目的:明确骨细胞中的Setd2对小鼠骨量的调控作用.方法:将Dmp1Cre与Setd2flox/flox小鼠配繁,构建在骨细胞中特异性敲除Setd2的小鼠(Setd2 cKO,Dmp1Cre;Setd 2flox/flox).采用免疫组织化学染色验证Setd2特异性敲除后,对成长至6月龄的同窝小鼠骨组织进行显微CT扫描.将CT进行三维重建,观察对照小鼠与Setd2 cKO小鼠鼠的皮质骨及松质骨骨量变化.利用骨形态学分析软件分析皮质骨及松质骨骨量变化,骨小梁数量、厚度及分离度等差异.利用番红O固绿染色观察Setd2 cKO小鼠与对照小鼠中呈蓝色的骨组织区域的变化.采用免疫组织化学染色观察Setd2特异性敲除后对组蛋白H3第36位赖氨酸位点的三甲基化修饰(trimethylation of lysine 36 on histone 3,H3K36me3)的影响.结果:免疫组织化学染色结果显示Setd2在骨细胞中被成功敲除,Setd2 cKO小鼠体型小于同窝对照小鼠,显微CT及骨形态学分析结果显示,6月龄时,与对照组小鼠相比,Setd2 cKO小鼠的皮质骨变薄,骨密度降低,骨小梁厚度减小,骨小梁数量减少,骨小梁分离度增加(P＜0.05).利用番红O固绿染色观察Setd2 cKO小鼠呈蓝色的骨组织区域比对照小鼠少,免疫组织化学染色结果显示与对照组小鼠相比,Setd2 cKO小鼠的H3K36me3的修饰出现缺失.结论:骨细胞中Setd2的缺失影响骨细胞的H3K36位点的三甲基化修饰,从而导致小鼠骨量减少.
Abstract
J Objective:To elucidate the regulatory impact of Setd2 within osteocytes on bone formation.Methods:Setd2flox/flox mice were crossed with the Dmp1Cre strain to generate Dmp1Cre,Setd2flox/flox mice.All mice analyzed were maintained on the C57BL/6 background.Immunohistochemical staining was used to confirm the specific dele-tion of Setd2 in osteocytes.Micro-CT and three-dimensional reconstruction were used to analyze cortical and trabec-ular bone mass variations in bone tissues from same-litter mice at 6 months of age.Differences in bone morphology parameters,such as trabecular number,thickness,and separation,were conducted using CT An software.Addi-tionally,safranine O/fast green staining was utilized to observe changes in the blue-stained bone tissue regions in Setd2 cKO and control mice.Immunohistochemical staining was used to observe trimethylation of lysine 36 on his-tone 3(H3K36me3).Results:With successful Setd2 deletion in osteocytes,Setd2 cKO mice exhibited smaller body size compared to littermate controls.Micro-CT and bone morphometry analysis revealed that,at 6 months of age,Setd2 cKO mice displayed thinner cortical bone,decreased bone density,reduced trabecular thickness,lower tra-becular number,and increased trabecular separation compared to control mice(P＜0.05).Safranine O/fast green staining further indicated a reduced blue-stained bone tissue area in Setd2 cKO mice compared to controls.Com-pared to the control group mice,there was a loss of H3K36me3 modification in Setd2 cKO mice.Conclusion:The loss of Setd2 in osteocytes affected the trimethylation modification of H3K36,resulting in a decrease in mouse bone mass.
维普
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