Abstract: Su Qianya, Lin Tong, Zhou Qiyan, Peng Lin Laser Department, Institute of Dermatology, Chinese Academy of Medical Sciences and Peking Union Medical College, Nanjing 210042, China Corresponding author: Lin Tong, Email: ddlin@hotmail.com 【Abstract】 Objective To evaluate the effect of Wnt5A gene over 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 plasmid group transfected with endotoxin-free pcDNA3.1（+） vector containing the Wnt5A gene by Lipo3000 in Opti-MEM medium. After the trans-fection, quantitative PCR （qPCR） was performed to measure the mRNA of Wnt5A, ras-related C3 botulinum toxin substrate 1 （Rac1）, filamentous actin （F-actin） and β-tubulin, Western blot analysis to determine the protein of Wnt5A, receptor tyrosine kinase like orphan receptor 2 （ROR2）, Rac1, F-actin and β-tubulin, and an immunofluorescence assay （IFA） to observe the of cytoskeletal proteins. Results qPCR showed significant differences in the mRNA 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 = 1 374.179, 112.576, 66.458, respectively, all P < 0.01）, but there was no significant difference in the mRNA of β-tubulin among the three groups （P > 0.05）. Additionally, the Wnt5A plasmid group showed significantly higher mRNA 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 （both P < 0.05）, but significantly lower protein of Rac1, ROR2 and F-actin （all P < 0.05）. However, no significant difference in β-tubulin protein 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 over of the Wnt5A gene in melanocytes can regulate the mRNA and protein of cytoskeletal proteins, make 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.