组织工程与重建外科杂志

组织工程与重建外科杂志 ›› 2016, Vol. 12 ›› Issue (2): 107-136.doi: 10.3969/j.issn.1673-0364.2016.02.005

• 论著 • 上一篇    下一篇

机械牵张刺激下大鼠皮肤再生的关键基因筛选

高宇，汪景，余庆雄，单圣周，李青峰   

  1. 上海交通大学医学院附属第九人民医院整复外科
  • 发布日期:2020-07-23

Key Gene Screening of Skin Regeneration Under Mechanical Stretch Stimulation in Rats

GAO Yu,WANG Jing,YU Qingxiong,SHAN Shengzhou,LI Qingfeng   

  • Published:2020-07-23
  • Contact: 国家自然科学基金(no.81230042)

摘要： 目的 明确机械牵张刺激下大鼠皮肤再生相关基因表达谱的变化,并筛选出其中的关键基因。方法 建立大鼠皮肤扩张模型,以未扩张皮肤为对照,以注水量不同分组取材。分别是:对照组(con)、扩张器最终注水60 mL组(exp60)、扩张器最终注水120 mL组(exp120)、扩张器最终注水180 mL组(exp180)。取材后一部分样本切片行免疫荧光染色,荧光标记增殖标记物Ki67,绘制皮肤细胞增殖曲线。另一部分样本行基因芯片检测,检测mRNA表达谱的变化,并利用t检验和差异表达的倍数变化(FC值)筛选出关键基因。筛选出皮肤再生的关键基因后,用PCR方法验证以确认该基因的表达量,及其与皮肤增殖之间的关系。结果 免疫荧光染色显示,增殖标记物Ki67在各组的表达量由高到低依次是:con、exp180、exp60和exp120。本研究筛选出不同机械牵张刺激下大鼠皮肤再生的11个差异基因,分别为:Myh1、Myh2、Myh3、Myh4、Car6、Pi15、Apt12a、Mt1a、Cthrc1、Rsad2和IGF-BP5。其中IGF-BP5和对照组的皮肤内表达相比为下调基因;PCR验证得出,IGF-BP5在各组的表达量由高到低依次是:exp120、exp60、exp180和con。结论 机械牵张刺激下,随着扩张时间及扩张量的增加,大鼠皮肤再生呈现早期皮肤细胞增殖加速,中期增殖活跃,后期增殖减缓的趋势;且IGF-BP5可能为影响机械牵张刺激下大鼠皮肤再生的关键基因。

关键词: 皮肤扩张, 机械牵张, 皮肤再生, 基因芯片

Abstract: Objective To determine the expression changes of skin regeneration-related key genes under mechanical stretch stimulation in rats and to screen key genes. Methods Skin expansion model was established and skin samples were harvested: the unexpanded control group (con), water-injected 60 mL group (exp60), water-injected 120 mL group (exp120) and water-injected 180 mL group (exp180). Immunohistochemical staining was implemented in the sliced the sample, stain-ing proliferation marker Ki67. According to that, the trend curve of Ki67 positive rate of samples was drawn. Gene Chip was also undertaken to reveal the gene expression changes during expansion process. T test and fold change of differential ex-pression were applied to screen key genes. Then PCR was used to confirm the relationship between the expression of the key gene and the proliferation of skin. Results Immunofluorescence staining showed that, the expression of the proliferation marker Ki67 descending order is con, exp180, exp60, exp120. This study screened 11 skin regeneration-related key genes of rats under different mechanical stretch stimulation: Myh1, Myh2, Myh3, Myh4, Car6, Pi15, Apt12a, Mt1a, Cthrc1, Rsad2, IGF-BP5. Compared to the expression of control group, IGF-BP5 was a down-regulated gene. PCR showed that the expres-sion of IGF-BP5 descending order was: exp120, exp60, exp180, con. Conclusion Under mechanical stretch stimulation, with the increasing time of expansion and amount of water injected, skin regeneration of rats shows accelerated proliferation at early stage, active proliferation in the mid-term and tendency to slow down the proliferation in the later period; IGF-BP5 may be a key regeneration-related gene of rats under mechanical stretch stimulation.

Key words: Skin expansion, Mechanical stretch, Skin regeneration, Gene chip

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