Effect Of ALA-PDT On Keratinocyte Proliferation And Apoptosis In Condyloma Acuminatum

Background and ObjecktiveCondyloma acuminatum(CA) is a common clinical sexually transmitted disease(STD) of the Department of Dermatology. This disease is caused by human papilloma virus(HPV) infection, with clinical manifestations of skin mucosal verrucous proliferator at the genitals and other body parts. The disease is featured by strong infectiousness, fast proliferation, and easy to relapse after treatment, etc. Currently, there are several clinical treatment options for this disease, such as laser treatment, cryotherapy and external medication, etc. Although with these methods the wart can be removed, while the HPV subclinical and latent infection can’t be eliminated. Therefore, it is easy to relapse after the treatment. 5-aminolevulinic acid photodynamic therapy(ALA-PDT) is a new method of treating CA. The treatment principle is to take advantage of the phototoxicity produced by the combination of the photosensitizer with the abnormal tissue(where the two are highly affiliative) under the irradiation of specific light source. Thus, the pathological cells are destroyed, without damaging the surrounding normal tissues. Such therapy produces little trauma, with lower relapse rate. However, currently, the systematic research on the precise effector mechanisms regarding the ALA-PDT treatment of CA is still insufficient. Furthermore, no studies have been reported regarding the effect of ALA-PDT on keratinocyte proliferation and apoptosis in CA tissues. This research aims to study the effect of ALA-PDT therapy on the keratinocyte proliferation and apoptosis in CA by observing the changes of the keratinocyte proliferation and apoptosis in CA tissues before and after the ALA-PDT treatment. Materials and Methods 1 Materials52 cases of CA were from the Dermatology & Venereology Clinic of the First Affiliated Hospital of Zhengzhou University, including 33 males and 19 females. Age of the patients were 18-60 years old, with an average of 33.15 years old; the course of disease was 15-120 days, with an average of 51.89 days; clinical manifestations were typical, detected as positive with acetic acid white, histopathological diagnosis was definite, confirmed as HPV infection with the in situ hybridization, meeting clinical and laboratory diagnostic criteria of CA. The patients before the treatment had not received any systematic and local treatment, with the exclusion of other systematic diseases. 2 MethodsThe ALT-PDT treatment was conducted to the 52 patients with CA who met the inclusion criteria. Before the treatment, the wart tissues were taken as the control group. One week later after the treatment, the wart tissues were taken as the experimental group. The Td T-mediated d UTP nick end labeling(TUNEL) and immunohistochemical SP method were applied respectively, to detect the positive expression of the keratinocyte proliferation related genes Ki-67 and apoptosis in the CA tissues.Results determination: Ki-67-positive cells were located in the nucleus with brown particles, and border of the nucleus was clear. Semi-quantitative grading was performed according to the percentage and staining intensity of the positive cells in the keratinocytes. Percentage of positive cells to total cells: 0 point:≤10%; 1 point:11% ~ 25%; 2 points:26% ~ 50%; 3 points:51% ~ 75%; 4 points:≥76%. Staining intensity scoring: not stained: 0 point; pale yellow: 1 point; yellow: 2 points, brown yellow: 3 points; the final score was obtained by multiplying the final score of the two scorings. 9-12 represents strongly positive(+++), 6-8 represents positive(++), 1-4 represents weakly positive(+), 0 represents negative. TUNEL positive cells were located in the nucleus with the brown particles, and nucleus pyknosis appeared; 5 high-power fields(x 400) with maximum amount of apoptotic bodies and apoptosis cells in the positive slices were selected by us, so as to account the amount of apoptotic bodies and apoptosis cells in 100 cells, and work out the apoptosis index(AI) with the mean value. Similar method is adopted to calculate the proliferation index. 3 Statistical treatmentSPSS17.0 statistical software was adopted to conduct data statistical analysis. Before and after the ALT-PDT treatment, χ2 test was adopted to compare the Ki-67 positive expression rates between the two groups; rank-sum test was applied to test the differences in expression strength. Before and after the ALT-PDT treatment, t test was applied to compare the apoptotic index and proliferation index between the two groups; the significant level =0.05, and P<0.05 for the difference was statistically significant Results 1 Expression of ki-67 in CA tissues before and after ALT-PDT treatmentki-67 positive coloration is mainly located in the nucleus, with brown particles and clear nucleus border. Among the 52 cases, there were 44 cases showing positive expression of ki-67 in the CA keratinocytes, with the positive expression rate of 84.61%(44/52), expression strength mostly at ++~+++; after the treatment, there were 22 cases showing positive expression of ki-67 in the CA keratinocytes, with the positive expression rate of 42.31%(22/52), and expression strength mostly at-~++. Before and after the treatment, the difference of positive expression rate between the two groups was statistically significant(c2=20.070, P<0.001); and the difference of positive expression strength between the two groups was statistically significant(H=28.94, P<0.001). before the treatment, the proliferation index of ki-67 was 80.26土 5.07%, and after the treatment, it was 42.68 土 3.06%; before and after the treatment, the difference of proliferation index between the two groups was statistically significant( P<0.001). 2 Expression of apoptotic cells in CA tissues before and after ALT-PDT treatmentTUNEL stained positive cells were mostly distributed at the middle and upper epidermal layer of the CA tissues, located in the nucleus, with brown particles. Before the ALT-PDT treatment, as to the visible apoptotic cells in CA keratinocytes, the expression strength was mostly at +~++, and the apoptotic index(AI) was(13.94 土2.35)% on average; after the treatment, as to the apoptotic cells in CA keratinocytes the expression strength was mostly at ++~+++, and the apoptotic index(AI) was(73.88 土 7.65)% on average; so, the difference of the apoptotic index between the two groups before and after the treatment was statistically significant(P<0.001). Conclusions1.After the ALA-PDT treatment, the positive expression rate, expression strength and proliferation index of ki-67 in CA keratinocytes were decreased, suggesting that ALA-PDT can inhibit the proliferation of keratinocytes cells as well as the proliferation of CA.2.After the ALA-PDT treatment, AI in CA keratinocytes was significantly higher than that before the treatment, suggesting that ALA-PDT can promote the apoptosis of keratinocytes, and facilitate the regression of the CA.3.To inhibit the keratinocyte proliferation and promote the apoptosis of keratinocytes is one of the effector mechanism of treating CA by ALA-PDT.