二次谐波结合双光子荧光成像方法观察人源胶原蛋白透皮吸收情况

摘要

目的:用二次谐波成像结合双光子荧光成像的方法观察人源胶原蛋白透皮吸收的情况.方法:将荧光标记的人源胶原蛋白(1 mg/mL)涂抹于小鼠表皮层经皮肤吸收1h后用背向二次谐波观察皮肤内胶原纤维作为真皮层定位标志,用双光子扫描共聚焦显微镜观察人源胶原蛋白透皮吸收深度,吸收方式.结果:二次谐波成像结合双光子荧光成像表明人源胶原蛋白透皮吸收1h后可观察到荧光信号沿着毛囊聚集,并有部分荧光分子由毛囊扩散至真皮层.结论:二次谐波可以更快速,更灵敏地检测皮肤中的胶原纤维,以此作为检测物质透皮吸收深度的定位标志,具有不受荧光信号干扰的优点.人源胶原蛋白可以沿着毛囊进入真皮层,并从毛囊中扩散至胶原纤维层从而补充皮肤中的胶原纤维.%Objective:Second harmonic generation imaging (SHG) and two-photon fluorescence imaging were combined to trace the transdermal absorption and distribution of Re-combinant human collagen (R-hc) in mice skin.Method:After applied the R-hc (1mg/mL) for 1 hour,the mice skin was cut off at 2 cm × 2 cm for further observation.The location of collagen fibers in the skin were observed by using the backwards SHG as the dermal layer markers.The process of transdermal absorption and observation of R-hc were traced by two photon microscopy.Result:The SHG combined with two-photon fluorescence imaging showed that the fluorescence signal of R-hc was gathered along the hair follicle and some fluorescent molecules diffused from the hair follicle to the dermis after transdermal absorption for 1 h.Conclusion:SHG can provide faster and more sensitive detection of skin collagen fibers,which can be used as the depth marker of the transdermal absorption of the test substance without interfering with the fluorescence signal.Human collagen can enter the dermis along the hair follicle,and replenish to the collagen fiber layer in the skin.