【作者】 宫智勇；

【导师】 邬堂春；

【作者基本信息】 华中科技大学 ， 劳动卫生与环境卫生学， 2006， 博士

【摘要】 当生物受到外界环境因素作用时，如高温、缺氧、毒物等，机体迅速启动高度程序化的热休克反应(heat shock response，HSR)，诱导合成许多热休克蛋白(heat shock proteins，Hsps)。Hsps可作为“分子伴侣”，促进新蛋白质的合成、折叠、装配和运输，参与某些特殊调节蛋白的激活，以及蛋白信号系统如类固醇激素的活化，并参与蛋白降解和受损蛋白质的清除或恢复．按分子量的不同，Hsps可分为许多家族，其中热休克蛋白70(HSP70)家族是机体内含量最丰富Hsps，占细胞总蛋白质含量的1～2％，主要包括诱导型Hsp70(即Hsp72)和组分型Hsc70(又称Hsp73)。许多研究表明，Hsp70的表达升高或降低，导致机体细胞抵御外来环境有害因素的能力变化。Hsp70升高对环境危害因素如紫外线导致细胞损伤具有保护作用，但抑制Hsp70对细胞功能产生严重损害。Hsp70对细胞起着保护作用。苯并(a)芘(benzo(a)pyrene，BaP)是一种化学致癌物，广泛存在于生产和生活环境中。进入体内的BaP可在体内代谢成活性代谢产物BPDE(benzo(a)pyrene diolepoxide)。BPDE具有亲电子性，可与DNA亲核位点共价结合形成加合物，导致DNA损伤。BaP作用产生的DNA损伤修复，主要是由核苷酸切除修复(nucleotide-excision repair，NER)途径来完成。参与DNA损伤NER修复的主要核苷酸切除修复酶有XPA-G(xeroderma pigmentosum group A-G)、ERCC1(excision repair cross complementation group 1)等。本室早先研究揭示，在有多环芳烃职业暴露史的焦炉工人中，Hsp70水平较高的个体淋巴细胞DNA损伤轻微，Hsp70较低的个体DNA损伤较严重。Hsp70作为一种重要的保护性蛋白，可能与BaP所致DNA损伤及损伤修复相关酶的合成有关。本研究采用细胞转染和槲皮素处理的方法，分别使肺腺癌细胞A549的Hsp70过表达或抑制，建立Hsp70过表达细胞株与Hsp70抑制细胞模型。采用单细胞琼脂糖凝胶电泳(single cell agarose gel electrophoresis，SCGE)、Western-blot等方法，观察在BaP作用下，未转染A549细胞DNA损伤和Hsp70表达的关系。并用单细胞琼脂糖凝胶电泳方法观察在BaP作用下Hsp70过表达和抑制细胞DNA损伤作用的变化，研究Hsp70在DNA损伤中的保护作用。用实时荧光定量RT-PCR方法，研究Hsp70在BaP致细胞主要核苷酸切除修复酶mRNA表达变化中的作用。探讨Hsp70在BaP所致细胞DNA损伤修复中的作用，为防治环境危害因素BaP对机体的危害作用提供科学依据。本研究共分三部分。第一部分：Hsp70过表达与Hsp70抑制细胞模型的建立本部分首先采用含hsp70基因cDNA的pcDNA3.0/hsp70重组质粒对A549细胞进行细胞转染，建立稳定过量表达Hsp70的细胞株。以空载体质粒pcDNA3.0转染A549细胞作载体对照。用G418筛选细胞，并对3个含hsp70基因的重组质粒转染成功的细胞克隆进行免疫荧光细胞化学染色分析。挑选Hsp70表达荧光强度最高的细胞克隆，与未转染A549细胞和空载体质粒pcDNA3.0转染成功细胞一起，采用Western-blot对其Hsp70表达水平进行分析。结果发现重组质粒转染细胞A549/hsp70的Hsp70蛋白表达明显高于肺腺癌A549细胞中的表达水平(P＜0.01)，空载体质粒转染细胞A549/pcDNA Hsp70的表达与肺腺癌A549细胞相比无明显改变，建立了Hsp70过表达细胞A549/hsp70。为了建立Hsp70抑制的细胞模型，本研究采用槲皮素处理A549细胞。槲皮素(quercetin)是一种广泛存在的生物黄酮类物质，有较强的抗氧化能力，可以抑制某些肿瘤细胞Hsp70的合成，使细胞Hsp70表达降低。本研究用50、100、150、200μmol/L槲皮素处理A549细胞，37℃培养6h后，用MTT实验和Western-blot方法分别检测A549细胞活性和Hsp70表达水平。结果发现，随槲皮素浓度的增加，A549细胞的细胞活性呈下降趋势，并呈现一定的剂量-反应关系。随着槲皮素浓度的加大，Hsp70表达逐渐受到抑制。当槲皮素浓度达到100、150和200μmol/L时，Hsp70表达显著降低。综合考虑槲皮素对A549细胞活性及Hsp70表达水平的影响，本研究用150μmol/L槲皮素处理A549细胞6h抑制Hsp70的表达作为Hsp70降低的细胞模型。第二部分：Hsp70在苯并(a)芘致细胞DNA损伤中的保护作用为了研究在BaP作用下，Hsp70在DNA损伤中的作用，本研究首先将未转染A549细胞暴露于不同浓度的BaP中，观察DNA损伤与Hsp70表达之间的关系。结果表明，随BaP染毒浓度的增加，A549细胞的细胞活性呈下降趋势，并呈现一定的剂量-反应关系；与未染毒细胞组相比，10、50、100μmol/LBaP染毒组，细胞活性显著性降低(P＜0.05)；各染毒组(1、5、10、50、100μmol/L BaP)A549细胞的DNA损伤明显增强(P＜0.05)，同时在较高剂量(10、50、100μmol/L)的BaP作用下，BaP代谢产物明显抑制了A549细胞Hsp70的表达。进一步分析A549细胞Hsp70表达与DNA损伤之间的关系，发现Hsp70表达水平和DNA损伤程度呈负相关。本研究结果表明，BaP的代谢产物有可能通过一定程度抑制Hsp70的表达而导致细胞活性及DNA的损伤。为进一步研究Hsp70在BaP致A549细胞DNA损伤中的保护作用，本研究采用第一部分建立的Hsp70过表达和抑制细胞模型，将不同Hsp70表达的细胞暴露于不同浓度的BaP，用MTT和单细胞琼脂糖凝胶电泳实验等方法，直接观察Hsp70在BaP致DNA损伤中的保护作用。结果表明，在BaP高浓度染毒组(50μmol/L、100μmol/L)，Hsp70过表达细胞A549/hsp70细胞活性明显高于未转染A549细胞(P＜0.05)，表明Hsp70水平升高有助于增强细胞对BaP危害作用的耐受能力；槲皮素处理细胞A549/Quercetin与A549细胞相比，BaP各浓度组的细胞活性无显著性改变，可能是槲皮素抗氧化作用特性减弱了抑制Hsp70对A549细胞活性的影响。研究结果还表明，与未转染A549细胞相比，各染毒剂量组Hsp70过表达细胞A549/hsp70的DNA损伤程度均显著性降(P＜0.05)；而槲皮素处理细胞A549/Quercetin，在BaP超过5μmol/L时，与相同BaP染毒剂量的A549细胞相比，DNA损伤程度呈显著性升高(P＜0.05)，可能是槲皮素致A549细胞Hsp70降低后，细胞DNA对环境危害因素BaP的耐受能力降低，对细胞DNA的危害加剧。这表明Hsp70对BaP所致细胞DNA损伤有明显的保护作用。当BaP浓度达到5μmol/L时，与未染毒A549/hsp70细胞相比，Hsp70过表达细胞中观察到明显DNA损伤。由此表明，当BaP作用过度时，Hsp70对BaP致A549细胞DNA损伤的保护作用降低，但Hsp70过表达细胞的BaP作用阈值浓度相对提高。第三部分：Hsp70在苯并(a)芘致细胞主要核苷酸切除修复酶mRNA水平变化中的作用以上实验结果表明，Hsp70对苯并(a)芘所致DNA损伤具有一定的保护作用，在BaP作用下Hsp70过表达细胞A549/Hsp70细胞活性明显高于A549细胞，DNA损伤程度明显低于A549细胞。但对于Hsp70如何发挥保护DNA损伤作用的机制仍不清楚。BaP作用产生的损伤，主要是由核苷酸切除修复途径来完成。Hsp70在苯并(a)芘致细胞主要核苷酸切除修复酶mRNA表达变化中是否具有一定作用呢?本研究应用实时荧光定量RT-PCR方法，检测BaP作用下，A549、Hsp70过表达细胞A549/hsp70和槲皮抑制Hsp70细胞A549/Quercetin主要核苷酸切除修复酶mRNA的变化，探讨Hsp70在苯并(a)芘致细胞主要核苷酸切除修复酶mRNA水平变化中的作用。XPA mRNA结果表明，与相同剂量BaP处理A549细胞组相比较，在10μmol/LBaP作用下，A549/pcDNA和A549/hsp70细胞XPA mRNA水平无明显变化，A549/Quercetin细胞XPA mRNA水平显著降低(P＜0.05)；在50μmol/LBaP作用时，A549/pcDNA XPA mRNA水平无明显变化，A549/hsp70、A549/Quercetin细胞XPA mRNA水平均显著降低(P＜0.05)；在100μmol/L BaP作用时，A549/pcDNA、A549/hsp70、A549/Quercetin细胞XPA mRNA水平均显著升高(P＜0.05)。XPC mRNA结果表明，与相同剂量BaP染毒A549细胞组相比较，在5μmol/LBaP作用时，A549/pcDNA细胞XPC mRNA水平显著性降低(P＜0.05)，A549/hsp70和A549/Quercetin细胞XPC mRNA水平显著性升高(P＜0.05)；在10μmol/LBaP作用时，A549/pcDNA细胞XPC mRNA水平无明显变化，A549/hsp70和A549/Quercetin细胞XPC mRNA水平均显著性升高(P＜0.05)；在100μmol/LBaP作用时，A549/pcDNA和A549/Quercotin细胞XPC mRNA水平无明显变化，A549/hsp70细胞XPC mRNA水平显著升高(P＜0.05)。XPB mRNA结果表明，与相同剂量BaP染毒A549细胞组相比较，在10μmol/L BaP作用下，A549/pcDNA和A549/Quercetin细胞XPB mRNA水平显著降低(P＜0.05)，A549/hsp70细胞XPB mRNA水平显著升高(P＜0.05)；在100μmol/L BaP作用下，A549/pcDNA细胞XPB mRNA水平显著降低(P＜0.05)，A549/hsp70和A549/Quercetin细胞XPB mRNA水平均显著升高(P＜0.05)。XPG mRNA结果表明，与相同BaP处理A549细胞相比较，在10μmol/L BaP作用下，A549/pcDNA、A549/hsp70和A549/Quercetin细胞XPG mRNA水平均显著降低(P＜0.05)；在100μmol/L BaP作用下，A549/pcDNA和A549/hsp70细胞XPG mRNA水平均无明显改变，A549/Quercetin细胞XPG mRNA水平显著降低(P＜0.05)。XPF mRNA结果表明，与相同BaP处理A549细胞组相比较，在1、5、10、50μmol/L BaP作用下，A549/pcDNA、A549/hsp70和A549/Quercetin细胞XPF mRNA水平均显著降低(P＜0.05)；在100μmol/L BaP作用下，A549/Quercetin细胞XPF mRNA水平显著升高(P＜0.05)。ERCC1 mRNA结果表明，与相同BaP处理A549细胞组相比较，在1、5、10、50μmol/L BaP作用下，A549/pcDNA、A549/hsp70和A549/Quercetin细胞ERCC1 mRNA水平均显著降低(P＜0.05)；在100μmol/L BaP作用下，A549/pcDNA、A549/hsp70细胞ERCC1 mRNA水平降低(P＞0.05)，A549/Quercetin细胞ERCC1 mRNA水平显著升高(P＜0.05)。综上所述，本研究结果表明：(1)采用含hsp70基因cDNA的pcDNA3.0/hsp70重组质粒进行细胞转染以及用150μmol/L槲皮素处理A549细胞6h抑制Hsp70的表达，可以分别建立Hsp70过表达和Hsp70抑制的细胞模型。(2)一定剂量的BaP作用24h，它的代谢产物可能通过一定程度抑制A549细胞的Hsp70表达而导致DNA损伤，Hsp70表达水平与DNA损伤程度呈负相关。(3)在BaP作用下，与未转染的A549细胞相比，Hsp70过表达细胞A549/hsp70细胞活性明显提高，DNA损伤程度显著性降低；而槲皮素处理细胞A549/Quercetin DNA损伤程度呈显著性升高。这表明升高Hsp70水平有助于增强细胞对BaP危害作用的耐受能力，对BaP所致细胞DNA损伤有明显的保护作用。(4) Hsp70可能在BaP致核苷酸切除修复酶XPC、XPB mRNA水平变化中具有一定作用。尽管本研究已得到一些初步结果，但如下几个方面的问题尚有待于进一步深入研究：1、运用siRNA技术，特异性抑制Hsp70的表达，深入研究Hsp70在DNA与修复中的作用机理。2、对于Hsp70在DNA损伤修复中的研究需更加深入，如探讨Hsp70是否与核苷酸切除修复酶结合而参与NER修复。3、如何利用Hsp70在DNA损伤与修复中的保护作用来保护呼吸系统等免受环境危害因素BaP的损伤等。更多还原

【Abstract】 Cells respond to a variety of environmental stresses such as heat, hypoxia, chemicals and other stressors by rapidly synthesizing a group of highly conserved proteins known as heat shock or stress proteins (Hsps). Hsps function as molecular chaperones, binding to the nascent polypeptides to assist proper refolding, assembling and translocating, and activating specific regulatory proteins including transcription factors, replication proteins and kinases. They also participate in protein signalling, including steroid hormone activation. They facilitate the misfolding peptides to repair or target damaged polypeptides for degradation. Hsps have a critical role in the recovery of cells from stress and in cytoprotection. According to the apparent molecular weight, Hsps are divided into many families. The 70kDa heat shock protein family (HSP70, mainly including inducible Hsp70 and constitutive Hsc70) is one of the most abundant of Hsps, 1-2% of total cellular protein. Many studies had indicated that Hsp70 expression was correlated to the living thing tolerant or susceptible ability to environmental harmful factors. In fact, specific increased expression of Hsp70 protected cells from ultraviolet radiation, and inhibition of Hsp70 produced profound impairment of cellular integrity. However few reports had showed whether the inducible Hsp70 plays a role in protection from DNA damage and DNA repair when cells exposed to harmful environmental factors, such as benzo(a)pyrene (BaP).Benzo(a)pyrene (BaP) is the prototype of a multitude of polycyclic aromatic hydrocarbons (PAHs), which are products of incomplete combustion of organic matter and are widespread in the environment. Considerable experimental evidence suggests that BaP has carcinogenic and mutagenic effects documented in human, animals and mammalian cell systems. Cytochrome P450 can oxidize BaP to the ultimate electrophilic metabolite benzo(a)pyrene diol epoxide (BPDE), which can bind covalent to DNA and thus result in DNA damage. Major types ofdamage induced by BaP are removed by nucleotide excision repair (NER). From our past researches, the results showed a significant negative correlation of Hsp70 levels with DNA damage in workers who exposed to coke-oven emissions. These emissions contain many toxic chemicals like PAHs. Hsp70, the major Hsp, may play a role in protecting cells from DNA damage and DNA repair when cells exposed to benzo(a)pyrene.In present study, we increased the level of Hsp70 expression in A549 cells (a human lung adenocarcinoma cell line) by transfecting A549 cells with a recombinant plasmid pcDNA3.0/hsp70 containing human hsp70 cDNA, and inhibited Hsp70 expression in A549 cells with quercetin treatment. A549 cells were transfected with a pcDNA3.0 plasmid containing the neomycin resistance gene but not hsp70 cDNA as vector control. After establishing the models of overexpression and inhibition of Hsp70 in A549 cells, we exposed these kinds of cells to different concentrations of BaP. We detected DNA damage with alkaline single-cell gel electrophoresis, and measured cell viability by MTT assay, and detected Hsp70 expression using Western blot, to investigate whether Hsp70 plays the role in the protection from DNA damage by BaP. Moreover, we detected mRNA levels of the major nucleotide excision repair (NER) factors with real time RT-PCR when above cells were exposed to BaP. Our results showed that Hsp70 plays the role in the protection from DNA damage by BaP, and Hsp70 has some effects on mRNA levels of major NER genes. These results have three parts.Part I Establishment of cell models of overexpression and inhibition of Hsp70The expression vector containing human hsp70 cDNA was kindly provided by Dr. Liu L. A549 cells were transfected with recombinant plasmid pcDNA3.0/Hsp70 with lipofectemine 2000 reagent according to the manufacturer’s instructions. After transfection, we selected the positive clones with antibiotic G418 (neomycin, 1000 μg/ml) for 4 weeks. Three positive clones were expanded and analyzed for the expression of Hsp70 by immunofluorescence staining. We chose the clone of the highest expression of Hsp70 for Western blot analysis. A control population of A549 cells was transfected with a pcDNA3.0 plasmid containing the neomycin resistance gene but not hsp70 cDNA. The results showed that A549 cells transfected with recombinant plasmid expressed obviously increased levels of Hsp70 compared withwild-type A549 and A549/pcDNA cells (transfected with empty plasmid pcDNA3.0). We developed a stably transfected A549 cell line having increased expression of human Hsp70 (A549/hsp70 cells).To establish the cell model of Hsp70 inhibition in A549 cells, we treated A549 cells with different concentrations of quercetin. The flavenoid quercetin has been shown to inhibit Hsp70 synthesis in a human colon carcinoma cell line after heat shock and in human monocyte-macrophages during erythrophagocytosis. We treated A549 cells with 50. 100, 150, 200umol/L quercetin at 37℃ for 6 h, then detected cell viability using MTT method. The results showed that cell viability decreased along with the increasing quercetin concentrations, from 50 100, 150 to 200umol/L. Compared with the group of normal culture, there was a significant decrease of cell viability in the group of 150 and 200μmol/L (P < 0.05) .And there was an obviously difference between the group of 150 and 200μmol/L (P < 0.05) .To observe the inhibition of Hsp70 by quercetin, we treated A549 cells with 50, 100, 150, 200μmol/L quercetin at 37℃ for 6 h, then at 42℃ water bath for 1h. Cells were cultured at 37℃ for 2h recovery. Normal cultured cells were as a normal control. Cells treated by 42℃ were as a positive control. Hsp70 levels were detected by western blot assay. Along with the increasing quercetin concentrations, from 50, 100, 150 to 200μmol/L, Hsp70 levels decreased. Compared with positive control, Hsp70 levels of A549 cells decreased significantly in the group of 100 (P < 0.05), 150 and 200umol/L (P < 0.01). There was no significant difference between 150μmol/L and 200μmol/L groups (P > 0.05). Taken together cell viability and Hsp70 inhibition of A549 cells exposed to different concentrations of quercetin, we chose the 150μmol/L quercetin treatment for cell model of Hsp70 inhibition.Part II Heat shock protein 70 protects cells against DNA damage caused by benzo(a)pyreneTo determine whether Hsp70 plays the role in the protection cells against DNA damage caused by BaP, we detected DNA damage and Hsp70 expression level of A549 cells exposed to the different concentration BaP groups. We investigated the relation between the degree of DNA damage and Hsp70 expression levels of A549 cells. Our results showed that cell viability of A549 cells exposed to BaP decreased along with the increase of doses. Comparedwith the control group (not exposed to BaP), cell viability of A549 cells decreased significantly at 10, 50, 100μM BaP groups (P < 0.01). We found the dose-dependent increases in DNA damage in A549 cells exposed to BaP. The OTM values in A549 cells exposed to 1, 5, 10, 50, 100μmol/L BaP all increased significantly compared with the group not exposed to BaP, respectively (P<0.01). We also found the dose-dependent decrease in Hsp70 expression in A549 cells exposed to BaP. Compared with the control group, Hsp70 expression level of A549 cells decreased significantly at 10, 50, 100μM BaP groups (P < 0.05). Moreover, we found a significant negative correlation of Hsp70 levels with DNA damage in A549 cells exposed to different concentrations of BaP. These results suggested that BaP inhibited inducible Hsp70, which might make A549 cells more susceptible to toxicity of BaP. To further confirm the effect of Hsp70 in the protection cells against DNA damage caused by BaP, we made use of the cell models of the first part, and exposed the cells with different expression levels of Hsp70 to the different concentrations of BaP for 24h. We detected DNA damage with alkaline single-cell gel electrophoresis, and measured the cell viability by MTT assay. Our results showed that A549/hsp70 cells had a significant increased in cell viability compared with A549 cells (P<0.05) in high concentrations of BaP (50μmol/L, 100μmol/L), and we found the dose-dependent increases in DNA damage in A549, A549/pcDNA, A549/hsp70 and A549/Quercetin cells exposed to BaP. The OTM values in A549, A549/pcDNA, A549/Quercetin cells exposed to 1, 5, 10, 50, 100μmol/L BaP all increased significantly compared with themselves control group cells (not exposed to BaP), respectively (P<0.05). The OTM values in A549/hsp70 cells exposed to 5, 10, 50, 100μmol/L BaP also increased significantly compared with the group not exposed to BaP (P<0.05). This indicated the threshold intensity of BaP in the transfected A549 cells overexpressing Hsp70 appeared to be shifted to high levels. Compared to A549 cells, OTM values in A549/hsp70 cells significantly decreased in all BaP treatment groups (P<0.05), but OTM values increased significantly in A549/Quercetin cells exposed to 5, 10, 50, 100μmol/L BaP, and no marked difference in A549/pcDNA cells. It was suggested Hsp70 protects cells against DNA damage caused by benzo(a)pyrene.Part III The effects of Hsp70 on mRNA level changes of major factors in nucleotide excision repair of the cells exposed to BaPThe before mentioned results have showed that Hsp70 is in protection cells from DNA damage caused by benzo(a)pyrene, but we have not known how Hsp70 plays a role in the DNA repair of cells exposed to BaP.BaP is a potent mutagen and carcinogen. Cytochrome P450 can oxidize BaP to the ultimate electrophilic metabolite benzo(a)pyrene diol epoxide (BPDE), which can bind covalent to DNA and thus result in DNA damage. Major types of damage induced by BaP are removed by nucleotide excision repair (NER). When the cells with different expression levels of Hsp70 were exposed to BaP, what happened to the major factors in nucleotide excision repair? In this study, we detected mRNA levels of major factors in NER of the cells with different expression levels of Hsp70 exposed to BaP by using real-time quantitative PCR, and studied the effects of Hsp70 in mRNA level changes of major factors in NER of the cells exposed to BaP. Our results showed that changes of XPA, XPC mRNA expression in the cells with different expression levels of Hsp70 are different when the cells were exposed to different concentrations of BaP. Compared with A549 cells exposed to BaP, the XPA mRNA levels of A549/hsp70 and A549/pcDNA cells had no significant change, but XPA mRNA levels of A549/Quercetin cells decreased significantly in 10μmol/L BaP groups (P<0.05). In 50μmol/L BaP groups, the XPA mRNA levels of A549/pcDNA cells had no significant change, but XPA mRNA levels of A549/hsp70 and A549/Quercetin cells decreased significantly compared with A549 cells(P<0.05). In 100μmol/L BaP groups, the XPA mRNA levels of A549/pcDNA, A549/hsp70 and A549/Quercetin cells increased significantly compared with A549 cells (P<0.05). Our results also showed that in 5μmol/L BaP groups, the XPC mRNA levels of A549/pcDNA cells decreased significantly (P<0.05), but XPC mRNA levels of A549/hsp70 and A549/Quercetin cells increased significantly compared with A549 cells (P<0.05). Compared with A549 cells, the XPC mRNA levels of A549/pcDNA cells had no significant change, but XPC mRNA levels of A549/hsp70 and A549/Quercetin cells increased significantly (P<0.05) in 10μmol/L BaP groups. In 100μmol/L BaP groups, the XPC mRNA levels of A549/pcDNA and A549/Quercetin cells had no significant change, but XPC mRNA levels of A549/hsp70 cells increased significantly compared with A549 cells (P<0.05).XPB is a subunit of TFIIH and plays a central role in NER. XPB and XPD exhibit DNA-dependent ATPase and helicase functions. XPB can unwind DNA in a 3’→5’ direction, and XPD in the opposite direction. In 10μmol/L BaP groups, the XPB mRNA levels of A549/pcDNA and A549/Quercetin cells decreased significantly (P<0.05), but XPB mRNA levels of A549/hsp70 cells increased significantly compared with A549 cells (P<0.05). Compared with A549 cells, the XPB mRNA levels of A549/pcDNA cells decreased significantly (P<0.05), but XPB mRNA levels of A549/hsp70 and A549/Quercetin cells increased significantly in 100μmol/L BaP groups (P<0.05).The XPG gene product and ERCC1-XPF complex are structure-specific endonucleases, which cleave a variety of artificial DNA substrates, including bubbles, splayed arms and stem-loops. In NER, XPG makes the 3’ incision and ERCC1-XPF makes the 5’ incision, consistent with their cleavage polarities. The XPG-mediated 3’ incision precedes the 5’ incision made by ERCC1-XPF. XPG has a structural function in the assembly of the NER DNA-protein complex. Unlike XPG, ERCC1-XPF does not appear to have an architectural function in the NER protein-DNA complex. In 10μmol/L BaP groups, the XPG mRNA levels of A549/pcDNA, A549/hsp70 and A549/Quercetin cells all decreased significantly (P<0.05) compared with A549 cells. There was no significant change in XPG mRNA levels of A549/pcDNA and A549/hsp70 cells, but there was a significant decreased in XPG mRNA levels of A549/Quercetin cells (P<0.05) in 100μmol/L BaP groups compared with A549 cells. ERCC1-XPF makes the 5’ incision consistent with their cleavage polarities in NER. It has an important role at cleavage stages of NER. In 1, 5, 10, 50μmol/L BaP groups, the XPF mRNA levels of A549/pcDNA, A549/hsp70 and A549/Quercetin cells all decreased significantly (P<0.05) compared with A549 cells. In 100μmol/L BaP groups, there was no significant change in XPF mRNA levels of A549/pcDNA and A549/hsp70 cells, but there was a significant increase in XPF mRNA levels of A549/Quercetin cells compared with A549 cells (P<0.05). In 1, 5, 10, 50μmol/L BaP groups, the ERCC1 mRNA levels of of A549/pcDNA, A549/hsp70 and A549/Quercetin cells all decreased significantly (P<0.05) compared with A549 cells. Compared with A549 cells, the ERCC1 mRNA levels of A549/pcDNA and A549/hsp70 cells decreased (P>0.05), but ERCC1 mRNA levels of A549/Quercetin cells increased significantly (P<0.05) in 100μmol/L BaP groups.In conclusion, our results suggested:(1) The experimental cell models of Hsp70 overexpression and inhibition could be established by transfected A549 cells with recombinant plasmid pcDNA3.0/Hsp70 and 150umol/L quercetin treatment for A549 cells.(2) BaP and its metabolic products inhibited inducible Hsp70 expression of A549 cells, which made A549 cells more susceptible to BaP. The results showed a negative correlation of Hsp70 levels with DNA damage in A549 cells exposed to BaP for 24h.(3) Hsp70 overexpression cells A549/hsp70 had a significant increased in cell viability compared with A549 cells (P<0.05) in high concentrations of BaP (50μmol/L, 100μmol/L). Compared to A549 cells, OTM values in A549/hsp70 cells significantly decreased in BaP treatment groups (P<0.05), and increased significantly in A549/Quercetin cells exposed to 5, 10, 50 100μmol/L BaP groups (P<0.05), but no marked difference in A549/pcDNA cells. These results suggested Hsp70 protects A549 cells against DNA damage caused by benzo(a)pyrene.(4) Under BaP treatment, Hsp70 may have effects on mRNA level changes of XPC and XPB in A549 cells. These possibly contribute to NER of DNA repair.Although some results have been received in this study, there still were some questions remained to be elucidated such as the special inhibition of Hsp70 using siRNA method, and whether Hsp70 having effects on NER, and how to protect human respiratory system against toxicity of BaP according to the character of Hsp70 expression.更多还原