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目的研究牙周组织改建过程中,牙周膜干细胞对破骨细胞形成的影响,初步探讨静压力和诱导因子对牙周膜干细胞调控破骨细胞形成的作用机制。方法有限稀释法克隆化培养牙周膜干细胞,密度梯度离心法分离脐血,收集单个核细胞。建立直接共培养和Transwell间接共培养体系。实验分组:A组,对照组(单纯共培养);B组,静压力组,共培养后第2天即对共培养细胞施加120 KPa压力,持续作用1 h后继续培养;C组,诱导因子组,培养液中加入诱导因子1,25二羟基维生素D3(1×10-8mol/L)和前列腺素E2(1×10-6mol/L)。采用倒置相差显微镜及TRAP染色,骨磨片染色等方法检测破骨样细胞(osteoclast-like cells,OLC)的形成及功能。结果各组第3、7天OLC计数比较结果显示,施加静压力后,直接共培养组OLC数量明显多于间接共培养组(P<0.01);而诱导因子组,直接共培养组OLC少于间接共培养组(P<0.01)。结论牙周膜干细胞能够促进脐血单个核细胞分化为破骨样细胞,且在静压力作用下,两种细胞直接接触时,这种调控作用更为明显。
Objective To study the effect of periodontal ligament stem cells on the formation of osteoclasts during periodontal remodeling and to explore the mechanism of action of static pressure and inducing factors on the regulation of osteoclasts by periodontal ligament stem cells. Methods The periodontal ligament stem cells were cloned by limiting dilution method. The cord blood was separated by density gradient centrifugation and the mononuclear cells were collected. Establish direct co-culture and Transwell indirect co-culture system. Group A, control group (simple co-culture); Group B, static pressure group, co-cultured on the first two days after the application of 120 KPa pressure on the co-cultured cells, continued for 1 h and then cultured; C group, induced factor In the culture medium, 1,25 dihydroxyvitamin D3 (1 × 10-8 mol / L) and prostaglandin E2 (1 × 10-6 mol / L) were added into the culture medium. The formation and function of osteoclast-like cells (OLCs) were detected by inverted phase contrast microscope, TRAP staining and bone fragment staining. Results OLC count comparison on the 3rd and 7th day in each group showed that after the static pressure was applied, the number of OLC in the direct co-culture group was significantly more than that in the indirect co-culture group (P <0.01); while in the induction factor group and direct co-culture group, Indirect co-culture group (P <0.01). Conclusions Periodontal ligament stem cells can promote the differentiation of umbilical cord blood mononuclear cells into osteoclast-like cells. Under the action of static pressure, the effect of this regulation is more obvious when the two kinds of cells are directly contacted.