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Experimental Study on Sites of Subcellular Photodamage by Photodynamic Therapy

Author: DaiWeiDe
Tutor: GuZuo；LiuFanGuang
School: PLA Postgraduate Medical School
Course: Internal Medicine
Keywords: Photodynamic effect Photosensitizer Hematoporphyrin monomethyl ether Intracellular distribution Fluorescence microscopy imaging Fluorescent probe
CLC: R318.51
Type: PhD thesis
Year: 2004
Downloads: 150
Quote: 1
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Abstract


Research purposes to compare the characteristics of three sets of fluorescence microscopy imaging system in the domestic novel photosensitizer HMME subcellular localization studies, the establishment of new subcellular distribution of real-time optical detection method for quantitative analysis methods and photosensitive damage sites, explore the photosensitizer subcellular distribution of impact factors. The methods and results of application of LSCM, CCD fluorescence microscopy imaging systems, the document ICCD fluorescence microscopy imaging system and fluorescence probe labeling technique, using the newly created cell - cell fluorescence intensity ratio method HMME qualitative and quantitative distribution within a single cell The study investigated different incubation times, cell type, and plasma protein binding status and other factors affect the to photosensitizer intracellular distribution. The probe DCF fluorescence image of the reactive oxygen species within the single-cell detection and analysis, combined with the cell probe applications, research HMME-PDT effect due to a single cell, the subcellular localization of ROS position. Dynamic process of subcellular localization and damage cell fluorescence in single cell morphology changes observed before and after the illumination process HMME-PDT damage sites. The results showed that: (1) the LSCM and CCD imaging system that can be taken to the the concentration 160μg/ml The HMME the fluorescence images, and ICCD imaging system simply HMME concentration of 5μg/ml, the fluorescence image characteristics were tested cytoplasm fluorescence intensity. high and uneven distribution, the the fluorescent weak nucleus area of ??the hollow phenomenon. Before two systems fluorescent probe image information display marked the mitochondria, lysosomes, endoplasmic reticulum and Golgi region average fluorescence intensity ratio (J 1 / J 2 < / sub> values) were significantly higher than the cells J 1 / J 2 values. The ICCD organelles probe fluorescence images spatially resolved not ideal. (2) With the incubation time, of the four organelles area in the A549 lung cancer cells within the J 1 / J 2 values ??are elevated and lysosomes increased range, and mouse pulmonary endothelial cells only lysosomal region showed an increasing trend in the remaining three cell area J 1 / J 2 values ??showed a downward trend; A549 cell lysosomes J 1 / J 2 values ??increased amplitude was significantly higher than that of rat lung endothelial small roar. (3) incubated for 2h 12h serum-free set of four organelles within HMME J 1 / J 2 value has increased to the mitochondria, endoplasmic reticulum and Golgi apparatus than significant, slightly increased lysosomal; the A1b group four organelles lysosomes J 1 / J 2 values ??higher than significant , the remaining three cells slightly elevated; the the four organelles the lysosome J LDL groups 1 / J 2 values ??increased more significantly Postgraduate Medical School, Dr. a summary of the dissertation Chinese forward, the remaining organelles JI / Jz elevated obviously greater than the Alb group. (4) Total cellular fluorescence light intensity decreased by 26.4% after exposure before irradiation; mitochondria, lysosomes, endoplasmic reticulum and Golgi apparatus in the light reduced 31.1%, 23.4%, 5.8% and 5%. Mitochondria were detected after the region in the case of pure light HMME presence DCF fluorescence enhancement. (5) mitochondrial probe Rh ') 123 damine fluorescence image morphological characteristics differences before and after PDT damage significantly; Can odamine 123 damage in PDT and then distributed in the nucleus area. Conclusion (1) LSCM CCD imaging system is limited to the detection sensitivity for weak fluorescent photosensitizer, applicable to its subcellular localization under high incubation concentration conditions. Both obtained experimental results are consistent: incubation 24h, HMME mitochondria, lysosomes, endoplasmic reticulum and Golgi apparatus in rat lung endothelial cells and are distributed almost into the nucleus. ICCD imaging system can be established in this subject are not limited incubation concentration conditions, to achieve the photosensitizer extremely weak fluorescence effective detection, but lower spatial resolution. (2) cell type and incubation time is an important factor photosensitizer subcellular distribution. With the extension of the incubation time, the organelles of A549 lung cancer cells absorb the photosensitizer capacity gradually enhanced especially lysosomal significantly; rat pulmonary endothelial cells, in addition to absorbing photosensitizer ability lysosomal outside the remaining three cell waning. (3) in a short time HMME mainly in the free state simple diffusion into the cell in the form of, mainly distributed to the Golgi apparatus, mitochondria, and other cell sites. With the prolongation of the incubation time, HMME and plasma protein binding and endocytosis to enter cells more easily through the cell membrane and LDL binding HMME the lysosomal absorption HMME ability. (4) light after the mitochondrial area HMME photobleaching rate; mitochondrial the regional produce a large number of singlet oxygen. (5) of HMME mediated PDT effects result in the subcellular level multilocus damage, mitochondrial and nuclear membranes may be sensitive to PDT sites: fluorescent probe labeling method is simple and reliable detection PDT damage subcellular sites.

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