發(fā)布時間：2018-06-06 21:27

  本文選題：放療 + 輻射敏感性��； 參考：《中國科學(xué)技術(shù)大學(xué)》2012年博士論文

【摘要】：微核產(chǎn)生與細胞中的DNA損傷密切相關(guān)。輻射引起細胞產(chǎn)生微核是一個普遍的現(xiàn)象。在腫瘤放療過程中，微核產(chǎn)生與許多腫瘤的輻射敏感性相關(guān)，因此微核率可用于評價放療預(yù)后。盡管如此，由于應(yīng)用傳統(tǒng)的細胞遺傳學(xué)技術(shù)我們無法對微核細胞命運做深入研究，因此微核作為評價細胞輻射敏感性的依據(jù)尚不清楚。 本部分研究中我們以鼻咽癌細胞為模型，，選用對輻射不敏感的CNE1細胞和對輻射敏感的CNE2細胞，考察X射線輻射引起的微核細胞的命運。由于這兩種細胞具有良好的形態(tài)及較慢的運動速度，因此有利于活細胞攝影追蹤。為在活細胞中研究微核，我們通過穩(wěn)定轉(zhuǎn)染用紅色熒光蛋白(mCherry)標記組蛋白H2B。通過長時間活細胞實時攝影記錄X射線誘導(dǎo)的微核細胞的行為。 通過分析活細胞實時攝影我們在微核細胞中有如下發(fā)現(xiàn)。首先，在X射線照射后對輻射敏感的細胞相對于對輻射不敏感的細胞會產(chǎn)生更多微核。同時，含有微核的細胞比不含有微核的細胞更容易發(fā)生細胞周期停滯和細胞死亡，并且細胞中所含有的微核越多，引起細胞周期停滯和細胞死亡的效應(yīng)就越強烈。當(dāng)微核細胞進入分裂時通常會以落后染色體（而非染色體橋）的方式將微核傳給子細胞。用人泛著絲粒探針進行熒光原位雜交顯示大約70%的由輻射引起的落后染色體及微核中均不含有著絲粒信號。最后，通過磷酸化H2AX和磷酸化p38熒光免疫染色我們發(fā)現(xiàn)微核細胞相對于不含微核的細胞含有更多的DNA損傷和更強的應(yīng)激激酶通路的活化。 綜上，我們的發(fā)現(xiàn)顯示微核的產(chǎn)生與DNA損傷程度相關(guān)，并最終影響細胞命運。揭示了微核細胞增殖能力減弱的原因。為將微核發(fā)生作為細胞輻射敏感性的指標提供了判斷依據(jù)和理論基礎(chǔ)。 細胞含有異常數(shù)目的染色體稱為非整倍體。非整倍體是引起人類自發(fā)流產(chǎn)和智力發(fā)育障礙的重要原因，同時也是腫瘤細胞的主要特性之一。在有絲分裂過程中微管與著絲粒形成的錯誤連接，尤其是merotelic連接（單個著絲粒被來自兩極的微管連接）導(dǎo)致分裂后期形成的落后染色體與形成非整倍體子細胞密切相關(guān)。同時，落后染色體可能形成微核而導(dǎo)致遺傳物質(zhì)丟失進而導(dǎo)致非整倍體形成。然而，落后染色體對非整倍體產(chǎn)生的貢獻尚存在爭議。對落后染色體及微核命運的推測大多基于對固定細胞的研究，落后染色體及微核在活細胞中的行為如何尚不清楚。 為了避免使用固定細胞帶來的不便，我們早期曾使用熒光蛋白標記染色體，在活細胞中初步研究了落后染色體、微核的形成及其存在對細胞命運的影響（如我們在第一部分中所討論的）。但是，用熒光蛋白標記所有染色體并不能在活細胞攝影時明確的追蹤落后染色體及微核的命運。為解答這一問題必須尋找新的實驗方案。 在本部分研究中，我們使用結(jié)腸直腸癌細胞HCT116作為研究落后染色體及微核的細胞。通過穩(wěn)定表達紅色熒光蛋白(mCherry)偶聯(lián)的組蛋白H2B，標記所有的染色體。在這些細胞中轉(zhuǎn)染GFP偶聯(lián)的大腸桿菌乳糖操縱子調(diào)控基因(LacRepressor, LacI)和大腸桿菌乳糖操縱子順式作用元件(LacOperator, LacO)。由于GFP-LacRepressor會結(jié)合在整合于單條染色體上的LacOperator序列，該染色體便被GFP特異標記。我們由此可在活細胞中追蹤這條被GFP特異標記的染色體。 我們借助活細胞實時攝影追蹤被GFP標記的由merotelic連接導(dǎo)致的落后染色體的命運。發(fā)現(xiàn)絕大多是落后染色體最終運動到正確的子細胞中并形成微核。這些在正確子細胞中的微核并不影響細胞的增殖。當(dāng)這些微核細胞進入分裂時幾乎所有微核中的遺傳物質(zhì)均能正常的凝集、正確的聚集在赤道板上，進而像主核中的染色體一樣分離、分配至兩個子細胞中，最終形成兩個不帶微核的子細胞。我們的研究顯示絕大多數(shù)落后染色體并不能導(dǎo)致非整倍體產(chǎn)生，并揭示了微核形成并不一定代表遺傳物質(zhì)丟失。
[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.
【學(xué)位授予單位】：中國科學(xué)技術(shù)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2012
【分類號】：R114

【參考文獻】

相關(guān)期刊論文 前1條

1 賀玉香;仲萍萍;嚴珊珊;劉莉;史弘流;曾木圣;夏云飛;;DNA-PK的活性與鼻咽癌細胞株CNE1/CNE2放射敏感性的關(guān)系(英文)[J];生理學(xué)報;2007年04期

本文編號：1988167