摘要
为了深入了解黄曲霉毒素B1对小鼠肝脏蛋白的影响,本研究之前我们已经采用双向电泳及质谱分析等方法来分析不同浓度的黄曲霉毒素B1对小鼠肝脏蛋白的影响。结果发现有35个蛋白质点变化差异显著,分析其中新出现和明显上调的5个蛋白质点以及缺失的和明显下调的7个蛋白质点。其中有5个蛋白属于引发小鼠肝脏发生病变甚至是癌变的蛋白,7个蛋白属于参与小鼠肝脏能量与物质代谢的蛋白,这些蛋白对小鼠肝脏都有一定的影响。因而推测,黄曲霉毒素B1可能通过作用于这12个蛋白质而进一步影响小鼠肝脏,使其发生病变甚至是癌变。
为排除上述实验中可能发生的错误,并在转录水平上探索这些基因的表达情况,我们利用荧光定量PCR对黄曲霉毒素诱导后的小鼠肝脏atp5b、idh3α和prxⅡ三个基因的mRNA量进行了相对定量。结果发现atp5b先增后减,idh3α有增加的趋势,prxⅡ则近似增加,这与双向电泳的结果一致。随后,我们将atp5b、idh3α和prxⅡ这三个基因构建到原核表达载体pET28a上,并转化到E.coli BL21(DE3)宿主菌中。通过100 mmol/L的IPTG诱导阳性菌,成功表达了ATP5B、IDH3α、PRXⅡ三个蛋白。最后,我们还将atp5b构建到哺乳动物细胞表达载体pCDNA3.1hisB上并成功插入了报告基因gfp。这些工作为揭示atp5b、idh3α和prxⅡ三个基因在黄曲霉毒素B1致癌中的作用奠定了基础。
In order to further understanding the effect of proteins induced by aflatoxin B1 in the mouse liver, in previous study, two-dimensional electrophoresis and mass spectrometry analysis methods were used to analyze different concentrations of Aflatoxin B1 on the impact of proteins in the mouse liver. The result showed that 35 protein spots changed significantly, of which 5 appeared protein and 7 disappeared proteins were analyzed. The results indicated that these 5 proteins maybe cause liver disease or even cancer, and 7 proteins were involved with the material and energy metabolism in the mouse live, thus all these proteins have a certain effect on the mouse liver. That is to say, Aflatoxin B1 might interact with these 12 proteins and finally affect the mouse liver, and ultimately lead to lesion or even cancer.
In order to excluding the err of the two-dimensional electrophoresis, we explored the mRNA variation of atp5b, idh3αand prxⅡby fluorescence quantitative PCR after infection. As we expected, the results were according with the protein analysis. Followingly, we inserted these three genes into the protokaryon expression vetor pET28a, and transformed them into the the host E.coli BL21 (DE 3), resepectively. After induced by 100 mmol/L IPTG, the positive bacteria successfully expressed these proteins. Finnally, the atp5b and reporter gene gfp were inserted into the mammal cell expression vector pCDNA3.1hisB. These work were helpful for revealing the function of atp5b, idh3αand prxⅡin hepatocarcinogenesis induced by Aflatoxin B1.
引文
[1]肖良,邢卫锋.黄曲霉毒素的危害与控制.世界农业, 2003, 287(3):40-42
[2]魏丽莉.黄曲霉毒素对食品的污染及防治措施.粮油加工, 2008, 9:86-89
[3]李洪,李爱军,董红芬,等.黄曲霉毒素的发生危害与防控方法.玉米科学, 2004, 12: 88-90, 94
[4]吴丹.黄曲霉毒素在粮食和食品中的危害及防治.粮食加工, 2007, 32(3):91-94
[5]杨建伯.真菌毒素与人类疾病.中国地方病学杂志, 2002, 1(21):4-10
[6]王凤荣,张祥宏,张振东,等.真菌及其毒素诱发肺癌的动物实验研究.北京大学学报(医学版), 2003, 1(35):1-8
[7]张艺兵,张鹏,宋小岩,等.真菌毒素的风险分析.食品工业科技, 2002, 1(23):7-13
[8] Wang JS, Groopman JD. DNA damage by mycotoxins [J]. Mutat Res, 1999 , 424(1-2) :167- 181
[9] Guengerichn FP , Johnson WW , Shimada T , et al. Activation and detoxication of aflatoxin B1[J]. Mutat Res, 1998, 402 (1-2):121-128
[10]姜丽娜,杨杰,李君文.黄曲霉毒素B1诱发肝癌的生物标记物检测技术.中国公共卫生, 2005, 21(1):114-115
[11] Denissenko MF, Cahill J, Koudriakova TB, et al. Quantitation and mapping of aflatoxin B1- induced DNA damage in genomic DNA using aflatoxin B1-8,9-epoxide and microsomal activation systems [J] . Mutat Res, 1999, 425(2):205-211
[12]龙喜带,唐月浩,曲德英,等.黄曲霉毒素B1毒性及其发挥与DNA修复(修复相关酶).右江民族医学院学报, 2006, 28(2):278-279
[13] Hoeijmakers JH. Genome maintenance mechanisms for preventing cancer [J]. Nature, 2001, 411(6835):366-374
[14] Qin LL, Su JJ, Li Y, Yang C, et al. Expression of IGF-II, p53, p21 and HBxAg in precancerous events of hepatocarcinogenesis induced by AFB1 and/ or HBV in tree shrews [J]. World J Gastroenterol, 2000, 6(1):138-139
[15]马韵,邓卓霖,罗虹,等.广西肝癌高低发区p53基因突变热点的对比研究[J].临床与实验病理学杂志, 1997, 13(4):302-304
[16]杜艳平,朱惠莲.黄曲霉毒素B1致癌机制及植物性化学物防治的研究进展.现代食品与药品杂志, 2006, 16(1):3-8
[17] Chan KT, Dennis PH, Maria LL, et al. In vitro aflatoxin B1 induced p53 mutations [J]. Canc Let, 2003, 199 (1):1-7
[18] Paul CT, Abdoulaye S, Kuang SK, et al. Absence of P53 Codon 249 mutations in young guinean children with high Aflatoxin exposure [J]. Canc Epid Bio & Pre, 2005, 14:2053-2055
[19]许真,金银龙.环境致癌剂与P53基因突变[J].卫生研究, 2004, 33(2): 239-243
[20] Su JJ, Ban KC, L i, Y, et al. Alteration of p53 and p21 during hepatocarcinogenesis in tree shrews [J]. World J Gas, 2004, 10(24): 3559-3563
[21]苏建家,李瑗,班克臣,等.动态研究HBV和AFB1诱发树鼩肝细胞癌过程中一些基因的表达[J].广西医科大学学报, 2001, 18:151-154
[22]班克臣,曹骥. Survivin在AFB1高暴露地区肝细胞癌中的表达及其临床意义[J].中国肿瘤临床, 2005, 32(11):614-617
[23] Duan XX, Ou JS, Li Y, et al. Dynamic expression of apoptosis-related genes during development of laboratory hepatocellular carcinoma and its relation to apoptosis [J].World J Gas, 2005, 11(30):4740-4744
[24] Henry SH, Bosch FX, Bowers JC, et al. Aflatoxin, hepatitis and worldwide liver cancer risks [J]. Adv Exp Med Biol, 2002, 504:229-233
[25] Kwak MK, Patricia AE, PatrickMD, et al. Role of phase enzyme induction in chemop rotection by dithiolethiones [J]. Mut Res, 2001, 480 (1) :305-315
[26]吴一迁,仇效坤,万曙光,等. HBVX转基因小鼠肝组织基因表达谱的微阵列研究[J].肿瘤, 2001, 21(4):235-238
[27] Zhang YJ , Ahsan H, Chen Y, et al. High Frequency of promoter hypermethylation of RASSF1A and p16 and its Relationship to aflatoxin B1-DNA adduct Levels in human hepatocellular carcinoma [J]. Mole Carc, 2002, 35: 85-92
[28]俞顺章,赵宁,资晓林,等.饮水中微囊藻毒素与我国原发肝癌关系的研究[J].中华肿瘤杂志, 2001, 23(2):96-99
[29]顾公望,周汉高.肝癌病因研究的共识.世界肿瘤杂志, 2002, 1(1):1-3
[30]秦雪,代智,崔杰峰,等.黄曲霉毒素B1诱发树鼩肝癌过程中的差异表达蛋白质分析及意义.中华检验医学杂志, 2006, 29(6):538-542
[31] Furger KA, Menon RK, Tuckl AB, et a1. The functional and clinical roles of osteopontin in cancer and metastasis. Curr Mol Med, 2001, 1(5):621-632
[32] Chang PL, Chambers AF. Transforming JB6 ceils exhibitenhanced integrin-mediated adhesion to osteopontin. Cell Biochem, 2000, 78(1):8-23
[33] Coppola D, Szabo M. Correlation of osteopontin protein expression andpathological stage across a wide variety of tumor histologies. Clin Cancer Res, 2004, 10(1):184-190
[34]曾丽霞,冯震博,苏建家,等.骨桥蛋白mRNA在黄曲霉毒素B1诱导大鼠肝细胞癌中的表达.广西医科大学学报, 2006, 23(2):216-218
[35] Wang Y, Mochida S, Kawashima R, et a1. Increased expression of osteopontin in activated Kupffer cells and hepatic macrophages during macrophage migration in propionibacterium acnestreated rat liver. J Gastroenterol, 2000, 35(9):696-701
[36] Shen HM, Ong CN. Mutations of the p53 tumor suppressor gene and ras oncogenes in aflatoxin hepatocarcinogenesis. Mutat Res, 1996, 366(1):23-44
[37] Gao X, Zhang YP, Mitchell DA, et a1. Identification of a ras-activated enhancer in the mouse osteopontin promoter and its interaction with a putative ETS-related transcription factor whose activity correlates with the metastaticpotential of the cel1. Mol Cell Biol, 1995, 15(1):476-487
[38] Teramoto H, Castellone MD, Malek RI, et al. Autocrine activation of an osteopontin-CD44-Rac pathway enhances invasion and transformation by H-RasV12. Oncogene, 2005, 24(3):489-501
[39] Ye QH, Qin LX, Forgues M, et a1. Predicting hepatitis B virus-positive metastatic hepatocellular carcinomas usinggene expression profiling and supervised machine learning. Nat Med, 2003, 9(4):416-423
[40] Lu Z, Ghosh S, Wan g Z, et a1. Downregulation of caveolin-l function by EGF leads to the loss of E-cadhefin increased transcriptional activity of beta-catenin and enhanced tumor cell invasion. Cancer Cell, 2003, 4:499-515
[41] Cui J, Zhou X, Liu L, et al. AIterations of beta-catenin and Tcf-4 instead of GSK-3beta contribute to activation of Wnt pathway in hepatocellular carcinoma. Chin Med J (Eng1), 2003, 116:l885-1892
[42] Logan CY, Nusse R. The Wnt signaling pathway in development and disease. Annu Rev Cell Dev Bio1, 2004, 20:78-810
[43]焦杨,班克臣,曹骥,等.黄曲霉毒素Bl诱发大鼠肝癌过程中β连环蛋白的变化.中华肝脏病杂志, 2007, l5(l):783-784
[44] Satoh S, Daigo Y, Furukawa, et al. AXINl mutations in hepatocellularcarcinomas and growth suppression in can cer cells by Virus-mediated transfer of AXIN1. Nat Genet, 2000, 24:245-250
[45] Devereux TR, Stem MC, Flake GP, et a1. CTNNBl mutations and beta-catenin protein accumulation in human hepatocellular carcinomas associated with high exposure to aflatoxin B1. Mol Carcinog, 2001, 3l: 68-73
[46] Morin PJ, Sparks AB, Korinek, et a1. Activation of beta-catenin Tcf signaling in colon cancer by mutations in beta-catenin or APC. Science, 1997, 275:l787-1790
[47] Miyoshi, Wao K, Nagasawa, et a1. Activation of the beta-catenin gene in primary hepatocellular carcinomas by somatic alterations involving exon3. Cancer Res, 1998, 58:2524-2527
[48]岳海英,黎丹戎,李瑗. PeroxiredoxinⅡ研究进展.医学研究杂志, 2007, 36(2):101-102
[49] Okazaki S, Naganuma A, Kuge S. Peroxiredoxin-mediated redox regulation of the nuclear localization of Yap1 a transcription factor in budding yeast. Antioxid Redox Signal, 2005, 7 (324):327-333
[50] Lee SC, Na YP, Lee JB. Expression of peroxiredoxin II in vascular tumors of the skin: A novel vascular marker endothelial cells. J Am Acad Dermatol, 2003, 49 (3):487-492
[51] Young DY, Young MC, Jong KP, et al. Synergistic effect of peroxiredoxin II antisense on cisplatininduced cell death. Exp Mol Med, 2002, 34(4):273-284
[52]秦雪,代智,崔杰峰,等.黄曲霉毒素B1诱发树鼩肝癌过程中的差异表达蛋白质分析及意义.中华检验医学杂志, 2006, 29(6):538-542
[53]刘萍,宋安萍,马丁.硫氧还蛋白过氧化物酶Ⅱ诱饵质粒的构建和检测.华中医学杂志, 2007, 31(6):467-469