本文選題：低劑量輻射 + 細(xì)胞增殖��； 參考：《復(fù)旦大學(xué)》2013年碩士論文

【摘要】：隨著電離輻射在醫(yī)學(xué)和工業(yè)中應(yīng)用的增加,低劑量輻射(LDR)也引起人們的高度重視,但有關(guān)LDR長(zhǎng)期暴露的效應(yīng)和機(jī)制尚不明確。LDR效應(yīng)與高劑量輻射(HDR)效應(yīng)不同,主要包括輻射超敏反應(yīng)、基因不穩(wěn)定性、輻射適應(yīng)性反應(yīng)和旁效應(yīng)等。除電離輻射外,環(huán)境中還存在許多化學(xué)污染物也危害著人類的健康。由于重金屬鎘(Cd)在工業(yè)上的廣泛應(yīng)用,其對(duì)人類健康的影響也受到廣泛關(guān)注。大量研究證實(shí)大劑量Cd和電離輻射均是重要的致癌因子,但有關(guān)長(zhǎng)期低劑量Cd和長(zhǎng)期LDR生物效應(yīng)的研究較少,而有關(guān)二者聯(lián)合效應(yīng)的研究更少。 眾所周知,DNA甲基化是表觀遺傳學(xué)研究最廣泛的機(jī)制之一,其在多種基礎(chǔ)生命活動(dòng)中均發(fā)揮重要調(diào)節(jié)作用。大量研究顯示,嚙齒類動(dòng)物和人類細(xì)胞株在暴露于電離輻射后其DNA甲基化模式均會(huì)發(fā)生改變,同時(shí),也有研究證實(shí)LDR相關(guān)效應(yīng)和風(fēng)險(xiǎn)本質(zhì)上可能與表觀遺傳學(xué)方面的調(diào)控有關(guān),尤其是DNA甲基化方面的調(diào)控,但機(jī)制尚不明確。另外,有研究證實(shí)全基因組高甲基化以及特異性基因高甲基化在Cd誘導(dǎo)多種細(xì)胞惡性轉(zhuǎn)化過(guò)程中也發(fā)揮重要作用。 本研究采用長(zhǎng)期低劑量γ射線及長(zhǎng)期低劑量Cd對(duì)人B淋巴母細(xì)胞(HMy2.CIR)進(jìn)行處理,研究二者對(duì)HMy2.CIR細(xì)胞的生物效應(yīng)及DNA甲基化在其中的作用。同時(shí),對(duì)LDR和Cd的交叉適應(yīng)性反應(yīng)(cross-AR)及相關(guān)機(jī)制進(jìn)行了研究。更深層次地理解長(zhǎng)期LDR及長(zhǎng)期低劑量Cd效應(yīng)的分子機(jī)制將有助于我們更好地對(duì)環(huán)境物理化學(xué)毒素對(duì)人體健康的影響進(jìn)行評(píng)估,也為人們正確防護(hù)環(huán)境理化毒素提供有力的實(shí)驗(yàn)依據(jù)。本研究?jī)?nèi)容分為以下三個(gè)部分： 第一部分：長(zhǎng)期低劑量Y射線對(duì)淋巴母細(xì)胞的生物學(xué)效應(yīng)及DNA甲基化在其中的作用 目的：探討長(zhǎng)期低劑量γ射線對(duì)人B淋巴母細(xì)胞(HMy2.CIR)增殖、輻射敏感性以及適應(yīng)性方面的影響,并簡(jiǎn)要探討DNA甲基化在其中的作用。 方法：選用可以顯著促進(jìn)細(xì)胞增殖的低劑量γ射線對(duì)HMy2.CIR細(xì)胞進(jìn)行照射,每周一、三、五照射,每次0.032Gy,連續(xù)照射4周(4w),并設(shè)非照射對(duì)照組。以微核形成實(shí)驗(yàn)檢測(cè)細(xì)胞DNA損傷,以CCK-8法檢測(cè)細(xì)胞增殖,以胞嘧啶延伸實(shí)驗(yàn)檢測(cè)細(xì)胞全基因組甲基化情況,用流式細(xì)胞術(shù)檢測(cè)細(xì)胞凋亡和γ-H2AX蛋白的表達(dá),以real-time RT-PCR法檢測(cè)基因cyclinD1、PCNA、bcl-2和bax的表達(dá),以Western blotting法檢測(cè)蛋白HP1和MeCP2的表達(dá)。 結(jié)果：長(zhǎng)期LDR可以顯著提高HMy2.CIR細(xì)胞的增殖率并誘導(dǎo)周期相關(guān)基因cyclinD1和增殖細(xì)胞核抗原PCNA表達(dá)增加。同時(shí),長(zhǎng)期LDR可以誘導(dǎo)淋巴母細(xì)胞對(duì)攻擊劑量(2Gy)電離輻射產(chǎn)生適應(yīng)性反應(yīng),與單純2Gy照射組相比,LDR+2Gy組細(xì)胞的凋亡率、微核形成率及γ-H2AX蛋白表達(dá)量均顯著降低。Real-time RT-PCR檢測(cè)發(fā)現(xiàn)長(zhǎng)期LDR處理細(xì)胞抗凋亡基因bcl-2表達(dá)升高而抑凋亡基因bax表達(dá)降低。胞嘧啶摻入實(shí)驗(yàn)檢測(cè)發(fā)現(xiàn)長(zhǎng)期LDR處理后全基因組DNA發(fā)生高甲基化,這種高甲基化伴隨甲基轉(zhuǎn)移酶1(DNMT1)、甲基化CpG結(jié)合蛋白2(MeCP2)以及異染色質(zhì)蛋白1(HP1)表達(dá)的升高。另外,用DNA甲基轉(zhuǎn)移酶抑制劑(5-aza-dC)處理細(xì)胞后發(fā)現(xiàn)長(zhǎng)期LDR誘導(dǎo)的細(xì)胞輻射適應(yīng)性反應(yīng)(RAR)消失。 結(jié)論：長(zhǎng)期LDR處理可通過(guò)改變基因表達(dá)而促進(jìn)細(xì)胞增殖,同時(shí)也可以通過(guò)改變細(xì)胞全基因組甲基化狀態(tài)及誘導(dǎo)凋亡抵抗進(jìn)而終誘導(dǎo)細(xì)胞產(chǎn)生RAR。 第二部分：低劑量鎘長(zhǎng)期暴露對(duì)淋巴母細(xì)胞增殖的影響及其分子途徑 目的：本部分著重研究低劑量Cd長(zhǎng)期暴露對(duì)人B淋巴母細(xì)胞(HMy2.CIR)增殖的影響,并探討周期抑制基因p16基因及DNA甲基化在其中的作用。 方法：以0、0.005、0.01、0.1μM濃度的氯化鎘(CdCl2)處理HMy2.CIR細(xì)胞,實(shí)驗(yàn)分為兩組：短期處理組和長(zhǎng)期處理組,短期組Cd處理48h,長(zhǎng)期組Cd處理3個(gè)月(3m)。采用細(xì)胞計(jì)數(shù)法檢測(cè)HMy2.CIR細(xì)胞增殖情況；以real-time RT-PCR法檢測(cè)DNMT1、DNMT3b和p16等基因表達(dá)；用胞嘧啶延伸實(shí)驗(yàn)檢測(cè)細(xì)胞全基因組甲基化狀態(tài)；用微核形成實(shí)驗(yàn)檢測(cè)細(xì)胞損傷；用甲基化特異性PCR(MSP)法檢測(cè)p16基因啟動(dòng)子的甲基化狀態(tài)。 結(jié)果：低劑量Cd短期暴露可以明顯促進(jìn)HMy2.CIR細(xì)胞增殖,而低劑量Cd長(zhǎng)期暴露促進(jìn)細(xì)胞增殖的作用更加顯著。Real-time RT-PCR檢測(cè)長(zhǎng)期低劑量Cd暴露細(xì)胞發(fā)現(xiàn)DNA甲基轉(zhuǎn)移酶DNMT1和DNMT3b表達(dá)升高,而周期抑制基因p16表達(dá)下降。長(zhǎng)期低劑量Cd暴露后全基因組發(fā)生高甲基化,且p16基因啟動(dòng)子也發(fā)生高甲基化改變。此外,用5-aza-dC處理低劑量Cd長(zhǎng)期暴露細(xì)胞可以顯著降低細(xì)胞增殖并恢復(fù)p16基因的表達(dá)。 結(jié)論：低劑量Cd長(zhǎng)期暴露通過(guò)誘導(dǎo)周期抑制基因p16啟動(dòng)子高甲基化而抑制該基因的表達(dá),并誘導(dǎo)HMy2.CIR細(xì)胞增殖加快,這可能是Cd致細(xì)胞惡性轉(zhuǎn)化的分子機(jī)制之一。 第三部分長(zhǎng)期低劑量γ射線與低劑量鎘誘導(dǎo)細(xì)胞交叉適應(yīng)性反應(yīng)及機(jī)制 目的：本研究主要探討長(zhǎng)期(4w) LDR與長(zhǎng)期(3m)低劑量Cd誘導(dǎo)人B淋巴母細(xì)胞(HMy2.CIR)交叉適應(yīng)性反應(yīng)(cross-AR),并探討DNA甲基化在其中的作用。 方法：長(zhǎng)期LDR組細(xì)胞每周一、三、五照射,每次0.032Gy,連續(xù)照射4w,并設(shè)非照射對(duì)照組。長(zhǎng)期低劑量Cd處理以0、0.005、0.01、0.1μM濃度的CdCl2連續(xù)處理細(xì)胞3m。在各組細(xì)胞受攻擊劑量CdCl2和γ射線處理后用微核形成實(shí)驗(yàn)檢測(cè)細(xì)胞適應(yīng)性反應(yīng)(AR)及cross-AR。 結(jié)果：長(zhǎng)期低劑量Cd能誘導(dǎo)HMy2.CIR細(xì)胞對(duì)隨后高劑量Cd及丫射線產(chǎn)生適應(yīng)性反應(yīng)及交叉適應(yīng)性反應(yīng)。當(dāng)攻擊劑量為50μM CdCl2時(shí),細(xì)胞表現(xiàn)出顯著的適應(yīng)性反應(yīng),但當(dāng)劑量增加到100μM CdCl2時(shí)并未誘導(dǎo)細(xì)胞產(chǎn)生顯著的適應(yīng)性反應(yīng)。同時(shí),我們發(fā)現(xiàn)不同濃度的低劑量Cd長(zhǎng)期暴露所誘導(dǎo)的適應(yīng)性反應(yīng)程度(MAR)不同,并具有一定的劑量-效應(yīng)關(guān)系。當(dāng)攻擊劑量為2Gy γ射線時(shí)細(xì)胞產(chǎn)生的MAR最顯著。長(zhǎng)期LDR處理同樣誘導(dǎo)HMy2.CIR細(xì)胞對(duì)隨后高劑量Cd及γ射線產(chǎn)生AR及cross-AR。當(dāng)攻擊劑量為50μM CdCl2時(shí),長(zhǎng)期LDR處理誘導(dǎo)細(xì)胞的MAR最為顯著,當(dāng)攻擊劑量為2Gyγ射線時(shí)細(xì)胞產(chǎn)生的MAR低于攻擊劑量為50μM CdCl2時(shí)的MAR,但當(dāng)攻擊劑量為100μM CdCl2時(shí),并未誘導(dǎo)細(xì)胞產(chǎn)生顯著的適應(yīng)性反應(yīng)。此外,用DNA甲基轉(zhuǎn)移酶5-aza-dC處理各組細(xì)胞后發(fā)現(xiàn)長(zhǎng)期低劑量Cd及長(zhǎng)期LDR誘導(dǎo)細(xì)胞的適應(yīng)性反應(yīng)均消失。 結(jié)論：長(zhǎng)期低劑量Cd及長(zhǎng)期LDR均能夠誘導(dǎo)HMy2.CIR細(xì)胞對(duì)隨后高劑量Cd及丫射線產(chǎn)生適應(yīng)性反應(yīng)。細(xì)胞產(chǎn)生MAR的大小與預(yù)處理劑量和攻擊處理劑量的大小密切相關(guān)。另外,DNA甲基化在二者誘導(dǎo)的交叉適應(yīng)性反應(yīng)中也發(fā)揮重要作用。
[Abstract]:With the increase in the application of ionizing radiation in medicine and industry, low dose radiation (LDR) has also aroused great attention. However, the effects and mechanisms of long-term exposure to LDR are not yet clear about the difference between the.LDR effect and the high dose radiation (HDR) effect, mainly including radiation hypersensitivity, gene instability, radiation adaptation response and side effects. In addition to radiation, there are many chemical contaminants in the environment that also harm human health. As heavy metal cadmium (Cd) is widely used in industry, its impact on human health is also widely concerned. A large number of studies have confirmed that large doses of Cd and ionizing radiation are important carcinogens, but the long-term low dose Cd and long-term LDR biological effects have been proved. There should be less research and less research on the joint effect of the two.
As is known to all, DNA methylation is one of the most extensive mechanisms of epigenetic research and plays an important role in a variety of basic life activities. A large number of studies have shown that the DNA methylation patterns of rodents and human cell lines are changed after exposure to ionizing radiation, and there are also studies to confirm the LDR related effects and The risk in essence may be related to epigenetic regulation, especially the regulation of DNA methylation, but the mechanism is not yet clear. In addition, some studies have confirmed that the high methylation of the whole genome and the hypermethylation of specific genes also play an important role in the process of Cd induced malignant transformation of various cells.
In this study, long term low dose gamma ray and long term low dose Cd were used to treat human B lymphoblastic (HMy2.CIR), and the effects of two people on the biological effects of HMy2.CIR cells and DNA methylation were studied. Meanwhile, the cross adaptation response (cross-AR) and related mechanisms of LDR and Cd were studied. The molecular mechanism of the long term low dose Cd effect will help us to better evaluate the effects of environmental physical and chemical toxins on human health, and provide a powerful experimental basis for the correct protection of environmental physical and chemical toxins. The contents of this study are divided into three parts:
Part one: the biological effects of long term low dose Y rays on lymphoblastoid cells and the role of DNA methylation.
Objective: To investigate the effect of long term low dose gamma ray on the proliferation, radiosensitivity and adaptability of human B lymphoblastic cell (HMy2.CIR), and to discuss the role of DNA methylation in it.
Methods: HMy2.CIR cells were irradiated with a low dose of gamma ray, which could significantly promote cell proliferation. Every Monday, three, five irradiated, 0.032Gy each time for 4 weeks (4W), and the non irradiation control group was set up. The cell DNA damage was detected by the micronucleus formation test, the cell proliferation was detected by CCK-8 method, and the whole gene was detected by the cytosine extension test. The expression of apoptosis and gamma -H2AX protein were detected by flow cytometry, and the expression of cyclinD1, PCNA, Bcl-2 and Bax were detected by real-time RT-PCR, and the expression of protein HP1 and MeCP2 was detected by Western blotting method.
Results: long term LDR can significantly increase the proliferation rate of HMy2.CIR cells and induce the increase of the expression of cyclinD1 and PCNA PCNA. Meanwhile, long-term LDR can induce the adaptive response of lymphoblastic cells to the attack dose (2Gy) ionizing radiation. Compared with the pure 2Gy irradiation group, the apoptosis rate and micronucleus of the LDR+2Gy group cells are micronucleus. The formation rate and the expression of gamma -H2AX protein were significantly reduced by.Real-time RT-PCR detection. The expression of anti apoptotic gene Bcl-2 in long-term LDR cells was increased and the expression of anti apoptotic gene Bax was reduced. Cytosine incorporation test found that the whole genome DNA occurred hypermethylation after long-term LDR treatment, and this hypermethylation was accompanied by methyltransferase 1 (DNMT1). The expression of methylated CpG binding protein 2 (MeCP2) and heterochromatin 1 (HP1) increased. In addition, after the treatment of cells with DNA methyltransferase inhibitor (5-aza-dC), the long-term LDR induced cell radiation adaptive response (RAR) was found to disappear.
Conclusion: long term LDR treatment can promote cell proliferation by changing gene expression, and can also induce RAR. to induce cell production by changing the whole genome methylation status and inducing apoptosis resistance.
The second part: the effect of low dose cadmium exposure on lymphoblastoid cell proliferation and its molecular pathways.
Objective: To study the effect of low dose Cd long-term exposure on the proliferation of human B lymphoblastic (HMy2.CIR), and to explore the role of the p16 gene and DNA methylation of the cyclical suppressor gene.
Methods: the HMy2.CIR cells were treated with cadmium chloride (CdCl2) with concentration of 0,0.005,0.01,0.1 mu M. The experiment was divided into two groups: short-term treatment group and long-term treatment group, short-term group Cd treatment 48h, long-term group Cd treatment for 3 months (3m). The cell count method was used to detect the proliferation of HMy2.CIR cells. Real-time RT-PCR method was used to detect the gene expression. The methylation of the whole genome of the cells was detected with cytosine extension test, and the cell damage was detected by micronucleus formation. Methylation specific PCR (MSP) method was used to detect the methylation status of the p16 gene promoter.
Results: low dose Cd short-term exposure could significantly promote the proliferation of HMy2.CIR cells, while low dose Cd long-term exposure promoted cell proliferation more significantly..Real-time RT-PCR detected the increase of DNA methyltransferase DNMT1 and DNMT3b expression in long term low dose Cd exposed cells, while the expression of cyclically inhibited gene p16 decreased. Long term low dose Cd exposure The whole genome was hypermethylation, and the promoter of p16 gene was also hypermethylation. In addition, 5-aza-dC treatment of low dose Cd long term exposed cells could significantly reduce cell proliferation and restore the expression of p16 gene.
Conclusion: low dose Cd long-term exposure can inhibit the expression of this gene by inducing hypermethylation of the promoter p16 promoter and induce the proliferation of HMy2.CIR cells, which may be one of the molecular mechanisms of Cd cell malignant transformation.
The third part is the cross adaptive response of long term low-dose gamma rays to low dose cadmium and its mechanism.
Objective: To investigate the cross adaptation response (cross-AR) of long-term (4W) LDR and long-term (3m) low dose Cd induced B lymphoblastoid (HMy2.CIR), and to explore the role of DNA methylation in it.
Methods: long term LDR group cells were irradiated every Monday, three and five, each time 0.032Gy, continuous irradiation of 4W, and set up a non irradiated control group. Long term low dose Cd treatment was used to treat cell 3m. in CdCl2 continuously with 0,0.005,0.01,0.1 micron M concentration, and the cell adaptation reaction (AR) and cros were detected by micronucleus formation in each group of cells after the attack dose CdCl2 and gamma ray treatment. S-AR.
Results: long term low dose Cd could induce HMy2.CIR cells to produce adaptive response and cross adaptation response to subsequent high dose Cd and X ray. When the attack dose was 50 M CdCl2, the cells showed significant adaptive response, but we did not induce a significant adaptive response when the dose increased to 100 u M CdCl2. It was found that the degree of adaptive response (MAR) induced by the low dose Cd exposure of different concentrations was different, and had a certain dose effect relationship. When the attack dose was 2Gy gamma ray, the MAR was the most significant. The long-term LDR treatment also induced the HMy2.CIR cells to produce AR and cross-AR. at a high dose of Cd and gamma rays at a dose of 5. When 0 mu M CdCl2, the MAR of the cells induced by long-term LDR treatment is the most significant. When the attack dose is 2Gy gamma ray, the MAR is lower than the attack dose of MAR when the attack dose is 50 mu M CdCl2. But when the attack dose is 100 mu M CdCl2, the cells do not induce the cells to produce a significant adaptive response. Long term low-dose Cd and long-term LDR induce the adaptive response of cells to disappear.
Conclusion: long term low dose Cd and long term LDR can induce the adaptive response of HMy2.CIR cells to the subsequent high dose Cd and the Cd ray. The size of MAR is closely related to the dose of preconditioning and the dose of attack. In addition, DNA methylation also plays an important role in the cross adaptation response induced by the two.
【學(xué)位授予單位】：復(fù)旦大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2013
【分類號(hào)】：R818.03

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本文編號(hào)：1927728