Ala Pdt - On Hela Caski Cell Proliferation And Apoptosis Mechanism Preliminarily

Cervical cancer is one of the most common gynecology malignant tumor. Each year about250,000women died of cervical cancer and about500,000new cases happened in the world. There is a clear relationship between the onset of cervical cancer and HPV infection, especially the high-risk HPV infection. About10%of women infected with HPV will keep in persistent HPV infection status. It is lack of effective methods to remove HPV infection. At present, there is no targeted drug for HPV infection. The effects of interferon and HPV prophylactic vaccine are also limited. We urgently need a useful treatment for the people with persistent HPV infection. Photodynamic therapy(PDT) has the advantage of less trauma, strong selectivity, good repeatability, less toxicity. And it is widely applied in the treatment of a variety of malignant tumors, but still used in the initial stage in gynecology diseases, especially in cervical disease. We select5-aminolevulinic acid (ALA) as our photosensitizer. This second generation photosensitizer can selectively accumulate in cells infected with virus,overcoming the disadvantage of long detention and evading light time and poor tissue selectivity happened in the first generation photosensitizer. By using SRB method, flow cytometry, real-time PCR and Western Blot on human cervical squamous and adenocarcinoma cell lines Caski and Hela treated by5-aminolevulinic acid based photodynamic therapy(ALA-PDT), this study aims to explore its mechanism of HPV infection elimination and induction of apoptosis, providing a basis for clinical treatment.Objects:1. To investigate the growth inhibition effect and acquire the optimal parameters of ALA-PDT in cervical cancer cell lines Hela and Caski.2. To analyze the influence of ALA-PDT on the expression of HPV18/16E6, E7gene mRNA in Hela and Caski cells and the ability in clear of HPV infection.3. To detect the effects of ALA-PDT on the protein expression of related apoptosis genes to explore the possible access to apoptosis. Methods:1. Detecting the effects of ALA-PDT on the proliferation of Hela and Caski cell lines by SRB method and selecting the optial parameters and compare the sensitivity of ALA-PDT.2. Indentifying the growth inhibition mechanism of Hela and Caski cell lines by observing cell apoptosis by flow cytometry.3. Detecting the effects of ALA-PDT on the expression of Casepase-3、 P53、HPV E6/E7mRNA in Heal and Caski cell lines by Real-time PCR.4. Indentifying the apoptosis mechanism of ALA-PDT by observing the expression of P53and cleaved Caspase-3protein detected by Western Blot.Results:1. The results of SRB method indicated that ALA-PDT significantly inhibited the growth of Hela and Caski cells. The cell survival rate between different ALA concentrations and different laser doses are all significantly different, and the same as treated by the same dose with different incubating concentration. While under the same concentration, the cell survival rate is also significantly different with different laser dose except that the concentration0.05mM. While ALA concentration increases from0.05-0.15mM for Hela cell line and from0.05-0.2mM for Caski cell line under the the laser dose of10J/cm2, the cell survival rate increased greatly. Hela cell line is more sensitive than Caski cell line to ALA-PDT.2. The data of FCM analysis demostrated that the apoptosis rate was significantly different between the control group and the ALA-PDT group. When concentration dose was low, the early apoptosis lead, while concentration increased up to1mM, late apoptosis took the most part and total apoptosis rate significantly increased.3. The results of Real-time PCR showed that, the expression of HPV E6/E7genes were all significant down-regulation in ALA-PDT groups in Hela and Caski cell lines, while Caspase-3、P53were all significant up-regulation versus control groups(all p<0.05).4. The results of Western blot showed that the expression of P53and cleaved Caspase-3protein were significant up-regulation in Hela and Caski cell lines versus control groups (all p<0.05).Conclusions:1. ALA-PDT significantly inhibited the growth of Hela and Caski cells. There was a relationship between dosage and effect at a certain range. Different cell lines had different sensitivity to ALA-PDT.2. ALA-PDT caused cell apoptosis. With the increase of ALA concentrations, apoptosis rate rose.3. ALA-PDT could get rid of HPV infection effectively.4. P53and Caspase-3may be the targeted genes on the apoptosis pathway of the influence of ALD-PDT on cervical cancer cells.