【作者】 吴赤蓬；

【导师】 钟雪云；

【作者基本信息】 暨南大学 ， 病理学与病理生理学， 2008， 博士

【摘要】 研究目的:研究低剂量NaNO₂能否诱导CHL细胞产生氧化损伤的适应性反应,即体外培养的CHL细胞先接触低剂量NaNO₂一段时间后,能否产生对大剂量NaNO₂的细胞毒性及遗传毒性的抵抗力,并阐明其剂量—效应关系。探讨聚ADP核糖聚合酶（polyADP-ribose polymerases,PARP-1）在低剂量NaNO₂诱导适应性反应形成过程中的作用。研究结果可为确定NaNO₂的安全接触剂量提供参考依据。研究方法:1、采用MTT试验观察NaNO₂对体外培养的中国仓鼠肺细胞（Chinese hamster lung cells,CHL cells）的细胞毒性作用,确定其最大无作用剂量（no observed active effect lever,NOAEL）,并以低于NOAEL的剂量预处理细胞6h,观察其对随后大剂量染毒的抵抗力。2、采用CHL细胞染色体畸变分析法、单细胞凝胶电泳试验及流式细胞仪检测细胞凋亡及细胞周期的方法研究低剂量NaNO₂诱导CHL细胞在遗传毒性作用方面的适应性反应及其剂量-反应关系。3、采用PARP-1的特异性抑制剂3-氨基苯甲酰胺（3-aminobenzamide,3-AB）抑制CHL细胞的PARP-1活性后,观察其对低剂量NANO₂诱导CHL细胞适应性反应的影响,从而探讨PARP-1蛋白在适应性反应形成过程中的作用。4、采用western-blot技术检测不同剂量NaNO₂作用6h后,CHL细胞PARP-1蛋白表达量的改变,从而进一步弄清PARP-1在适应性反应中的作用。研究结果:1、MTT试验结果显示,1.52mg/L～40mg/L的NaNO₂作用于CHL细胞24h,可以促进细胞增殖,其细胞存活率高于正常对照组,表现出明显的hormesis效应;0.0005mg/L～0.01mg/L的NaNO₂作用6h,可诱导CHL细胞产生对1g/L大剂量NaNO₂细胞毒性作用的适应性反应;但是,低剂量NaNO₂预处理不能诱导CHL细胞对更高剂量（如5g/L）的NaNO₂产生适应性反应。2、CHL细胞染色体畸变试验的结果显示,当NaNO₂的剂量高于128mg/L时即可诱导CHL细胞染色体畸变率的增加,染色体畸变类型有断裂、交换、双着丝粒、环状染色体、多倍体等。0.5mg/L～2.5mg/L的NaNO₂预处理CHL细胞6h,则可使后续1.5g/L大剂量NaNO₂染毒导致的染色体畸变率的增加幅度明显降低,表现出一定程度的适应性反应。在采用3AB抑制PARP-1的活性的情况下,可观察到适应性反应被阻断。3、单细胞凝胶电泳试验的结果显示,剂量高于200mg/L的NaNO₂作用于CHL细胞18h,可导致细胞DNA的明显损伤,表现为细胞拖尾率的增加,拖尾程度加重,与正常对照组相比,其差异有统计学意义。0.01mg/L～0.1mg/L的NaNO₂预处理CHL细胞6b,可减轻随后1g/L大剂量NaNO₂染毒造成的DNA的损伤程度,表现出明显的适应性反应。在低剂量NaNO₂预处理CHL细胞6h后,采用3AB抑制PARP-1的活性,不会阻断适应性反应的产生;而在低剂量预处理CHL细胞之前,即用3AB抑制PARP-1的活性,则可以阻断适应性反应的产生。4、对细胞凋亡率及细胞周期的观察结果显示,剂量高于1.5g/L的NaNO₂作用于CHL细胞18h,可导致细胞凋亡率的明显升高,细胞发生明显的G₁期阻滞,S期及G₂期细胞比例减少,细胞的DNA相对含量及增殖指数均降低,表明细胞的DNA受到了较为严重的损伤;以0.01mg/L～0.1mg/L的NaNO₂预处理CHL细胞6h,可明显降低随后1.5g/L大剂量NaNO₂染毒导致的细胞凋亡率的增加,并使G₁期的阻滞现象明显减轻,S期及G₂期细胞比例增加,细胞的DNA相对含量及增殖指数均有所上升,表明细胞对DNA损伤的修复能力增强。3AB可抑制低剂量NaNO₂诱导的DNA损伤修复能力,表现为S期细胞比例的明显减少,DNA相对含量及细胞增殖指数的下降;但3AB不会抑制低剂量NaNO₂诱导的促凋亡作用。5、0.1mg/L的NaNO₂作用于CHL细胞6h后,通过Western-blot试验可观察到分子量为116KD的PARP-1蛋白条带显色变浅,推测这一现象与PARP-1通过磷酸化的ERK₂途径被激活,发生自身聚ADP核糖基化作用,形成大量聚ADP核糖聚合体,从而使其分子量发生改变有关。1g/L剂量组的蛋白条带也变浅,原因可能是1g/L大剂量的NaNO₂导致CHL细胞DNA较严重的损伤,从而也使其PARP-1蛋白经DNA损伤途径大量被激活,形成聚ADP核糖聚合体的缘故。结论:1、0.01mg/L～0.1mg/L剂量范围的NaNO₂可诱导CHL细胞对1.0g/L和1.5g/L剂量NaNO₂染毒所致的损伤作用的适应性反应;低剂量NaNO₂不能诱导CHL细胞对更高剂量（如5g/L）NaNO₂的毒性作用的适应性反应。2、低剂量NaNO₂具有毒物刺激作用（即hormesis效应）,其对CHL细胞的hormeticzone的剂量范围并不与其能诱导适应性反应的剂量范围重叠,适应性反应的剂量范围低于hormetic zone的下限。据此可以认为,hormesis效应是NaNO₂对CHL细胞的遗传物质造成损伤的预警信号,能诱导适应性反应的剂量才是真实的安全剂量。3、PARP-1蛋白在低剂量NaNO₂诱导CHL细胞氧化损伤适应性反应的形成过程中具有关键性的作用。当低剂量NaNO₂作用于CHL细胞时,CHL细胞的PARP-1很可能通过磷酸化的ERK₂激活途径被激活,从而增强细胞DNA损伤的修复能力,并促使不可修复的细胞发生凋亡。PARP-1蛋白是细胞DNA损伤修复时所必需的蛋白,如果其活性被抑制,则DNA损伤的修复过程也将被抑制;但PARP-1蛋白却不是细胞凋亡所必需的蛋白,抑制PARP-1的活性,并不能阻止细胞发生凋亡,PARP-1很可能只是细胞凋亡的起始因素之一,需要通过激活其下游的其它凋亡相关基因而发挥作用。4、本研究的受试生物体为中国仓鼠肺细胞,由于受到种属特性的限制,以上结论尚不能外推于人。有必要进一步对整体动物或人的体细胞进行研究。更多还原

【Abstract】 Research Aim:To study the problem if low dose NaNO₂ can induce the adaptive response of cultured Chinese hamster lung cells（CHL cells） to oxidative damage caused by higher dose NaNO₂ and how the dose-effect relationship is.Try to explain the function of polyADP-ribose polymerases（PARP-1） in the adaptive response.Methods:1.The MTT-test was used to detect the cytotoxic effect of NaNO₂ in CHL cells.And the highest dose in which NaNO₂ cannot harm the cells would be found.This dose is also called "no observed active effect lever,NOAEL".The adaptive response of CHL cells to cytotoxicity of NaNO₂ induced by NaNO₂ pretreatment with its dose below the NOAEL and its dose-response relationship were observed.2.The adaptive response of CHL cells to the genotoxicity of NaNO₂ and its dose-response relationship was observed by the methods of the chromosomal aberration test,single cell gel electrophoresis technique and flow cytometry of apoptosis and cell cycle.3.3-aminobenzamide（3AB） is the specific inhibitor of PARP-1.After the activity of PARP-1 was inhibited by 3AB,it was observed whether the adaptive response induced by low dose NaNO₂ pretreatment could be blocked.And the function of PARP-1 was revealed according to the research results.4.The expression of PARP-1 protein was tested with western-blot test when CHL cells exposing to NaNO₂ of different doses.Results:1.MTT test reveals that NaNO₂ can enhance proliferation of cells when the doses are between 1.52mg/L and 40mg/L.The survival rates of CHL cells are higher than that of the normal control group.And the hormesis effect can be observed.NaNO₂ can induce the adaptive response of CHL cells to the cytotoxicity of 1g/L NaNO₂ when the cells were pretreated with the doses between 0.0005mg/L and 0.01mg/L for 6 hours.But the pretreatment with low dose NaNO₂ cannot induce the adaptive response of CHL cells to much higher dose NaNO₂（such as 5g/L）.2.Chromosomal aberration test reveals that NaNO₂ can increase the rate of chromosome aberration in CHL cells when the exposing doses are higher than 128mg/L.The types of chromosome aberration include break,chromatid exchange,dicentric chromosome,ring chromosome and polyploid.When CHL cells are pretreated with doses between 0.5mg/L and 2.5mg/L for 6 hours,NaNO₂ can induce the adaptive response to chromosome or chromatid aberration caused by 1.5g/L NaNO₂.And 3AB can inhibit the adaptive response induce by low dose NaNO₂.3.NaNO₂ can cause DNA damage in CHL cells when the doses are higher than 200mg/L and the treatment time is 18 hours.After CHL cells are treated with low dose（0.01mg/L～0.1mg/L） NaNO₂ for 6 hours,NaNO₂ can induce the adaptive response to DNA damage caused by 1g/L NaNO₂.3AB can block the adaptive response of DNA damage caused by low dose NaNO₂ before the pretreatment of the cells.But after 6 hours of low dose NaNO₂ pretreatment,3AB cannot block the adaptive response induced by NaNO₂.4.NaNO₂ can enhance apoptosis of CHL cells,cause the blockage of the cells in G₁ stage,and decrease the percentage of cell numbers in S and G₂ stages,and make the DNA related concentrations(DNA_RC) and the cell proliferation index（PI） decreasing.That indicates the cells suffer from severe DNA damage when they are exposed to 1.5g/L NaNO₂ for 18 hours.The adaptive response of apoptosis can be induced by pretreatment cells for 6 hours with low dose NaNO₂（0.01mg/L～0.1mg/L）.When the activity of PARP-1 is inhibited by 3AB,the function of DNA repair is blocked,but the actions related with apoptosis is not affected.5.Western-blot test reveals that the strap of PARP-1 protein with 116KD molecular weight become more pale when CHL cells exposing to NaNO₂ of 0.1mg/L group for 6 hours than that of other groups.It can be known that the NaNO₂ dose of 0.1mg/L is the optimal dose to induce adaptive response from the above other tests results.This also indicates that PARP-1 has a close relationship with adaptive response induced by low dose NaNO₂.Because low dose NaNO₂ can activate PARP-1 through the phosphorylated ERK₂ route,and change PARP-1 itself to polyADP-ribose（PAR） polymers,the molecular weight of PARP-1 protein is also changed.The strap of 116KD PARP-1 protein of 1g/L group is also more pale,but this can only be related with severe DNA damage caused by 1g/L NaNO₂.Conclusion:1.NaNO₂ pretreatment of low dose between 0.01mg/L and 0.1mg/L.can induce adaptive response of cultured CHL cells to oxidative damage caused by NaNO₂ of 1.0g/L or 1.5g/L.But to much higher dose NaNO₂（5g/L）,low dose NaNO₂ pretreatment cannot induce the adaptive response. 2.Low dose NaNO₂ can trigger hormesis effect in CHL cells.But the hormetic zone is much higher than the dose range that can provoke adaptive response.It indicates that hormosis may be a precaution signal of DNA damage.And only the much lower dose under the lowest limit of hormetic zone that can induce adaptive response is the real safe dose.3.PARP-1 is a critical factor in the formation of adaptive response induced by low dose NaNO₂. PARP-1 can probably be activated through the phosphorylated ERK₂ route when CHL cells are treated with low dose NaNO₂.Then the function of DNA repair is enhanced,and meanwhile,the apoptosis procedure is also provoked in the cells with their DNA damaged severely.The DNA repair function of PARP-1 is crucial to the adaptive response.But the apoptosis mediated by PARP-1 is not the only way.PARP-1 only is one of initiated factors of apoptosis procedure probably.The function of PARP-1 may be related with the activation of its downstream relative genes.4.In this research,the receptive biosystem is cultured Chinese hamster lung cells.The above conclusions cannot be extrapolated to human conditions because of the differences between the species.Such problem should be further studied in animal vivo or in other human somatic cells.更多还原