| Part Ⅰ: A Single-center retrospective study of hemoporfin photodynamic therapy for port wine stains in paedatric patientsObjective: To investigate the clinical efficacy and safety of hemoporfin photodynamic therapy for port wine stain in paediatric patients.Methods: A retrospective analysis was performed on 439 children who received hemoporfin photodynamic therapy in Children’s Hospital of Chongqing Medical University from July 2017 to January 2020.The patients were classified according to age,type,location,lesion area and treatment sessions,etc.The lesions’ blanching degree and adverse events were registered before and after treatment.Results: The effective response(regression>20%)was 95.2%,and74.3% of the children showed at least great improvement(regression≥60%).The efficacy of red and pink lesions was better than that of purple lesions(P<0.05).The therapeutic effect of face and neck was better than that of trunk and limbs(P<0.05).There was a cumulative effect with increasing treatment sessions.No obvious systemic adverse reactions or photosensitivity were found.Short-term local adverse reactions included pain(95%),swelling(90%),purpura(80%),and scab(6%).The recovery period ranged from 1 to 4 weeks,and 2% of children developed permanent scar.Conclusion: The overall efficacy of hemoporfin photodynamic therapy in children with port wine stain is good,and there was little systemic adverse reactions.So it can be recommended as the first choice over pulsed dye laser,especially for large size lesions.Part Ⅱ: Pathological changes of dermal capillaries before and after hemoporfin photodynamic therapy with different drug-light intervals in children with port wine stainsObjective: To clarify the pathological characteristics and relative proteins expressed in signal transduction pathways of flat and nodular port wine stain before and after treatment with different drug-light intervals,so as to provide theoretical basis for exploring the feasibility of treatment.Methods: A total of 11 patients with nodular port wine stain were enrolled in the study.3 in control group(untreated);4 in PDT-A group(hemoporfin constant speed 5min pump illumination)and 4 in PDT-B group(hemoporfin constant speed 5min pump illumination).2 months after treatment,Nodules and adjacent erythema tissues of the same child were obtained.Then tissues were fixed with formaldehyde overnight,dehydrated and embedded,HE staining was performed and the expression of VEGF,CD31 and SMA was detected by immunohistochemistry.The expression of p-PI3 K in lesions was detected by Western blot.Results: Compared with the control group,the dilated vessels in the superficial dermis of flat and nodules port wine stains were reduced in both groups after photodynamic therapy with different drug-light intervals.There was no obvious expression of VEGF in the erythema and nodule lesions in the treated and untreated groups,but the expression of p-PI3 K protein in the nodule lesions was increased compared with that in the flat lesions,and the expression was reduced after PDT.There was no obvious expression of VEGF in the flat and nodule tissuse before and after treatment.However,the expression of p-PI3 K protein was detected in nodular tissue,and decreased after treatment in both PDT group.Conclusion: Both PDT group can reduce the dilated vessels in the superficial dermis.In addition,the negative expression of VEGF indicated that HMME-PDT could achieve a permanent therapeutic effect.However,the expression of p-PI3 K in nodules was increased compared with that in flat spots,indicating that the activation of PI3 K pathway may play a role in the progression of skin lesions from erythema to nodule,and HMME-PDT can reduce the expression of this protein to a certain extentPart Ⅲ: The effects of PDT on apoptosis and autophagy of HUVECs with different drug-light intervals in children with port wine stainsObjective: To observe the time of hemoporfin absorbed by vascular endothelial cells and the damage degree to the cells with different drug-light intervals,and to further explore the possible molecular mechanisms.Methods: HUVECs were isolated and cultured by enzyme digestion method.The cells were divided into six groups: Control group(withou irradiation or/and hemoporfin);only irradiation group(4 J/cm~2);only hemoporfin group A(5ug/ml 30min);only hemoporfin group A(10ug/ml5min);PDT-A group(5ug/ml 20min+4 J/cm~2);PDT-B group(10ug/ml3min+4 J/cm~2).CCK-8 was used to detected the proliferation of cells in each group.Morphological changes of cells were observed under the inverted microscope.Cell apoptosis was detected by flow cytometry.Cell migration was observed by wound healing and tube form assay.Bioinformatics analysis was used to explore the possible mechanism of photodynamic therapy,and relative gene and protein expression were detected by PCR and Western.Results: The positive rate of umbilical vein endothelial cells isolated by enzyme digestion was 100% identified by specific marker CD31.The living cells decreased gradually with the increase of the concentration of hemoporfin with the same dose and incubation time.Besides,the living cells decreased with the the extension of incubation time at the same dose and concentration.Flow cytometry and CCK-8 showed that PDT-A group had the same cell survival rate as PDT-B group,and the two groups had the same inhibition of migration and tube formation.CDKN1 A m RNA,VEGF m RNA,Cleaved Casepase3 and autophagy protein LC3 expression were up-regulated and p-PI3 K protein expression was down-regulated in photodynamic group after 24h(p < 0.05).Conclusion: With the same dose of light,the living cells decreased with the increase of incubation concentration of hemoporfin or the extension of incubation time.The cells incubated at high concentration for a short time and low concentration for a long time had the same cell mortality,cell migration and tube formation.The underline mechanisms of hemoporfin photodynamic may be induce cell apoptosis and autophagic death via PI3K/AKT pathway and P53 pathway.Part Ⅳ: Comparison of efficacy and safety as well as the blood concentration of hemoporfin with different drug-light intervalsObjective: To investigate the safety and efficacy of children with port wine stain of photodynamic therapy with the different drug-light intervals,and to monitor the blood concentration of hemopofin during the treatment.Methods: The patients were divided into two groups:PDT-A(Hemoporfin was intravenous infusion a constant speed for 5min and then irradiated)and PDT-B(Hemoporfin was intravenous infusion at a constant speed for 20 min,and irradiated 10 min later).The efficacy and side effects of the two groups were observed by clinical randomized controlled study,and the blood concentration of hemoporfin was analyzed by LC-MS.Results: With the same irradiation dose,66.7% of patients in the PDT-A group and 20.7% in the PDT-B group showed great improvement(regression>50%),there was significant difference between the two groups(P<0.05).The incidence of swelling and purpura in both groups were 100%,and the scabby rate in the 5min group was 45.9%,but they were scattered and thin.Only one child(8.3%)developed a scab with a diameter greater than 2cm.After the scab fell off,the skin texture showed temporary slight atrophic,but the changes were gradually relieved with time.The scabby rate in the 20 min group was 3.4%,no thick scab appeared.There was no permanent scar occurred in both groups.The peak time of hemoporfin blood concentration in the two groups was 5min and 20 min respectively,that is,both occurred at the end of the infusion.The blood concentration in the PDT-A group was maintained at 41.06-105.92ug/m L during the irradiation,and that of the PDT-B group was maintained at 41.06-78.71ug/m L.There was no statistical difference in the minimal concentration between the two groups,but the peak concentration of PDT-A group was significantly higher than that of PDT-B group(P < 0.05).Conclusion: With the same dose of irradiation,the efficacy and short-term side effects in group A were higher than those in group B,but there were no systemic adverse reactions and permanent scars in both groups.It indicates the feasibility and safety of short-term infusion of hemoporfin in photodynamic therapy of paediatric patients with port wine stains. |
PDF Full Text Request