【摘要】 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-γ mRNA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-γ mRNA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。更多还原

【Abstract】 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-q PCR 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 sh NC-MSC group, the number of lipid droplets and the m RNA expression levels of ADI and PPAR-γ were 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.更多还原