长期低剂量辐射与镉暴露的细胞效应及DNA甲基化在其中的作用

发布时间：2018-05-24 04:44

本文选题：低剂量辐射 + 细胞增殖　；参考：《复旦大学》2013年硕士论文

【摘要】：随着电离辐射在医学和工业中应用的增加,低剂量辐射(LDR)也引起人们的高度重视,但有关LDR长期暴露的效应和机制尚不明确。LDR效应与高剂量辐射(HDR)效应不同,主要包括辐射超敏反应、基因不稳定性、辐射适应性反应和旁效应等。除电离辐射外,环境中还存在许多化学污染物也危害着人类的健康。由于重金属镉(Cd)在工业上的广泛应用,其对人类健康的影响也受到广泛关注。大量研究证实大剂量Cd和电离辐射均是重要的致癌因子,但有关长期低剂量Cd和长期LDR生物效应的研究较少,而有关二者联合效应的研究更少。众所周知,DNA甲基化是表观遗传学研究最广泛的机制之一,其在多种基础生命活动中均发挥重要调节作用。大量研究显示,啮齿类动物和人类细胞株在暴露于电离辐射后其DNA甲基化模式均会发生改变,同时,也有研究证实LDR相关效应和风险本质上可能与表观遗传学方面的调控有关,尤其是DNA甲基化方面的调控,但机制尚不明确。另外,有研究证实全基因组高甲基化以及特异性基因高甲基化在Cd诱导多种细胞恶性转化过程中也发挥重要作用。本研究采用长期低剂量γ射线及长期低剂量Cd对人B淋巴母细胞(HMy2.CIR)进行处理,研究二者对HMy2.CIR细胞的生物效应及DNA甲基化在其中的作用。同时,对LDR和Cd的交叉适应性反应(cross-AR)及相关机制进行了研究。更深层次地理解长期LDR及长期低剂量Cd效应的分子机制将有助于我们更好地对环境物理化学毒素对人体健康的影响进行评估,也为人们正确防护环境理化毒素提供有力的实验依据。本研究内容分为以下三个部分：第一部分：长期低剂量Y射线对淋巴母细胞的生物学效应及DNA甲基化在其中的作用目的：探讨长期低剂量γ射线对人B淋巴母细胞(HMy2.CIR)增殖、辐射敏感性以及适应性方面的影响,并简要探讨DNA甲基化在其中的作用。方法：选用可以显著促进细胞增殖的低剂量γ射线对HMy2.CIR细胞进行照射,每周一、三、五照射,每次0.032Gy,连续照射4周(4w),并设非照射对照组。以微核形成实验检测细胞DNA损伤,以CCK-8法检测细胞增殖,以胞嘧啶延伸实验检测细胞全基因组甲基化情况,用流式细胞术检测细胞凋亡和γ-H2AX蛋白的表达,以real-time RT-PCR法检测基因cyclinD1、PCNA、bcl-2和bax的表达,以Western blotting法检测蛋白HP1和MeCP2的表达。结果：长期LDR可以显著提高HMy2.CIR细胞的增殖率并诱导周期相关基因cyclinD1和增殖细胞核抗原PCNA表达增加。同时,长期LDR可以诱导淋巴母细胞对攻击剂量(2Gy)电离辐射产生适应性反应,与单纯2Gy照射组相比,LDR+2Gy组细胞的凋亡率、微核形成率及γ-H2AX蛋白表达量均显著降低。Real-time RT-PCR检测发现长期LDR处理细胞抗凋亡基因bcl-2表达升高而抑凋亡基因bax表达降低。胞嘧啶掺入实验检测发现长期LDR处理后全基因组DNA发生高甲基化,这种高甲基化伴随甲基转移酶1(DNMT1)、甲基化CpG结合蛋白2(MeCP2)以及异染色质蛋白1(HP1)表达的升高。另外,用DNA甲基转移酶抑制剂(5-aza-dC)处理细胞后发现长期LDR诱导的细胞辐射适应性反应(RAR)消失。结论：长期LDR处理可通过改变基因表达而促进细胞增殖,同时也可以通过改变细胞全基因组甲基化状态及诱导凋亡抵抗进而终诱导细胞产生RAR。第二部分：低剂量镉长期暴露对淋巴母细胞增殖的影响及其分子途径目的：本部分着重研究低剂量Cd长期暴露对人B淋巴母细胞(HMy2.CIR)增殖的影响,并探讨周期抑制基因p16基因及DNA甲基化在其中的作用。方法：以0、0.005、0.01、0.1μM浓度的氯化镉(CdCl2)处理HMy2.CIR细胞,实验分为两组：短期处理组和长期处理组,短期组Cd处理48h,长期组Cd处理3个月(3m)。采用细胞计数法检测HMy2.CIR细胞增殖情况；以real-time RT-PCR法检测DNMT1、DNMT3b和p16等基因表达；用胞嘧啶延伸实验检测细胞全基因组甲基化状态；用微核形成实验检测细胞损伤；用甲基化特异性PCR(MSP)法检测p16基因启动子的甲基化状态。结果：低剂量Cd短期暴露可以明显促进HMy2.CIR细胞增殖,而低剂量Cd长期暴露促进细胞增殖的作用更加显著。Real-time RT-PCR检测长期低剂量Cd暴露细胞发现DNA甲基转移酶DNMT1和DNMT3b表达升高,而周期抑制基因p16表达下降。长期低剂量Cd暴露后全基因组发生高甲基化,且p16基因启动子也发生高甲基化改变。此外,用5-aza-dC处理低剂量Cd长期暴露细胞可以显著降低细胞增殖并恢复p16基因的表达。结论：低剂量Cd长期暴露通过诱导周期抑制基因p16启动子高甲基化而抑制该基因的表达,并诱导HMy2.CIR细胞增殖加快,这可能是Cd致细胞恶性转化的分子机制之一。第三部分长期低剂量γ射线与低剂量镉诱导细胞交叉适应性反应及机制目的：本研究主要探讨长期(4w) LDR与长期(3m)低剂量Cd诱导人B淋巴母细胞(HMy2.CIR)交叉适应性反应(cross-AR),并探讨DNA甲基化在其中的作用。方法：长期LDR组细胞每周一、三、五照射,每次0.032Gy,连续照射4w,并设非照射对照组。长期低剂量Cd处理以0、0.005、0.01、0.1μM浓度的CdCl2连续处理细胞3m。在各组细胞受攻击剂量CdCl2和γ射线处理后用微核形成实验检测细胞适应性反应(AR)及cross-AR。结果：长期低剂量Cd能诱导HMy2.CIR细胞对随后高剂量Cd及丫射线产生适应性反应及交叉适应性反应。当攻击剂量为50μM CdCl2时,细胞表现出显著的适应性反应,但当剂量增加到100μM CdCl2时并未诱导细胞产生显著的适应性反应。同时,我们发现不同浓度的低剂量Cd长期暴露所诱导的适应性反应程度(MAR)不同,并具有一定的剂量-效应关系。当攻击剂量为2Gy γ射线时细胞产生的MAR最显著。长期LDR处理同样诱导HMy2.CIR细胞对随后高剂量Cd及γ射线产生AR及cross-AR。当攻击剂量为50μM CdCl2时,长期LDR处理诱导细胞的MAR最为显著,当攻击剂量为2Gyγ射线时细胞产生的MAR低于攻击剂量为50μM CdCl2时的MAR,但当攻击剂量为100μM CdCl2时,并未诱导细胞产生显著的适应性反应。此外,用DNA甲基转移酶5-aza-dC处理各组细胞后发现长期低剂量Cd及长期LDR诱导细胞的适应性反应均消失。结论：长期低剂量Cd及长期LDR均能够诱导HMy2.CIR细胞对随后高剂量Cd及丫射线产生适应性反应。细胞产生MAR的大小与预处理剂量和攻击处理剂量的大小密切相关。另外,DNA甲基化在二者诱导的交叉适应性反应中也发挥重要作用。
[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.
【学位授予单位】：复旦大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R818.03

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