Notch信号通路对婴幼儿血管瘤间充质干细胞成脂分化的影响

Effects of Notch signaling pathway on the adipogenic differentiation of infantile hemangioma-derived mesenchymal stem cells

目的研究Notch信号通路对婴幼儿血管瘤间充质干细胞(Hem-MSCs)成脂分化的影响及其可能机制。方法选取2021年1至6月南京医科大学附属儿童医院烧伤整形科手术切除的10例婴幼儿血管瘤标本(男6例,女4例,年龄2至6个月,平均3.5个月)。用贴壁筛选法从增生期血管瘤中分离Hem-MSCs,通过流式细胞术进行鉴定。鉴定成功后进行成脂诱导。使用实时荧光定量PCR检测Hem-MSCs成脂诱导14 d后Notch1、Jagged1及Hes1基因表达情况。在Hem-MSCs培养基中分别加入Notch信号通路抑制剂DAPT、PI3K/AKT信号通路激动剂740Y-P及溶剂DMSO,再进行成脂诱导。将细胞分为6组:空白对照组、成脂诱导组、成脂诱导+DMSO组、成脂诱导+DAPT组、成脂诱导+740Y-P组、成脂诱导+DAPT+740Y-P组。成脂诱导7 d后,蛋白质印迹法检测成脂诱导+DMSO组、成脂诱导+DAPT组、成脂诱导+DAPT+740Y-P组p-PI3K、PI3K、p-AKT、AKT蛋白表达情况;成脂诱导14 d后,实时荧光定量PCR检测上述6组细胞中成脂分化关键转录因子中PPARγ和C/EBPα基因表达情况;油红O染色鉴定成脂效果。两组间比较采用t检验,多组间比较采用方差分析,组内比较采用SNK-q检验。P<0.05为差异有统计学意义。结果Notch1、Jagged1及Hes1在Hem-MSCs成脂诱导过程中表达逐渐降低,差异均有统计学意义(t_(Notch1)=8.99,P_(Notch1)=0.008;t_(Jagged1)=9.49,P_(Jagged1)=0.007;t_(Hes1)=7.74,P_(Hes1)=0.015)。在Hem-MSCs成脂诱导过程中加入Notch信号通路抑制剂DAPT后,PI3K/AKT信号通路相关蛋白表达降低,差异有统计学意义(q_(p-PI3K)=4.78、P_(p-PI3K)=0.014,q_(p-AKT)=5.04、P_(p-AKT)=0.010);相关成脂指标表达增高(q_(油红O)=6.07,P_(油红O)=0.003;q_(PPARγ)=17.34,P_(PPARγ)<0.001;q_(C/EBPα)=14.8,P_(C/EBPα)<0.001),差异均有统计学意义。加入PI3K/AKT信号通路激动剂740Y-P后,相关成脂指标无明显变化,差异均无统计学意义(q_(油红O)=1.82,P_(油红O)=0.786;q_(PPARγ)=0.97,P_(PPARγ)=0.981;q_(C/EBPα)=1.98,P_(C/EBPα)=0.654)。同时加入DAPT和740Y-P后,相关成脂指标表达降低(q_(油红O)=5.22,P_(油红O)=0.013;q_(PPARγ)=9.78,P_(PPARγ)<0.001;q_(C/EBPα)=16.74,P_(C/EBPα)<0.001)结论抑制Notch信号通路可以通过降低PI3K/Akt信号通路活性促进Hem-MSCs向脂肪细胞分化。

Objective To study the effects and potential mechanisms of Notch signaling pathway on the adipogenic differentiation of infantile hemangioma-derived mesenchymal stem cells.Methods From January 2021 to June 2021,10 infantile hemangioma specimens(6 males and 4 females,aged 2 months to 6 months,average age 3.5 months)were collected from the Department of Burns and Plastic Surgery of the Children’s Hospital of Nanjing Medical University.Hem-MSCs were isolated from hemangioma in the proliferating phase by adherent screening and identified by flow cytometry.Adipogenic induction was performed after successful identification.Real-time quantitative fluorescence PCR was used to detect the expression of Notch1,Jagged1 and Hes1 genes after 14 days of adipogenesis induction of Hem-MSCs.The Notch signaling pathway inhibitor DAPT,PI3K/Akt signaling pathway agonist 740Y-P and solvent DMSO were added to Hem-MSCs,and adipogenesis induction was performed.The cells were divided into six groups:control group,adipogenic induction group,adipogenic induction+DMSO group,adipogenic induction+DAPT group,adipogenic induction+740Y-P group,and adipogenic induction+740Y-P+DAPT group.After 14 days of adipogenic induction,real-time quantitative fluorescence PCR was used to detect the expression of PPARγand C/EBPαgenes in the key transcription factors of adipogenic differentiation in the above six groups.Oil red O staining was applied to identify the adipogenic differentiation capabilities;after adipogenic induction for 7 days,the expression levels of p-PI3K,PI3K,p-AKT,and AKT proteins in the adipogenic induction+DMSO group,adipogenic induction+DAPT+740Y-P group were detected by western blotting.In comparing the two groups,an unpaired Student’s t-test was used to assess the differences,and analysis of variance was applied for the analysis of the mean values among multiple groups.and P<0.05 was considered statistically significant.Results The expression levels of Notch1,Jagged1 and Hes1 decreased gradually during the adipogenic induction of Hem-MSCs,and the differences were statistically significant(t_(Notch1)=8.99,P_(Notch1)=0.008;t_(Jagged1)=9.49,P_(Jagged1)=0.007;t_(Hes1)=7.74,P_(Hes1)=0.015).After the addition of Notch signaling pathway inhibitor DAPT during the adipogenic induction of Hem-MSCs,the expression of proteins associated with PI3K/AKT signaling pathway decreased,and the difference was statistically significant(q_(p-PI3K)=4.78,P_(p-PI3K)=0.014;q_(p-AKT)=5.04,P_(p-AKT)=0.010)and the expression of adipogenesis indexes increased(q_(Oil red O)=6.07,P_(Oil red O)=0.003;q_(PPARγ)=17.34,P_(PPARγ)<0.001;q_(C/EBPα)=14.8,P_(C/EBPα)<0.001).There was no significant change in adipogenesis indexes after addition of PI3K/Akt signaling pathway agonist 740Y-P(q_(Oil red O)=1.82,P_(Oil red O)=0.786;q_(PPARγ)=0.97,P_(PPARγ)=0.981;q_(C/EBPα)=1.98,P_(C/EBPα)=0.654).The expression of adipogenesis indexes decreased after adding DAPT and 740Y-P(q_(Oil red O)=5.22,P_(Oil red O)=0.013;q_(PPARγ)=9.78,P_(PPARγ)<0.001;q_(C/EBPα)=16.74,P_(C/EBPα)<0.001).Conclusion The inhibition of Notch signaling pathway can promote the adipogenic differentiation of Hem-MSCs by reducing the activity of PI3K/Akt signaling pathway.