HSPB8在棒曲霉毒素引起的人胚肾细胞毒性中的作用及调控机理研究

Patulin, a secondary metabolites mainly produced by Penicillium and Aspergillus, has shown to be toxic in animals, carcinogenic, mutagenic and teratogenic, and kidney is one of the target organs. The small heat shock protein HSPB8 that located in the mitochondrial matrix has chaperone-like, kinase activity, pro- or anti apoptotic and life-span prolonging effects, being involoved in the thermol-tolerance, oxidation-resistance and antitumor. In our previous studies, it was found that HSPB8 gene to be up-regulated 615, 739 and 607-fold under 0.25μM PAT stress at 3,10 and 24 hour respectively in human embryo kidney cells HEK293. In order to understand of the role and mechanism of HSPB8 on the cytotoxicity in human embryo kidney cells induced by patulin, in our experiment, we fist overexpressed HSPB8 cDNA gene into HEK293 by gene transfection and G418 screening, and meanwhile inhibited the expression of HSPB8 gene of HEK293 by RNA interference technology. On that basis, through the detections of cell damage,apoptosis signals and mitochondrial functions as well as the gene expression profiling using the burgeoning digital gene expression tag profile (DGE) method, the effects of HSPB8 on HEK293 were observed. The studies should provide potent scientific basis for the explaination of molecular mechanism of the human cells under PAT stress and the corresponding food safety issues, in the meantime, it should provide reliable scientific guarantee for the biological treatment field and human health.

棒曲霉毒素（PAT）是一种有毒的真菌代谢产物，具有致癌、致畸、致突变等毒理作用，肾脏是其靶器官之一。定位于线粒体基质的热休克蛋白HSPB8具有分子伴侣、蛋白激酶、促凋亡/抗凋亡、延长生物寿命等功能，与生物耐热、抗氧化、抗肿瘤相关。我们在前期初步研究发现人胚肾细胞HEK293中HSPB8基因受PAT诱导超高表达，本研究拟通过构建人HSPB8过表达重组质粒，转染细胞，获得稳定高表达HSPB8的细胞株；同时利用RNA干扰技术，构建慢病毒RNA干扰载体，特异地将HSPB8基因沉默，从而使其功能丧失或表达量降低。在此基础上，通过细胞损伤、凋亡信号、线粒体功能、数字基因表达谱检测，观察HSPB8对PAT所致细胞损伤及相关信号通路的影响，综合分析其调控机制。该研究可为解释人体细胞对PAT应激反应的分子机制及相应食品安全问题提供有力的科学基础，同为生物治疗领域和人体健康提供可靠的科学保障。

棒曲霉毒素（patulin，PAT）主要是由青霉和曲霉产生的一种有毒的次生代谢产物，具有致癌、致畸、致突变等毒理作用，已经成为影响食品安全性的重大问题。热休克蛋白HSPB8具有分子伴侣、蛋白激酶、促凋亡/抗凋亡、延长生物寿命等生物学功能，与生物耐热、抗氧化、抗肿瘤相关。前期初步研究发现人胚肾细胞HEK293的HSPB8基因受PAT诱导高表达，为深入探讨HSPB8在PAT致胚肾细胞毒性中的调控作用，实验拟通过构建人HSPB8过表达重组质粒，转染HEK293细胞，获得稳定表达HSPB8蛋白的细胞株；同时利用RNA干扰技术，构建慢病毒RNA干扰载体，特异地将HSPB8基因沉默，从而使其功能丧失或表达量降低。在此基础上，通过细胞凋亡、线粒体功能、数字基因表达谱测定，观察HSPB8对PAT所致细胞损伤及相关信号通路的影响，综合分析其调控机制。初步结果表明：HSPB8过表达能够有效缓解PAT引起的细胞损伤，而HSPB8基因敲除则促进了PAT引起的细胞毒性。 HSPB8对PAT毒性作用的调控可能和线粒体氧化磷酸化（特别是复合体I和复合体V），caspase凋亡信号级联信号途径以及蛋白、核酸的损伤修复有关。该研究可为解释人体细胞对PAT应激反应的分子机制及相应食品安全问题提供有力的科学基础，同时为生物治疗领域和人体健康提供可靠的科学保障。