脂肪干细胞对D-半乳糖致裸鼠皮肤老化的拮抗作用及对皮肤恢复功能的影响

Antagonistic Effect of Adipose-derived Stem Cells on Skin Aging Induced by D-galactose in Nude Mice and its Effect on Skin Recovery

目的:本文通过研究脂肪干细胞(Adipose stem cells,ASCs)在D-半乳糖诱导裸鼠皮肤老化的拮抗作用及其对皮肤恢复功能的影响,旨在探讨抗衰老机制并为临床上抗衰老疗法提供新的思路。方法:选择SPF级裸鼠40只,随机分为4组:对照组、D-半乳糖+磷酸盐缓冲液(PBS)组、D-半乳糖+ASCs组与D-半乳糖+氨基胍(AG)组,每组10只。对D-半乳糖+PBS组,D-半乳糖+ASCs组和D-半乳糖+AG组的裸鼠的背部进行皮下注射1 000mg/kg D-半乳糖,每天1次,持续8周。2周之后,3组小鼠背部分别注射PBS缓冲液(0.5ml)、ASCs(1×10^6/ml)和AG(100mg/kg),均注射至真皮层,每天1次,4周后处死。取每组皮肤组织,采用硫代巴比妥酸法测定丙二醛(MDA),黄嘌呤氧化酶法测定超氧化物歧化酶(SOD),ELISA法测定晚期糖基化终末产物(AGEs)、总胶原蛋白、Ⅰ型胶原蛋白及Ⅲ型胶原蛋白水平。通过HE染色和马松染色比较各组皮肤组织真皮层厚度及胶原分布比,采用免疫组化测定各组CD31及血管内皮生长因子(VEGF)表达情况。结果:四组裸鼠的体重与模型构建前并无显著差别。对照组裸鼠表现为充满活力,反应敏捷,并且皮肤富有弹性,粪便无明显臭味;衰老小鼠模型皮肤逐渐变弱,变薄,失去弹性,并患有便秘,且排泄物发臭;而经ASCs或AG处理的小鼠裸鼠在处理后情况有所改善。相较于对照组,D-半乳糖+PBS组裸鼠SOD、总胶原蛋白、CD31、VEGF、Ⅰ型胶原蛋白及Ⅲ型胶原蛋白水平显著降低,MDA及AGEs表达量显著增加,而通过ASCs或AG处理的裸鼠各指标水平均有所改善。HE及马松染色结果显示,相较于对照组,D-半乳糖+PBS组裸鼠真皮层相对较薄,胶原分布比明显降低,而经ASCs或AG处理的裸鼠鼠真皮层有所增厚,胶原分布比显著上升。结论:ASCs移植对D-半乳糖诱发衰老的裸鼠的自由基与糖基化水平具有良好的拮抗作用,为ASCs的移植抵抗肌肤衰老的理论提供了实验依据。

Objective To investigate the anti-aging mechanism and provide new ideas for clinical anti-aging therapy by studying the antagonism of adipose-derived stem cells (ASCs) in D-galactose-induced skin aging in nude mice and its effect on skin recovery.Methods Forty-one SPF nude mice were randomly divided into 4 groups:control group,D-galactose +phosphate buffer(PBS) group,D-galactose+ASCs group and D-galactose+aminopurine(AG) Group,10 in each group.The back of D-galactose+PBS group,D-galactose+ASCs group and D-galactose +AG group were injected subcutaneously with 1 000mg/kg D-galactose once a day for 8 weeks.After 2 weeks,the mice in the three groups were injected with PBS buffer (0.5ml),ASCs (1×10^6/ml),and AG (100mg/kg),respectively,and injected into the dermis layer once a day,and sacrificed 4 weeks later.For each group of skin tissues,malondialdehyde (MDA) was determined by thiobarbituric acid method,superoxide dismutase (SOD),Collagen,type Ⅰ collagen and type Ⅲ collagen were determined by xanthine oxidase method,and advanced glycation end products(AGEs) were determined by ELISA.The dermis thickness and collagen distribution ratio of skin tissue were compared by HE staining and Masson staining.The expression of CD31 and vascular endothelial growth factor(VEGF) in each group were determined by immunohistochemistry.Results The body weight of the four groups of nude mice was not significantly different from that before the model was constructed.The nude mice in the control group showed vigorousness,rapid response,and the skin was elastic,and the feces had no obvious odor.The skin of the aging mouse model gradually weakened,thinned,lost elasticity,suffered constipation,and the excrement smelled.Mouse nude mice treated with ASCS or AG improved after treatment.Compared with the control group,the SOD、CD31、VEGF、Collagen,type Ⅰ collagen and type Ⅲ collagen levels of D-galactose+PBS group was significantly decreased,and the expression levels of MDA and AGEs were significantly increased,while the levels of all the indexes of nude mice treated by ASCS or AG were improved.The results of HE and Masson staining showed that the dermis layer of D-galactose+PBS group was relatively thin compared with the control group,and the collagen distribution ratio was significantly decreased,while the dermis layer of nude mice treated with ASCS or AG increased.Thick,collagen distribution ratio increased significantly.Conclusion The transplantation of ASCs has a good antagonistic effect on the free radical and glycosylation level of D-galactose-induced aging nude mice,and provides an experimental basis for the theory of ASC transplantation against skin aging.