| Photodynamic therapy(PDT) of tumors is based on the tumor-specific accumulation of a photosensitizer in target cells, often a porphyrin derivative, which are damaged by reactive oxygenin termediates generated on irradiation with lightin which the wavelengths match the photosensitizer absorption spectrum.Hematoporphrin monomethyl ether (HMME) is a new-generation porphyrin-related photosensitizer for PDT. Experimental studies and clinical trials have shown HMME is a promising photosensitizer for PDT. However, the effect of HMME-PDT on MCF-7 is still not known, and the mechanisms of HMME-PDT induced cancer cell apoptosis are not completely defined.The research will use the new-generation photosensitizer HMME and 632.8nm He-Ne laser to study the photodynamic effects on MCF-7 with different concentration of HMME and illumination energy.CCK-8 was used to assay cell inhibition rates. The cells morphology will be observed using fluorescent microscope and electron transmission microscope. The rates of apoptosis and necrosis will be examined using flow cytometry.Mehtods:1.Cell inhibition assayCells were seeded into a 96-well culture plate at 1×10~5 cells/ml in 100μl culture medium without FBS. After growing overnight, cells were incubated with HMME freshly diluted with RPMI 1640 medium for 2h at 37°C in the dark. Then add full medium and subsequently irradiated with 632. 8nm light using a He-NeLaser. Cells were cultured for another 24h at 37°Cin the dark. Then10μl CCK-8 was added to each well and incubated for 3h at 37°C. The optical density(OD) of each well was immediately read on an ELISA reader at 450nm. The inhibition rate was given as the percent of treated cells absorbance from control cells.This assay involved PDT groups and control groups. The PDT groups included five groups of different HMME concentration which is 5μg/ml(group A), 10μg/ml (group B), 20μg/ml(group C),40μl/ml(group D), 80μg/ml (group E). And these five groups were respectively irradiated at a fluences of 1.2J/cm~2(group a), 2.4J/cm~2(group b), 4.8J/cm~2(group c), 9.6J/cm~2(group d), 19.2J/cm~2(group e) at room temperature. The control groups involved HMME groups which were treated with 5μg/ml, 10μg/ml, 20μg/ml,40μg/ml,80μg/ml only and laser groups which were irradiated at the fluence of 1. 2J/cm2, 2.4J/cm~2, 4.8J/cm~2, 9.6J/cm~2, 19.2J/cm~2 and a group treated with nothing. Each group included 4 wells.2.Histology observingCells were seeded into 35-mm dishes. After growing overnight cells were cultured with 10μg/ml HMME for 2h and then irradiated with 632. 8nm Laser at a fluence of 2.4J/cm~2. 24 hours later, cells were stained with Hoechst 33342 or collected with cell scraper and fixed by valeric aldehyde and osmic acid respectively for fluorescence or transmission microscopy.3.Flow cytometry detectingCells were seeded into 35-mm dishes. The PDT process was the same as histology observing. 24hours later, the cells of two groups were collected respectively and then assayed using flow cytometry.Results:1.The groups treated only with HMME or laser had no statistical significances compared with the control group. It indicated that HMME or laser only had no inhibiton effect on MCF-7 cells.2. HMME-PDT which involved HMME(5μg/ml, 10μg/ml, 20μg/ml, 40μg/ml and 80μg/ml) and 632. 8 nm He-Ne Laser (1.2J/cm~2, 2.4J/cm~2, 4.8J/cm~2, 9. 6J/cm2 and 19.2J/cm~2) could inhibit the proliferation of MCF-7 cells. And the inhibiton rates are positive to the PDT concentration within a certain range.3.The results decteted using transmission, fluorescence microscopy and flow cytometry showed that HMME-PDT could induce cell death through apoptosis or necrosis.4. For the MCF-7 cells raised outside body, when the concentration of HMME is 40μg/ml, only very small irradiation energy can produce a higher rate of cell proliferation, When the irradiation energy live up to 4. 8J/cm2, higher inhibiton effect can be achieved with lower HMME concentration. |
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