牙周膜单克隆细胞间成骨异质性及组蛋白乙酰转移酶影响细胞交流的研究

Effects of cell-to-cell communication and histone acetyltransferase on the change of osteogenic differentiation ability among single-cell clones from healthy periodontium with heterogeneity of osteogenic differentiation abilities

目的 探讨成骨分化能力存在差异的正常牙周膜单克隆细胞之间信息交流对单克隆细胞成骨分化能力的影响,以及组蛋白乙酰转移酶在其中的作用.方法 通过有限稀释法获取正常牙周膜来源的单克隆细胞,采用间接共培养法在6孔板铺有一定成骨能力的单克隆细胞,设置空白对照组、成骨弱组及成骨强组,分别对应Transwell小室不加细胞、加成骨能力弱和成骨能力强的细胞,每组设置3个复孔,间接共培养4 d后移除Transwell小室进行成骨诱导,实时荧光定量PCR（quantitative real-time PCR,qPCR）及茜素红染色检测3组下层细胞成骨诱导后成骨能力的改变,蛋白质印迹法检测下层细胞组蛋白H3的乙酰化水平,qPCR比较组蛋白乙酰转移酶表达的改变.结果 流式细胞术检测结果显示牙周膜单克隆细胞阳性表达间充质干细胞表面标志物CD29、CD90、CD105和CD146,表达率分别为（99.80±0.02）%、（99.36±0.18）%、（99.41±0.05）%和（95.10±2.11）%;阴性表达CD31和CD34,表达率分别为（0.29±0.11）%和（0.22±0.13）%.茜素红及油红O染色显示单克隆细胞具有成骨成脂分化能力.碱性磷酸酶和茜素红染色结果显示同一个体牙周膜来源的不同单克隆细胞间存在成骨差异.间接共培养实验显示成骨强和弱的单克隆细胞成骨相关基因骨钙蛋白（分别为14.24±5.60、4.78±2.90）及Runt相关转录因子2（Runt-related transcription factor 2,RUNX2）mRNA表达量（分别为2.75±1.44、1.61±0.44）均显著高于空白对照组（骨钙蛋白与RUNX2分别为1.00±0.47和1.00±0.39）（P〈0.05）;成骨强组骨钙蛋白及RUNX2 mRNA相对表达量均显著高于成骨弱组（P〈0.05）.蛋白质印迹法结果显示,成骨强及成骨弱组组蛋白H3乙酰化水平[分别为（0.76±0.09）和（0.54±0.12）]均显著高于空白对照组（0.30±0.04）（P〈0.05）.qPCR结果显示组蛋白乙酰转移酶中KAT6A变化最显著,趋势与组蛋白H3的乙酰化水平趋势相同.结论 正常牙周膜来源的单克隆细胞间成骨分化能力存在差异,成骨能力强的单克隆细胞可能通过促进其他细胞KAT6A上调影响其组蛋白乙酰化水平,促进其成骨分化.

Objective To investigate the effect of cell-to-cell communication amongst single-cell clones from healthy periodontium with different osteogenic differentiation potentials on change of osteogenic differentiation capabilities and the role histone acetyltransferase partaken in this process. Methods In order to research the change of osteogenic differentiation ability via cell-to-cell communication, indirect co-culture method was used by placing two single-cell clones with different osteogenesis potentials in each of the 6-well plates. Blank control, weak and strong osteogenic groups were set up, corresponding to Transwell chambers with blank, cells of weak osteogenesis ability and cells of strong osteogenesis ability, respectively. Each group was made in triplicate. After co-culture for four days, Transwell chamber was removed. Quantitative real-time PCR （qPCR） and alizarin red staining were employed to detect the change of osteogenic differentiation ability. The acetylation level of H3 was measured by using Western blotting. Histone acetyltransferases were detected by qPCR. Results Single-cell clones were ensured from mesenchymal stem cells by flow cytometer, the positive expression of CD29, CD90, CD105, CD146 was （99.80 ± 0.02）%, （99.36 ± 0.18）%, （99.41 ± 0.05）% and （95.10 ± 2.11）%, respectively. And CD31 and CD34 expression were （0.29±0.11）%and （0.22±0.13）%, respectively. Alizarin red and oil red O staining confirmed that single-cell clones had the abilities of adipogenesis and osteogenesis. Alkaline phosphatase （ALP） and alizarin red staining indicated that different single-cell clones were heterogeneity in osteogenesis differentiation. Indirect co-culture indicated that the mRNA expression of osteocalcin （OCN） were 14.24 ± 5.60 and 4.78±2.90, respectively and Runt-related transcription factor 2 （RUNX2） were 2.75±1.44 and 1.61± 0.44, respectively, in strong and weak osteogenic groups. They were significantly higher compared to the blank group （the mRNA expression of OCN and RUNX2 were 1.00±0.47 and 1.00±0.39, respectively）. The expression of OCN and RUNX2 were also higher in strong osteogenic group than that in weak osteogenic group （P〈0.05）. The mean gray level of the acetylation of H3 in strong osteogenic group （0.76 ± 0.09） and weak osteogenic group （0.54±0.12） were also higher than that in the blank group （0.30±0.04）（P〈0.05）. qPCR results showed that KAT6A in strong osteogenic group exhibiting higher expression （P〈0.05） compared to weak osteogenic group and the blank group, which were corresponding to the changes of acetylation levels. Conclusions Single-cell clones from healthy periodontium showed heterogeneity in osteogenic differentiation abilities. Single-cell clones with strong osteogenesis abilities had an advantage over others by promoting others＇ osteogenesis differentiation and this change mediated by cell-to-cell communication might be caused by modulating KAT6A to affect the acetylation level of histone.