颌骨骨髓基质细胞对牙周膜细胞生物学特性的影响

Inductive effects of canine maxillary bone marrow stromal cells onbiological features of canine periodontal ligament cells

关键词:  颌骨骨髓基质细胞  牙周膜细胞  共培养  条件培养液

Key words:  maxillary bone marrow stromal cells  periodontal ligament cells  co-culture  condi-tioned medium

摘要:

目的观察间接共培养犬颌骨骨髓基质细胞(canine maxillary bone marrow stromal cells, cM-BMSCs)对犬牙周膜细胞(canine periodontal ligament cells, cPDLCs)生物学特性的影响。方法 原代培养犬颌骨骨髓基质细胞和犬牙周膜细胞。MTT法检测犬牙周膜细胞增殖速率；利用Transwell结构建立犬颌骨骨髓基质细胞与犬牙周膜细胞共培养体系；qPCR法检测犬牙周膜细胞矿化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；Western印迹法检测Runx2和OCN蛋白的表达变化。构建牙片/细胞膜片复合体植入裸鼠皮下。结果 两种细胞体外均贴壁生长,呈纺锤状外形；颌骨骨髓基质细胞间接共培养下对牙周膜细胞增殖有抑制作用；共培养组牙周膜细胞ALP活性高于对照组；qPCR和Western印迹法均能检测到牙周膜细胞Runx2、OCN的表达,共培养组牙周膜细胞的Runx2和OCN的表达均高于对照组。经过颌骨骨髓基质细胞条件培养液诱导后的牙周膜细胞与牙本质片复合形成了排列更加有序的牙周膜/牙骨质样结构,单纯的牙周膜细胞膜片不能新生类似的牙周组织复合结构。结论犬颌骨骨髓基质细胞间接共培养情况下可能会限制牙周膜细胞的增殖,促进牙周膜细胞向成骨样细胞分化。

Abstract:

Objective To investigate the effect of canine maxillary bone marrow stromal cells (cM-BMSCs) on biological features of canine periodontal ligament cells (cPDLCs). Methods Primary cM-BMSCs were co-cultured with cPDLCs, the cell proliferation was examined by MTT method. Total mRNA and protein were extracted at d0, d3 and d7, the expression of ALP, Runx2 and OCN mRNAs was detected by qPCR; the protein levels of Runx2 and OCN were measured by Western-blotting. cM-BMSCs conditioned medium (cM-BMSCs-CM) was used to mimic the environment of maxilla and to induce the differentiation of cPDLCs. A cell sheetstrategy was chosen for the construction of periodontal tissue in vivo. Results Both cPDLCs and cM-BMSCs showed spindle-like morphology. Conditioned medium of cultured maxillary bone marrow stromal cells retarded the proliferation of cPDLCs. The expressions of ALP, Runx2 and OCN mRNAs were up-regulated in co-cultured group; and the protein levels of Runx2 and OCN in test group were higher than those in control group. cPDLCs treated by the cM-BMSCs-CM produced a cementum/PDL-like structure; however, cPDLCs alone failed to form the similar cementum/PDL-like structure in nude mice. Conclusion Conditioned medium of cM-BMSCs may hinder the proliferation of cPDLCs and promote the differentiation of cPDLCs.