热刺激条件下血管内皮细胞的形态变化及凋亡研究

发布时间：2018-04-30 10:51

本文选题：热疗 + 肿瘤　；参考：《上海交通大学》2007年博士论文

【摘要】： 热疗作为一种治疗肿瘤的全新疗法,得到了广大研究者广泛的关注。但是已有的研究都局限于热疗或者热疗结合其他疗法对肿瘤细胞的损伤,血管内皮层作为肿瘤生长中营养和氧气供应的主要通道一直被忽视。随着抗血管新生疗法的问世,血管内皮细胞在肿瘤治疗中受到越来越多的重视。以新生血管为靶点,抑制肿瘤生长,阻断肿瘤的营养来源和转移通道已成为近年来国内外研究的热点。本文主要从细胞和分子水平研究热刺激过程中血管内皮细胞形态改变与血管渗透性变化的关系,探讨热疗对血管内皮细胞的损伤,并探索引起其改变的机理。在临床上热疗可以增强化疗的疗效,但是对于引起此效果的机理却很少有系统性的研究。我们课题组已有的研究工作表明热疗通过增大血管的通透性,促进抗肿瘤药物在肿瘤部位释放从而增强其疗效。本课题进一步研究了热刺激引起细胞形态改变与微丝骨架变化的关系,通过体外加热血管内皮细胞发现加热后细胞形态变圆,细胞之间的间隙变大,同时细胞之间的连接消失。通过对细胞微丝骨架的固定染色,发现加热后,细胞内的微丝骨架发生了巨大的变化,有序的网状结构消失,微丝聚集体散布在细胞内。同时,我们将GFP-actin质粒转染血管内皮细胞后建立单克隆细胞系,利用该细胞系及激光共聚焦荧光显微镜对血管内皮细胞进行实时观察,研究了加热过程中血管内皮细胞细胞骨架的实时变化。结果表明,细胞微丝骨架的变化与其形态以及细胞之间连接的变化几乎是同步发生的,说明了血管内皮细胞形态以及细胞之间连接发生变化的内在原因是细胞内微丝骨架受热发生了解聚。热疗结合化疗除了对肿瘤细胞有杀伤作用外,其对血管内皮细胞的直接杀伤作用也是热疗增强疗效的重要机制。我们进一步研究了热刺激以后血管内皮细胞的损伤,结果显示血管内皮细胞在45℃加热以后,细胞的表面出现了大量凋亡小体。我们又用荧光双染色的方法定量分析了血管内皮细胞在不同温度下细胞存活率、凋亡率和坏死率随时间变化的情况,结果表明血管内皮细胞死亡的模式与温度的高低密切相关:当温度低于45℃时,细胞的死亡模式以发生凋亡为主,而当温度大于45℃时,细胞的死亡模式以坏死为主。已有的研究发现热刺激以后肿瘤环境下的血管内皮细胞比正常血管内皮细胞死亡率更高,对热的刺激更加敏感。我们通过进一步的实验证明温度处于43-45℃时,肿瘤环境下的血管内皮细胞凋亡率要大于正常血管内皮细胞。除了本研究所涉及的热刺激引起血管内皮细胞微丝骨架以及细胞发生凋亡、坏死的研究,已有的一些研究还对热刺激过程中血管内皮细胞胞内钙离子浓度以及热激蛋白(HSP)水平变化等细胞水平的现象进行了研究,但这些单因素的研究始终无法从全局和系统的角度探讨热刺激对细胞的影响。为此,我们采用了大规模基因芯片的方法检测受热刺激血管内皮细胞后基因表达水平的变化,发现在温热和高热刺激的条件下都有大量的基因表达发生上调或者下调。随后又通过正交分类以及基因语义学(Gene Ontology)等分析方法,将统计上有基因显著聚集的功能子类挑选出来,随后将这些功能子类的基因与已经报道的实验现象进行详细地对比分析,找出了其中一些生理现象可能的分子机制。其中包括细胞结构相关蛋白基因,如DNA结合蛋白,骨架蛋白等;还有一些是细胞功能相关蛋白基因,如细胞信号转导相关蛋白,还有细胞凋亡相关蛋白等。将这些现象与基因水平的变化联系起来,为进一步研究这些生理现象找到了线索。结合我们的实验结果,我们认为在热疗促进药物的热靶向输送研究中,热疗除了引起血管内皮细胞骨架解聚改变细胞形态而达到抗肿瘤药物在肿瘤部位大量渗透外,还通过细胞凋亡的方式引起肿瘤血管的破坏,因此热疗具有双重的疗效,一方面促进了药物的渗透,另一方面也造成了肿瘤血管的破坏,这两方面的作用同时增强了热疗结合化疗的抗肿瘤效果。本课题的开展具有重要的理论价值,从理论上解释了临床上热疗能够加强抗肿瘤药物在肿瘤部位靶向释放以及热疗通过直接杀伤血管内皮细胞达到杀死肿瘤组织的机制。同时热刺激以后血管内皮细胞基因表达的研究可以较系统地解释受热刺激以后血管内皮细胞发生的部分生理现象。同时本课题具有重要的实践价值,本研究在解释临床研究基础上又进一步提出改进的治疗方法,不仅为热疗的研究打下了坚实的基础理论,而且具有重要的临床指导意义。
[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.

【学位授予单位】：上海交通大学
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R73-3;R329

【引证文献】