Experimental Study Of Photodynamic Effect With HMME On Human Osteosarcoma Cell Line MG-63 In Vitro

Osteosarcoma mostly occurs in the young people and its malignant degree is high.In the treatment aspect,surgery is the mainly method,combining with chemotherapy and radiotherapy.However, the effect of chemotherapy is not obvious, the complications resulted from radiotherapy are quite many.It usually result in recurrence and metastasis,and the prognosis is not good.Photodynamic therapy (PDT) is an effective therapeutic method widely used for various of localized tumors.PDT includes localized or systematic injection with photosensitizer,as the photosensitizer selectively accumulates in the tumor site, suitable wave length (500-800nm) of the visible light was used to illuminate the tumor.Under the circumstance of oxygen,photosensitizer may induce photodynamic response(typeâ… response and typeâ…¡response) and creat varieties of cytotoxic materials(for instance singlet oxygen, reactive oxygen species),which induce necrosis and apoptosis of the diseased region.Photodynamic therapy with hematoporphyrin monomethyl ether(HMME) is widely applied in the basic research and clinical treatment of esophagus cancer, carcinoma of urinary bladder, breast cancer.However, research on the photodynamic therapy with hematoporphyrin monomethyl ether in osteosarcoma is still few. ObjectiveTo observe the photochemotherapeutic effect of HMME with 630nm visible light on MG-63 cell in vitro. MethodsGroups of experiments:A .experimental group(HMME with illumination);B.photosensitizer control group(HMME only);C.illumination control group(illumination only);D.blank control group(without HMME either illumination)1.The OD₄₉₂ values and surviving ratio of MG-63 cells under different concentrations of HMME and different light energy of PDT,using MTT assay After being treated with different doses(0,0.5,1,2,4,8μg/ml)of HMME for 4 hours and irradiated with different optical doses(1.5,3,6,12J/cm²)of laser,the OD values and survival rates of MG-63 cells were measured by MTT assay2.Observation of cells after HMME-PDTAfter being treated with different doses(1,2,4μg/ml)of HMME for 4 hours and irradiated with 6J/cm² optical dose of laser, cell morphological changes were observed by microscope.3.After being treated with different doses(1,2,4μg/ml)of HMME for 4 hours and irradiated with 6J/cm² optical dose of laser,mechanisms of cell death during PDT was determined by Annexin V/PI double staining technique and analyzed by flow cytometry.4. After being treated with different doses(1,2,4μg/ml)of HMME for 4 hours and irradiated with 6J/cm² optical dose of laser,the cell cycles of MG-63 were measured with flow cytometry.Results1.It has no obvious photochemotherapeutic effect of MG-63 cells while using only HMME or only illumination.There is no obvious cytotoxic effect While the concentration of HMME is below 2μg/ml and the light energy is below 6J/cm².When it reaches the parameters,it results in obvious cytotoxic effect and we believe HMME is 2μg/ml and the light energy is 6J/cm² is the effective dosage of HMME-PDT.2.The cells of Blank control group grow in good condition,the cells were spindle or polygonal.The cells of PDT group became round and the spindle-shaped structure and polygonal structure reduced,the cells'size became smaller,part of the cells became necrosis.When HMME is 4μg/ml,the cells'nucleus after PDT has shrunk firmly, the cytoblastema nearly disappear.3.After Annexin V-FITC/PI double dyeing,the results showed that after PDT,most of the cells show Annexin V(+)/PI(+),which includes non-viable apoptotic cell and necrosis cells.Few cells show Annexin V(+)/PI(-),which includes viable apoptotic cell.Rare cells show Annexin V(+)/PI(-),which includes injured cells during collection.4.After HMME-PDT,compared with the blank control group,the percentage of G0G1 period decrease slightly , the percentage of S period has no obvious change and the percentage of G2M period increase obviously in PDT group.And the level of the change in G2M has a relationship with the dosage of HMME. ConclusionsHMME has an obvious photochemotherapeutic effect on MG-63 cells.The effect relies on the concentration of HMME and the light energy. HMME is 2μg/ml and the light energy is 6J/cm² is the effective dosage of HMME-PDT.HMME-PDT induces G2M period block to get cell apoptosis.