BMP-2,BGP mRNA表达量及ALP活性的变化对大鼠骨髓间充质干细胞体外诱导成骨细胞的影响

BMP-2,BGP mRNA Expression Quantity and ALP Active Change to Rat Bone Marrow Mesenchymal Stem Cell in Vitro Induction Osteoblasts Influence

目的建立骨髓间充质干细胞(BMSCs)体外分离培养体系,探讨BMSCs诱导条件下骨向分化能力和骨形成蛋白-2(BMP-2)、骨钙素(BGP)mRNA表达量及ALP活性的变化对成骨细胞增殖分化的影响。方法采用密度梯度离心法和贴壁培养法分离大鼠BMSCs,观察细胞形态学特征;应用成骨诱导体系诱导BMSCs向成骨细胞进行分化;通过细胞形态学观察、组织化学染色鉴定,酶联免疫分析仪和RT-PCR分别定量检测7 d和14 d ALP活性及BMP-2,BGP mRNA表达。结果用密度梯度离心法结合贴壁培养法能获得高纯度的BMSCs;经骨向诱导7 d和14 d后,ALP和矿化结节染色阳性;骨向诱导组:14 d ALP活性82±1.0 U/L,mRNA表达BMP-2为6.04±0.02,BGP为3.21±0.02,分别较7 d(33±2.0 U/L,1.40±0.01,1.1±0.01)显著增高(P<0.01)。未诱导组:ALP活性低(12±1.0 U/L),BMP-2,BGP mRNA未表达,无矿化结节形成。结果显示:随培养时间延长,BMP-2增高的同时ALP活性及BGP表达量也明显增高,细胞形态发生改变并向成骨方向分化,表明与形成成骨细胞有关。结论将BMSCs成功诱导分化为成骨细胞,并建立了体外分离培养体系。提示BMP-2,BGP mRNA表达量的增加及ALP活性增高,与细胞骨向分化具有较高的相关性。

Objective To establish a system of isolating and cultivation of bone mesenchymal stem cells （BMSCs） from the bone marrow in vitro and to study the ability of osteoinductive induced by BMSCs. The effect of the expression of BMP-2, BGPmRNA and the changes of ALP activity on proliferation and differentiation of osteoblast were also studied. Methords Rat bone marrow stromal cells （BMSCs） were isolated by the methods of density centrifugation and adhesive culture. Cell morphology was observed,BMP were induced into ostoblast by the mechanism of osteoinductive. The ALP activity and the exp ression of BMP-2,BGP mRNA were detected quantitatively by observation of cell morphology,histochemical staining,enzyme immunoassay and RT-PCR on the 7d and 14d. Results High pure BMSCs was obtained by the methods of density centrifugation combined with adhesive culture. Both ALP activity and mineraliztion node staining were positive in bmscs after osteogenic differentiation 7d and 14d. osteogenic differentiation group ：the activity of ALP（82± 1.0 U/L）, the expression of BMP-2（6.04±0.02）,BGP mRNA（3. 21±0. 02） on the 14 d were significantly higher than that on the 7d. Uninduced group ：the activity of ALP was low. No expression of BMP-2 ,BGP mRNA,no formation of the mineralized nodules either. The results showed that ：along with prolongation of the culture time ,the expression of BMP-2 became higher. The ALP activity and the expression of BGP mRNA were higher also. Cells showed changes in morphology differentiating into osteoblasts. All these indicate ：there was a relation between the formationg of osteoblast. Conclusion BMSCs could have been induced to osteoblast successfully and a system of isolating and cultivation in vitro was established. It is suggested that osteoinductive has a high relativity with the increase of the ALP activity and the higher expression of the BMP-2,BC-PmRNA.