Zou Lijin, Lv Nonghua, Feng Wenzhou
Burn Center, the First Affiliated Hospital of Nanchang University, Nanchang Jiangxi, 330006, PR China.
Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi. 2007 Nov;21(11):1222-7.
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, 3-month-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 x 10(-8) mmol/L dexamethasone (Dex), 10 mmol/L beta-glycerophosphate (beta-GP), 82 microg/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 alpha1 (Cbfalpha1), osterix, ALP, collagen I (Col I ), osteonectin (ON) and osteocalcin (OC) increased gradually. Cbfalpha1, 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 ostcogenic conditions.
目的：观察猪骨髓间充质干细胞（MSCs）成骨分化过程中的特征及相关基因表达。 方法：从6头3月龄、体重约50 kg的长白猪的胫骨近端骨髓腔中抽取骨髓。采用Ficoll密度梯度离心结合贴壁培养法分离并纯化MSCs。将第1代MSCs在含1×10⁻⁸ mmol/L地塞米松（Dex）、10 mmol/Lβ-甘油磷酸钠（β-GP）、82 μg/ml抗坏血酸（Asc）和10%灭活胎牛血清（FBS）的DMEM中培养21天。将MSCs在基础DMEM中培养作为对照。通过显微镜观察细胞形态。使用荧光染料SYBR green I检测法检测细胞增殖。通过碱性磷酸酶（ALP）组织化学染色、定量钙沉积和实时PCR技术评估成骨分化。 结果：原代MSCs的特征：第1天，大多数细胞呈现圆形且漂浮的造血细胞。去除非贴壁细胞后第3天观察到由成纤维样细胞组成的集落，第7天集落生长至不同大小。原代培养中发生了13次群体倍增。成骨分化：在成骨诱导过程中，MSCs的细胞形态从成纤维形状变为立方形。与基础培养基相比，成骨培养基对细胞增殖无影响（P>0.05）。第14天，ALP染色呈强阳性。第21天钙沉积明显增加（P<0.01）。此外，核心结合因子α1（Cbfalpha1）、osterix、ALP、Ⅰ型胶原（ColⅠ）、骨连接蛋白（ON）和骨钙素（OC）的mRNA水平逐渐升高。Cbfalpha1、ON和ALP基因在成骨分化早期增加。与第7天相比，第21天osterix和OC显著增加（P<0.05）。ColⅠ在第14天增加（P<0.05）。 结论：通过密度梯度离心从骨髓中收获的猪MSCs能够在体外进行成骨分化。成骨分化潜能依赖于在成骨条件下该谱系特异性基因的上调。
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