犬骨髓基质细胞矿化不同阶段对犬牙周膜干细胞增殖和分化的影响

Effects of canine bone marrow stromal cells after different stages of mineralization on the differentiation, proliferation of canine periodontal ligament stem cells

目的观察矿化不同阶段犬髂骨骨髓基质细胞(iliac bone marrow stromal cells,I-BMSCs)条件培养液对犬牙周膜干细胞(canine periodontal ligament stem cells,PDLSCs)增殖和分化的影响,为分析骨髓基质细胞调控牙周膜干细胞的生物学行为奠定基础。方法体外培养犬骨髓基质细胞和流式细胞术分选纯化PDLSCs。取第三代PDLSCs,分别加入髂骨骨髓基质细胞矿化条件培养液(iliac bone marrow stromal cells conditioned me-dium,I-BMSCs-CM)行间接共培养PDLSCs。Control组:单纯的未诱导的PDLSCs,用含15%FBS的DMEM培养液常规培养;实验组为I-BMSCs-CM组、I-BMSCs-CM-10ds组、I-BMSCs-CM-15ds组。I-BMSCs-CM组:I-BMSCs诱导的PDLSCs;I-BMSCs-CM-10ds组:成骨诱导10 d的I-BMSCs诱导的PDLSCs;I-BMSCs-CM-15ds组:成骨诱导15 d的I-BMSCs诱导的PDLSCs。MTT法绘制细胞增殖曲线;qRT-PCR检测PDLSCs成骨分化相关基因的表达:特异AT序列结合蛋白2(special AT-rich sequence binding protein 2,Satb2)、核心结合因子2(runt-related tran-scription factor 2,Runx2)、骨钙素(osteocalcin,OCN);Western blot检测Satb2、Runx2和OCN蛋白表达变化。结果PDLSCs呈长梭形,表达波形丝蛋白,具备成骨及脂向分化能力。生长曲线显示I-BMSCs能促进PDLSCs的增殖,I-BMSCs-CM-15ds促进PDLSCs增殖最为明显(F=342.8,P=0.017)。qRT-PCR和Western blot均能检测到PDLSCs的Satb2、Runx2和OCN的表达,第7天时实验组的PDLSCs的Satb2、Runx2和OCN的表达均高于对照组。I-BMSCs-CM-15ds上调PDLSCs的Satb2、Runx2、OCN蛋白表达最为显著,分别是Control组的3.04倍(F_(Satb2)=24.48,P=0.014)、5.1倍(F_(Runx2)=12.25,P <0.001)和3.67倍(F_(OCN)=18.35,P=0.022)。结论矿化程度较高的I-BMSCs更能促进PDLSCs增殖和向成骨样细胞分化,提示终末分化骨细胞可能诱发了PDLSCs的成骨分化。

Objective To investigate the inductive effects of canine periodontal ligament stem cells （PDLSCs） co-cultured with canine disparate differentiating-period iliac bone marrow stromal cells （I-BMSCs）. Methods Purified PDLSCs were isolated by in vitro culture of cBMSCs and flow- cytometl7. Third-generation PDLSCs were obtained, and conditioned culture medium derived fromiliac bone marrow stromal cells （I-BMSCs-CM） was added as indicated for co-culture of PDLSCs. As the control group, pure uninduced PDLSCs were routinely cuhtued in DMEM cuhure medium containing 15%FBS. The experimental groups included the I-BMSCs-CM, I-BMSCs-CM-10ds and I-BMSCs-CM-15ds groups. The I-BMSCs-CM group consisted of PDLSCs induced by I-BMSCs, the I-BMSCs-CM-10ds group consisted of PDLSCs induced by I-BMSCs after osteogenic induction for 10 days, and the I-BMSCs-CM-15ds group consisted of PDLSCs induced by I-BMSCs after osteogenic induction for 15 days. The cocultured PDLSCs w-ere examined via the MTF assay. Total mRNA and protein were prepared at 3 and 7 days. The mRNA expression levels of runt-related tran-scription factor 2（Runx2）, special AT-rich sequence binding protein 2（Satb2） and osteocalcin （OCN） were measured by qRT-PCR. The protein expression levels of Satb2, Runx2 and OCN were detected by Western blot. Results The PDLSCs showed a spindle-like mollohology. While the BMSC-conditioned media increased PDLSCs proliferation, the media conditioned by BMSCs allowed to differentiate for 15 days （I-BMSCs-CM-15days） significantly enhanced PDLSCs proliferation （F= 342.8, P = 0.017）. The expression levels of the analyzed genes were upregulated in the coculture groups, and the protein expression levels of Sath2, 1Flunx2 and OCN were higher in the test groups than in the control group at 7 days. At the protein level, I-BMSCs-CM-15days upregulated the expression of Satb2 by 3.04-fold （Fso,b2 = 24.48, P = 0.014）, 1Runx2 by 5.1-fold （FRomax2 = 12.25, P 〈 0.001）, and OCN by 3.67-fold （FOCN = 18.35, P = 0.022）. Con-elusion The conditioned medium of I-BMSCs may enhance the proliferation of PDLSCs, and that of terminally differen-tiated bone cells probably triggered the osteogenesis of PDLSCs, suggesting important implications for periodontal engi-neering.