Study On Morphology Change And Apoptosis Of Human Endothelial Cells Under Heat Shock

As a new method of cancer therapy, heat treatment has been concerned by researchers most widely. But in basic research area, most studies paid attention to the damage of tumor cells by heating or heating combined other cancer therapy method. Endothelial cells, the major channels providing nutrition and oxygen for tumor tissues, were almost ignored. As the anti-agiogenesis therapy came out, Endothelial cells were paid more attention. Disrupting endothelial cells and cutting off the nutrition source to kill tumor cells became a hot area for tumor therapy. Our study mainly concerned on relationship between the shape of endothelial cells and the permeability of blood vessels in cellular and molecular level, and the mechanism of damage to endothelial cells induced by heat was dicussed.Hyperthermia can increase the effects of chemo-therapy in clinical, but there is no systematic theory to explain it. Previous study of our group has proved that hyperthermia can increase the release of anti-tumor drugs in the tumor tissues by increasing the permeability of blood vessels. In this thesis, the relationship between cell shape and the microfilament cytoskeleton under heat shock was studied. The results showed that endothelial cells became round, junctions between cells disappeared and cells gaps enlarged when endothelial cells were heated, then the microfilaments of endothelial cells were fixed and dyed, and the observation found that microfilaments changed much more after heat shock: ordered network of microfilaments disappeared, in stead, congeries of actins spreaded in the cells. Meanwhile, endothelial cells were transfected with GFP-actin combined plasmid and stable cell lines were established. Real-time change of endothelial cells were observed during heat treatment using this cell line and the laser scanning microscopy, and the results showed that the morphology of endothelial cells changed simultaneously with the microfilament cytoskeleton. The results explained the de-polymerization of microfilament under heat shock was the intrinsic reason for the morphology and the cell gap change of endothelial cells.Besides combination with chemotherapy, heat can take effects in clinical through direct killing of endothelial cells. The damage of endothelial cell after heat shock was studied, and blebs were found on the surface of endothelial cells after 45℃heat shock. Then double dye method was used to calculate ratio of apoptosis, necrosis and survive statistically after different temperature heat shock. The results showed the temperature could strongly affect the death mode of endothelial cells. When the temperature was lower than 45℃, the main death mode was apoptosis; When the temperature was higher than 45℃, the main death mode was necrosis. Previous study showed that tumor endothelial cells were more sensitive to heat than normal endothelial cells. Our further experiment showed that the apoptosis ratio of tumor endothelial cells was higher than normal endothelial cells between the temperature of 43-45℃.Our study showed heat shock could change the cytoskeleton of endothelial cells and induce apoptosis or necrosis. And other studies proved that intra-cellular calcium and hsps changes of endothelial cells under heat shock. But these single-factor studies could not explain the cellular response to heat shock at whole and systematic level. Huge-scale gene chip was applied to probe the gene change of endothelial cells after heat shock. The result showed that many genes up-regulated or down-regulated after heat shock at both temperatures. Orthogonal analysis and gene ontology analysis were used to find out statistically significant functional gene groups. These functional significant gene groups and previous reported phenomenon were compared carefully, and possible molecular mechanisms of some phenomenon were found. Including structure related genes, such as DNA protein and cytoskeleton protein etc. The others were functional genes, such as signal transduction related protein and apoptosis related protein etc. Relation between genes and physiological phenomenon was the clue for further research. This research concerned on change of endothelial cell under heat shock at molecular level, partially explained some reported physiological phenomenon.Base on our results, we found the enhancement of the nanoparticle anti-tumor drug carrier delivery by local hyperthermia induced. Heat could not only inducing de-polymerization of microfilaments, which further inducing shape change of endothelial cells and leaking out of anti-tumor drugs in tumor areas, but also inducing the damage to tumor blood vessels. So heat treatment had double effects, the first one was inducing leaking out of drugs, the other was damaging to tumor blood vessels. These double effects increased anti-tumor effects of heat combined chemo-therapy.Our research had very important theory value. It explained the mechanism of heat inducing drug-targeted delivery at the tumor area and direct damaging to endothelial cells. Mechanism of some reported phenomenon was revealed by systematically analyzing change of gene expressions after heat shock. Meanwhile, our research had important practice value. The results provided some possible method to improve the clinical therapy. This research provided better base of further theory study, and had very important clinical value.