【摘要】：背景及目的: DNA聚合酶β(DNA polymeraseβ,DNApolβ)的功能主要是在堿基切除修復(fù)(base excision repair,BER)的過(guò)程中填補(bǔ)單核苷酸缺口。在細(xì)胞的正常分化以及DNA的正常復(fù)制與修復(fù)中,DNApolβ在細(xì)胞內(nèi)是低水平表達(dá)的。當(dāng)DNApolβ突變、表達(dá)及功能出現(xiàn)異常時(shí),可以導(dǎo)致細(xì)胞自身突變率增加、遺傳不穩(wěn)定性的發(fā)生以及對(duì)一些抗癌藥物的耐受。因此研究DNApolβ對(duì)腫瘤的發(fā)生、發(fā)展及對(duì)抗癌藥物的耐受性問(wèn)題都有重大意義。我省林州市是食管癌高發(fā)區(qū),其糧食中提取出的互隔交鏈孢毒中分離出有七種毒素,其中互隔交鏈孢酚(Alternariol,AOH)和互隔交鏈單甲醚(Alternariol methyl ether,AME)已被證實(shí)有較強(qiáng)的致癌性,能夠損傷DNA,誘發(fā)DNApolβ表達(dá)增高。究竟引起DNApolβ表達(dá)增高是經(jīng)由哪條通路,如果阻斷這條通路是否可以逆轉(zhuǎn)DNApolβ表達(dá)增高的趨勢(shì)成為關(guān)心的焦點(diǎn)。信號(hào)通路的有關(guān)研究表明,在致癌物烷化劑N-甲基-N'-硝基-N-亞硝胍(MNNG)處理細(xì)胞中cAMP含量、PKA活性和CREB133位絲氨酸殘基磷酸化程度都有一過(guò)性升高,提示MNNG誘使DNApolβ升高是通過(guò)cAMP-PKA-CREB途徑介導(dǎo)。本實(shí)驗(yàn)旨在探討另一主要代謝產(chǎn)物互隔交鏈孢霉素(Altenuene,ALT)對(duì)DNApolβ表達(dá)的影響以及是否是由cAMP—PKA—CREB的信號(hào)轉(zhuǎn)導(dǎo)通路介導(dǎo)。 方法: 1.采用適宜的低濃度ALT處理小鼠胚胎成纖維細(xì)胞(NIH3T3),利用免疫細(xì)胞化學(xué)、RT-PCR及Western Blotting方法測(cè)定ALT對(duì)細(xì)胞中DNApolβmRNA以及蛋白表達(dá)的影響。 2.采用ALT作用與NIH3T3細(xì)胞,通過(guò)放射免疫方法測(cè)定細(xì)胞中cAMP的含量;Western Blotting、免疫細(xì)胞化學(xué)等方法檢測(cè)細(xì)胞中PKA催化亞基及p-CREB蛋白水平的表達(dá),觀察cAMP-PKA-CREB信號(hào)通路是否被激活。 3.利用PKA阻斷劑H89預(yù)處理細(xì)胞后,再觀察ALT對(duì)PKA、p-CREB及DNApolβ表達(dá)的影響。 4.實(shí)驗(yàn)組與對(duì)照組數(shù)據(jù)的比較用SPSS11.0軟件進(jìn)行分析,兩兩均數(shù)間用t檢驗(yàn),多樣本均數(shù)間采用方差分析,α=0.05為檢驗(yàn)水準(zhǔn)。 結(jié)果: 1.采用15μmol/LALT處理細(xì)胞后,實(shí)驗(yàn)組與陽(yáng)性對(duì)照組細(xì)胞的DNApolβmRN以及蛋白含量與溶劑對(duì)照組相比升高。(P＜0.05) 2.分別采用0、5、10、15、20μmol/LALT處理細(xì)胞30分鐘后,利用~(125)Ⅰ標(biāo)記的放射免疫法測(cè)定細(xì)胞中cAMP含量,發(fā)現(xiàn)細(xì)胞cAMP水平隨著ALT的濃度增加而增加,與溶劑對(duì)照組比較差異有統(tǒng)計(jì)學(xué)意義。(P＜0.05) 3.采用15μmol/LALT處理細(xì)胞后,實(shí)驗(yàn)組與陽(yáng)性對(duì)照組細(xì)胞的PKA催化亞基含量與溶劑對(duì)照組相比升高,加上H89先行阻斷后,PKA催化亞基比單純加ALT時(shí)升高程度有所下降。(P＜0.05) 4.采用15μmol/L ALT處理細(xì)胞后,實(shí)驗(yàn)組與陽(yáng)性對(duì)照組細(xì)胞的p-CREB蛋白含量與溶劑對(duì)照組相比升高,加上H89先行阻斷后,p-CREB比單純加ALT時(shí)升高程度有所下降。(P＜0.05) 結(jié)論: 1.證實(shí)了互隔交鏈孢霉素可以誘導(dǎo)NIH3T3細(xì)胞的DNApolβ的表達(dá)上調(diào)。 2.NIH3T3細(xì)胞面對(duì)一定濃度的ALT損傷作用時(shí),細(xì)胞內(nèi)的cAMP含量、PKA催化亞基及p-CREB表達(dá)均有一過(guò)性增強(qiáng)。提示ALT處理細(xì)胞后,DNApolβ表達(dá)上調(diào)是經(jīng)由cAMP-PKA-CREB途徑介導(dǎo)的。而在NIH3T3細(xì)胞中加入PKA抑制劑H89阻斷PKA下游的激活后再加入ALT,,檢測(cè)到p-CREB及DNApolβ表達(dá)水平有所降低,提示可以利用阻斷信號(hào)轉(zhuǎn)導(dǎo)通路的方法干預(yù)DNApolβ的異常表達(dá)。
[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.
【學(xué)位授予單位】：鄭州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類(lèi)號(hào)】：R363

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本文編號(hào)：2152811