摘要
目的比较幼龄1型糖尿病(T1DM)模型小鼠和幼龄正常小鼠脂肪来源干细胞(ADSC)的成脂分化能力,为阐明T1DM发病机制提供理论基础和实验依据。方法3周龄C57BL/6J小鼠,连续5 d ip给予小剂量(50 mg·kg^(-1))链脲佐菌素(STZ)制备幼龄T1DM小鼠模型;正常对照组ip给予等体积柠檬酸缓冲液;7 d后连续3 d小鼠尾静脉采血检测空腹血糖水平,空腹血糖均值≥16.7 mmol·L^(-1)认为幼龄小鼠T1DM建模成功。建模成功14 d后,取皮下脂肪和性腺周围脂肪组织,用组织酶消化法培养小鼠ADSC,倒置显微镜下观察细胞形态;用流式细胞术检测细胞表面标志物Sca-1、CD29、CD90、CD31、CD34、CD45、主要组织相容性复合体(MHC)Ⅱ和CD11b。选取第3代(P3代)ADSC进行成骨和成脂诱导分化,分别用油红O染色和碱性磷酸酶染色检测细胞的成骨和成脂诱导分化能力,实时荧光定量PCR检测ADSC成骨关键转录因子骨钙素(Ost)和Runt相关转录因子2(Runx2)及成脂关键转录因子CCAAT增强子结合蛋白α(CEBPα)和过氧化物酶体增殖物激活受体γ(PPARγ)mRNA表达。结果成功建立幼龄T1DM小鼠模型,并从T1DM模型小鼠脂肪组织分离获得ADSC,细胞形态呈成纤维样、贴壁生长,高表达CD29,CD90和Sca-1,低表达CD45,CD34,MHCⅡ,CD11b和CD31。幼龄T1DM模型小鼠P3代ADSC成骨诱导分化后,碱性磷酸酶染色阳性,Ost和Runx2 mRNA表达显著增高(P<0.01);成脂诱导分化后,油红O染色可见ADSC脂滴明显增多(P<0.01),PPARγ和CEBPαmRNA表达显著增高(P<0.01)。与正常对照组相比,幼龄T1DM模型小鼠ADSC脂滴数增多(P<0.01)且增大,PPARγ和CEBPαmRNA表达亦显著增高(P<0.01)。结论幼龄T1DM模型小鼠ADSC体外成脂分化能力增强。
OBJECTIVE To compare the adipogenic differentiation ability of adipogenic stem cells(ADSCs)in young type 1 diabetes mellitus(T1 DM)model mice and normal young mice in order to help elucidate the pathogenesis of T1 DM.METHODS Three-week-old C57 BL/6 J mice received low-dose intraperitoneal injection of streptozotocin(STZ)(50 mg·kg^(-1))for 5 d to establish a young T1 DM mouse model.The normal control group received intraperitoneal injection of citric acid buffer at the same time.Blood glucose levels were detected after three days of tail vein blood collection after seven days of STZ treatment.The STZ-induced young T1 DM model was considered successful when the blood glucose level was above 16.7 mmol·L^(-1).Fourteen days after successful modeling,the subcutaneous fat and perigonadal fat were digested by tissue enzyme to culture mouse ADSCs respectively.ADSC morphology was observed under a microscope.Cell surface markers Sca-1,CD29,CD90,CD31,CD34,CD45,major histocompatibility complex(MHC)Ⅱand CD11 b were detected by flow cytometry.ADSCs of P3 generation were induced to differentiate into adipocytes and osteocytes.Oil Red O staining and alkaline phosphatase staining were used to detect the adipogenic and osteogenic differentiation ability,respectively.Quantitative real-time PCR was used to detect osteogenic key transcription factors osteocalcin(Ost)and Runt-associated transcription factor 2(Runx2)as well as adipogenic key transcription factors CCAAT enhancer binding proteinα(CEBPα)and peroxisome proliferator-activated receptorγ(PPARγ)in ADSCs of T1 DM model and normal control groups.RESULTS A young T1 DM mouse model was established.ADSCs were isolated from adipose tissue of young T1 DM model mice.Morphologically,the ADSCs were of long spindle-like shape and vortice-like adherent growth.The ADSCs showed high expressions of CD29,CD90 and Sca-1,but low expressions of CD45,CD34,MHCⅡ,CD11 b and CD31.After osteogenic differentiation,the alkaline phosphatase staining result of ADSCs of P3 generation in T1 DM young model mice was positive,and the mRNA expressions of Ost and Runx2 were also significantly increased(P<0.01).Moreover,Oil Red O staining was performed on ADSCs of P3 generation in T1 DM young mice after adipogenic induction,and lipid droplets were increased and became larger than in the normal group.The mRNA expressions of PPARγand CEBPαwere also significantly increased compared with the normal group(P<0.01).CONCLUSION The adipogenic differentiation ability of ADSCs in young T1 DM model mice is enhanced in vitro.
作者
张金霞
刘伟江
苏菲娅
张晗
于丰实
白海涛
袁福临
刘元林
李雪
王洋
郑荣秀
张毅
ZHANG Jin-xia;LIU Wei-jiang;SU Fei-ya;ZHANG Han;YU Feng-shi;BAI Hai-tao;YUAN Fu-lin;LIU Yuan-lin;LI Xue;WANG Yang;ZHENG Rong-xiu;ZHANG Yi(Department of Pediatrics,General Hospital of Tianjin Medical University,Tianjin 300052,China;Institute of Radiation Medicine,Academy of Military Medical Sciences,Beijing 100850,China)
出处
《中国药理学与毒理学杂志》
CAS
北大核心
2021年第5期345-352,共8页
Chinese Journal of Pharmacology and Toxicology
基金
国家重点研发计划(2016YFC1000305)
天津市卫计委重点攻关研究项目(16KG123)
天津市自然科学基金(17JCZDJ36400)
天津市科技局科学技术普及项目(18KPHDSF00140)。
关键词
幼龄小鼠
1型糖尿病
脂肪干细胞
成脂分化
young mice
type 1 diabetes mellitus
adipose-derived stem cells
adipogenic induction
