高表达诱导型热休克蛋白70对甲醛所致损伤的体外试验研究

Effects of overexpression of heat shock protein 70 on the damage induced by formaldehyde in vitro

目的研究诱导型热休克蛋白70（heat shock proteinTO，Hsp70）在甲醛所致损伤中的保护作用。方法人支气管上皮细胞human bronehi Mepithelium，HBE）转染含有hsp70基因的质粒，以增高Hsp70的表达，转染载体质粒的细胞和正常培养的细胞作为对照，分别为Hsp70高表达组（HBE／hsp70）、转染对照组（HBE／pcDNA）和对照组（HBE），并对3组细胞Hsp70的表达量进行鉴定。HBE／hsp70组和HBE组在接受不同浓度甲醛（0,0．39、1．56、6．25mmol/L）染毒4h后，检测各组细胞谷胱甘肽（GSH）和丙二醛（MDA）含量以及DNA-蛋白质交联（DNA—protein crosslink，DPC）。结果细胞转染含有hsp70基因的质粒后，Hsp70蛋白表达量与HBE组相比增高约80％。HBE／hsp70组GSH含量先增高，在甲醛浓度为0．39和1．56mmol/L时分别为141．0、119．6mg／gpro，与HBE组（分别为75．8、56．7mg／gpro）比较，差异有统计学意义（P〈0．01），但当甲醛浓度增高到6．25mmol/L时，GSH含量降低。而HBE组细胞的GSH含量随着甲醛浓度的增高一直呈下降的趋势。HBE／hsp70组、HBE组细胞MDA含量均随甲醛浓度的升高而增加，且HBE／hsp70组细胞MDA含量一直低于HBE组，在甲醛浓度为1．56mmol／L时MDA含量为0．088μmol／gpro，与HBE组（0．138μmol／gpro）相比，差异有统计学意义（P〈0．05），甲醛浓度为6．25mmo]／L时，HBE／hsp70组细胞MDA含量为0．140μmo]／gpro，与HBE组（0．289μmol／gpro）相比，差异有统计学意义（P〈0．01）。HBE／hsp70组、HBE组细胞DPC百分含量均随甲醛浓度的升高而增加，且HBE／hsp70组一直低于HBE组，在甲醛浓度为0．39mmol／L时，HBE／hsp70组DPC含量为3．94％，HBE组为6．25％，差异有统计学意义（P〈0．01），甲醛浓度为1．56mmol／L时，HBE／hsp70组DPC含量为11．86％，HBE组为20．89％，差异有统计学意义（P〈0．05）。结论Hsp70可降低甲醛对体外支气管上皮细胞造成的损伤。

Objective To investigate the protective role of inducible beat shock protein 70 （Hsp70） against damage induced by formaldehyde. Methods Human bronchial epithelium （HBE） cells were transfected with plasmid harboring hsp70 gene to increase the protein expression level. HBE cells transfected with pcDNA3.1 plasmid were used as transfection control and HBE cells cultured at normal condition served as control. Three groups were marked as HBE/hsp70, HBE/pcDNA and HBE. Hsp70 expression levels of 3 groups were detected. The cells of HBE/hsp70 and HBE groups were exposed to different concentrations of formaldehyde （0,0.39,1.56,6.25 mmol/L） for 4 h. The contents of GSH and MDA were measured, and KCl- SDS method was applied to measure DNA-protein crosslink （DPC）. Results Hsp70 level in HBE/hsp70 group increased by 80% compared with HBE group. GSH contents in HBE/hspT0 group significantly increased and were 141.0, 119.6 mg/gpro at 0.39, 1.56 mmol/L, respectively （P〈0.01）, as compared with HBE group. However, it decreased when formaldehyde concentration increased to 6.25 mmol/L. While GSH content in HBE group remained decreasing. MDA contents in HBE/hsp70 and HBE group increased with formaldehyde. MDA content in HBE/hsp70 was 0.088μmol/gpro and significantly lower than that （0.138 μmol/gpro） in HBE group （P〈0.05） when formaldehyde concentration was 1.56 mmol/L, At the formaldehyde dose of 6.25 mmol/L MDA content in HBE/hsp70 was 0.140 μmol/gpro which was significantly lower than that （0.289 μmol/gpro ） in HBE group （P〈0.01）. DPC% in two groups increased with formaldehyde. At the formaldehyde dose of 0.39 mmol/L, DPC% in HBE/hsp70 group was 3.94% which was significantly lower than that （6.25%） in HBE group （P〈 0.01）. At the formaldehyde dose of 1.56 mmol/L, DPC% in HBE/hsp70 group was 11.86% which was significantly lower than that （20.89%） in HBE group （P〈0.05）. Conclusion Hsp70 can reduce formaldehyde-induced damages in human bronchial epithelium cells in vitro.