Study Of Apoptotic Mechanism Induced By5-Aminolevulinic Acid-Photodynamic Therapy In Keratinocytes Of HPV16E7Transfection

Objective1. To establish keratinocytes cell line that can stably express of HPV16E7protein. The cell line is observed as HPV infection models to study of apoptotic mechanism induced by5-aminolevulinic acid-photodynamic therapy.2. To explore the optimal ALA incubation time for hpv infectious diseases and the best experimental data of ALA PDD diagnosis for HPV infectious diseases.3. To investigate the mechanism of apoptosis pathway induced by ALA-PDT in keratinocyteof HPV infection.It would be significant for further promote the ALA-PDT used to treat carriers subclinical and latent HPV infection Patients.Method1. HPV16E7gene was amplified from CaSki cells by RT PCR. The pcDNA3.1(+) plasmid was constructed with EcoRⅠ and NotⅠ and transfected into HaCaT cells with lipofectamine2000, the stable transfectants screened by G418. Growth curve of the HaCaT and transfected cells was determined by cck8assay and cell morphological change was observed by transmission electron microscope.2. The HaCaT/E7cells were incubated with optimal concentration1mM ALA (preliminary results). The PpIX fluorescence intensity of cells within continuous eight hours was detected by Biotek microplate reader. To explore the optimal ALA incubation time for HPV infectious diseases and the best experimental data of ALA PDD diagnosis for HPV infectious diseases. 3. Apoptotic cell was studied by Annexin V-FITC/PI through flow cytometry assay.We observed the proportion ofapoptosis and necrosis on the HaCaT/E7at different time points (8consecutive hours)after the same light dose (8J/cm2) ALA-PDT and the same time Point (3hours) after different light doses (OJ/cm,4J/cm,8J/cm,10J/cm2,12J/cm2,16J/cm2and20J/cm2) ALA-PDT.4. Flow cytometry analysis was used to detect variations in Mitochondrial Memb-rane Potential(MMP) by using the lipophilic cation5,5’,6,6’-tetrachloro-1,1’,3,3’-tetraethy-benzimidazol-carbocyanine iodide(JC-l).5. Western blot technique was employed to determine the release of cytochrome c and the expression of caspase-9/3before and after ALA-PDT.6. Semi-quantitative RT-PCR and Real time PCR were used to evaluate the mRNA expression of caspase-9/3in HaCaT/E7cell lines.Results1. HaCaT/HPV16E7cell line stably expressing HPV16E7protein was successful-y established.western blotting detected the expression of HPV16E7protein w-hile reverse transcription polymerase chain reaction (RT-PCR) amplification pr-oducts detected the expression of specific297bp HPV16E7mRNA band by agarose gel electrophoresis. There is no significant morphological changes afte-r transfection.HaCaT/E7cells showed significant growth advantage.2. The study of protoporphyrin IX (PpIX) fluorescence kinetics on cells treated by ALA.HaCaT, HaCaT/E7and HaCaT/pcDNA3.1three groups of cells, PpIX peak time was5hours. There was s statistically significantly increased compared with the control group at3hours (P<0.05). HaCaT/E7group PpIX fluorescence intensity is generally higher than in control HaCaT group, HaCaT/pcDNA3.1group showed the highest fluorescence intensity.The PpIX fluorescence intensity of three groups cells showed a statistically significant Differences at1hour(P<0.05).3. Flow cytometry showed the percentage of apoptosis and necrosis increased with light dose. Increase of apoptotic cells were detected in a time-depengdent manner.4. The fraction of HaCaT/E7cells with the disrupted membrane potential was sig-nificantly higher after ALA combined with irradiation with time-dependence.L-SCM showed morphological changes of HaCaT/E7cell after ALA-PDT with t-he time.ALA-PDT may alter mitochondrial function of the HaCaT/E7cell.This changes was earlier than the morphological alteration and Annexin V.5. Semi-quantitative comparison of the results from Western blot,we found the release of cytochrome c and the expression of caspase-9and caspase-3and their activatied fragments were significantly increased after ALA-PDT was applied and the peak time was3hours after the treatment.The optimal light dose is8J/cm2.6. Real time RT-PCRhave shown that ALA-PDT do not influence Caspase-3, Caspase-9gene expression in HaCaT/E7cells (P<0.05).Conclusions1. HaCaT/HPV16E7cell line stably expressing HPV16E7protein was successfully established. Given that the lack of animal models supporting HPV infection, the cell line can provide an alternative platform for the investigation of HPV infection and pathogenesis.2. The optimal ALA incubation time for HPV infectious diseases is3-5hour and the time for ALA PDD of HPV infectious diseases is1hour.3. ALA-PDT cause HPV infected cells death by two ways of apoptosis and necrosis.Small light doses mainly induce apoptosis while large light dose induce necrosis with time-dependence.4. ALA-PDT may alter mitochondrial function of the HaCaT/E7cell,resulting in loss of MMP.These changes were earlier than the morphological alteration and Annexin V.5. ALA-PDT can upregulate Caspase-9, Caspase-3protein expression in HaCaT/HPV16E7cells This demonstrates that ALA-PDT induced apoptosis of HPV inf ected kcratinocytes mainly through the mitochondrial pathway. 6. ALA-PDT on HaCaT/HPVI6E7cells Caspase-3, Caspase-9gene expression level was not influenced, ALA-PDT do not influence Caspase-3, Caspase-9gene ex-pression in HaCaT/HPV16E7cells indicating the mitochondrial apoptosis pathwa-y mediated only at the protein level but not gene level.