it was tested no false positive results. We successfully established quantitative standard curve by plasmid standards constructed by EV71 strain, the detection limit of the standard curve was 105 copies /μl, the slope rate of the curve was -3.474 with an intercept in Y axis was 53.67, the data showed a strong linear relationship with a R2 at 0.999, the efficiency of PCR amplification in our method was 94%. After viral titrated culture the TCID50 of EV71 strain was based on to construct sensitivity standard curve, the lowest concentration of our system was 5.0 TCID50, the slope rate of the curve was -3.230 with an intercept in Y axis was 16.36, the R2 of the standard curve was 0.991, and the amplification efficiency of our method was 104.0%. It proved that we constructed standard curves with a high correlation coefficient in each concentration. Amplification on templates was completely in PCR reactions. On detection of clinical specimens, 89 stool samples from suspected enterovirus infection patients were tested by conventional RT-PCR then by our real time RT-PCR. The result of conventional RT-PCR detected 56 positive hand-foot-and-mouth diseases (HFMD), the detection rate was 62.92%. By using real time RT-PCR it detected 72 positive, real time RT-PCR has a detection rate much higher than conventional RT-PCR, the detection rate was 70.9%. It proved the two methods have a high coincidence at 79.78%. It can be applied to routine screening work on suspected enterovirus infection patients in clinical. Conclusions: We have successfully developed a Real Time RT-PCR to rapid detect enterovirus using universal primers. It is triumphing in high sensitivity, good specificity, high efficiency, strong stability, accuracy and not labor-consuming. When screening clinical specimens it has a high detection rate and a good coincidence, proved that it can be used to rapid detect enterovirus, applied to routine screening specimens from suspected enterovirus infection in clinical.
端粒酶是逆转录酶,由RNA和蛋白质组成的核糖蛋白酶,它主要有三个组成成分:端粒酶RNA组分(telomerase RNA component, TERC)、端粒酶相关蛋白(telomerase-associated protein, TELPI)和端粒酶逆转录酶催化蛋白亚单位(telomerase reverse transcriptase, TERT)。端粒酶能合成新的端粒DNA,添加到染色体末端,来补偿端粒的丢失、粘连、重组阻止端粒的缩短,使细胞逃脱程序性死亡。在大多数体细胞和正常组织中,端粒酶活性不表达或低水平表达,生殖细胞、胚胎细胞、造血干细胞、骨髓细胞、肿瘤、癌组织中高表达。它使端粒,染色体末端重复DNA系列呈帽子结构维持染色体稳定性,与细胞衰老、分化、肿瘤形成有关,维持长度并跨过生物钟,进行分裂,细胞衰老、凋亡、永生化过程中激活端粒酶高表达,引起肿瘤和癌症,并且表达水平同肿瘤恶化程度正相关。端粒酶激活是肿瘤形成的必经途径,它是肿瘤具特异性和普遍性的效应标志物和早期分子生物学标志物。
硒具有营养作用,在体内发挥消除自由基、抗氧化等作用。营养剂量硒具有激活端粒酶活性、延长端粒、延长细胞寿命的功能。它在肿瘤的发生发展过程中起预防作用,它可以抗衰老、抑制肿瘤、癌症。
苯对细胞产生毒性,我们采用动物实验构建模型对其进行毒性评价。选用健康昆明小鼠,随机分成几组,分别设不同剂量组用苯对其进行染毒,并设不同剂量苯联合硒组进行处理,同时设阴性对照和试剂对照。染毒后观察变化,收获外周血淋巴细胞,采用端粒重复扩增分析法(telomere repeat amplification protocol assay, TRAP)法检测其端粒酶活性。分析苯及硒对淋巴细胞端粒酶活性的影响,观察两者的相关性。鉴于此,我们研究了苯对小鼠淋巴细胞端粒酶活性的毒性影响及硒的调节作用,进一步了解苯致肿瘤及癌症的机制,旨在从端粒系统获得新的苯暴露早期效应生物学标志物和易感性标志物。
第一部分苯对小鼠淋巴细胞端粒酶活性的影响及硒的调节作用
目的:本部分通过研究苯对小鼠淋巴细胞端粒酶活性的影响及硒的调节作用,评价端粒酶作为生物标志物对苯致癌症的分子机制的影响,旨在获得新的苯暴露早期效应生物学标志物和易感性标志物。方法:选择6~8周龄的健康雄性昆明小鼠,25-30g左右,40只随机分成8组,每组5只。分别设100.0 mg/kg苯、200.0 mg/kg苯及400.0 mg/kg苯染毒组,200.0 mg/kg苯+ 0.75 mg/kg亚硒酸钠、200.0 mg/kg苯+ 1.5 mg/kg亚硒酸钠及200.0 mg/kg苯+ 3.0 mg/kg亚硒酸钠处理组,阴性对照组及试剂对照组。对小鼠采用腹腔注射染毒,连续5天,末次染毒48h后,眼球摘除取外周血,分离淋巴细胞。用TRAP-ELISA法定量分析淋巴细胞端粒酶活性,在前引物P1-TS的作用下,端粒酶先以自身为模板合成端粒DNA,然后在前引物P1-TS和后引物P2作用下对TTAGGG结构进行PCR特异性扩增。取5μl PCR产物进行变性,再与地高辛标记的探针进行杂交,加入到生物素标记的微孔板中,最后加入TMB底物,在450nm和690nm波长处读取溶液的OD值,A450-A690的值即我们的端粒酶活性值。比较不同组间的端粒酶活性差异,分析苯对端粒酶的毒性作用和硒的调节作用,利用统计软件分析实验数据。结果:阴性对照和试剂对照呈现微弱的端粒酶活性表达,活性值均在正常范围内。各剂量苯处理组与阴性对照和试剂对照相比,端粒酶活性阳性,均值范围均升高,其中100.0 mg/kg苯组有统计学差异(P<0.01),说明苯刺激了端粒酶的表达。与阴性对照和试剂对照相比,各剂量苯联合硒作用组端粒酶活性均增高,其中200.0 mg/kg苯+ 0.75 mg/kg亚硒酸钠组有显著性差异(P<0.001),三组活性值均为阳性。同时,与相应的200.0 mg/kg苯组相比,端粒酶活性均增加,说明硒激活了端粒酶活性。结论:本实验中,活体不同浓度苯作用均增高端粒酶活性表明小鼠淋巴细胞得到激活和增殖,端粒酶可能是苯刺激淋巴细胞的一种敏感的早期效应标志物。硒可以上调小鼠淋巴细胞端粒酶活性。说明端粒酶是潜在的苯暴露致肿瘤的早期效应生物标志物。
第二部分荧光定量RT-PCR快速检测重要肠道病毒的研究
目的:利用荧光定量RT-PCR技术建立一种快速检测重要肠道病毒的方法。方法:从GeneBank下载相应肠道病毒的基因系列,利用TaqMan技术,根据肠道病毒基因的相对保守序列参考国外文献分别设计一对共引物及相应的TaqMan探针。首先检测荧光定量RT-PCR的特异性,以PolioⅠ、Ⅱ、Ⅲ、EV71、CA16和CA24标准株作为阳性标准,轮状病毒、诺沃克病毒为阴性标准,并设阴性对照。对反应体系及反应条件进行优化。其次建立荧光定量RT-PCR的标准曲线,以EV71为标准株,用通用引物构建质粒标准品,测定OD值定量后,十倍倍比稀释标准品(108-104拷贝/μl)并用建立的方法构建定量标准曲线。同时,以EV71为标准株进行病毒效价滴定后作为参考株,根据TCID50十倍倍比稀释至8个浓度,并设置阴性对照用建立的方法构建灵敏度标准曲线。最后,收集2006年深圳市疑似肠道病毒感染的病人粪便标本共69份,用通用引物进行传统的RT-PCR检验,再用荧光定量RT-PCR进行检验,验证其应用价值。结果:所有对照经我们建立的体系检验,该方法具有高度的特异性,PolioⅠ、PolioⅡ、PolioⅢ、EV71、CA16和CA24阳性标准均观察到荧光信号增强,轮状病毒、诺沃克病毒和阴性对照均未观察到荧光信号增强,说明无假阳性结果。阳性标准和阴性标准间无交叉反应。我们用EV71标准株构建的质粒标准品,建立的定量标准曲线最低检测限为105拷贝/μl,曲线斜率为-3.474,在Y轴截距为53.67,R2=0.999,PCR扩增效率为94%。用EV71标准株进行病毒效价滴定后的TCID50,建立了灵敏度标准曲线,本体系检测到的最低浓度值为5.0 TCID50,曲线斜率为-3.230,在Y轴截距为16.36,R2=0.991,PCR扩增效率为104.0%。说明我们建立的标准曲线各个浓度的相关系数非常好,PCR反应对模板的扩增很完全。临床标本验证中,89份肠道病毒疑似病例的粪便标本用建立的荧光RT-PCR和传统的RT-PCR进行检测,普通RT-PCR检出56份手足口病阳性,检出率为62.92%,我们建立的荧光RT-PCR检出72份阳性结果,检出率为80.9%,荧光RT-PCR检出率显著高于普通RT-PCR,两者符合率为79.78%。说明荧光RT-PCR可以用于日常疑似肠道病毒感染的临床标本的筛检工作。结论:我们成功建立了快速检测重要肠道病毒共引物的荧光定量RT-PCR方法,该方法具有敏感性高、特异性好、效率高、稳定性好、准确、简单等优点,筛检标本检出率及符合性好,可以作为肠道病毒的快速检测方法,用于疑似肠道病毒感染的日常临床标本筛检工作。
Benzene Hydrocarbon is a kind of aromatic hydrocarbon, mainly achromatous or pale yellow transparent and evaporable oleaginous liquid, rich in aromatic odor, they including Benzene, Toluene, Xylene and so on. Benzene Hydrocarbon can lead to inhibiting of bone marrow and immune system, mainly cause hematotoxicity, can result in cytotoxicity and genotoxicity. They can give rise to many kinds of biological effects, cause hematopoietic cells apoptosis and influence hepatocytes and progenitor cells replication. They inhibit cytokine secretion, influence on T lymphocytes function and variation of its subpopulation in peripheral blood. They give rise to peripheral blood hemogram variation, cause lymphocytes and erythrocytes reduction, lead to micronuclei. They induce cells apoptosis, mutation and replication, resulting in DNA breakage, leading to lymphocytes aberration in structural and other aspects, cause chromosome damage, including aneuploidy, chromosome deletion, chromosome translocation, and so on. After benzene was absorbed in human bodies, it was transported into liver, after activated and metabolized under the mediation by cytochrome P-450 monooxygenase, their metabolin hydroquinone, Phenol, intermediates were distributed to blood, Kidney, Spleen, Thymus Gland and Bone Marrow Hematopoiesis system. They targeted at lymphocytes and myeloid cells, injured chromosome, they lead to Hematologic disease and carcinogenesis. It can cause Aplastic Anemi, Acute Myeloid Leukaemia (AML), Hemangioma, Myelodysplastic Syndrome (MDS), Thymic Lymphoma, Chronic Lymphadenoma, Chronic Myelogenous Leukemia (CML) and other Hematogenic Neoplasma. Benzene is showed to be a carcinogen cause multi-organ neoplasma. Benzene and its metabolite effect preferential on specific non-lethal chromosome, elimination of telomere has been reported to be a selective chromosome, it is a sensitive chromosome preferential effect on and damage in the chromosome will be detected more selectively. Researches on benzene ranged from its genotoxicity, chromosome aberration and carcinogenesis mechanism. Recent work on benzene toxicity has provided insight into its gene level from chromosome damage.
The telomerase, is a ribonucleoprotein comprised by RNA and protein that functions as a reverse transcriptase, it consists of three components, telomerase RNA component (TERC), telomerase-associated protein (TELPI) and catalytic subunit that possesses reverse transcriptase activity (TERT). Telomerase uses its RNA component as a template for the addition of G-rich repeats to the 3’end of telomeric DNA, thereby compensating for the telomere loss, fusion, and preventing homologous recombination to avoid shortening associated with cell division. This escape cells from apoptosis. Telomerase activity is undetectable or express low level in most somatic cells and normal tissues, but activated and expressed high level in germ cell, embryonic cell, hemopoietic stem cell, myeloid cell and most carcinoma such as neoplasma and hematologic malignancies. Telomeres, the protective caps of eukaryotic chromosomes are the physical ends of eucaryotic chromosomes, prevent the chromosome from end-to-end fusion, recombination, and degradation, which lead to chromosome instability and cell death. The gradual loss of telomere repeats in cells appears to be a major component of cell senescence and tissue ageing, limit cellular proliferation and develop to tumor. Telomerase function as elongating telomeric RNA and bypass crisis, allowing indefinite replicating to immortal cells. Cells senescence, apoptosis and immortalization resulting in critical telomere shortening, telomerase is activated and then triggering the carcinogenesis and neoplasma, telomerase activity is strong expressed in 80~90% of human cancers, telomerase activity rise with the acuteness and aggressiveness of tumor and carcinoma. It is well accepted that the re-activation of telomerase is the common final pathway of malignant tumor occurrence and telomerase plays an important role in tumorigenesis. Telomerase is expressed to detect tumors as a specific and popular effect biomarker and early biomarker. Since regulation of telomerase activity and telomere length are critical in maintaining stability of chromosome and benzene is known for its tumorigenesis in human cells, alterations in telomerase activity and telomere may be associated with the chromosomal damage exerted by benzene.
Selenium, a trace element, is nourished to cells, it functions nutrition by antioxidant effects and clearing free radical on human bodies. Low dose selenium can activate telomerase activity, enlongate telomere length and increase cells life span. It has protection role on preventing tumor initiation, development, meanwhile, it has prevention of ageing, specific cancers and antitumorigenic effects in postinitiation phases of cancer.
Benzene is toxic to cells after metabolized by enzymes, therefore, we select animals to construct models to observe its toxicity and appreciate influence performance. Healthy Kunming mice were selected to perform on, they are randomly divided into several groups. We designed different benzene treated groups with different doses to exert on mice, defined different doses of benzene combined with selenium to treat on mice, meanwhile, negative control and reagent control was established. After treated with different groups, we observed the indications, obtained peripheral blood then isolated lymphocytes, and then TRAP (telomere repeat amplification protocol assay) was used to detect telomerase activities in lymphocytes. According to the telomerase activity expression in different treated groups, we observed their different influence on telomerase activity, analyzed benzene and selenium treated groups varied on influence, we found out their correlation. In the present paper, we shed light on the telomerase activity changes by benzene toxic influence on mice lymphocyte and the regulation function of selenium in vivo, and to further understand the molecular mechanism of benzene induced neoplasma and tumorigenesis, aim to find out a new biomarker of the early effect and biomarker of susceptibility for benzene exposure from telomerase system.
PartⅠEffects of benzene on mice lymphocytes telomerase activity and regulations of selenium in vivo
Objective: We researched on the telomerase activity changes by benzene toxic influence on mice lymphocytes and the regulation function of selenium in vivo, and to further understand the molecular mechanism of benzene induced neoplasma and tumorigenesis, aim to find out a new biomarker of the early effect and biomarker of susceptibility for benzene exposure from telomerase system. Methods: 40 male healthy Kunming mice aged from 6~8 weeks, weighted from 25-30g were randomly divided into 8 groups, with 5 animals in each group. Three benzene treated groups were designed with different doses, separately they were 100.0 mg/kg benzene, 200.0 mg/kg benzene and 400.0 mg/kg benzene; three benzene combined with selenium treated groups were established with different doses, they were 200.0 mg/kg benzene + 0.75 mg/kg selenium, 200.0 mg/kg benzene + 1.5 mg/kg selenium and 200.0 mg/kg benzene + 3.0 mg/kg selenium individually; two control groups were also set up, they were negative control and reagent control. Abdominal injection was adopted to treat mice, injection lasted for 5 days in succession, after last injection and 48h later, 1 ml heparinized PBMC was obtained from eyeball removal, lymphocytes were then isolated by kit. To quantitative detect telomerase activity, TRAP-ELISA Kit was used to assay. In the first step, telomerase adds telomeric repeats (TTAGGG) to the 3’end of the biotinlabeled synthetic P1-TS-primer. In the second step, primers P1-TS and P2 were used by PCR to amplify elongation products with telomerase 6 nucleotide increments. Then, after PCR, 5μl PCR product was denatured and hybridized to a digoxigenin-(DIG)-labeled probe, the mixture was immobilized to a streptavidin-coated microplate, finally, the probe was visualized by virtue of peroxidase metabolizing TMB to form a colored reaction product. The absorbance of the samples at 450 nm was measured with a reference wavelength of 690 nm. The telomerase activity was valued by A450nm– A690nm units. Telomerase activity differences were compared between different groups, effects of benzene on telomerase and regulations of selenium were analyzed. Data was analyzed with one-way ANOVA by SPSS13.0. Results: The telomerase activity of lymphocytes from negative control group and reagent control group located in normal range which was judged as negative. It indicated that normal mononuclear cells do have demonstrated a low but marginal level telomerase activity. Among lymphocytes from mice treated with benzene at different doses, the telomerase activity was positive in all groups. The mean values were increased and higher than negative control and reagent control groups, at the dose of 100.0 mg/kg it was significantly increased compared to control groups with a P<0.01. It indicating that benzene activated lymphocytes telomerase expression. For cases of benzene combined with selenium at different concentrations, the telomerase activity was positive in all groups, the mean values were higher than negative control and reagent control groups. Compared with 200 mg/kg benzene, they were much increased, at the dose of 200 mg/kg benzene + 0.75 mg/kg selenium it was significantly enhanced with a P<0.05. Compared with negative control and reagent control groups, they were remarkably increased in all groups, at the dose of 200 mg/kg benzene + 0.75 mg/kg selenium it has a significance of P<0.01. It demonstrated that selenium activated telomerase activity. Conclusions: It is presented in our paper that, telomerase activity is increased after treated by benzene at different concentrations demonstrated that mice lymphocytes are activated and proliferated in vivo, telomerase activity might be a sensitive early biomarker of lymphocytes proliferation by benzene. Selenium can up-regulate mice lymphocytes telomerase activity. It is suggested that telomerase can be used as a biomarker of the early effect for benzene exposure to induce neoplasma and tumorigenesis.
PartⅡRapid detection of Enterovirus by Real Time Quantitative RT-PCR
Objective: To develop a method of rapid detection of Enterovirus by using Real Time Quantitative RT-PCR. Methods: Enterovirus gene sequences were downloaded from Genebank, using TaqMan technique, we designed a set of universal primers and TaqMan probe according to the highly reserved sequence of enterovirus gene and foreign literature. Firstly, the specificity of real time RT-PCR was detected. PolioⅠ, PolioⅡ, PolioⅢ, EV71, CA16 and CA24 standard virus were used as positive standards, Rotavirus and Norwalk virus were used as controls, a set of negative control was established to examine specificity. Then primers, probe and mixture concentrations and reactive condition were optimized. Secondly, standard curves of real time RT-PCR were established. Use EV71 as standard, we constructed plasmid standard by common primers. After quantified by detected absorbance at 260nm and 280nm, standard plasmid was serially diluted by ten-fold (108-104 copies/μl) and detected by real time RT-PCR we developed to construct quantitative standard curve. Meanwhile, a titrated viral culture from the EV71 strain was used as standard to detect sensitivity curve, according to the TCID50 the cultured virus stock was serially diluted ten-fold from 10 to 107 times in sterile water, real time RT-PCR we developed was used to construct sensitivity curve by setting a negative control. Lastly, to test the clinical specimens, we collected 2006 suspected enterovirus infection patients’feces samples in Shenzhen city, a total of 89. The specimens were tested by conventional RT-PCR using common primers, then we used real time RT-PCR to detect the samples. Compared with the two results, we testified the real time RT-PCR application in clinical diagnoses. Results: The real time RT-PCR we developed has high specificity, PolioⅠ、PolioⅡ、PolioⅢ、EV71、CA16 and CA24 positive standards had fluorescence signal increased, Rotavirus, Norwalk virus and negative control didn’t observed fluorescence value, it was tested no false positive results. We successfully established quantitative standard curve by plasmid standards constructed by EV71 strain, the detection limit of the standard curve was 105 copies /μl, the slope rate of the curve was -3.474 with an intercept in Y axis was 53.67, the data showed a strong linear relationship with a R2 at 0.999, the efficiency of PCR amplification in our method was 94%. After viral titrated culture the TCID50 of EV71 strain was based on to construct sensitivity standard curve, the lowest concentration of our system was 5.0 TCID50, the slope rate of the curve was -3.230 with an intercept in Y axis was 16.36, the R2 of the standard curve was 0.991, and the amplification efficiency of our method was 104.0%. It proved that we constructed standard curves with a high correlation coefficient in each concentration. Amplification on templates was completely in PCR reactions. On detection of clinical specimens, 89 stool samples from suspected enterovirus infection patients were tested by conventional RT-PCR then by our real time RT-PCR. The result of conventional RT-PCR detected 56 positive hand-foot-and-mouth diseases (HFMD), the detection rate was 62.92%. By using real time RT-PCR it detected 72 positive, real time RT-PCR has a detection rate much higher than conventional RT-PCR, the detection rate was 70.9%. It proved the two methods have a high coincidence at 79.78%. It can be applied to routine screening work on suspected enterovirus infection patients in clinical. Conclusions: We have successfully developed a Real Time RT-PCR to rapid detect enterovirus using universal primers. It is triumphing in high sensitivity, good specificity, high efficiency, strong stability, accuracy and not labor-consuming. When screening clinical specimens it has a high detection rate and a good coincidence, proved that it can be used to rapid detect enterovirus, applied to routine screening specimens from suspected enterovirus infection in clinical.
引文
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1. K. Kay-Yin Lai, Linda Cook, Sharon Wendt, et al. Evaluation of Real-Time PCR versus PCR with Liquid-Phase Hybridization for Detection of Enterovirus RNA in Cerebrospinal Fluid. Journal of Clinical Microbiology, 2003, 41(7): 3133~3141.
2. Monique Nijhuis, Noortje van Maarseveen, Rob Schuurman, et al. Rapid and Sensitive Routine Detection of All Members of the Genus Enterovirus in Different Clinical Specimens by Real-Time PCR. Journal of Clinical Microbiology, 2002, 40(10): 3666~3670.
3. Caroline E. Corless, Malcolm Guiver, Raymond Borrow, et al. Development and Evaluation of a‘Real-Time’RT-PCR for the Detection of Enterovirus and Parechovirus RNA in CSF and Throat Swab Samples. Journal of Medical Virology,2002, 67: 555~562.
4. G. J. Zoll, W. J. G. Melchers, H. Kopecka, et al. General Primer-Mediated Polymerase Chain Reaction for Detection of Enteroviruses: Application for Diagnostic Routine and Persistent Infections. Journal of Clinical Microbiology, 1992, 30(1): 160~165.
5. Schm idt NJ, L ennette EH, Ho HH. An apparently new enterovirus isolated from patients with disease of the central nervous system. J Infect Dis, 1974, 129: 304~309.
6. McMinn P, Lindsay K, Perera D, et al. Phylogenetic analysis of enterovirus 71 strains isolated during linked epidemics in Malaysia, Singapore, and Western Australia. J Virol, 2001, 75(16): 7732~7738.
7. Tsan-Chi Chen, Guang-Wu Chen, Chao Agnes Hsiung, et al. Combining Multiplex Reverse Transcription-PCR and a Diagnostic Microarray To Detect and Differentiate Enterovirus 71 and Coxsackievirus A16. Journal of Clinical Microbiology, 2006, 44(6): 2212~2219.
8. Dale W. Griffin, Kim A. Donaldson, John H. Paul, et al. Pathogenic Human Viruses in Coastal Waters. Clinical Microbiology Reviews, 2003, 16(1): 129~143.
9. S.E. Capaul, M. Gorgievski-Hrisoho. Detection of enterovirus RNA in cerebrospinal fluid (CSF) using NucliSens EasyQ Enterovirus assay. Journal of Clinical Virology, 2005, 32: 236~240.
10. Nahla Mohamed, Amal Elfaitouri, Jan Fohlman, et al. A sensitive and quantitative single-tube real-time reverse transcriptase-PCR for detection of enteroviral RNA. Journal of Clinical Virology, 2004, 30: 150~156.
11. Carita Savolainen-Kopra. Molecular Epidemiology of Human Rhinoviruses. Publications of the National Public Health Insitute, A2/2006.
12. Saskia A. Rutjes, Ronald Italiaander, Harold H. J. L. van den Berg, et al. Isolation and Detection of Enterovirus RNA from Large-Volume Water Samples by Using the NucliSens miniMAG System and Real-Time Nucleic Acid Sequence-BasedAmplification. Applied and Environmental Microbiology, 2005, 71(7): 3734~3740.
13. Jed A. Fuhrman, Xiaolin Liang, Rachel T. Noble. Rapid Detection of Enteroviruses in Small Volumes of Natural Waters by Real-Time Quantitative Reverse Transcriptase PCR. Applied and Environmental Microbiology, 2005, 71(8): 4523~4530.
14. Kellogg J. Schwab, Ricardo De Leon, Mark D. Sobsey. Concentration and Purification of Beef Extract Mock Eluates from Water Samples for the Detection of Enteroviruses, epatitis A Virus, and Norwalk Virus by Reverse Transcription-PCR. Applied and Environmental Microbiology, 1995, 61(2): 531~537.
15. Morteza Abbaszadegan, Mary S. Huber, Charles P. Gerba, et al. Detection of Enteroviruses in Groundwater with the Polymerase Chain Reaction. Applied and Environmental Microbiology, 1993, 59(5): 1318~1324.
16. Amvrosieva TV, Titov LP, Mulders M, et al. Viral water contamination as the cause of aseptic meningitis outbreak in Belarus. Cent Eur J Public Health, 2001, 9(3): 154~7.
17. Beller M, Ellis A, Lee SH, et al. Outbreak of viral gastroenteritis due to a contaminated well. JAMA, 1997, 278(7): 563~8.
18. H?fliger D, Hübner P, Lüthy J. Outbreak of viral gastroenteritis due to sewage-contaminated drinking water. Int J Food Microbiol, 2000, 54(1-2): 123-6.
19. A. M. Hauri, M. Schimmelpfennig, M. Walter-domes, et al. An outbreak of viral meningitis associated with a public swimming pond. Epidemiology and Infection, 2005, 133: 291~298.
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