摘要
目的:探讨姜黄素联合脂肪间充质干细胞(ADSCs)促进大鼠皮肤创面愈合的效应及相关信号通路的研究。方法:用酶消化法分离和培养大鼠ADSCs,流式细胞仪鉴定干细胞表面抗原。取活性良好的第3代细胞,不同浓度(0、2.5、5、10、20、40μmol/L)的姜黄素作用于ADSCs,通过CCK8法测定干细胞的存活率,筛选姜黄素最适宜浓度。40只雄性SD大鼠切除背部直径1 cm全层皮肤,建立皮肤创面模型,按照不同治疗方法分为模型对照组、干细胞组、姜黄素20μmol/L组、姜黄素20μmol/L联合干细胞组,测量记录各组0、3、7、14 d的皮肤创面愈合率。治疗后7 d ELISA法检测大鼠血清成纤维细胞生长因子(FGF)、血管内皮生长因子(VEGF)、转化生长因子-β(TGF-β)、白细胞介素-1β(IL-1β)、IL-6、肿瘤坏死因子α(TNF-α)含量。治疗后14 d HE染色观察各组大鼠皮肤创面情况,Masson染色观察胶原纤维含量,免疫荧光观察干细胞数量及分布情况。荧光定量PCR检测各组大鼠皮肤创面组织内Wnt4、β-catenin mRNA表达。结果:流式细胞仪鉴定ADSCs表面抗原CD44、CD90阳性,CD34阴性。CCK8法检测姜黄素在20μmol/L时对干细胞增殖无明显毒性作用。与模型对照组比较,干细胞组、姜黄素20μmol/L组、姜黄素20μmol/L联合干细胞组的皮肤创面愈合率、胶原纤维面积百分比、血清生长因子FGF、VEGF、TGF-β明显升高(P<0.05),血清炎症因子IL-1β、IL-6、TNF-α明显降低(P<0.05),Wnt4、β-catenin mRNA表达明显上调(P<0.05),与姜黄素20μmol/L联合干细胞组比较,姜黄素组FGF、VEGF、TGF-β含量、胶原纤维面积明显降低,IL-1β、IL-6、TNF-α含量明显升高,Wnt4、β-catenin mRNA明显下调(P<0.05或P<0.01)。姜黄素20μmol/L联合干细胞组皮肤创面中的ADSCs荧光表达明显高于干细胞组(P<0.05),ADSCs主要分布在真皮层和表皮基底层。结论:姜黄素联合ADSCs可促进大鼠皮肤创面愈合,提高移植后干细胞的存活率,可能与刺激相关生长因子分泌,减少炎症因子含量,上调Wnt4/β-catenin相关信号通路有关。
Objective:To investigate the effect of curcumin combined with adipose derived mesenchymal stem cells(ADSCs)on promoting skin wound healing in rats and the related signal pathways.Methods:ADSCs of rats were isolated and cultured by enzyme digestion method the antigen was identified by flow cytometry.The third generation cells with good activity were treated with curcumin at different concentrations(0,2.5,5,10,20,40μmol/L).The the survival rate of stem cells was determined by CCK8 method to screen the optimal concentration of curcumin.Forty male SD rats were used to establish skin wound model by removing 1 cm full-thickness skin on the back.According to different treatment methods.,rats were divided into model group,stem cell group,20μmol/L curcumin group and 20μmol/L curcumin stem cell+stem cell group.Wound healing rates of each group were measured and recorded at 0,3,7 and 14 days.At 7 days after treatment,the serum levels of fibroblast growth factor(FGF),vascular endothelial growth factor(VEGF),transforming growth factor-β(TGF-β),interleukin-1β(IL-1β),interleukin-6(IL-6)and tumor necrosis factor-α(TNF-α)were detected by ELISA.At 14 days after treatment,HE staining was used to observe the wound surface of rats in each group,Masson staining was used to observe the content of collagen fiber,and immunofluorescence was used to observe the number and distribution of stem cells,the expression levels of Wnt4 andβ-catenin mRNA in skin wound tissues of rats in each group were detected by fluorescence quantitative PCR.Result:The ADSCs surface antigen CD44,CD90 positive and CD34 negative were identified by flow cytometry.CCK8 method showed that curcumin at 20μmol/L had no obvious toxic effect on stem cell proliferation.Compared with model group,the wound healing rate,collagen fiber area percentage,serum FGF,VEGF and TGF-βcontents in stem cell group,20μmol/L curcumin group and 20μmol/L curcumin combined with stem cell group were increased(P<0.05),the serum levels of inflammatory factors IL-1β,IL-6 and TNF-αwere decreased(P<0.05),the expression levels of Wnt4 andβ-catenin mRNA were up regulated(P<0.05).Compared with 20μmol/L curcumin combined with stem cell group,the serum contents of FGF,VEGF and TGF-β,the collagen fiber area percentage were significantly decreased,the serum levels of IL-1β,IL-6 and TNF-αwere increased,while the expression levels of Wnt4 andβ-catenin mRNA were down regulated(P<0.05 or P<0.01).Compared with stem cell group,the Fluorescent expression of ADSCs in skin would was up regulated in 20μmol/L curcumin group+stem cell group(P<0.05).ADSCs were mainly distributed in the dermis and the basal layer of the epidermis.Conclusion:Curcumin combined with ADSCs can promote skin wound healing in rats,improve the survival rate of stem cells after transplantation,which may be related to stimulating the secretion of related growth factors,reducing the content of inflammatory cytokines,and activating Wnt4/β-catenin related signaling pathway.
作者
张璐
李悦
李晓鲁
吴诗惠
龚晓丽
全云云
田媛
岳倩华
赵军宁
蓝海
Zhang Lu;Li Yue;Wu Shihui;Gong Xiaoli;Quan Yunyun;Tian Yuan;Yue Qianhua;Zhao Junning;Li Xiaolu(Sichuan Academy of Chinese Medicine Sciences,Sichuan Institute for Translational Chinese Medicine,Translational Chinese Medicine Key Laboratory of Sichuan Province,Chengdu 610041)
出处
《中药药理与临床》
CAS
CSCD
北大核心
2022年第1期41-47,共7页
Pharmacology and Clinics of Chinese Materia Medica
基金
四川省省级公益性科研院所基本科研业务费专项(编号:A-2020N-20)
中医药科研平台建设(编号:510000_01_111685)
四川省科技厅重点研发计划(重大科技专项)(编号:20ZDYF2222)
四川省中医药管理局科学技术研究专项(编号:2020JC0110)
