壳多糖酶3样蛋白1对人脐带来源间充质干细胞成脂和成骨分化的调控作用

Adipogenic and osteogenic differentiation in human umbilical cord-derived mesenchymal stem cells regulated by chitinase--like protein

目的探讨壳多糖酶3样蛋白1(CHI3L1)对人脐带来源间充质干细胞(hUC-MSC)成脂和成骨分化的调控作用。方法复苏并培养hUC-MSC,流式细胞术检测细胞表面标志物CD14,CD34,CD45,CD73,CD90和CD105;成脂和成骨诱导分化后,油红O染色和碱性磷酸酶(ALP)染色检测体外成脂和成骨分化能力,实时荧光定量PCR(RT-qPCR)检测成脂和成骨关键转录因子mRNA表达,即脂肪酶(ADI)和过氧化物酶体增殖物激活受体γ(PPAR-γ)mRNA表达及ALP和骨桥蛋白(OPN)mRNA表达。利用慢病毒载体构建稳定敲低CHI3L1的hUC-MSC(shCHI3L1-MSC)和对照hUC-MSC(shNC-MSC),同上鉴定其生物学特征。shCHI3L1-MSC和shNC-MSC体外成脂诱导分化后,油红O染色检测细胞内脂滴数目,RT-qPCR检测ADI和PPAR-γmRNA表达;成骨诱导分化后,ALP染色检测ALP染色面积,RT-qPCR检测ALP和OPN mRNA表达。结果培养的hUC-MSC高表达CD73,CD90和CD105,低表达或不表达CD14,CD34和CD45;成骨诱导分化后,与未诱导组相比,诱导组ALP活性增加,ALP和OPN mRNA表达显著增加(P<0.01);成脂诱导分化后,经油红O染色,诱导组出现大量红色脂滴,ADI和PPAR-γmRNA表达显著增加(P<0.01)。敲低CHI3L1基因,hUC-MSC表面标志物、成脂和成骨分化能力生物学特性未发生改变。shNC-MSC和shCHI3L1-MSC成脂和成骨诱导分化后,shCHI3L1-MSC诱导组脂滴数及ADI和PPAR-γmRNA表达均显著高于shNC-MSC(P<0.01),ALP活性及ALP和OPN mRNA表达均显著低于shNC-MSC(P<0.01),提示hUC-MSC敲低CHI3L1基因后成脂分化能力增强,成骨分化能力降低。结论CHI3L1参与hUC-MSC体外成脂和成骨分化调控。

OBJECTIVE To explore the role of chitinase-3-like protein 1(CHI3L1)in regulating the adipogenic and osteogenic differentiation abilities of human umbilical cord-derived mesenchymal stem cells(hUC-MSCs).METHODS hUC-MSCs were thawed and cultured.MSC surface markers CD14,CD34,CD45,CD73,CD90,and CD105 were detected by flow cytometry.The abilities of hUC-MSCs to differentiate into adipocytes and osteocytes were proved by oil red O staining and alkaline phosphatase(ALP)staining,respectively.The expressions of adipogenic and osteogenic key transcription factors were examined by real-time quantitative PCR(RT-qPCR).The lentiviral vector was used to construct hUC-MSCs with stable knockdown of CHI3L1 gene(shCHI3L1-MSCs)and the control group hUCMSCs(shNC-MSCs).The effect of CHI3L1 on adipogenic differentiation of hUC-MSCs was investigated by in vitro induction experiments.The differences of lipid droplet numbers and ALP activity between shCHI3L1-MSC and shNC-MSC groups were detected by oil red O staining and ALP staining,respectively,and the key transcription factors of adipogenic and osteogenic differentiation were assessed by RT-qPCR.RESULTS The cultured hUC-MSCs showed high expressions of CD73,CD90 and CD105,and low or no expressions of CD14,CD34 or CD45.CHI3L1 knockdown did not affect the expressions of hUC-MSC surface markers.After osteogenic differentiation,the activity of ALP and the mRNA expression of osteogenic differentiation markers ALP and osteopontin(OPN)increased significantly in the induced group compared with the cell control group(P<0.01).After adipogenic differentiation,oil red O staining showed a large number of red lipid droplets in the induced group.RT-qPCR results showed that adipsin(ADI)and peroxisome proliferator activated receptor-γ(PPAR-γ)mRNA expressions were significantly increased in the induced group(P<0.01),suggesting that the hUC-MSCs whose CHI3L1 gene was knocked down remained capable of osteogenic and adipogenic differentiation.Meanwhile,compared with the shNC-MSC group,the number of lipid droplets and the mRNA expression levels of ADI and PPAR-γwere significantly increased(P<0.01),but ALP activity and the mRNA expression levels of OPN and ALP were significantly decreased(P<0.01)in the shCHI3L1-MSC group,indicating that hUC-MSCs with knockdown of CHI3L1 enhanced the adipogenic differentiation ability but weakened the osteogenic differentiation abibity.CONCLUSION CHI3L1 is involved in the regulation of adipogenic and osteogenic differentiation of hUC-MSCs in vitro.