多聚嘧啶序列结合蛋白在骨髓间充质干细胞成骨分化过程中的作用

Polypyrimidine tract binding protein regulates the osteogenic differentiation of bone marrow mesenchymal stem cells

目的明确多聚嘧啶序列结合蛋白(polypyrimidine tract binding protein,PTB)在正常人和骨质疏松大鼠骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)成骨分化中的作用及其机制。方法收集中国医学科学院整形外科医院收治的4例齿槽嵴裂患儿术后废弃骨髓血,原代分离培养人BMSCs并诱导分化为成骨细胞,RT-PCR和蛋白质印迹法检测PTB在诱导分化7 d和14 d的表达水平;应用shRNA技术对人BMSCs中PTB的表达进行敲减,茜素红染色和荧光定量PCR评价其对成骨分化的影响;体外分别应用浓度为10 ng/ml和40 ng/ml的肿瘤坏死因子α(TNF-α)处理人BMSCs,荧光定量PCR和茜素红染色检测其对PTB表达及成骨分化能力的影响;流式细胞术检测人BMSCs经PTB敲减后细胞内活性氧(ROS)含量。10只6个月龄雌性SD大鼠采用随机数字表法随机分为卵巢切除骨质疏松模型组和假手术组,每组5只,荧光定量PCR检测其BMSCs中PTB的表达变化。各项定量数据以均值±标准差表示,2组间比较应用t检验,P<0.05为差异具有统计学意义。结果BMSCs成骨诱导分化7 d和14 d PTB表达显著升高,敲减PTB并成骨诱导14 d后,成骨标志基因RUNX2和ALP相对表达量显著下降,分别为对照组的0.74±0.15和0.29±0.18倍(t=3.490、P=0.039,t=7.983、P=0.004),且茜素红染色阳性的矿化结节形成减少。10 ng/ml TNF-α(t=3.528,P=0.039)和40 ng/ml TNF-α(t=4.306,P=0.023)均下调BMSCs中PTB表达,并抑制成骨分化。敲减PTB后BMSCs细胞内ROS含量(720.7±129.7)较对照组(1204.0±300.1)下降,差异具有统计学意义(t=4.872,P=0.040)。相比于假手术组(0.00166±0.00018),卵巢切除骨质疏松模型大鼠BMSCs中PTB的相对表达量(0.00125±0.00012)显著降低(t=3.217,P=0.032)。结论PTB的表达可促进BMSCs成骨分化,PTB敲减介导的ROS含量下调是其调控BMSCs成骨分化的一个重要机制。

Objective To clarify the role of polypyrimidine tract binding protein(PTB)in osteogenic differentiation of bone marrow mesenchymal stem cells(BMSCs)and explore its mechanism.Methods The bone marrow blood of 4 patients with alveolar cleft treated in Plastic Surgery Hospital of Chinese Academy of Medical Science was collected.Human BMSCs were cultured and induced to osteoblasts,RT-PCR and Western blotting were applied to detect the expression of PTB at different time points.PTB expression in BMSCs was knocked down using shRNA,alizarin red S staining and real-time PCR were applied to evaluate its effect on osteogenic differentiation.Human BMSCs were also treated in vitro with TNF-α,an inflammatory factor closely related to osteoporosis,and PTB expression and osteogenic differentiation were detected by Real-time PCR and alizarin red S staining.The rat osteoporosis model was established and the expression of PTB in the BMSCs was detected by Real-time PCR.Flow cytometry was used to detect intracellular ROS in human BMSCs after PTB knockdown.All quantitative data were displayed as mean±standard deviation,student t test was applied for the comparison between the two groups and the difference was statistically significant when P<0.05.Results PTB expression increased during osteogenic differentiation of BMSCs in vitro.Compared with the control,the fold changes of relative expressions of osteogenic gene RUNX2 and ALP in BMSCs with PTB knockdown were 0.74±0.15 and 0.29±0.18,respectively,and the decreases were statistically significant(t=3.490,P=0.039;t=7.983,P=0.004).Consist with that,the formation of mineralized nodules decreased after PTB was knocked down.TNF-αat both 10 ng/ml(t=3.528,P=0.039)and 40 ng/ml(t=4.306,P=0.023)inhibited PTB expression and osteogenic differentiation of human BMSCs.The relative expression of PTB in BMSCs from ovariectomized rat osteoporosis models(0.00125±0.00012)was significantly lower than the sham-operated group(0.00166±0.00018)(t=3.217,P=0.032).Compared with the control group(1204.0±300.1),ROS content in BMSCs with PTB knockdown(720.7±129.7)significantly decreased(t=4.872,P=0.040).Conclusions PTB promotes the osteogenic differentiation of BMSCs,and PTB knockdown mediated down-regulation of ROS content is an important mechanism for its regulation of osteogenic differentiation of BMSCs.