In Vitro Study Of Safety And Efficacy Of 5-aminolevulinic Acid Photodynamic Therapy In The Treatment Of HPV-related Infections

Background:Photodynamic therapy(PDT)has a history of more than 100 years.It has been widely used in the treatment of diseases in many fields.The mechanism of PDT is that photosensitizers selectively concentrate in target cells and activated byappropriate wavelength of light,with the participation of oxygen and generate reactive oxygen species(ROS),which induces the destruction of protein and DNA in cells and causes cell apoptosis or necrosis and rarely causes damage to surrounding tissues.5-aminolevulinic acid(ALA)has become a widely used photosensitizer for its small molecular weight and easy penetration into the coneum.Since Hermann von Tappeiner[1]use fluorescent sensitizers as diagnostic and therapeutic agents in dermatology,PDT has become a safe and effective treatment for condyloma acuminatum,acne,rosacea,sebaceous gland hyperplasia,Bowen’s disease,superficial basal cell carcinoma and other diseases[2-4].Condyloma acuminatum is one of the indications of photodynamic therapy.It is a common sexually transmitted disease caused by human papillomavirus(HPV)infection.The incidence of condyloma acuminatum is only minor to gonorrhea.Traditional treatment methods will inevitably damage the surrounding normal tissues while destroying the lesion.Unlike traditional treatment,ALA-PDT can preserve normal structure and can be reused to eradicate lesions when it’s necessary.Its effectiveness and low side effects make it increasingly widely used in the treatment of condyloma acuminatum.HPV is species-specific and human is its only host and there is no animal model of HPV and it can’t be cultured in vitro.Therefore,researches on HPV are focused on HPV infected cells.In this study,human cervical cancer cell line(Hela)was used as a cell model of HPV infection and human keratinocyte(HaCaT)as a normal control cell.The safety and efficacy of ALA-PDT in the treatment of HPV-related infection were analyzed at the cytological level.Purpose:1.To compare the effects of 5-aminolevulinic acid photodynamic therapy on proliferation and apoptosis of Hela and HaCaT cells;2.To explore the relationship between the photodynamic effect and the amount of PpIX and ROS in cells;3.To verify the safety and effectiveness of 5-aminolevulinic acid photodynamic therapy in the treatment of HPV infection-related diseases at the cytological level.Methods:1.Two kinds of cells:Hela and HaCaT;2.A photosensitizer:ALA;3.Two kinds of experimental cells were divided into four groups:(1)control group;(2)irradiation group;(3)ALA group;(4)ALA-PDT group;4.Evaluation of photodynamic efficacy:CCK8 method was used to measure the proliferation activity of cells in each group,and flow cytometry was used to detect the apoptotic ratio after treatment.5.The changes of cell morphology before and after ALA-PDT treatment were observed by inverted microscope,the production of protoporphyrin(PpIX)was observed by inverted fluorescence microscope after incubation with different concentrations of ALA,and the level of ROS was detected by ROS Assay kit.Results:1.ALA-PDT inhibited the proliferation of Hela and HaCaT in a dose-dependent manner at low concentrations(<2 mmol/1),When the concentration of ALA was more than 2 mmol/1,the photodynamic effect would not be increased by increasing the concentration of ALA;2.ALA-PDT inhibited the proliferation of Hela cells more obviously than HaCaT cells;3.After 0.5 mmol/1 ALA-PDT,the apoptotic rate of Hela cells was higher than that of HaCaT cells;4.The amount of PpIX and ROS was dose-dependent at low concentration(<2 mmol/1),When the concentration of ALA was more than 2 mmol/1,the production of PpIX and ROS would not be increased by increasing the concentration of ALA.5.The photodynamic effect is related to the amount of PpIX and ROS produced in cells.Conclusion:This study confirms that 5-aminolevulinic acid photodynamic therapy inhibits the both proliferation of Hela and HaCaT cells and is more effective in the inhibition of Hela.This phenomenon may be due to much more PpIX produced in Hela than that in HaCaT,and the more reactive oxygen species produced after irradiation,which can induce more cell necrosis and apoptosis.This study demonstrated that 5-aminolevulinic acid photodynamic therapy has a strong inhibitory effect on HPV-infected cells,and has little effect on normal cells meanwhile.