|
SONODYNAMICS (sonodynamic therapy, SDT) for the Japanese scholar Umemura et photodynamic therapy was first proposed on the basis of an anti-tumor methods and theories. The therapy with certain strength and frequency ultrasound irradiation tumor site, with sound-sensitive agent in the tumor tissue-specific targeting of enrichment and with its strong ultrasonic tissue penetration, and thus the tumor tissue irreversible damage to achieve anti-tumor effect. First used sonosensitizer is hematoporphyrin (haematoporphyrin derivative, HpD) and porphyrins, protoporphyrin (protoporphyrin Ⅸ, Pp Ⅸ) belongs to a porphyrin compounds, has been shown that the combined effects of ultrasound and its obvious The anti-tumor effect. The past on Pp Ⅸ-SDT antitumor studies focused on induction of apoptosis, in recent years, with the alternative way of programmed cell death - autophagy death deepening of the study, autophagy in the anti-tumor The role has become a hot research. Our previous studies in SDT-induced apoptosis was observed in parts of autophagy morphological characteristics, and found that there are a variety of anti-tumor SDT cell death mode. In this thesis, experimental parameters by studying certain conditions SDT MCF-7 human breast cancer cell damage effects, and its mechanism of action was discussed, you can supplement and improve the antitumor SDT theory for the clinical treatment of SDT to provide new ideas and countermeasures. This thesis to the Ministry of Education Research Fund for the Doctoral Program \In previous studies, based on in vitro selection of human breast cancer cell line MCF-7 cell model is, through a variety of cellular and molecular biology research techniques and methods to explore the dynamic parameters of a certain sound treatment of human breast cancer MCF- 7 induced cell killing effects and molecular mechanisms of autophagy, the current experimental results obtained are as follows: 1. protoporphyrin Ⅸ at 0-400 nmol / L concentration range of its fluorescence intensity had a good linear relationship, pp Ⅸ in MCF -7 cells metabolic process is dynamic, concentration of 0.5μM, 1μM, 2.5μM protoporphyrin Ⅸ in MCF-7 cells were incubated for 2.5 h after enrichment has reached its maximum. 2 breast cancer MCF-7 Protoporphyrin Ⅸ colocalization with mitochondria found protoporphyrin Ⅸ by red fluorescence emitted after excitation with mitochondrial markers of probes (MitoTracker Green) green fluorescence have a good overlap, Pp Ⅸ co-localized with mitochondria occurred, suggesting the role of mitochondria may be a major target for SDT. 3 simple Pp Ⅸ, simple ultrasound and ultrasound combined with Pp Ⅸ of MCF-7 cell injury study found that when protoporphyrin Ⅸ concentration of less than 2.5μM, its on MCF-7 cells almost no injury, Pp Ⅸ concentration of 2.5μM, the the cell has a slight injury, compared with the control group significantly different, protoporphyrin Ⅸ when the concentration was increased to 5μM, produced a significant inhibitory effect, Pp Ⅸ of MCF-7 cells injury showed concentration-dependent effect ; intensity of 1 W/cm2, 2 W/cm2, 3 W/cm2, 4 W/cm2 sonication MCF-7 cells, we found that cell viability according to the intensity of sound increases as the decrease of the light intensity of the sound sensitivity threshold of 3 W/cm2; intensity of 3 W/cm2 ultrasound combined with 2.5μM of Pp Ⅸ effect on MCF-7 cells, showing a significant synergistic damage effects. 4.Pp Ⅸ-SDT role in MCF-7 morphological changes after results showed that compared with the control group, simply Pp Ⅸ group cell damage is not obvious, simple ultrasound decreased ability of adherent cells, floating cells increased, SDT group cell number and morphology changed significantly, cellular debris increased, compared with the ultrasound group and drug group alone serious injury. 5 by flow cytometry analysis of mitochondrial membrane potential and found that ultrasound combined with protoporphyrin Ⅸ group of MCF-7 cell mitochondrial membrane potential greater impact. Compared with the control group, simply protoporphyrin Ⅸ little effect on mitochondrial membrane potential. Ultrasound group and ultrasound combined with Pp Ⅸ group of mitochondrial membrane potential was decreased cell numbers were increased, and the ultrasound activation Pp Ⅸ group was significantly higher than that of ultrasound group. 6 through autophagy specific dye acridine orange detection autophagy observed ultrasonic activation Pp Ⅸ group relative to the control group, there are a large number of acidic cytoplasm autophagic vacuoles appeared, suggesting that SDT can induce human breast cancer cell line MCF -7 autophagic activity increased. 7 immunoblotting (Western Blotting) detect autophagy-related proteins, and found that SDT treatment group LC3 autophagy marker molecules by the type Ⅰ to Ⅱ type conversion increased, and autophagy-related proteins Vps34, Cathepsin B, UVRAG expression, etc. were significantly increased, suggesting that ultrasound combined with Pp Ⅸ MCF-7 cells induced autophagy occurs.
|