| Objective: The key factor of photodynamic therapy was photosensitizer. CDHS801 was a chlorophyll derivative, and was classified as the second-generation photosensitizer. In clinical practice, it has been prescribed to the patients who suffered from superficial bladder transitional cell carcinoma, and has cured a lot of patients. But its mechanism of treatment was unknown, and we know little how and why it killed tumor cells. Our experiments aimed to investigate the bio-distribution of CDHS801 in the bladder carcinoma cells and the mechanism it killed tumor during photodynamic therapy. What we got in this paper would found a firm and scientific foundation for CDHS801 based photodynamic therapy on bladder tumor. Methods: 1 Bladder transitional cell carcinoma T24 cells were incubated with 25 μg/ml CDHS801 for 6 hours, then incubated with a mitochondria specific fluorescent dye . T24 cells were investigated under confocal laser scanning microscope. Excited by laser, Both CDHS801 and mitochondria probe can fluoresce. We can know the subcellular localization of CDHS801 by means of its fluorescence. 2 After incubated with CDHS801, T24 cells were irradiated by laser of 650 nm wave-length, the influence on proliferation of T24 cells was studied by MTT methods. 3 To determine the apoptosis of T24 cell induced by CDHS801-based photodynamic therapy, inverted phase contrast microscope, flow cytometry, PI (Propidium iodide) and Hoechst-33258 fluorescent staining, TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP Nick End Labeling) and electron microscope were used. By evaluating the morphological and ultra-structural changes of the cells and nuclei, we determined if the cells apoptosized. 4 By detecting the protein expression of Bcl-2, Bax, Caspase-9, Caspase-3 and cytoplastic protein Cytochrome C, the mechanism of apoptosis induced by CDHS801-based photodynamic therapy were analyzed. Meantime, the changes of apoptosis-related gene Bcl-2, Bax mRNA were investigated by real-time quantitative PCR. 5 We injected live T24 cells into the subcutaneous space of nude mice and successfully made the animal model of bladder carcinoma. After CDHS801-based photodynamic therapy, changes of morphology and ultrastructure of the tumor tissues were investigated by HE staining, TUNEL andelectron microscope to illuminate the mechanism of photodynamic therapy. Results: 1 Incubated with 25 u g/ml CDHS801 for 6 hours, CDHS801 localized in the cytoplasm of the T24 cells, in the peri-nuclear area, little in the cell nuclear area, mainly in the mitochondria. 2 Incubated with CDHS801 for 6 hours, irradiated by 20mW/cm2 laser of 650 ran wavelength for 5 minutes, over half of the cells were killed. The relative inhibitory rate of PDT on T24 cells elevated along with the increase of the concentration of CDHS801. 3 T24 cells of the PDT group became smaller, round, detached from each other, some floated in the medium. The cytoplasm condensed. The chromatin stained by Hoechst-33258 became condensed, margined and abnormally fragmented, chromatins and nuclei of T24 cells broke. The results of flow cytometry indicated the ability of forward scatter light in the cells of the PDT group decreased and "an apoptotic peak" occurred in the sub-Gl zone. Detected by an immunohistochemistry method TUNEL, brown yellow pigments occurred in the nuclei of the cells in the PDT group. Electron microscope showed the chromatins of the PDT cells became condensed and margined. None of the observations occurred in the other three control groups. 4 Our results of Western blotting showed that cytochrome C was released from the mitochondria into the cytoplasm in the cells of the PDT group and the expression of the Bcl-2 protein decreased significantly, Bax protein changed little. The Caspase^ Caspase-9 were activated. The results of Real-time Quantitative PCR indicated the expression of the apoptotic related gene Bax mRNA was up-regulated and the Bcl-2 mRNA remained unchanged. 5 The bladder tumors of the transplanted nude mice of the PDT group showed signs of edema, the microvessels of the tumor demonstrated signs of dilation, congestinon, erythrostasis, occlusion and thrombosis. Most tumor cells were clearly pyknotic and nuclei segmented and disappeared. The results of TUNEL showed the chromatins of the PDT group broke and segmented and brown yellow pigments occurred. Electron microscope indicated that mitochondria became dilated and edematous, chromatin became condensed and apoptotic body occurred.Conclusion: 1 Incubated with bladder carcinoma T24 cells, CDHS801 localized in the cytoplasm, mainly in the mitochondria. 2 After incubated with CDHS801, irradiated by 650nm laser, most T24 cells were eliminated. 3 CDHS801 based photodynamic therapy could induce the apoptosis of the T24 cells. 4 CDHS801 based photodynamic therapy induced the apoptosis of T24 cells by means of mitochondrion quick apoptotic pathway. 5 CDHS801 based photodynamic therapy...
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