摘要： 目的：研究犬髂骨骨髓基质细胞( iliac bone marrow stromal cells, I-BMSCs)和犬颌骨骨髓基质细胞（maxilla bone marrow stromal cells，M-BMSCs）对犬牙周膜细胞（periodontal ligament cells，PDLCs）增殖和成骨分化的影响，了解不同部位来源的骨髓基质细胞对牙周膜细胞调控作用的差异性。方法：原代培养犬髂骨骨髓基质细胞、颌骨骨髓基质细胞和牙周膜细胞。分别建立犬髂骨和颌骨来源的骨髓基质细胞与牙周膜细胞的Transwell共培养体系；制备骨髓基质细胞条件培养液培养牙周膜细胞，MTT法检测牙周膜细胞生长曲线；利用实时荧光定量PCR法检测牙周膜细胞成骨相关基因核心结合因子2（Runx2）、碱性磷酸酶（ALP）、骨钙素（OCN）的变化；Westernblot法检测牙周膜细胞Runx2和OCN蛋白的表达变化。结果：髂骨骨髓基质细胞条件培养液能促进牙周膜细胞的增殖；颌骨骨髓基质细胞条件培养液对牙周膜细胞增殖有抑制作用；QPCR检测到共培养组牙周膜细胞Runx2、OCN、ALP基因表达高于对照组，Westernblot检测到共培养组牙周膜细胞的Runx2和OCN的蛋白表达高于对照组，且颌骨骨髓基质细胞诱导牙周膜细胞成骨分化效果更显著。结论：犬颌骨骨髓基质细胞条件培养液可能会抑制牙周膜细胞的增殖，相较于髂骨骨髓基质细胞，颌骨骨髓基质细胞促进牙周膜细胞成骨分化效果更显著，颌骨骨髓基质细胞和牙周膜细胞共同作为种子细胞可能更有利于牙周组织再生。

关键词: 髂骨骨髓基质细胞；  , 颌骨骨髓基质细胞；  , 牙周膜细胞；  , 共培养

Abstract: Objective: The aim of this study was to compare osteoblastic differentiation and capacity of bone marrow stromal cells (BMSCs) derived from canine mandible (M-BMSCs) vs BMSCs derived from canine iliac bone (I-BMSCs), and discuss whether the proliferation and differentiation of periodontal ligament cells (PDLCs) are associated with the diverse osteogenic potentials in vitro. Methods: Two 12-month-old male beagle dogs (weight of 15 kg) were obtained. Bone marrow stromal cells from maxilla and iliac crest and periodontal ligament cells were isolated and collected independently. BMSCs were cultured and formed single colonies recognized as passage 0(P0) BMSCs, which passed to P2-3 for subsequent experiments. P2-3 PDLCs in group 1 were cultured in ɑ-MEM conditioned media, P2-3 PDLCs in group 2 were co-cultured with M-BMSCs, P2-3 PDLCs in group 3 were co-cultured with I-BMSCs. The MTT colorimetric assay was used to assess PDLCs proliferation and viability. Total mRNA and protein were extracted at 0, 3, 7 days respectively. To compare the  in vitro osteogenic differentiation ability of M-BMSCs and I-BMSCs incubated with PDLCs, a real-time QPCR assay, Western-blot assay, ALP activity assay,  Runx2 and OCN immunofluorescence assay were performed. Results: In conditioned medium, the I-BMSCs enhanced the proliferation of PDLCs. On the contrary, the M-BMSCs retarded the proliferation of PDLCs. The expression of ALP, Runx2, and OCN gene in the co-cultured group were up-regulated. The protein levels of Runx2 and OCN in group 2 and group 3 were higher than that of group 1, especially in the induction of maxilla bone marrow stromal cells. Conclusion: Conditioned medium of M-BMSCs may retard the proliferation of PDLCs but enhance the osteogenic differentiation of PDLCs.

Key words: iliac bone marrow stromal cells, maxilla bone marrow stromal cells, periodontal ligament cells, co-culture

中图分类号: 

• R782.2

• R318