贝美前列素激活Wnt/β-环连蛋白信号通路对小鼠重构毛囊毛发生长的影响

Bimatoprost promotes hair growth of reconstructed hair follicles in mice through activation of the Wnt/β-catenin signaling pathway

目的探讨贝美前列素(BimP)对小鼠背部重构毛囊毛发生长的影响。方法自1日龄的新生C57BL/6J鼠皮肤分离培养表皮细胞和真皮细胞,用硅胶小室法在Balb/c-nu小鼠背部重构毛囊,术后将18只Balb/c-nu小鼠随机分为BimP组、米诺地尔组和对照组,每组各6只;2周后在小鼠背部的细胞移植区分别外涂0.03%BimP 100μl、2%的米诺地尔溶液100μl和生理盐水100μl,每天涂药1次,连续2周;观察局部毛发生长情况;取小鼠背部移植区皮肤的新生毛发测量其长度;组织学观察新生毛囊的数目与生长周期;用实时荧光定量PCR和Western印迹法分别检测Wnt家族分泌蛋白3a(Wnt3a)、淋巴细胞增强因子(LEF1)、β-环连蛋白和Frizzled跨膜受体蛋白7(Frizzled7)mRNA和蛋白表达水平。免疫荧光检测毛囊细胞β-环连蛋白的分布与表达。结果BimP组毛干长度高于对照组[(0.57±0.07)比(0.36±0.05)cm,P<0.01],BimP组与米诺地尔组差异无统计学意义;BimP组毛囊细胞Wnt3a(2.73±0.17比1.00±0.14,P<0.01)、LEF1(1.71±0.12比1.00±0.19,P<0.01)、β-环连蛋白(2.37±0.21比1.00±0.11,P<0.01)和Frizzled7(2.62±0.15比1.00±0.18,P<0.01)mRNA表达水平均高于对照组;Western印迹结果相同,BimP组毛囊细胞Wnt3a(1.44±0.21比1.00±0.13,P<0.05)、LEF1(1.36±0.15比1.00±0.09,P<0.05)、β-环连蛋白(1.60±0.13比1.00±0.16,P<0.01)和Frizzled7(1.52±0.15比1.00±0.21,P<0.05)蛋白表达水平均高于对照组。免疫荧光染色发现β-环连蛋白在BimP组和米诺地尔组新生毛囊的毛球细胞和皮脂腺细胞强表达,而对照组几乎未见荧光染色存在。结论BimP可通过激活经典Wnt/β-环连蛋白信号通路促进小鼠重构毛囊的毛发生长。

Objective To investigate effect of Bimatoprost(BimP)on growth of reconstructed hair follicles in recipient nude mice.Methods Primary epidermal and dermal cells were isolated from newborn C57BL/6J mice(1-day-old)skins,and the reconstructed hair follicles was implanted in the dorsal skin of Balb/c-nu nude mice using a silicon chamber protocol,then,the 18 nude mice were randomly divided into control group,BimP group and minoxidil group,with 6 mice in each group.After 2 weeks,topical treatment was applied to the grafted area of the nude mice by 2%minoxidil 100μl,0.03%BimP 100μl and saline 100μl,respectively,once daily for 2 weeks.At day 14 after treatment,the mice were euthanized to measure the length of dorsal hair,and the number and hair cycle of the reconstructed follicles was observed histologically.The total mRNA and proteins expression of Wnt3a,LEF1,β-catenin and Frizzled7 were determined by qPCR and Western Blotting.The distribution and expression ofβ-catenin in the reconstructed follicles was detected by immunofluorescence staining.Results As compared to the control group,the BimP group had thicker and longer hair[(0.57±0.07)vs(0.36±0.05)cm,P<0.01],no significant difference was seen between the BimP and minoxidil group.The mRNA expression levels of Wnt3a(2.73±0.17 vs 1.00±0.14,P<0.01)、LEF1(1.71±0.12 vs 1.00±0.19,P<0.01)、β-catenin(2.37±0.21vs 1.00±0.11,P<0.01)and Frizzled7(2.62±0.15vs 1.00±0.18,P<0.01)were significantly increased in BimP group compared with the control group.Western Blotting showed the same results,the protein expression levels of Wnt3a(1.44±0.21vs 1.00±0.13,P<0.05)、LEF1(1.36±0.15 vs 1.00±0.09,P<0.05)、β-catenin(1.60±0.13 vs 1.00±0.16,P<0.01)and Frizzled7(1.52±0.15 vs 1.00±0.21,P<0.05)in BimP group were higher than those in control group,and the difference was statistically significant.Immunofluorescence staining showed thatβ-catenin was strongly expressed in hair bulb cells and sebaceous gland cells of reconstructed hair follicles in BimP group and minoxidil group,whereas barely seen in the control group.Conclusion BimP directly promotes growth of reconstructed hair follicles in mice by activating canonical Wnt/β-catenin signaling pathway.