ABSTRACT
Objective To optimize experimental conditions for transfecting primary human melanocytes with Wnt5A-specific siRNA,and to establish the Wnt5A gene silencing model.Methods Primary human melanocytes (PHM)were cultured in vitro.Positive control GAPDH-siRNAs at different concentrations of 0,20.0,33.3,40.0 and 60.0 nmol/L were transfected into PHM by using liposomes.Realtime fluorescence-based quantitative PCR (qPCR) was performed to select the optimal concentration of siRNA with the highest transfection efficiency.Three Wnt5A-siRNAs including Wnt5A-siRNA-793,Wnt5A-siRNA-943 and Wnt5A-siRNA-1743 were constructed and transfected into PHM separately,and qPCR was conducted to select the most specific Wnt5A-siRNA.Then,the most specific Wnt5A-siRNA was transfected into PHM,and the total mRNA and total protein were extracted after 24-,48-and 72-hour treatment.qPCR and Western blot analysis were performed to determine the optimal time with the highest transfection efficiency.Results There were significant differences in the mRNA expression of GAPDH among cells transfected with GAPDH-siRNAs at different concentrations of 0,20.0,33.3,40.0 and 60.0 nmol/L (1.009 ± 0.161,0.086 ± 0.010,0.140 ± 0.016,0.285 ± 0.095,0.012 ± 0.007 respectively;F =69.469,P ＜ 0.05).Additionally,the 60-nmol/L GAPDH-siRNA group showed the lowest GAPDH mRNA expression (all P ＜ 0.05),as well as the best transfection efficiency.The mRNA expression of Wnt5A differed in the Wnt5A-siRNA-793 group,Wnt5A-siRNA-943 group,Wnt5A-siRNA-1743 group and the blank control group (0.331 ± 0.010,2.229 ± 0.029,0.078 ± 0.006 and 1.000 ± 0.024 respectively;F =7 006.094,P ＜ 0.05),and the Wnt5A-siRNA-1743 group showed the lowest Wnt5A mRNA expression (all P ＜ 0.05),as well as the best transfection efficiency.The mRNA expression of Wnt5A differed among the Wnt5A-siRNA-1743 group after 24-,48-and 72-hour treatment and the blank control group (0.396 ± 0.002,0.026 ± 0.008,0.131 ± 0.079,1.025 ± 0.276 respectively;F =29.215,P ＜ 0.05),so did the protein expression of Wnt5A (112.798 ± 0.218,77.765 ± 0.415,30.540 ± 0.219,130.025 ± 0.158 respectively;F =79 122.889,P ＜ 0.05).Moreover,the Wnt5A mRNA expression was significantly lower in the Wnt5A-siRNA-1743 group at 48 hours compared with that at 24 and 72 hours and the blank control group (all P ＜ 0.05),while the Wnt5A protein expression was significantly lower in the Wnt5A-siRNA-1743 group at 72 hours compared with that at 24 and 48 hours and the blank control group (all P ＜ 0.05).Conclusion The siRNA-mediated Wnt5A gene silencing model of human melanocytes is established successfully.
ABSTRACT
Objective To optimize experimental conditions for transfecting primary human melanocytes with Wnt5A-specific siRNA,and to establish the Wnt5A gene silencing model.Methods Primary human melanocytes (PHM)were cultured in vitro.Positive control GAPDH-siRNAs at different concentrations of 0,20.0,33.3,40.0 and 60.0 nmol/L were transfected into PHM by using liposomes.Realtime fluorescence-based quantitative PCR (qPCR) was performed to select the optimal concentration of siRNA with the highest transfection efficiency.Three Wnt5A-siRNAs including Wnt5A-siRNA-793,Wnt5A-siRNA-943 and Wnt5A-siRNA-1743 were constructed and transfected into PHM separately,and qPCR was conducted to select the most specific Wnt5A-siRNA.Then,the most specific Wnt5A-siRNA was transfected into PHM,and the total mRNA and total protein were extracted after 24-,48-and 72-hour treatment.qPCR and Western blot analysis were performed to determine the optimal time with the highest transfection efficiency.Results There were significant differences in the mRNA expression of GAPDH among cells transfected with GAPDH-siRNAs at different concentrations of 0,20.0,33.3,40.0 and 60.0 nmol/L (1.009 ± 0.161,0.086 ± 0.010,0.140 ± 0.016,0.285 ± 0.095,0.012 ± 0.007 respectively;F =69.469,P ＜ 0.05).Additionally,the 60-nmol/L GAPDH-siRNA group showed the lowest GAPDH mRNA expression (all P ＜ 0.05),as well as the best transfection efficiency.The mRNA expression of Wnt5A differed in the Wnt5A-siRNA-793 group,Wnt5A-siRNA-943 group,Wnt5A-siRNA-1743 group and the blank control group (0.331 ± 0.010,2.229 ± 0.029,0.078 ± 0.006 and 1.000 ± 0.024 respectively;F =7 006.094,P ＜ 0.05),and the Wnt5A-siRNA-1743 group showed the lowest Wnt5A mRNA expression (all P ＜ 0.05),as well as the best transfection efficiency.The mRNA expression of Wnt5A differed among the Wnt5A-siRNA-1743 group after 24-,48-and 72-hour treatment and the blank control group (0.396 ± 0.002,0.026 ± 0.008,0.131 ± 0.079,1.025 ± 0.276 respectively;F =29.215,P ＜ 0.05),so did the protein expression of Wnt5A (112.798 ± 0.218,77.765 ± 0.415,30.540 ± 0.219,130.025 ± 0.158 respectively;F =79 122.889,P ＜ 0.05).Moreover,the Wnt5A mRNA expression was significantly lower in the Wnt5A-siRNA-1743 group at 48 hours compared with that at 24 and 72 hours and the blank control group (all P ＜ 0.05),while the Wnt5A protein expression was significantly lower in the Wnt5A-siRNA-1743 group at 72 hours compared with that at 24 and 48 hours and the blank control group (all P ＜ 0.05).Conclusion The siRNA-mediated Wnt5A gene silencing model of human melanocytes is established successfully.
ABSTRACT
Objective To compare the efficacy and safety of two intense pulsed light (IPL) devices for the treatment of facial photoaging.Methods A randomized split-face clinical trial was conducted,and 30 female subjects with facial photoaging were enrolled and randomized to receive treatment with Lumenis One on one half of the face and BBL on the other facial side,once every 3-5 weeks for 5 sessions.Each subject was followed up before the first treatment (the first interview),4 weeks after the third treatment (the second interview),4 weeks after the fifth treatment (the third interview) and 8 weeks after the fifth treatment (the fourth interview).During each follow-up period,global scores for photoaging (GSP) were used to evaluate the photoaging degree on the whole face,a 4-level grading method was applied to evaluate the improvement degree of 5 photoaging signs on each facial side,including wrinkles,skin texture,pigmented spots,telangiectasia and skin tightening,and the visual analogue scale (VAS) to assess pain induced by treatment.After the last treatment,self-assessment on the degree of satisfaction with therapeutic effects was conducted in subjects.Comparisons in the GSP and improvement scores between the two facial sides were conducted by repeated measures analysis of variance (ANOVA).Results A total of 26 subjects completed all the treatments and follow-up.Evaluation of the whole face showed that the GSP significantly decreased from 3.19 ± 0.75 before the first treatment to 2.15 ± 0.83 at 4 weeks after the third treatment (P ＜ 0.01).At 4 and 8 weeks after the fifth treatment,the GSP decreased to 1.85 ± 0.88 and 1.85 ± 0.97 respectively,and no significant difference was observed between the two GSPs (P ＞ 0.01).Evaluation of each facial side showed that improvement scores of skin texture,pigmented spots,telangiectasia and skin tightening on the two facial sides all increased at first and then decreased over the treatment time (Ftime =18.75,10.25,12.83,15.73,respectively,all P ＜ 0.05),and the improvement scores significantly increased at 4 weeks after the fifth treatment compared with those at 4 weeks after the third treatment (all P ＜ 0.017).There were no significant differences in the improvement scores of skin texture,telangiectasia and skin tightening between the third and the fourth interview,but the improvement score of pigmented spots decreased slightly at 8 weeks after the fifth treatment compared with that at 4 weeks after the fifth treatment (P ＜ 0.017).During the whole treatment period,no evident improvement was observed in wrinkles (Ftime =3.17,P ＞ 0.05),and improvement scores of 5 photoaging signs did not differ between the Lumenis One-treated side and BBL-treated side (all P ＞ 0.05).In addition,the VAS pain score was significantly lower in the BBL-treated side than that in the Lumenis One-treated side (4.62 ± 1.54 vs.5.80 ± 1.74,t =2.87,P ＜ 0.05).Most subjects were satisfied with the therapeutic effects (88.46％,23/26).Conclusion Both Lumenis One and BBL can be applied to treat facial photoaging safely and effectively,and improve signs of photoaging such as pigmented spots and skin texture,but the degree of pain on the BBL-treated side is milder than that on the Lumenis One-treated side.
ABSTRACT
Objective To evaluate the effect of Wnt5A gene overexpression on cytoskeletal proteins of melanocytes after the plasmid containing the Wnt5A gene is transfected into primary melanocytes.Methods In vitro cultured primary human melanocytes were divided into three groups:blank control group receiving no treatment,negative control group transfected with endotoxin-free pcDNA3.1 (+)empty vector by Lipo3000 in Opti-MEM medium,Wnt5A plasnid group transfected with endotoxin-free pcDNA3.1 (+) vector containing the Wnt5A gene by Lipo3000 in Opti-MEM medium.After the transfection,quantitative PCR (qPCR) was performed to measure the mRNA expression of Wnt5A,ras-related C3 botulinum toxin substrate 1 (Rac1),filamentous actin (F-actin) and β-tubulin,Western blot analysis to determine the protein expression of Wnt5A,receptor tyrosine kinase like orphan receptor 2 (ROR2),Rac1,F-actin and β-tubulin,and an immunofluorescence assay (IFA) to observe the expression of cytoskeletal proteins.Results qPCR showed significant differences in the mRNA expression of the Wnt5A gene and its downstream genes Rac1 and F-actin among the Wnt5A plasmid group,negative control group and blank control group (F =1374.179,112.576,66.458,respectively,all P ＜ 0.01),but there was no significant difference in the mRNA expression of β-tubulin among the three groups (P ＞ 0.05).Additionally,the Wnt5A plasmid group showed significantly higher mRNA expression of Wnt5A,Rac1 and F-actin compared with the blank control group and negative control group (all P ＜ 0.05).As Western blot analysis revealed,compared with the blank control group and negative control group,the Wnt5A plasmid group showed significantly higher Wnt5A protein expression (both P ＜ 0.05),but significantly lower protein expression of Rac 1,ROR2 and F-actin (all P ＜ 0.05).However,no significant difference in β-tubulin protein expression was observed among the three groups (P ＞ 0.05).IFA showed no obvious difference in the fluorescence intensity of β-tubulin or F-actin between the Wnt5A group and the two control groups,but melanocytes showed larger size and increased number of dendrites,and the cytoskeleton changed dramatically with varying fluorescence intensity of F-actin,fuzzy texture,fractured or locally clustered tonofilaments in the Wnt5A group.Conclusion The overexpression of the Wnt5A gene in melanocytes can regulate the mRNA and protein expression of cytoskeletal proteins,nake melanocytes larger and more dendritic,and cause changes in the cytoskeleton,which may facilitate the transportation of melanosomes,and participate in the occurrence of hyperpigmented diseases.