微核及微核细胞命运的研究

发布时间：2018-06-06 21:27

本文选题：放疗 + 辐射敏感性　；参考：《中国科学技术大学》2012年博士论文

【摘要】：微核产生与细胞中的DNA损伤密切相关。辐射引起细胞产生微核是一个普遍的现象。在肿瘤放疗过程中，微核产生与许多肿瘤的辐射敏感性相关，因此微核率可用于评价放疗预后。尽管如此，由于应用传统的细胞遗传学技术我们无法对微核细胞命运做深入研究，因此微核作为评价细胞辐射敏感性的依据尚不清楚。本部分研究中我们以鼻咽癌细胞为模型，，选用对辐射不敏感的CNE1细胞和对辐射敏感的CNE2细胞，考察X射线辐射引起的微核细胞的命运。由于这两种细胞具有良好的形态及较慢的运动速度，因此有利于活细胞摄影追踪。为在活细胞中研究微核，我们通过稳定转染用红色荧光蛋白(mCherry)标记组蛋白H2B。通过长时间活细胞实时摄影记录X射线诱导的微核细胞的行为。通过分析活细胞实时摄影我们在微核细胞中有如下发现。首先，在X射线照射后对辐射敏感的细胞相对于对辐射不敏感的细胞会产生更多微核。同时，含有微核的细胞比不含有微核的细胞更容易发生细胞周期停滞和细胞死亡，并且细胞中所含有的微核越多，引起细胞周期停滞和细胞死亡的效应就越强烈。当微核细胞进入分裂时通常会以落后染色体（而非染色体桥）的方式将微核传给子细胞。用人泛着丝粒探针进行荧光原位杂交显示大约70%的由辐射引起的落后染色体及微核中均不含有着丝粒信号。最后，通过磷酸化H2AX和磷酸化p38荧光免疫染色我们发现微核细胞相对于不含微核的细胞含有更多的DNA损伤和更强的应激激酶通路的活化。综上，我们的发现显示微核的产生与DNA损伤程度相关，并最终影响细胞命运。揭示了微核细胞增殖能力减弱的原因。为将微核发生作为细胞辐射敏感性的指标提供了判断依据和理论基础。细胞含有异常数目的染色体称为非整倍体。非整倍体是引起人类自发流产和智力发育障碍的重要原因，同时也是肿瘤细胞的主要特性之一。在有丝分裂过程中微管与着丝粒形成的错误连接，尤其是merotelic连接（单个着丝粒被来自两极的微管连接）导致分裂后期形成的落后染色体与形成非整倍体子细胞密切相关。同时，落后染色体可能形成微核而导致遗传物质丢失进而导致非整倍体形成。然而，落后染色体对非整倍体产生的贡献尚存在争议。对落后染色体及微核命运的推测大多基于对固定细胞的研究，落后染色体及微核在活细胞中的行为如何尚不清楚。为了避免使用固定细胞带来的不便，我们早期曾使用荧光蛋白标记染色体，在活细胞中初步研究了落后染色体、微核的形成及其存在对细胞命运的影响（如我们在第一部分中所讨论的）。但是，用荧光蛋白标记所有染色体并不能在活细胞摄影时明确的追踪落后染色体及微核的命运。为解答这一问题必须寻找新的实验方案。在本部分研究中，我们使用结肠直肠癌细胞HCT116作为研究落后染色体及微核的细胞。通过稳定表达红色荧光蛋白(mCherry)偶联的组蛋白H2B，标记所有的染色体。在这些细胞中转染GFP偶联的大肠杆菌乳糖操纵子调控基因(LacRepressor, LacI)和大肠杆菌乳糖操纵子顺式作用元件(LacOperator, LacO)。由于GFP-LacRepressor会结合在整合于单条染色体上的LacOperator序列，该染色体便被GFP特异标记。我们由此可在活细胞中追踪这条被GFP特异标记的染色体。我们借助活细胞实时摄影追踪被GFP标记的由merotelic连接导致的落后染色体的命运。发现绝大多是落后染色体最终运动到正确的子细胞中并形成微核。这些在正确子细胞中的微核并不影响细胞的增殖。当这些微核细胞进入分裂时几乎所有微核中的遗传物质均能正常的凝集、正确的聚集在赤道板上，进而像主核中的染色体一样分离、分配至两个子细胞中，最终形成两个不带微核的子细胞。我们的研究显示绝大多数落后染色体并不能导致非整倍体产生，并揭示了微核形成并不一定代表遗传物质丢失。
[Abstract]:Micronucleus is closely related to DNA damage in cells. Radiation induced micronucleus is a common phenomenon. Micronucleus is associated with radiosensitivity of many tumors during tumor radiotherapy, so micronucleus rate can be used to evaluate the prognosis of radiotherapy. The nuclear cell fate has been studied in depth, so the basis of micronucleus as a radiosensitivity assessment is unclear.
In this part, we use nasopharyngeal carcinoma cells as a model to investigate the fate of micronucleus cells caused by radiation insensitive CNE1 cells and radiation sensitive CNE2 cells. Because these two cells have good morphology and slow motion speed, they are beneficial to the tracing of living cells. In micronucleus, we recorded the behavior of micronucleus induced by X ray through a long time live cell by stable transfection by using the red fluorescent protein (mCherry) labeled histone H2B. in a long time live cell.
By analyzing live cell real-time photography, we have the following findings in micronuclear cells. First, the cells that are sensitive to radiation after X ray irradiation produce more micronucleus than those that are insensitive to radiation. Meanwhile, cells with micronucleus are more likely to have cell cycle stagnation and cell death than cells without micronucleus, and cells are more likely to occur. The more micronucleus contained in the cell, the stronger the effect of cell cycle stagnation and cell death. When micronucleus cells divide into division, micronuclei are usually transmitted to subcells in the way of backward chromosomes (rather than chromosome bridges). Fluorescence in situ hybridization shows about 70% of the backward chromosomes caused by radiation. No centromeric signal was contained in micronucleus. Finally, we found that micronucleus cells have more DNA damage and stronger activation of stress kinase pathway than those without micronucleus by phosphorylated H2AX and phosphorylated p38 fluorescent immunostaining.
To sum up, our findings show that the production of micronucleus is related to the degree of DNA damage and ultimately affects cell fate. The cause of the weakening of micronucleus cell proliferation is revealed, which provides a basis for judging the occurrence of micronucleus as an indicator of cell radiosensitivity.
The chromosomes containing abnormal numbers of cells are called aneuploidy. Aneuploidy is an important cause of spontaneous abortion and mental retardation. It is also one of the main characteristics of tumor cells. In the process of mitosis, the wrong connection between microtubules and centromere formation, especially the merotelic connection (single centromere is from the two poles. Microtubule connections lead to a close correlation between the backward chromosomes formed in the late division and the formation of aneuploidy. At the same time, the backward chromosomes may form micronucleus which lead to the loss of genetic material and lead to aneuploidy. However, the contribution of the backward chromosomes to the aneuploidy is still controversial. The fate of the backward chromosomes and micronucleus. Most of the speculation is based on the study of fixed cells, and how the behavior of backward chromosomes and micronucleus in living cells is unclear.
In order to avoid the inconvenience of using fixed cells, we used a fluorescent protein to mark chromosomes early and initially studied the backward chromosomes in living cells, the formation of micronucleus and their influence on cell fate (as discussed in the first part). However, the use of fluorescent protein to mark all chromosomes is not in living cells. In photography, we should clearly track the fate of the backward chromosomes and micronucleus. In order to solve this problem, we must find a new experimental scheme.
In this part of the study, we used colorectal cancer cell HCT116 as a cell to study the backward chromosomes and micronucleus. All chromosomes were labeled by the stable expression of the histone coupled with the red fluorescent protein (mCherry) H2B. In these cells, the GFP coupled Escherichia coli operon regulation gene (LacRepressor, LacI) was transfected into these cells. E. coli lactose operon CIS (LacOperator, LacO). Because GFP-LacRepressor will bind to the LacOperator sequence integrated on a single chromosome, the chromosome is specifically labeled by GFP. We can trace this GFP specific chromophore in living cells.
We used live cell real-time photography to track the fate of the backward chromosomes marked by the merotelic connection by GFP. It is found that most of the backward chromosomes eventually move into the correct subcells and form micronucleus. These micronucleus in the correct subcells do not affect the cell proliferation. When these micronucleus cells enter division, almost all of them are divided. The genetic material in all the micronucleus can be agglutinated properly, correctly aggregated on the equatorial plate, and then separated from the chromosomes in the main nucleus and allocated to two subcells to form two subcells without micronucleus. Our study shows that most of the backward chromosomes do not cause aneuploidy and reveal micronucleus. Formation does not necessarily represent the loss of genetic material.
【学位授予单位】：中国科学技术大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R114

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