体外诱导家猪骨髓间充质干细胞向成骨细胞分化

CHARACTERISTICS OF OSTEOBLASTIC DIFFERENTIATION IN MESENCHYMAL STEM CELLS FROM PORCINE BONE MARROW IN VITRO

目的观察家猪骨髓间充质干细胞(marrow mesenchymal stem cells,MSCs)在体外诱导条件下成骨分化的特征及相关基因的表达。方法选取3月龄雌性长白猪6头,无菌条件下于胫骨近端抽取骨髓15ml。采用密度梯度离心法并根据细胞贴壁特性对MSCs进行分离、纯化,倒置相差显微镜观察原代细胞生长情况,于培养第7天计算MSCs百分比含量及群体倍增值。将第1代细胞置于含1×10-8mmol/L地塞米松(dexamethasone,Dex)、10mmol/Lβ-磷酸甘油(β-glycerophosphate,β-GP)和82μg/ml抗坏血酸(ascorbic acid,Asc)的成骨诱导培养液中培养21d,作为实验组;DMEM培养液中培养作为对照组。分别行细胞形态学观察、碱性磷酸酶(alkaline phosphatase,ALP)组织化学染色、钙沉积和细胞增殖测定,采用实时定量PCR分析成骨分化的相关基因表达。结果原代MSCs特征:培养第1天,有核细胞大部分由悬浮的圆形血源性细胞组成;第3天换液弃除非贴壁细胞,MSCs克隆开始形成,细胞呈成纤维细胞样生长;第7天,镜下观察到大小不一的克隆。细胞在培养后12～14d基本长满,原代细胞群体倍增值平均为13。MSCs成骨分化:诱导培养14d实验组细胞形态由成纤维细胞样变成立方体样,而对照组细胞始终保持成纤维细胞样。培养5d,对照组细胞计数为11723±4040,实验组为10276±5513,二者差异无统计学意义(P>0.05)。与对照组相比,实验组诱导培养14d,ALP染色呈强阳性,21d钙沉积明显增加(P<0.01)。实验组MSCs成骨相关基因:核心结合因子α1(corebinding factorα1,Cbfα1)、osterix、ALP、型胶原、骨连接素(osteonectin,ON)、骨钙素(osteocalcin,OC)表达逐渐增强;Cbfα1、ALP、ON在分化早期增加明显;与第7天比较,第21天osterix和OC基因表达明显上调(P<0.05);第14天,型胶原表达也上调(P<0.05)。结论密度梯度离心法分离的猪MSCs,在体外诱导条件下能通过上调分化特异基因表达向成骨细胞分化。

Objective To observe the characteristics and related gene expression of osteoblastic differentiation in porcine bone marrow mesenchymal stem cells （MSCs） during. Methods Bone marrow from 6 landrace pigs, 3month-old about 50 kg, was aspirated from the medullary cavity of the proximal tibia. The MSCs were isolated, and purified by Ficoll density gradient centrifugation combined with adherent culture method. The MSCs from passage 1 were cultivated in DMEM with 1 × 10 s mmol/L dexamethasone （Dex）, 10 mmol/L β-glycerophosphate （13-GP）, 82 μg/ml ascorbic acid （Asc） and 10% inactivated fetal bovine serum （FBS） up to 21 days. The MSCs were cultivated in basic DMEM as a control. Cell morphology was observed by microscope. Cell proliferation was tested by using the fluorescent dye SYBR green I measurement. Osteoblastic differentiation was evaluated by alkaline phosphatase （ALP） histochemical staining, quantitative calcium deposit, and real-time PCR technology. Results Characterization of primary MSCs： At day 1, most cells depicted round and floating hematopoietic cells. Colonies consisting of fibroblastlike cells were observed at day 3 after removal of non-adherent cells, colonies grew to various sizes at day 7. Thirteen population doublings took place in primary culture. Osteoblastic differentiation： During osteogenic stimulation, cellular morphology of MSCs changed from a fibroblastic shape to a cubical form. Cell proliferation had no impact in osteogenic medium compared to basic medium （P〉0.05）. At day 14, ALP staining presented strong positive. Calcium deposit pronouncedly increased at day 21 （P〈0.01）. Furthermore, the mRNA levels of core binding factor al （Cbfαl）,osterix, ALP, collagen I （Col I ）, osteonectin （ON） and osteocalcin （OC） increased gradually. Cbfαl, ON and ALP genes increased at early stage of osteoblastic differentiation. Osterix and OC at day 21 were significantly increased when compared with that at day 7 （P〈0.05）. Col I was increased at day 14 （P〈0.05）. Conclusion Porcine MSCs harvested from bone marrow by density gradient centrifugation are capable of osteoblastic differentiation in vitro. The potential of osteoblastic differentiation relies upon upregulation of genes specific to this lineage under the osteogenic conditions.