A Study Of The Influence Of Wnt5A Gene Silencing And Overexpression On The Function Of Melanocytes

Section 1 A Study of the influence of Wnt5A gene silencing on the function of melanocytesObjective:To study the influence of Wnt5A gene silencing on the cytoskeleton and tyrosinase of melanocytes, by the means of Wnt5A-specific siRNA transfecting primary human melanocytes in vitro. To preliminarily explore the influence of non-classic Wnt pathway on cytoskeleton and melanogenesis.Method:Primary human melanocytes were cultured in vitro, and transfected with Wnt5A-specific siRNA by lipofectamine in order to determine optimal experimental protocol (including dosage, siRNA and timing). Then the transfection efficiency of Wnt5A was tested by the means of qPCR and Western-Blot, as well as the expression of mRNA and protein of ROR2、 Racl、F-Actin、β-Tubulin and TYR. In the mean time, the cytoskeleton of melanocytes was observed through immunof luorescence. Independent sample T test and one-way ANOVA were used for statistical analysis of the difference among groups.Result:After transfection, mRNA expression of Wnt5A was lower statistically than blank group and negative control group, and the transfection efficiency was 91.0%; mRNA expression of Racl was higher than negative control group. There was no statistical difference of mRNA expression of F-Actin、β-Tubulin and TYR among groups. Protein expression of Wnt5A was lower statistically than blank group and negative control group, and the transfection efficiency was 86.7%; Protein expression of TYR and β-Tubulin was 2.6 times and twice as much as blank group and negative control group respectively; Protein expression of ROR2、F-Actin was slightly lower than blank group and negative control group, while Racl was almost equal among groups. No obvious change was observed in cytoskeleton through immunofluorescence.Conclusion:The modeling of Wnt5A-specific siRNA transfecting primary human melanocytes in vitro was established successfully. After transfection, protein expression of TYR and β-Tubulin were elevated, and no obvious change was observed in cytoskeleton.Section2 A Study of the influence of Wnt5A gene overexpression on the function of melanocytesObjective:To study the influence of Wnt5A gene overexpression on the cytoskeleton and tyrosinase of melanocytes by the means of Wnt5A plasmid transfecting primary human melanocytes in vitro. To preliminarily explore the influence of non-classic Wnt pathway on cytoskeleton and melanogenesis.Method:Primary human melanocytes were cultured in vitro, and transfected primary human melanocytes with Wnt5A plasmid by lipofectamine in order to determine optimal experimental protocol by the means of qPCR and Western-Blot. Then the transfection efficiency of Wnt5A was tested by the means of qPCR and Western-Blot, as well as the expression of mRNA and protein of R0R2、 Racl、F-Actin、 β-Tubulin and TYR. The cytoskeleton of melanocytes was observed through immunofluorescence. Independent sample T test and one-way ANOVA were used for statistical analysis of the difference among groups.Result:After transfection, mRNA expression of Wnt5A gene was 800 times higher than blank group and negative control group, and the difference was statistically significant; mRNA expression of Racl、 F-Actin and TYR gene was higher than blank and negative control group, and the difference was statistically significant; There was no statistical difference of mRNA expression of β-Tubulin among groups. Protein expression of Wnt5A was 4.2～5.4 times as much as blank group and negative control group, so it could be inferred as a successful transfection; Protein expression of TYR was 3 times as much as blank group and negative control group; Protein expression of Racl、 F-Actin was 33.3% as much as blank group and negative control group; Protein expression of β-Tubulin was 1.4 times as much as blank group and negative control group. Melanocytes were oberserved to become larger in size and have more dendrites through immunofluorescence, and the cytoskeleton had significant changes, which included that the fluorescence intensity was uneven, the texture of F-Actin was unclear, stress fibers fell apart and clustered in local.Conclusion:The modeling of Wnt5A plasmid transfecting primary human melanocytes in vitro was established successfully. After melanocytes were transfected with Wnt5A plasmid, mRNA and protein expression of TYR was significantly elevated, which could promote melanogenesis. mRNA expression of Racl and F-Actin was elevated, protein expression was reduced, protein expression of β-Tubulin was elevated, and melanocytes were oberserved to become larger in size and have more dendrites and changed cytoskeleton, which altogether could help melanocytes transfer melanin and might play a role in hyperpigmentation diseases.