摘要
目的:探讨微囊藻毒素与人体健康的关系,深入研究微囊藻毒素毒作用的机制,检测微囊藻毒素的抗TMV活性。
方法:(1)在福清市和永泰县部分乡镇进行2002～2005年回顾性以恶性肿瘤为主的死因调查,对其水体微囊藻毒素污染和其它污染物含量进行检测,分析恶性肿瘤死亡率和水体微囊藻毒素污染间关系。同时采用病例对照研究,收集福州市各大医院肝癌确诊的新发病例,并选择与病例在性别、年龄、民族和居住地相匹配的同期入院的非肿瘤患者为对照。采用统一编制的调查表进行问卷调查。采用PCR-RFLP法对调查对象的血标本进行XRCC3、ERCC1基因多态检测。采用广义线性模型分析饮水类型与其它环境因素、与基因多态性在肝癌发生中的交互作用。(2)按照两阶段致癌理论建立微囊藻毒素促癌实验动物模型,利用HE染色、γ-GT染色来判断动物模型建立情况,并应用免疫组化、RT-PCR、PCR-SSCP、分子克隆、基因测序等技术来研究微囊藻毒素促癌过程中对癌基因和抑癌基因表达与调控影响。(3)采用叶碟法对微囊藻纯毒素(MC-LR)抑制TMV复制活性进行测定。
结果:(1)福清与永泰水体中微囊藻毒素阳性检出率为28.4%。沟塘水微囊藻毒素阳性率最高为41.7%,不同水体类型的阳性率从高到低的顺序为:沟塘水>河水>自来水>井水,且差别有统计学意义(P<0.05);福清微囊藻毒素检测阳性率为46.5%,显著高于永泰的阳性率11.1%;福清恶性肿瘤的标化死亡率是永泰的2倍,其中肝癌、胃癌、食管癌、肠癌、肺癌、膀胱癌标化死亡率福清均高于永泰。(2)饮沟塘水、肝炎病毒感染、食用霉变食物是福州地区肝癌发生的危险因素。饮沟塘水与其他因素间存在交互作用。饮沟塘水与肝炎病毒感染之间交互作用为超相加模型,与食用霉变食物之间交互作用为超相乘模型,三个环境因素交互作用为超相加模型。ERCC1基因8092位点突变能增加患肝癌的危险度,与饮沟塘水的交互作用为次相乘模型,说明遗传因素与环境因素间存在交互作用,基因突变会增加个体对环境暴露的易感性。(3)微囊藻毒素LR(MCLR)在促大鼠肝癌过程中能增加γ-谷氨酰转肽酶(γ-GT)阳性率,二乙基亚硝胺(DEN)+微囊藻纯毒素(MC)组γ-GT染色的阳性率为100%,而DEN对照组的阳性率22.22%,两者差别有统计学意义(P<0.05)。MCLR可提高反映染色体损伤畸变情况的指标-微核率。DEN+MC组微核率为13‰,显著高于其他各组(P<0.05)。(4)MCLR在促大鼠肝癌过程中能增加大鼠肝脏BCL-2、PCNA基因蛋白表达强度,同时降低大鼠肝脏Bax、p21WAF1基因蛋白表达强度。Bcl-2与PCNA蛋白表达强度在DEN+MC组分别为0.0377和0.0088,显著高于表达强度分别为0.0205和0.0033的DEN对照组(P<0.05);Bax与p21WAF1蛋白表达强度在DEN+MC组分别为0.0073和0.0058,显著低于表达强度为0.0244和0.034的DEN对照组(P<0.05)。(5)MCLR在促大鼠肝癌过程中能显著增加大鼠肝脏Bcl-2和p53mRNA表达强度。这两种基因在DEN+MC组表达强度分别为2.244和2.5719,显著高于其他各组(P<0.05)。而Bax、p21WAF1、p16的mRNA表达强度各组间差别无统计学意义(P>0.05)。(6)微囊藻毒素可引起p53基因突变,本次实验在DEN+MC组筛查出4例突变阳性者,该组突变率为26.6%(4/15)。测序结果显示:突变分别位于密码子268(TTT→CTT)3例,编码的氨基酸由苯丙氨酸(Phe)→亮氨酸(Leu);位于密码子283(GAG→GAA)1例,但编码的氨基酸没有改变。Bax基因未筛查到突变。(7)微囊藻毒素对TMV抑制作用不强,最大抑制率只为46.03%,且抑制作用不随浓度增加而加强。
结论:(1)水体富营养化引起水体藻类毒素污染可能与福清高恶性肿瘤死亡率有关。(2)饮沟塘水是福州地区肝癌发生的危险因素。饮沟塘水与其他因素间存在交互作用。(3)微囊藻毒素对肝癌的发生具有强的促进作用。微囊藻毒素可在基因水平、转录水平、翻译水平以及翻译后水平影响癌基因与抑癌基因的表达,而且在促癌过程中微囊藻毒素对不同的基因调控机制影响有所不同,最终达到蛋白表达的影响,促进肝癌的发生。所以调节与细胞增殖和凋亡相关的癌基因和抑癌基因表达是MCLR促癌过程的重要机制。(4)微囊藻毒素TMV抑制作用不强。
Objective To study the relationship between microcystins and human health, and to explore in depth the role and molecular mechanism of mocrocystins in hepatocarcinogenesis and anti-TMV activities of mocrocystins in vitro.
Methods (1) We carried out an investigation on the microcystins water pollution and cause of death in Fuqing and Yongtai, and conducted a case-control study including newly diagnosed 388 hepatocellular carcinoma (HCC) and 388 hospital controls. These controls without history of malignant tumor were selected during the same period and matched to each case by gender, age, nationality and inhabited areas. The genotypes of XRCC3 and ERCC1 were analyzed with PCR-RFLP. General Relative Risk Regression Models were applied to analyze the interaction between drinking and other environmental factors, and the interaction between drinking and genes in hepatocarcinogenesis. (2) The two-stage-medium-term theory was applied to the establishment of the animal model. The promoting effect of MC-LR on liver tumor was evaluated with the Albertγ-GT methods and micronucleus test. During the tumor-promoting course, the effects of MC-LR on the regulation and expression of the oncogenes and suppressor genes were studied with immunohistochemical technique, RT-PCR, PCR-SSCP, Molecular Cloning and Sequencing. (3) anti-TMV activities of mocrocystins was detected by leaf-disc method.
Results (1) The positive reaction rate of microcystins in Fuqing and Yuntai was 28.4%. The positive reaction rate of microcystins in pond-ditch, river, shallow, and well were 41.7%, 29.4%, 13.6%, 8.0%, respectively; The positive reaction rate of microcystins in Fuqing was 46.5%, which was significantly higer than that in Yongtai 11.1%(P<0.05); The standard cancer mortality in Fuqing was double higher than that in Yongtai. The standard mortality rate of the esophagus cancer, the stomach cancer, the liver cancer, the intestines cancer, the lung cancer, and the urinary bladder cancer in Fuqing were higher than in Yongtai. (2) Logistic regression analysis showed that drinking pond-ditch, eating moldy food and hepatitis B were the risk factors of hepatoma cancer in fuzhou; The analysis of interaction showed that drinking pond-ditch appeared super-additive interaction with hepatitis B, super-multiplicative interaction with eating moldy food, and super-additive interaction with hepatitis B and eating moldy food. The point mutation in ERCC1-8092 increased the risk of hepatoma cancer in Fuzhou. Super-additive interaction was observed between genetic polymorphism of ERCC1-4533 and drinking pond-ditch. (3) MCLR can enhance the positive reaction ofγ-GT which is a preneoplasm marker. The positive reaction rate ofγ-GT in DEN+MC group is 100%, significantly higher than that of the DEN control group (22.22%, P<0.05). MCLR can significantly enhance the micronucleus ratio, which indicates the condition of chromosome aberration. The micronucleus ratio in DEN+MC group is 13‰, significantly higher than that in the other groups (P<0.05). (4) MCLR can increase the expression of Bcl-2 and PCNA and decrease the protein expression of Bax and p21WAF1. The intension of Bcl-2 and PCNA protein expression in DEN+MC group are 0.0377 and 0.0088, respectively, significantly higher than those in DEN control group (P<0.05). The intensions of Bax and p21WAF1 protein expression in DEN+MC group are significantly lower than those in DEN control group (P<0.05). The intension values of the two genes in DEN+MC group are 0.0073 and 0.0058, respectively(P<0.05)。(5) The mRNA expression of Bcl-2 and p53 are significantly increased by MCLR. The intension of Bcl-2 and p53 mRNA expression in DEN+MC group are 2.244 and 2.5719, respectively, significantly higher than those in the other groups (P<0.05). The mRNA expression of Bax, p21WAF1 and p16 are not significantly different between DEN+MC group and the other groups (P>0.05). (6) The point mutation in exon 8 of p53 gene was detected in the promotion of liver tumor by MCLR. p53 gene mutations were found in 26.6 % of cases in DEN+MC group(4/15). There was no p53 gene mutation found in other groups. The results of cloning and sequencing indicated that three cases showed 268th condon T→C missense mutation, and this mutation made Phe change to Leu in p53 protein. There was one case of mutation with a G→A tranvertion, located at condon283, and this mutation made no change of p53 protein. No Bax gene mutation was detected during the tumor-promoting course. (7) MCLR inhibited TMV multiplication in N. tabacum var.K326 weakly. The highest inhibition rate against Tobacco Mosaic Virus infection in Nicotiana glutinosa was only 46.03%.
Conclutions (1) The high mortality rate of cancer in Fuqing is positively correlated with the microcystins pollution. (2) Drinking pond-ditch was a risk factor of hepatoma cancer in Fuzhou, and act synergistically with other risk factors in hepatocelluar carcinogenesis. (3) MCLR can strongly promote liver tumor and affect the regulation and expression of the oncogenes and suppressor genes in different levels. The expression change of oncogenes and suppressor genes possibly plays an important role in the promotion of liver tumor by MCLR. (4) MCLR inhibited TMV multiplication in N. tabacum var.K326 weakly.
引文
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