【作者】 赵劲风；

【导师】 张阳德；

【作者基本信息】 中南大学 ， 外科学， 2009， 博士

【摘要】 目的:脱嘌呤/脱嘧啶位点（AP site）是DNA损伤最常见的形式,通常由碱基切除修复途径修复,而APE1是该修复途径的一个重要成分;Bcl₂是细胞内一重要的肿瘤蛋白,它的致癌活性因抑制多种DNA修复途径引起,包括碱基切除修复途径,Bcl₂能抑制NNK诱导剂的DNA损伤后产生AP位点的修复,但其机理仍不清楚,为了探索Bcl₂抑制DNA损伤AP位点的修复的分子机理,进行Bcl₂与APE1是否直接相互作用的研究。方法:Western Blotting检测Bcl₂、APE1和XRCC1蛋白的表达情况,单细胞电泳、定量检测AP位点来观察DNA损伤程度,免疫沉淀检测Bcl₂蛋白和APE1蛋白,APE1蛋白和XRCC1蛋白相互作用,免疫荧光染色检测Bcl₂、APE1蛋白的细胞分布情况及其变化,亚细胞结构分离提取蛋白观察Bcl₂蛋白在细胞核及线粒体中表达水平及其变化,APE1核酸内切酶活性测定观察Bcl₂对APE1核酸内切酶活性的影响及其Bcl₂的BH结合域的关系。Bcl₂SiRNA干扰技术观察Bcl₂内源性表达被抑制后对APE1核酸内切酶活性的影响。粒的H1299细胞中AP位点数量也在下降,但比前一组慢,仍维持在较高水平,说明去掉NNK后未转染Bcl₂质粒的H1299细胞中DNA损伤在逐步修复,而转染野生型Bcl₂质粒的H1299细胞中DNA损伤后AP位点的修复受到抑制。NNK处理1小时后两组H1299细胞的细胞核均有拖尾现象,说明NNK可以使H1299细胞中DNA发生损伤;去掉NNK诱导剂后24小时观察发生未转染H1299细胞中DNA的细胞核未有拖尾现象现象,说明DNA损伤已得到修复,但另一组细胞中仍有部分细胞核有拖尾,说明DNA损伤仍有部分未得到修复,再次说明Bcl₂蛋白对NNK诱导的DNA损伤后的修复有抑制作用。用H₂O₂和NNK诱导DNA损伤后,免疫沉淀可以检测到Bcl₂蛋白,说明Bcl₂蛋白和APE1蛋白可以直接相互作用;免疫荧光染色可以看到红色的Bcl₂蛋白和绿色的APE1蛋白在细胞核中积聚,叠加以后细胞核中出现橙色斑点,说明Bcl₂和APE1蛋白在细胞核中直接相互作用。NNK处理1小时后,H460细胞的细胞核中Bcl₂蛋白开始增加,3小时后达到最高,而线粒体中Bcl₂蛋白没有变化,说明Bcl₂蛋白在DNA损伤诱导下在H460细胞核积聚,核中Bcl₂蛋白的增加并不是从线粒体中转移过来的。APE1蛋白在所有肺癌细胞中均有较好表达,但内源性Bcl₂蛋白仅在H69和H460肺癌细胞中表达。同位素法检测所有肺癌细胞中APE1的活性,发现H69和H460细胞中APE1活性明显比其它肺癌细胞中APE1活性低,说明有内源性Bcl₂蛋白表达能抑制APE1的活性。用转染野生型Bcl₂和缺失突变型△BH₁、△BH₂、△BH₃、△BH₄质粒的H1299细胞作免疫共沉淀发现仅野生型Bcl₂组能检测到Bcl₂蛋白,而其它缺失突变型组未能检测到Bcl₂蛋白,说明Bcl₂蛋白和APE1蛋白直接相互作用与Bcl₂的四个结合域BH₁、BH₂、BH₃和BH₄都有关。对转染野生型Bcl₂和缺失突变型Bcl₂质粒的H1299细胞中APE1核酸内切酶活性定量检测,发现野生型组APE1核酸内切酶活性低,而缺失突变型各组APE1核酸内切酶活性高;可以推断野生型Bcl₂对APE1蛋白核酸内切酶活性有抑制作用,这种抑制作用与野生型Bcl₂蛋白的四个结合域都有关,Bcl₂蛋白的BH结合域的任一缺失都会导致Bcl₂对APE1蛋白核酸内切酶活性和AP位点修复的抑制能力消失。各组APE1核酸内切酶活性定量检测结果野生型组APE1核酸内切酶活性明显低于缺失突变型各组。将纯化的野生型Bcl₂和BH缺失型△BH₁、△BH₂、△BH₃、△BH₄蛋白进行体外试验,发现结果与体内试验结果一致。将Bcl₂siRNA转染到H460细胞组中发现Bcl₂蛋白表达很低,而对照组Bcl₂蛋白的表达水平未有改变;同位素法检测APE1核酸内切酶活性结果发现Bcl₂siRNA三组APE1核酸内切酶活性升高,从而可以推断Bcl₂蛋白可以抑制APE1核酸内切酶活性。H1299肺癌细胞和转染野生型Bcl₂质粒的H1299肺癌细胞中APE1和XRCC1两种DNA损伤修复蛋白均有比较高水平的表达,而Bcl₂蛋白仅在转染野生型Bcl₂质粒的H1299细胞中出现;DNA损伤诱导剂NNK处理后,免疫共沉淀试验发现未转染的H1299细胞组中检测XRCC1蛋白,并且NNK处理后XRCC1增多,转染野生型Bcl₂质粒的H1299细胞组中虽然也能检测到XRCC1,并且NNK处理后XRCC1也增多,但明显比前两组少很多,说明Bcl₂蛋白可以干扰APE1和XRCC1DNA修复复合物的形成。将不同浓度的纯化Bcl₂蛋白进行体外试验,发现随着纯化Bcl₂蛋白浓度的升高,检测到的XRCC1越来越少,说明纯化的野生型Bcl₂蛋白体外能够抑制APE1/XRCC1的修复复合物的形成,从而抑制DNA损伤后AP位点的修复。结论:1.Bcl₂能够抑制NNK和H₂O₂诱导的DNA损伤后产生AP位点的修复。2.当NNK诱导DNA损伤时细胞核中Bel₂表达水平增加,但不是从线粒体中转移过来的。3.Bcl₂通过它的结合域与APE1直接相互作用下调APE1活性,有助于延缓DNA修复,造成遗传不稳定和肿瘤发生。4.Bcl₂蛋白能在体内外离解DNA修复复合物APE1/XRCC1,从而抑制DNA损伤后AP位点的修复。更多还原

【Abstract】 Ojective:Apurinic/apyrimidinic（AP） or abasic sites are the most common form of DNA damage.AP sites are primarily repaired by the DNA base excision repair（BER） pathway.APE1/Ref-1 （apurinic/apyrimidinic endonuclease 1/redox effect factor-1）is the essential enzyme in the BER pathway.Interestingly,APE1 is a multifunctional protein that not only is a DNA repair enzyme,but also functions as a redox factor maintaining transcription factors.Bcl2 is a major cellular oncogenic protein,it’s oncogenic activity may result from its inhibitory effects on multiple DNA repair pathways including BER pathway.Bcl2 potently suppresses the repair of NNK-induced abasic sites of DNA lesions.However,the mechanism（s） is not fully understood.In order to understand the mechanism（s） that Bcl2 suppresses the repair of NNK-induced abasic sites of DNA lesions,testing for a direct interaction between Bcl2 and APE1 will be researched.Methods:The expression levels of Bcl2,APE1 and XRCC1 proteius will be detected with Western Blotting.Single cell gel electrophoresis and AP site counting will be used to test the DNA damage. Co-immunoprecipitation will be used to analysis the interactions between Bcl₂ and APE1,APE1 and XRCC1.The cellular distributious of Bcl₂ and APE1 proteins will be tested by immunofluorescence staining.Subcellular fractionation will be used to extract the Bcl₂ protein in nucleus and mitochdria to test their expression levels and changes.AP oligonucleotides assay for APE1 endonuclease activity will be used to observe Bcl₂’s effect the APE1.Oendonuclease activity and velationship between Bcl₂’s BH domaius and APE1 endonuclease activity.Bcl₂ siRNA technology will be used to test the effect of APE1 endonuclease activity after the expression of endogenous Bcl₂ will be silent.Results:Many AP sites are produced by NNK-induced DNA damage.After NNK was washed,AP sites in H1299 cells with no Bcl2 plasmid transfected are fall-off,APsites in H1299 cells with wild type（WT）Bcl2 plasmid also decreased,but slower than first group, maintaining the high level.It explains that after washing NNK,AP sites DNA damage in H1299 cells with no Bcl2 plasmid was repaired,the repair of AP site DNA damage H1299 cells with WT Bcl2 plasmid was inhibited.After treates one hour with NNK,the nucleuses of both two H1299 cells groups have trails.It shows that NNK may induce DNA damage in H1299 cells.After washes NNK for 24 hours,the nucleuses of H1299 cells with no Bcl2 plasmid have no trails.It explained that the DNA damage has been repaired.Still have trails,it show that pare of DNA damage is not repaired.It explains again that Bcl2 protein can suppress NNK-induced DNA damage repair.After H₂O₂ and NNK induced DNA damage,Bcl2 protein can be detected with co-immunoprecipitation,It explains Bcl2 and APE1 protein can directly interaction.Red Bcl2 protein and green APE1 protein are observed to accumulate in nucleus.Via immunofluorescence staining.After emergeing,there are some orange spot in nuclus.It explains that Bcl2 and APE1 protein can directly interaction in nucleus. After treated for one hour,Bcl2 protein in H460 nucleus starts increase. After three hours,it will be highest.But Bcl2 protein in mitochondrium has no change.It imports that increased nuclear Bcl2 may not result from a movement from mitochondria into nucleus.APE1 protein can be expressed in all lung cancer cell lines,but endogenous Bcl2 protein is only expressed in H69 and H460 cells.APE1 nuclease activity in H69 and H460 cells is lower than others.It shows that Bcl2 protein can inhibit APE1 nuclease activity.Co-immunoprecipitation in H1299 cells with WT Bcl2 plasmid and deletion mutation△BH₁、△BH₂、△BH₃、△BH₄ plasmid was found that only WT Bcl2 group can be detested Bcl2 protein,but others can not.It explains that the direct interaction between Bcl2 and APE1 proteins is concerned with BH₁、BH₂、BH₃ and BH₄ domains.Assay for APE1 nuclease activity found APE1 nuclease activity of WT group is lower than others.It will be concludes that WT Bcl2 protein can inhibit APE1 nuclease activity.The inhibitory effect is related with four BH domains.Purified WT Bcl2 protein and△BH₁、△BH₂、△BH₃、△BH₄ protein have been tested in vitro.The result is same as that in vivo.When Bcl2 siRNA was transfected in H460 cells,the express level of Bcl2 protein is much low. But there is no change in control group.It was found that the APE1 activities in three Bcl2 siRNA gronps are increased.It show Bcl2 protein can inhibit APE1 nuclease activity. APE1 and XRCC1 DNA damage repain proteins can be expnessed in H1299 and H1299 with WT Bcl2 plasmid lung cells.But Bcl2 protein can only be detected in H1299 cells with WT Bcl2 plasmid.After NNK induced DNA damage,co-immunoprecipitation test can detect XRCC1 protein increased in H1299 cells.But XRCC1 protein in H1299 cells with WT Bcl2 plasmid is lower.It shows that Bcl2 can dissociate the DNA repair complex APE1/XRCC1 in vivo.The different concentration purified Bcl2 protein is tested the same experience in vitro. The result is same as in vivo.Conclusion:1.Bcl2 suppresses the repair of NNK or H₂O₂-induced AP site DNA lessions.2.The expression level of nuclear Bcl2 are increased when NNK induced DNA damage,increased nuclear Bcl2 may not result from a movement from mitochondria into nucleus.3.Bcl2 downregulates APE1 activity via its direct interaction with APE1 at the BH domains,which may contribute retardation of DNA repair,genetic instability and tumorigenesis.4.Bcl2 can dissociate the DNA repair complex APE 1-XRCC1 in vivo and in vitro to inhibite the repair of NNK or H₂O₂-induced AP site DNA lessions.更多还原