诱导型热休克蛋白70降低甲醛生物学损伤的作用和机制研究

Functions of inducible heat shock protein 70(Hsp70), such as regulating production of reductive glutathione, strengthening resistance to oxidative damage and promoting DNA repair, related not only to formaldehyde(FA) metabolism but also to the induction and repair of biologic damage caused by FA. It was found that Hsp70 synthesis in primary spermatocytes and spermatids increased when FA induced damage in the testicular tissue of rats. What was the function of Hsp70 in the process, however, remained unknown. Therefore, applicants carried out corresponding research and found that cells overexpressed Hsp70 had less oxidative and genetic damages than those of the control cells after exposure to FA, but the mechanism warranted further investigation. So, in this scientific research item, using human bronchial epithelium (HBE) as research object, we firstly plan to investigate the function of Hsp70 when HBE exposes to FA. By transfecting plasmid harboring hsp70 gene or RNA interfering (RNAi) sequence targeting hsp70 gene into HBE, we establish hsp70 overexpressed, hsp70 knocked-out and corresponding control HBE and then measure the oxidative and genetic damages in these cell groups to find whether there is a difference in the induction and repair of damages caused by FA, thus to approve that Hsp70 play roles in the alleviation of FA toxicity. Then, we compare the expression level, location and activity of FA metabolic and DNA repair enzymes in different cell groups, and further explore the interaction between Hsp70 and FA metabolic or DNA repair enzymes, thus to interpret the mechanism how Hsp70 could alleviate the biologic damages caused by FA. In this way, we try to offer scientific evidence for the further elucidation of Hsp70 function and the prevention of occupational injury of FA.

诱导型热休克蛋白70（Hsp70）调控谷胱甘肽的生成、抵抗氧化性损伤、促进DNA修复的功能分别和甲醛的代谢、所致损伤、损伤的修复有一定关联。研究发现甲醛导致损伤时，Hsp70的表达增加，但其作用尚不清楚。申请者前期研究发现高表达Hsp70细胞的甲醛氧化性损伤和遗传损伤程度均低于对照细胞，但机制未知。本项目拟以Hsp70在甲醛染毒后的作用为切入点，以人支气管上皮细胞为研究对象，利用质粒转染和RNA干扰技术，建立Hsp70高表达、低表达和相应的对照细胞组，比较细胞组间甲醛所致损伤和损伤修复的差异，证实Hsp70具有降低甲醛生物学损伤的作用；比较细胞组间甲醛代谢酶和DNA修复酶的表达量、定位和活性的差异，进一步运用免疫共沉淀、激光共聚焦技术分析Hsp70与甲醛代谢酶、DNA修复酶之间的交互作用和共定位关系，阐述可能的作用机制，为Hsp70功能的进一步阐明和甲醛所致职业损伤的预防提供科学依据。

甲醛是一种用途广泛的化工产品，在我国有着覆盖面很广的职业接触人群。2004年，国际癌症研究机构(IARC)把甲醛归为人类明确致癌物（Ⅰ类），和鼻咽癌、鼻窦癌、白血病等的发生有关。.甲醛经呼吸道、消化道和皮肤进入人体后，绝大部分甲醛脱氢酶（formaldehyde dehydrogenase, FDH也是alcohol dehydrogenase，ADH）为主的代谢酶作用下转化成甲酸而解毒。进入人体的甲醛如果逃脱了代谢解毒就会产生一系列的生物学损害，如对蛋白质、脂质和DNA造成氧化性损伤，使生物大分子结合形成交联甲醛造成的上述损伤分别进入不同的修复过程。DNA发生氧化性碱基损伤后，通过碱基切除修复（base excision repair, BER）途径修复，DNA-蛋白质交联（DNA protein crosslinks，DPC）的修复机制目前还不十分清楚，研究报道表明与核苷酸切除修复(nucleotide excision repair, NER)、同源重组 (homologous recombination, HR)、范科尼贫血（Fanconi anemia, FA）修复途径有关。 .Hsp70由位于人类6号染色体的HSP70-1、HSP70-2基因编码，它不仅和减小有害因素造成的损伤有关，而且还参与了损伤的修复过程。Hsp70可以增强谷胱甘肽过氧化物酶(Glutathione peroxidase, GPx)和谷胱甘肽还原酶(glutathione reductase, GR)的活性，并且和FA修复途径中的重要蛋白FANCC、BER途径中的多种修复蛋白有交互作用。.本研究为了进一步证实Hsp70在甲醛染毒过程发挥了生物学保护作用，以人支气管上皮细胞（HBE）为研究对象，采用基因过表达和RNA干扰技术，建立Hsp70高表达细胞组、Hsp70降低表达细胞组、对照细胞组和正常培养细胞组。用不同浓度甲醛染毒，发现Hsp70表达水平不同的细胞组间氧化性损伤（MDA的含量）存在明显差别，进一步检测到GR酶活性在组间存在差异，GSH含量在组间也存在差异，这样的结果提示Hsp70在甲醛染毒时候，通过增加GSH含量和促进GR酶的活性从而降低了甲醛造成的氧化性损伤。在DPC的形成方面，我们的结果显示仅在较高剂量时(大于500uM)时，高表达Hsp70的细胞才出现比其他组细胞更