游离毛囊体外培养模型构建及形态学检查的实验研究

Experimental study on constructing the model of human hair follicle in vitro and morphology inspection

目的:构建游离毛囊体外培养模型,观察离体培养人头皮毛囊形态变化。方法:在Williams E无血清培养基中,建立离体人头皮毛囊培养模型;通过倒置显微镜观察毛囊的形态变化;目镜测微器测量毛囊长度;3H-TdR 掺入检测毛囊DNA合成率;组织学检测用冰冻切片光镜观察和电镜切片透射电镜观察。结果:毛囊有不同程度的生长,随时间后移形态结构由清晰逐渐变为模糊;冰冻切片HE染色后在光学显微镜下可清楚分辨出毛囊的结构;培养3天毛囊的内外根鞘中可见凋亡细胞,内皮根鞘较外皮根鞘明显多见。结论:在离体培养人头皮毛囊时,Williams E无血清培养基是合适的选择,该模型也是筛选促进或抑制毛发生长药物的良好模型;离体培养毛囊组织学检测用冰冻切片可以简化步骤;透射电镜可以用来观察离体培养的毛囊的形态和超微结构的变化,也可以用来协助观察细胞凋亡的状况。目镜测微器测量毛囊长度简单可行,辅以3H-TdR 掺入则更为客观。

Objective This study was carried out to construct the model of human hair follicle in vitro and observe morphologic change of the hair follicle. Methods We established the model of human hair follicle in vitro in Williams E serum-free medium. We can continuously observe morphologic change of hair follicle under an invert microscope. The length of hair follicle was measured by eyepiece micrometer and DNA synthesis rate of human hair follicle in vitro by measuring the rates of incorporation of 3H-TdR.Histology form was observed by Cryo-section and transmission electron microscope. Results We found the human hair follicle in vitro have different degree elongation,whose constitution transform clouding followed by time. We can clearly tell the difference constitution of hair follicle under the light microscope after the Cryo-section was stained with HE.With transmission electron microscope observe hair follicle culture in vitro 3 days later,we found there are apoptosis cells in inner root sheath and outer root sheath. Furthermore there are more apoptosis cells in inner root sheath than in outer root sheath. Conclusion When we culture human hair follicle in vitro, Williams E serum-free medium is a suitable choice.This model is a fine model also, which is used for screening drug that may accelerate or inhibit the hair growth.Cryo-section can simplify procedure of paraffin section in histology detection of culture human hair follicle in vitro.We can observe the ultrastructural change of human hair follicle in vitro by transmission electron microscope,which can assist to observe apoptosis cells. The eyepiece micrometer is a simple tool for measuring length of hair follicle in vitro.Assisted by measuring the rates of incorporation of 3H-TdR, the results are further objective.