互隔交链孢霉素经cAMP-PKA-CREB途径激活DNA聚合酶β表达的研究
发布时间：2018-07-29 13:23
【摘要】：背景及目的: DNA聚合酶β(DNA polymeraseβ,DNApolβ)的功能主要是在碱基切除修复(base excision repair,BER)的过程中填补单核苷酸缺口。在细胞的正常分化以及DNA的正常复制与修复中,DNApolβ在细胞内是低水平表达的。当DNApolβ突变、表达及功能出现异常时,可以导致细胞自身突变率增加、遗传不稳定性的发生以及对一些抗癌药物的耐受。因此研究DNApolβ对肿瘤的发生、发展及对抗癌药物的耐受性问题都有重大意义。我省林州市是食管癌高发区,其粮食中提取出的互隔交链孢毒中分离出有七种毒素,其中互隔交链孢酚(Alternariol,AOH)和互隔交链单甲醚(Alternariol methyl ether,AME)已被证实有较强的致癌性,能够损伤DNA,诱发DNApolβ表达增高。究竟引起DNApolβ表达增高是经由哪条通路,如果阻断这条通路是否可以逆转DNApolβ表达增高的趋势成为关心的焦点。信号通路的有关研究表明,在致癌物烷化剂N-甲基-N'-硝基-N-亚硝胍(MNNG)处理细胞中cAMP含量、PKA活性和CREB133位丝氨酸残基磷酸化程度都有一过性升高,提示MNNG诱使DNApolβ升高是通过cAMP-PKA-CREB途径介导。本实验旨在探讨另一主要代谢产物互隔交链孢霉素(Altenuene,ALT)对DNApolβ表达的影响以及是否是由cAMP—PKA—CREB的信号转导通路介导。 方法: 1.采用适宜的低浓度ALT处理小鼠胚胎成纤维细胞(NIH3T3),利用免疫细胞化学、RT-PCR及Western Blotting方法测定ALT对细胞中DNApolβmRNA以及蛋白表达的影响。 2.采用ALT作用与NIH3T3细胞,通过放射免疫方法测定细胞中cAMP的含量;Western Blotting、免疫细胞化学等方法检测细胞中PKA催化亚基及p-CREB蛋白水平的表达,观察cAMP-PKA-CREB信号通路是否被激活。 3.利用PKA阻断剂H89预处理细胞后,再观察ALT对PKA、p-CREB及DNApolβ表达的影响。 4.实验组与对照组数据的比较用SPSS11.0软件进行分析,两两均数间用t检验,多样本均数间采用方差分析,α=0.05为检验水准。 结果: 1.采用15μmol/LALT处理细胞后,实验组与阳性对照组细胞的DNApolβmRN以及蛋白含量与溶剂对照组相比升高。(P＜0.05) 2.分别采用0、5、10、15、20μmol/LALT处理细胞30分钟后,利用~(125)Ⅰ标记的放射免疫法测定细胞中cAMP含量,发现细胞cAMP水平随着ALT的浓度增加而增加,与溶剂对照组比较差异有统计学意义。(P＜0.05) 3.采用15μmol/LALT处理细胞后,实验组与阳性对照组细胞的PKA催化亚基含量与溶剂对照组相比升高,加上H89先行阻断后,PKA催化亚基比单纯加ALT时升高程度有所下降。(P＜0.05) 4.采用15μmol/L ALT处理细胞后,实验组与阳性对照组细胞的p-CREB蛋白含量与溶剂对照组相比升高,加上H89先行阻断后,p-CREB比单纯加ALT时升高程度有所下降。(P＜0.05) 结论: 1.证实了互隔交链孢霉素可以诱导NIH3T3细胞的DNApolβ的表达上调。 2.NIH3T3细胞面对一定浓度的ALT损伤作用时,细胞内的cAMP含量、PKA催化亚基及p-CREB表达均有一过性增强。提示ALT处理细胞后,DNApolβ表达上调是经由cAMP-PKA-CREB途径介导的。而在NIH3T3细胞中加入PKA抑制剂H89阻断PKA下游的激活后再加入ALT,,检测到p-CREB及DNApolβ表达水平有所降低,提示可以利用阻断信号转导通路的方法干预DNApolβ的异常表达。
[Abstract]:Background and purpose:
The function of DNA polymerase beta (DNA polymerase beta, DNApol beta) is mainly to fill the single nucleotide gap in the process of base excision repair (base excision repair, BER). In normal differentiation of the cells and in the normal replication and repair of DNA, DNApol beta is a low level in the cell. When the DNApol beta mutation, the expression and function are abnormal, In order to increase the mutation rate of cell itself, the occurrence of genetic instability and tolerance to some anticancer drugs, the study of DNApol beta is of great significance to the occurrence, development and tolerance of cancer drugs. Linzhou is a high incidence area of esophageal cancer in our province, and seven of the isolated septum from the grain is isolated from the food. Alternariol, AOH and Alternariol methyl ether (AME) have been proved to have strong carcinogenicity, which can damage DNA and induce the increase of the expression of DNApol beta. Which pathway is caused by the increase of DNApol beta expression, if blocking this pathway can reverse the increase of DNApol beta expression The trend of the signal pathway has shown that the cAMP content in the carcinogen N- methyl -N'- nitrosanidine (MNNG) treated cells, the PKA activity and the degree of phosphorylation of the CREB133 site of the serine residues are elevated, suggesting that MNNG induced DNApol beta rise is mediated by cAMP-PKA-CREB pathway. The effect of Altenuene (ALT) on the expression of DNApol beta and whether it is mediated by the signal transduction pathway of cAMP - PKA - CREB.
Method:
1. the mouse embryonic fibroblast (NIH3T3) was treated with a suitable low concentration of ALT, and the effects of ALT on the expression of DNApol beta mRNA and protein in the cells were measured by immunocytochemistry, RT-PCR and Western Blotting.
2. ALT and NIH3T3 cells were used to determine the content of cAMP in cells by radioimmunoassay; Western Blotting, immunocytochemistry and other methods were used to detect the expression of PKA subunits and p-CREB protein levels in the cells, and to observe whether the cAMP-PKA-CREB signaling pathway was activated.
3. after pretreatment with PKA antagonist H89, the effect of ALT on the expression of PKA, p-CREB and DNApol beta was observed.
4. the comparison between the experimental group and the control group was compared with the SPSS11.0 software, and the 22 was tested by t. The variance analysis was used among the various numbers, and the alpha =0.05 was the test level.
Result:
1. after treated with 15 mol/LALT, the DNApol beta mRN and protein content of the experimental group and the positive control group increased compared with the solvent control group (P < 0.05).
2. after 30 minutes of 0,5,10,15,20 mu mol/LALT treated cells respectively, the radioimmunoassay of ~ (125) I labeled cells was used to determine the content of cAMP in the cells. It was found that the level of cAMP increased with the increase of the concentration of ALT, and the difference was statistically significant compared with the solvent control group (P < 0.05).
3. after the treatment of 15 mol/LALT cells, the content of the PKA subunit in the experimental group and the positive control group was higher than that in the solvent control group, and the PKA catalyzed the increase of the subbasis of the subunit with the ALT. (P < 0.05).
4. after the treatment of 15 mol/L ALT cells, the content of p-CREB protein in the experimental group and the positive control group was higher than that in the solvent control group, and the increase of p-CREB was lower than that of the pure ALT. (P < 0.05).
Conclusion:
1. it was confirmed that the cross septate can induce the up regulation of DNApol beta expression in NIH3T3 cells.
When 2.NIH3T3 cells were exposed to a certain concentration of ALT damage, the content of cAMP in the cells and the expression of PKA catalyzed subunits and p-CREB were enhanced. The up-regulation of DNApol beta expression was mediated by cAMP-PKA-CREB pathway. The addition of PKA inhibitor to NIH3T3 cells H89 blocked the activation of the downstream PKA. The expression level of p-CREB and DNApol beta decreased, suggesting that the abnormal expression of DNApol beta can be interfered by blocking signal transduction pathway.
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R363
本文编号：2152811
[Abstract]:Background and purpose:
The function of DNA polymerase beta (DNA polymerase beta, DNApol beta) is mainly to fill the single nucleotide gap in the process of base excision repair (base excision repair, BER). In normal differentiation of the cells and in the normal replication and repair of DNA, DNApol beta is a low level in the cell. When the DNApol beta mutation, the expression and function are abnormal, In order to increase the mutation rate of cell itself, the occurrence of genetic instability and tolerance to some anticancer drugs, the study of DNApol beta is of great significance to the occurrence, development and tolerance of cancer drugs. Linzhou is a high incidence area of esophageal cancer in our province, and seven of the isolated septum from the grain is isolated from the food. Alternariol, AOH and Alternariol methyl ether (AME) have been proved to have strong carcinogenicity, which can damage DNA and induce the increase of the expression of DNApol beta. Which pathway is caused by the increase of DNApol beta expression, if blocking this pathway can reverse the increase of DNApol beta expression The trend of the signal pathway has shown that the cAMP content in the carcinogen N- methyl -N'- nitrosanidine (MNNG) treated cells, the PKA activity and the degree of phosphorylation of the CREB133 site of the serine residues are elevated, suggesting that MNNG induced DNApol beta rise is mediated by cAMP-PKA-CREB pathway. The effect of Altenuene (ALT) on the expression of DNApol beta and whether it is mediated by the signal transduction pathway of cAMP - PKA - CREB.
Method:
1. the mouse embryonic fibroblast (NIH3T3) was treated with a suitable low concentration of ALT, and the effects of ALT on the expression of DNApol beta mRNA and protein in the cells were measured by immunocytochemistry, RT-PCR and Western Blotting.
2. ALT and NIH3T3 cells were used to determine the content of cAMP in cells by radioimmunoassay; Western Blotting, immunocytochemistry and other methods were used to detect the expression of PKA subunits and p-CREB protein levels in the cells, and to observe whether the cAMP-PKA-CREB signaling pathway was activated.
3. after pretreatment with PKA antagonist H89, the effect of ALT on the expression of PKA, p-CREB and DNApol beta was observed.
4. the comparison between the experimental group and the control group was compared with the SPSS11.0 software, and the 22 was tested by t. The variance analysis was used among the various numbers, and the alpha =0.05 was the test level.
Result:
1. after treated with 15 mol/LALT, the DNApol beta mRN and protein content of the experimental group and the positive control group increased compared with the solvent control group (P < 0.05).
2. after 30 minutes of 0,5,10,15,20 mu mol/LALT treated cells respectively, the radioimmunoassay of ~ (125) I labeled cells was used to determine the content of cAMP in the cells. It was found that the level of cAMP increased with the increase of the concentration of ALT, and the difference was statistically significant compared with the solvent control group (P < 0.05).
3. after the treatment of 15 mol/LALT cells, the content of the PKA subunit in the experimental group and the positive control group was higher than that in the solvent control group, and the PKA catalyzed the increase of the subbasis of the subunit with the ALT. (P < 0.05).
4. after the treatment of 15 mol/L ALT cells, the content of p-CREB protein in the experimental group and the positive control group was higher than that in the solvent control group, and the increase of p-CREB was lower than that of the pure ALT. (P < 0.05).
Conclusion:
1. it was confirmed that the cross septate can induce the up regulation of DNApol beta expression in NIH3T3 cells.
When 2.NIH3T3 cells were exposed to a certain concentration of ALT damage, the content of cAMP in the cells and the expression of PKA catalyzed subunits and p-CREB were enhanced. The up-regulation of DNApol beta expression was mediated by cAMP-PKA-CREB pathway. The addition of PKA inhibitor to NIH3T3 cells H89 blocked the activation of the downstream PKA. The expression level of p-CREB and DNApol beta decreased, suggesting that the abnormal expression of DNApol beta can be interfered by blocking signal transduction pathway.
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R363
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