POSTN基因调控人脐带间充质干细胞向成骨细胞分化的实验研究

The ability of periostin(POSTN)gene to regulate osteogenic differentiation of human umbilical cord-derived mesenchymal stem cells

目的 探讨骨膜蛋白调控人脐带来源间充质干细胞(hUC-MSC)向成骨细胞分化的作用。方法 培养并鉴定hUC-MSC,利用慢病毒载体构建稳定敲低POSTN基因的hUC-MSC(sh-POSTN-MSC)和对照组hUC-MSC(sh-NC-MSC),流式细胞术检测细胞表面标志物,油红O和碱性磷酸酶(ALP)染色比较两组细胞体外成脂和成骨诱导分化能力,实时荧光定量PCR(qRT-PCR)检测成脂分化关键转录因子和成骨分化标志物表达的差异。结果 培养的sh-NC-MSC和sh-POSTN-MSC表面标志物均为CD73^(+)/CD90^(+)/CD105^(+)/CD14^(-)/CD34^(-)/CD45^(-),POSTN基因敲低后不影响hUC-MSC表面标志物的表达。成脂诱导后,sh-NC-MSC和sh-POSTN-MSC经染色均观察到红色脂滴出现。qRT-PCR结果显示,与自分化组相比,诱导组的脂肪酶(ADI)和过氧化物酶体增殖物激活受体γ(PPARγ)表达均明显增加(P<0.001)。成骨细胞诱导后,sh-NC-MSC和sh-POSTN-MSC经染色均观察到胞内ALP活性;qRT-PCR结果显示,二者诱导组成骨细胞标志物骨桥蛋白(OPN)表达均显著增加(P<0.001,P<0.01),提示sh-NC-MSC和sh-POSTNMSC具有成脂、成骨分化能力;同时,与sh-NC-MSC成骨诱导组相比,sh-POSTN-MSC成骨诱导组的胞内ALP活性明显降低,OPN和ALP的mRNA相对表达均显著降低(P<0.001)。结论 敲低POSTN基因的hUC-MSC成骨诱导分化能力降低,POSTN参与调控hUC-MSC的体外成骨分化作用。

Objective To explore the role of periostin(POSTN)in regulating osteogenic differentiation ability of human umbilical cord-derived mesenchymal stem cells(hUC-MSCs). Methods hUC-MSCs were cultured and identified.The lentiviral vector was used to construct hUC-MSCs with stable knockdown of POSTN gene(sh-POSTN-MSCs)and the control group hUC-MSCs(sh-NC-MSCs). Cell surface markers were detected by flow cytometry. sh-NC-MSCs and sh-POSTN-MSCs were induced to differentiate into adipocytes and osteocytes. The ability of sh-NC-MSCs and sh-POSTNMSCs to differentiate into adipocytes and osteocytes was measured by oil red O staining and alkaline phosphatase(ALP)staining,respectively. The expressions of adipogenic and osteogenic key transcription factors were examined by quantitative real-time PCR(qRT-PCR). Results The cultured sh-NC-MSCs and sh-POSTN-MSCs surface markers were CD73^(+)/CD90^(+)/CD105^(+)/CD14^(-)/CD34^(-)/CD45^(-). POSTN gene knockdown did not affect the expressions of surface markers in hUC-MSCs. The appearance of red lipid droplets was observed in both sh-NC-MSCs and sh-POSTN-MSCs with adipogenic-inducing differentiation,and the expressions of adipocytes differentiation transcription factors ADI and PPAR γ were both significantly increased by qRT-PCR(P<0.001). A significant increase in ALP activity was displayed in both sh-NC-MSCs and sh-POSTN-MSCs with osteogenic-inducing differentiation. Compared with the self-differentiated groups,the expression of OPN was significantly increased in the sh-NC-MSCs and sh-POSTN-MSCs osteogenic-inducing groups(P<0.001).These results suggested that sh-NC-MSCs and sh-POSTN-MSCs were capable of adipogenic-inducing differentiation and osteogenic-inducing differentiation. Meanwhile,compared with sh-NC-MSCs,the ALP activity was significantly decreased in the sh-POSTN-MSCs in the osteogenic-inducing group according to staining results. Also,the expressions of OPN and ALP were significantly decreased by qRT-PCR. Conclusion The POSTN gene is involved in regulating the osteogenic differentiation of hUC-MSCs in vitro.