1型糖尿病脂肪来源间充质干细胞成骨分化的实验研究

Osteogenic differentiation of adipose derived-stem cells in type 1 diabetes mellitus

目的探究1型糖尿病小鼠脂肪来源间充质干细胞的成骨分化能力。方法10只21~28日龄C57BL/6雄鼠随机分为两组,糖尿病组小鼠(n=5)腹腔注射链脲佐菌素溶液(STZ)诱导小鼠1型糖尿病(T1DM)模型,正常组小鼠(n=5)注射柠檬酸钠缓冲液作为对照,连续注射5 d,1周后监测小鼠随机血糖,连续测量3 d。分别提取两组小鼠腹股沟和性腺脂肪,分离培养脂肪间充质干细胞(ADSC),通过细胞形态、流式细胞术检测细胞表型,体外多向诱导分化鉴定细胞特性。为进一步比较两组细胞成骨分化能力的差异,取第3代(P3代)两组ADSC进行体外成骨诱导分化,分别在诱导的第3、5、7天进行碱性磷酸酶(ALP)染色,实时荧光定量PCR(qPCR)和Western印迹检测细胞成骨分化关键转录因子表达。结果T1DM组小鼠连续3 d随机血糖≥16.7 mmol/L,并出现多饮、多食、多尿及体重增长缓慢的典型表现;两组小鼠脂肪组织来源的培养细胞均呈梭形纤维样、贴壁漩涡状生长;流式细胞术结果显示,两组细胞高表达CD29、CD90,低表达或不表达CD11b、CD34、CD45、MHC-Ⅱ;体外诱导分化结果显示,二者均具有体外成脂和成骨分化能力,证实分别获得T1DM小鼠和正常小鼠脂肪来源的间充质干细胞。两组ADSC体外成骨诱导细胞ALP染色结果表明,与NC-ADSC组相比,在诱导的第3、5、7天,T1DM-ADSC组ALP活性低;qPCR结果显示调控成骨分化关键基因RUNX2、OCN、ALP、BMP2的表达降低;Western印迹结果显示,T1DM-ADSC与NC-ADSC相比,成骨分化关键转录因子RUNX2表达下降,而成脂细胞分化关键转录因子CEBPα表达升高。结论T1DM小鼠来源ADSC体外成骨分化能力显著降低。

Objective To investigate the osteogenic differentiation capacity of adipose derived-stem cells(ADSCs)in mice with type 1 diabetes mellitus(T1DM).Methods Ten C57BL/6 mice were randomly divided into two groups.Streptozotocin(STZ)was administrated to C57BL/6 mice through peritoneal injection to induce a T1DM model(n=5),while the normal control group(NC group,n=5)was given the same volume of sodium citrate buffer injection.After one week,the random blood glucose of the mice was monitored and measured for 3 consecutive days.ADSCs were isolated and cultured from groin and gonad fat in two groups.Cell characteristics were identified by cell morphology,immunophenotype,and induced differentiation assay.The key transcription factors in adipogenic and osteogenic differentiation were measured by real-time quantitative PCR(qPCR)and Western blotting.To study the difference in the osteogenic differentiation ability between the two groups,the third passage ADSCs(P3)in the two groups were used for osteogenic differentiation assay in vitro.Alkaline phosphatase(ALP)staining was performed to measure the osteogenic capacity at 3,5 and 7 d,respectively,and the expression of osteogenic differentiation related genes were detected by qPCR.Results In the T1DM group,the random blood glucose of the mice was found to be≥16.7 mmol/L for 3 consecutive days.Typical symptoms of diabetes,including polydipsia,polyphagia,polyuria and slowed weight growth occurred in T1DM mice induced by STZ.Cultured cells from murine adipose tissue in the T1DM group and NC group were spindle-shaped and showed adherent vortex growth.Flow cytometry results showed that CD29 and CD90 were highly expressed,but the expressions of CD11b,CD34,CD45 and MHC-Ⅱwere lower or not expressed.Moreover,these cells presented adipogenic and osteogenic differentiation ability.These results demonstrated that ADSCs from T1DM mice and normal mice had been isolated.In vitro osteogenic differentiation assay results showed that the ALP activities in T1DM-ADSCs were decreased at 3,5 and 7 d,compared with NC-ADSCs.Furthermore,the mRNA expression levels of RUNX2,OCN,ALP and BMP2 in T1DM-ADSCs were significantly reduced.Western blotting results revealed that the RUNX2 expression was decreased,while CEBPαwas increased in T1DM-ADSCs.Conclusion ADSCs isolated from T1DM mice display weak osteogenic differentiation ability.