Chinese Journal of Tissue Engineering Research ›› 2022, Vol. 26 ›› Issue (25): 3993-3998.doi: 10.12307/2022.404
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Wang Ao, Chu Hongshang, Wu Hongguang, Li Ke, Liu Huijuan
Received:
2021-04-07
Accepted:
2021-05-14
Online:
2022-09-08
Published:
2022-01-26
Contact:
Liu Huijuan, MD, Associate researcher, Master's supervisor, Bio-X Institutes, Shanghai Jiao Tong University, Shanghai 200240, China
About author:
Wang Ao, Master, Bio-X Institutes, Shanghai Jiao Tong University, Shanghai 200240, China
CLC Number:
Wang Ao, Chu Hongshang, Wu Hongguang, Li Ke, Liu Huijuan. Expression of leptin receptor in mouse skin and the role of leptin receptor-positive cells in skin development and wound healing[J]. Chinese Journal of Tissue Engineering Research, 2022, 26(25): 3993-3998.
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2.1 瘦素受体在小鼠皮肤中的谱系示踪 首先将LepR-Cre小鼠与Rosa26a-tdTomato小鼠杂交,构建LepR-Cre; tdTomato小鼠[25-26]。取2月龄LepR-Cre; tdTomato小鼠背部皮肤,进行冰冻切片观察Tomato阳性细胞在皮肤中的分布情况,结果显示Tomato阳性细胞存在于小鼠皮肤的真皮乳头、真皮层和真皮白色脂肪层中,见图1A。由于真皮乳头、真皮层和真皮白色脂肪层都属于皮肤间质,由此表明瘦素受体主要标记小鼠皮肤中的间质。Vimentin是一个常用的间质标记物,Perilipin是白色脂肪中脂肪细胞的常用标记物。Vimentin以及Perilipin免疫荧光染色结果显示Tomato阳性细胞可以与少量Vimentin阳性细胞共标,且在Tomato阳性细胞中Vimentin阳性细胞占比为(27.37±5.39)%,见图1B。Tomato阳性细胞与部分Perilipin阳性细胞共标,且共标比例为(25.26±4.54)%,见图1C。由此推测瘦素受体所标记的细胞类群很有可能是皮肤间充质干细胞中的一个谱系。"
2.2 LepR-Cre; tdTomato小鼠皮肤细胞表面标记物流式分析 谱系示踪实验和免疫荧光染色显示,瘦素受体可能标记皮肤间充质干细胞中的一个亚群。在研究中鉴别一类细胞是否属于间充质干细胞,常常采用流式细胞表面标记物分析。该研究将2月龄LepR-Cre; tdTomato小鼠背部皮肤消化得到分散的细胞,使用经典的间充质干细胞表面标记物和其他间质标记物进行流式分析,结果显示Tomato阳性细胞CD11b、CD45、CD31呈阴性表达(0.67%, 0.97%, 2.60%)。Tomato阳性细胞表达典型的间充质干细胞标记物CD29、CD73、Sca-1(23.7%,47.1%,50.3%),且间质标记物PDGFRα、PDGFRβ、CD34、CD51呈阳性表达(24.9%,33.3%,61.3%,86.6%),见图2。之前的谱系示踪实验发现瘦素受体标记皮肤真皮白色脂肪层,而以往研究发现脂肪组织来源间充质干细胞高度表达CD34,这与流式鉴定的结果相吻合。以上结果表明,瘦素受体谱系细胞符合间充质干细胞的特征,并且还表达一些骨髓间充质干细胞标记物和其他常见的间质标记物,由此说明皮肤中瘦素受体阳性细胞属于一类间充质干细胞。"
2.3 瘦素受体谱系细胞参与皮肤创伤愈合的进程 已知皮肤中的间充质干细胞在创伤愈合中发挥着关键作用,既然瘦素受体可以标记皮肤中的一类间充质干细胞,那么瘦素受体所标记的细胞谱系可能会参与到皮肤损伤修复的进程。因此,为了进一步探究瘦素受体谱系细胞在小鼠皮肤创伤愈合中的作用,该研究利用LepR-Cre; tdTomato小鼠构建小鼠背部直径为5 mm的皮肤全层切除创伤模型(此模型小鼠7 d创伤可以基本愈合)。在对小鼠皮肤打孔后的0 d(未损伤的正常皮肤)以及2,4,6 d分别取伤口部位皮肤,固定包埋进行冰冻切片,结果显示被Tomato红色荧光所标记的瘦素受体阳性细胞在伤口边缘及伤口中央都有分布,瘦素受体阳性细胞在伤口边缘部位增殖并迁移到伤口中央,并且细胞数量随时间逐渐增多,见图3A。通过免疫荧光染色染Vimentin,部分瘦素受体阳性细胞可以与Vimentin共标,即一部分瘦素受体阳性细胞以成纤维细胞的形式参与损伤修复,见图3B。免疫荧光染色显示在创伤愈合进程中瘦素受体谱系细胞表达p-S6,见图3C,在损伤修复过程中瘦素受体谱系细胞的mTOR信号通路被激活。小鼠皮肤损伤后的谱系示踪结果表明,在皮肤创伤愈合后瘦素受体标记的间质细胞被激活,通过增殖和迁移参与真皮层的重建,即瘦素受体谱系细胞参与皮肤损伤后的修复。"
与对照组相比,敲除组小鼠的体型、体质量及生理状态无明显差异且发育正常。取2月龄对照组和敲除组小鼠背部均处于静息期的皮肤,进行组织固定包埋石蜡切片,经苏木精-伊红染色并借助图像软件测量统计,结果显示敲除组小鼠真皮层和真皮白色脂肪层厚度增加,见图4A。Perilipin免疫荧光染色显示敲除组小鼠Perilipin所标记的白色脂肪细胞数量增多,见图4B。免疫荧光染色结果显示敲除组小鼠真皮层中p-S6表达高于对照组小鼠,见图4C。此外,免疫荧光染色结果显示敲除组小鼠p-Erk阳性细胞比例明显高于对照组,见图4D,4E。Western blot检测结果也显示,与对照组相比,敲除组小鼠S6磷酸化水平和Erk磷酸化水平均明显上调,见图4F。以上结果表明,瘦素受体阳性细胞中敲除Tsc1后影响小鼠皮肤的发育,mTOR信号通路和MAPK信号通路激活,真皮层和真皮白色脂肪层结构受到影响。"
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