| Background and Objection:Cervical cancer is one of the commom gynecological malignancies of female, which is caused by Human Papillomavirus (HPV). One of the most common symptoms of cervical cancer is abnormal vaginal bleeding, but in some cases there may be no obvious symptoms until the cancer has progressed to an advanced stage. Treatment usually consists of surgery (including local excision) in early stages,and chemotherapy and/or radiotherapy in more advanced stages of the disease. Photodynamic therapy has been proven a promising treated modalities of cervical cancer.PDT process involves the photosensitiser localized in tumor tissue and irradiation of the tumor site by visible light of specific wavelength, to produce singlet oxygen and other reactive oxygen species, leading to photodamage in cancer cells and cancer destruction. As contrast to conventional therapy such as operation and chemotherapy, PDT has the advantages as minimally invasive, little toxicity, high selectivity, wide applicability, well repeatability. It is used as pamper treatment, maintaining features and important organ function and might cure precancerous lesion. Nowadays, PDT has been used in curing some malignant tumor, and become a new method of tumor therapy gradually. Choosing optimal parameters of PDT properly, complete cure of early cervical cancer and a good therapeutic result in advanced stage of the disease can be frequently achieved. However, in clinical application, the optimal parameters has not been established that the effects for cervical patients were instability.The tumor cell death by PDT is induced via apoptosis and/or necrosis, depending on various conditions, such as tumor cell type, laser irradiation dose, photosensitizer concentration and subcellular localization, etc. It has been reported that PDT with mitochondria-localizing photosensitizers, such as 5-aminolevulinic acid (ALA) and MAL, can induce rapid cell death via apoptosis and change the express of apoptotic related gene and protein. Expression of some genes related with apoptosis pathway, is involved and plays an important role in this photodynamic process. However, their roles in MAL-PDT for cervical cancer have still not been fully investigated.MAL is one of second-generation photosensitizers, and has been approved to be used in clinical trial by the USA FDA since 2004. MAL is an endogenous material and a precursor of heme in living cells, thus the induced protoporphyrin ? (Pp?) can be employed as a photosensitizer in PDT. The MAL-produced Pp? induces a short-lasting phototoxicity about 24 hours, much shorter compared with that induced by photo frin?In the first part of this paper, laser medicine and photodynamic therapy were introduced; In the second part, the effects of MAL-PDT on cervical cancer cells in vitro was investigated, while optimal parameters and mechanism of MAL-PDT for cervical cancer were explored; In the third part, a physical model of PDT was built in cervical cancer tissue by Monte Carlo method.Methods and materialsThe human cervical cancer Hela cells were routinely cultured and then treated with MAL for different time and followed by irradiation of light. The PDT-induced phototoxicity of the cells was determined by MTT assay. Cells at various concentrations with MAL at a fixed time followed by irradiation of light, at a fixed concentration subjected to various doses of light, MAL only and light only were also determined by MTT assay. Use the MTT assay to detect MAL-PDT effect on the human cervical cancer Hela cells:The experiment was divided into two groups: the control group and PDT group. Human cervical cancer Hela cells were incubated in vitro with MAL with different incubating concentration (0.00625?0.0125? 0.025?0.05?0.1?0.2?0.4?0.8?1.6mmol/L) and different incubating time (for 1?3?6?8?12hour), and then irradiated under 18 J/m2 saturated laser dose, meanwhile make other tests that human cervical cancer Hela cells were incubated in vitro with different MAL incubating concentration(0.00625?0.0125?0.025?0.05?0.1? 0.2?0.4?0.8?1.6mmol/L) and then irradiated under five different laser dose (3? 6?9?18?27J/cm2), and survival rate of each concentration was measured by MTT assay after 24 hours'incubation. The software of statistics was spssl3.0. The results of experiment were expressed by the way of meanąSD. Survival rate of each group, was analyzed by the way of General linear Models of Univariate.Morphological change of the human cervical cancer Hela cells after MAL-PDT was observed by optical microscopy.Physical model of PDT on cervical cancer was established, and the light distribution in cervical cancer tissues was simulated with Monte Carlo method. The function relation of photodynamic dose with light dose, photosensitizer concentration, oxygen concentration and the photobleaching rate was established.Results:MAL-PDT significantly inhibited the growth of human cervical cancer Hela cells. The cell survival rate between different MAL concentration and five different laser dose and under the same laser dose with different incubating concentration were all significant different, all P<0.01.While under the same concentration, the cell survival rate was also significant different with different laser dose except that the concentration was 0.05 mmol/L and above. The cell survival rate was the lowest as MAL concentration was 0.05 mmol/L and the laser dose was 18J/m2. While MAL concentration increases from 0.05 mmol/L to 1.6 mmol/L, the cell survival rate was not decrease as the laser dose increase from 18J/m2 to 27J/m2. The killing effect was the best as MAL concentration wais 0.05 mmol/L and the laser dose was 18 J/m2. The cell survival rate between different MAL concentration and five different incubating time and under the same incubating time with different MAL concentrations were all significant different, all P<0.01. While under the same concentration, the cell survival rate was also significant different with different incubating time except that the concentration was 0.05 mmol/L and above. The cell survival rate was the lowest as MAL concentration was 0.05 mmol/L and the incubating time was 6 hours. While MAL concentration increases from 0.05 mmol/L to 1.6 mmol/L, the killing effect was not enhance as the incubating time increase from 6 hours to 12 hours.After MAL-PDT, microscopy with staining detected typical apoptotic changes (condensed chromatin, shrunken nuclei and high blue fluorescence by staining) and necrotic cells in the PDT-treated Hela cells. In contrast, few apoptotic and necrotic cells were observed in the cells of control group.Physical model of PDT on cervical cancer was established, and the light distribution in cervical cancer tissues was simulated with Monte Carlo method. The function relation of photodynamic dose with light dose, photosensitizer concentration, oxygen concentration and the photobleaching rate was established.ConclusionMAL-PDT significantly inhibites the growth of Hela cells. Under the special light source, the laser dose, the kinds of photosensitizers, the photosensitizers incubating concentration and the incubating time, have main effects to MAL-PDT. Under the specific incubating time(6h), the cell survival rate is the lowest as MAL concentration is 0.5mmol/L and the laser dose is 18J/m2. While MAL concentration increases from 0.5mmol/L to 1.6mmol/L, the cell survival rate reach a plateau as the laser dose increase from 18J/m2 to 27J/m2. Under specific laser dose(18 J/m2.), the cell survival rate is the lowest as MAL concentration is 1.6mmol/L and the incubating time is 6 hours. While MAL concentration increases from 0.5mmol/L to 1.6mmol/L, the killing effect also reach a plateau as the incubating time increase from 6 hours to 12 hours.Physical model of PDT on cervical cancer is established, and the light distribution in cervical cancer tissues is simulated with Monte Carlo method. The function relation of photodynamic dose with light dose, photosensitizer concentration, oxygen concentration and the photobleaching rate is established.. |
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