本文選題：熱療 + 腫瘤��； 參考：《上海交通大學(xué)》2007年博士論文

【摘要】： 熱療作為一種治療腫瘤的全新療法,得到了廣大研究者廣泛的關(guān)注。但是已有的研究都局限于熱療或者熱療結(jié)合其他療法對腫瘤細(xì)胞的損傷,血管內(nèi)皮層作為腫瘤生長中營養(yǎng)和氧氣供應(yīng)的主要通道一直被忽視。隨著抗血管新生療法的問世,血管內(nèi)皮細(xì)胞在腫瘤治療中受到越來越多的重視。以新生血管為靶點(diǎn),抑制腫瘤生長,阻斷腫瘤的營養(yǎng)來源和轉(zhuǎn)移通道已成為近年來國內(nèi)外研究的熱點(diǎn)。本文主要從細(xì)胞和分子水平研究熱刺激過程中血管內(nèi)皮細(xì)胞形態(tài)改變與血管滲透性變化的關(guān)系,探討熱療對血管內(nèi)皮細(xì)胞的損傷,并探索引起其改變的機(jī)理。 在臨床上熱療可以增強(qiáng)化療的療效,但是對于引起此效果的機(jī)理卻很少有系統(tǒng)性的研究。我們課題組已有的研究工作表明熱療通過增大血管的通透性,促進(jìn)抗腫瘤藥物在腫瘤部位釋放從而增強(qiáng)其療效。本課題進(jìn)一步研究了熱刺激引起細(xì)胞形態(tài)改變與微絲骨架變化的關(guān)系,通過體外加熱血管內(nèi)皮細(xì)胞發(fā)現(xiàn)加熱后細(xì)胞形態(tài)變圓,細(xì)胞之間的間隙變大,同時(shí)細(xì)胞之間的連接消失。通過對細(xì)胞微絲骨架的固定染色,發(fā)現(xiàn)加熱后,細(xì)胞內(nèi)的微絲骨架發(fā)生了巨大的變化,有序的網(wǎng)狀結(jié)構(gòu)消失,微絲聚集體散布在細(xì)胞內(nèi)。同時(shí),我們將GFP-actin質(zhì)粒轉(zhuǎn)染血管內(nèi)皮細(xì)胞后建立單克隆細(xì)胞系,利用該細(xì)胞系及激光共聚焦熒光顯微鏡對血管內(nèi)皮細(xì)胞進(jìn)行實(shí)時(shí)觀察,研究了加熱過程中血管內(nèi)皮細(xì)胞細(xì)胞骨架的實(shí)時(shí)變化。結(jié)果表明,細(xì)胞微絲骨架的變化與其形態(tài)以及細(xì)胞之間連接的變化幾乎是同步發(fā)生的,說明了血管內(nèi)皮細(xì)胞形態(tài)以及細(xì)胞之間連接發(fā)生變化的內(nèi)在原因是細(xì)胞內(nèi)微絲骨架受熱發(fā)生了解聚。 熱療結(jié)合化療除了對腫瘤細(xì)胞有殺傷作用外,其對血管內(nèi)皮細(xì)胞的直接殺傷作用也是熱療增強(qiáng)療效的重要機(jī)制。我們進(jìn)一步研究了熱刺激以后血管內(nèi)皮細(xì)胞的損傷,結(jié)果顯示血管內(nèi)皮細(xì)胞在45℃加熱以后,細(xì)胞的表面出現(xiàn)了大量凋亡小體。我們又用熒光雙染色的方法定量分析了血管內(nèi)皮細(xì)胞在不同溫度下細(xì)胞存活率、凋亡率和壞死率隨時(shí)間變化的情況,結(jié)果表明血管內(nèi)皮細(xì)胞死亡的模式與溫度的高低密切相關(guān):當(dāng)溫度低于45℃時(shí),細(xì)胞的死亡模式以發(fā)生凋亡為主,而當(dāng)溫度大于45℃時(shí),細(xì)胞的死亡模式以壞死為主。已有的研究發(fā)現(xiàn)熱刺激以后腫瘤環(huán)境下的血管內(nèi)皮細(xì)胞比正常血管內(nèi)皮細(xì)胞死亡率更高,對熱的刺激更加敏感。我們通過進(jìn)一步的實(shí)驗(yàn)證明溫度處于43-45℃時(shí),腫瘤環(huán)境下的血管內(nèi)皮細(xì)胞凋亡率要大于正常血管內(nèi)皮細(xì)胞。 除了本研究所涉及的熱刺激引起血管內(nèi)皮細(xì)胞微絲骨架以及細(xì)胞發(fā)生凋亡、壞死的研究,已有的一些研究還對熱刺激過程中血管內(nèi)皮細(xì)胞胞內(nèi)鈣離子濃度以及熱激蛋白(HSP)水平變化等細(xì)胞水平的現(xiàn)象進(jìn)行了研究,但這些單因素的研究始終無法從全局和系統(tǒng)的角度探討熱刺激對細(xì)胞的影響。為此,我們采用了大規(guī)模基因芯片的方法檢測受熱刺激血管內(nèi)皮細(xì)胞后基因表達(dá)水平的變化,發(fā)現(xiàn)在溫?zé)岷透邿岽碳さ臈l件下都有大量的基因表達(dá)發(fā)生上調(diào)或者下調(diào)。隨后又通過正交分類以及基因語義學(xué)(Gene Ontology)等分析方法,將統(tǒng)計(jì)上有基因顯著聚集的功能子類挑選出來,隨后將這些功能子類的基因與已經(jīng)報(bào)道的實(shí)驗(yàn)現(xiàn)象進(jìn)行詳細(xì)地對比分析,找出了其中一些生理現(xiàn)象可能的分子機(jī)制。其中包括細(xì)胞結(jié)構(gòu)相關(guān)蛋白基因,如DNA結(jié)合蛋白,骨架蛋白等;還有一些是細(xì)胞功能相關(guān)蛋白基因,如細(xì)胞信號轉(zhuǎn)導(dǎo)相關(guān)蛋白,還有細(xì)胞凋亡相關(guān)蛋白等。將這些現(xiàn)象與基因水平的變化聯(lián)系起來,為進(jìn)一步研究這些生理現(xiàn)象找到了線索。 結(jié)合我們的實(shí)驗(yàn)結(jié)果,我們認(rèn)為在熱療促進(jìn)藥物的熱靶向輸送研究中,熱療除了引起血管內(nèi)皮細(xì)胞骨架解聚改變細(xì)胞形態(tài)而達(dá)到抗腫瘤藥物在腫瘤部位大量滲透外,還通過細(xì)胞凋亡的方式引起腫瘤血管的破壞,因此熱療具有雙重的療效,一方面促進(jìn)了藥物的滲透,另一方面也造成了腫瘤血管的破壞,這兩方面的作用同時(shí)增強(qiáng)了熱療結(jié)合化療的抗腫瘤效果。 本課題的開展具有重要的理論價(jià)值,從理論上解釋了臨床上熱療能夠加強(qiáng)抗腫瘤藥物在腫瘤部位靶向釋放以及熱療通過直接殺傷血管內(nèi)皮細(xì)胞達(dá)到殺死腫瘤組織的機(jī)制。同時(shí)熱刺激以后血管內(nèi)皮細(xì)胞基因表達(dá)的研究可以較系統(tǒng)地解釋受熱刺激以后血管內(nèi)皮細(xì)胞發(fā)生的部分生理現(xiàn)象。同時(shí)本課題具有重要的實(shí)踐價(jià)值,本研究在解釋臨床研究基礎(chǔ)上又進(jìn)一步提出改進(jìn)的治療方法,不僅為熱療的研究打下了堅(jiān)實(shí)的基礎(chǔ)理論,而且具有重要的臨床指導(dǎo)意義。
[Abstract]:As a new therapy for cancer treatment, hyperthermia has received extensive attention from the vast majority of researchers. However, previous studies have been limited to hyperthermia or thermotherapy combined with other therapies for tumor cells. The vascular endothelium has been neglected as the main channel for nutrition and oxygen supply in tumor growth. With anti angiogenic therapy In recent years, vascular endothelial cells have been paid more and more attention in the treatment of tumor. It has become a hot spot in recent years to study the growth of neovascularization, inhibit the growth of tumor and block the tumor's nutrient sources and transfer channels. This paper mainly studies the morphological changes of vascular endothelial cells and blood vessels during the process of hot spur stimulation from the cell and molecular level. To explore the mechanism of hyperthermia on vascular endothelial cells and explore the mechanism of changes.
Clinical hyperthermia can enhance the efficacy of chemotherapy, but there is little systematic study of the mechanism that causes this effect. Our research team has already studied the effect of hyperthermia by increasing the permeability of blood vessels, promoting the release of antitumor drugs at the tumor site and enhancing its efficacy. The relationship between cell morphologic changes and the change of microfilament skeleton was found by heating vascular endothelial cells in vitro. It was found that the cell morphology became round, the gap between cells became larger and the connections between cells disappeared. By staining the cell microfilament skeleton, it was found that after heating, the microfilament skeleton in the cell had a great change and ordered. The reticular formation disappeared and the microfilament aggregates were scattered in the cells. At the same time, we transfected the GFP-actin plasmid into the vascular endothelial cells to establish a monoclonal cell line. The cell lines and laser confocal fluorescence microscopy were used to observe the endothelial cells in real time. The real-time changes in the cytoskeleton of the vascular endothelial cells during the heating process were studied. The results showed that the changes in the cellular microfilament skeleton and the changes in the connection between cells almost synchronously occur, indicating that the internal cause of the changes in the morphology of the endothelial cells and the changes in the connection between cells is the depolymerization of the intracellular microfilament skeleton.
The direct killing effect of the hyperthermia combined with chemotherapy on the tumor cells is also an important mechanism for the heat treatment to enhance the therapeutic effect. We further studied the damage of vascular endothelial cells after heat stimulation. The results showed that the surface of the vascular endothelial cells appeared to be apoptotic on the surface of the cells after heating at 45. We also quantitatively analyzed the cell survival rate, apoptosis rate and necrosis rate of vascular endothelial cells at different temperatures. The results showed that the mode of vascular endothelial cell death was closely related to the temperature of vascular endothelial cells. When the temperature was below 45, the death pattern of the cells was mainly apoptosis. When the temperature is more than 45, the death pattern of the cell is mainly necrosis. The previous study found that the vascular endothelial cells in the tumor environment were more sensitive to the heat stimulation than the normal vascular endothelial cells after the heat stimulation. We proved that the temperature was at 43-45 degrees centigrade, and the vascular endothelium in the tumor environment was proved by further experiments. The rate of cell apoptosis is greater than that of normal vascular endothelial cells.
In addition to the research involved in this study, some studies have been carried out to study the cellular level of intracellular calcium ion concentration and the changes of HSP levels during the process of heat stimulation, but these single factors have been studied. We have always been unable to explore the effects of thermal stimulation on cells from a global and systematic perspective. To this end, we used a large-scale gene chip to detect changes in the gene expression level after the heat stimulation of vascular endothelial cells, and found that a large number of gene expressions were up or down under the conditions of warm and high heat stimulation. Through the analysis of over orthogonal classification and genetic semantics (Gene Ontology), the functional subclasses with significant aggregation of genes are selected, and then the genes of these functional subclasses are compared with the reported experimental phenomena in detail, and some possible molecular mechanisms of these physiological phenomena are found, including cell junctions. Related protein genes, such as DNA binding protein, skeleton protein, and cell function related protein genes, such as cell signal transduction related proteins, and apoptosis related proteins, linked these phenomena with the changes of gene level, and found clues for further study of these physiological phenomena.
In combination with our experimental results, we believe that in the study of thermally targeted drug delivery, hyperthermia, in addition to causing the depolymerization of vascular endothelial cells to change the morphology of the endothelial cells to achieve a large amount of infiltration of antitumor drugs in the tumor site, also causes the destruction of the blood vessels of the tumor by the way of cell apoptosis, thus thermotherapy has a dual treatment. Effects, on the one hand, promote the penetration of drugs, on the other hand, also cause the destruction of tumor vessels. These two aspects also enhance the antitumor effect of hyperthermia combined with chemotherapy.
The development of this topic has important theoretical value. It has theoretically explained that clinical thermo therapy can strengthen the target release of antitumor drugs at the tumor site and the mechanism of killing tumor tissue by direct killing vascular endothelial cells by heat therapy, and the research on gene expression of vascular endothelial cells can be systematically solved after thermal stimulation. The partial physiological phenomenon of vascular endothelial cells after heat stimulation. Meanwhile, this topic has important practical value. In this study, on the basis of the interpretation of clinical research, the improved treatment method is further proposed, which not only lays a solid foundation theory for the research of thermotherapy, but also has important clinical significance.

【學(xué)位授予單位】：上海交通大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2007
【分類號】：R73-3;R329

【引證文獻(xiàn)】

相關(guān)碩士學(xué)位論文 前1條

1 徐迅;冠心病、糖尿病腎病及乳腺癌相關(guān)基因的初步研究[D];華東理工大學(xué);2011年

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本文編號：1824271