不同浓度的大黄素对人乳牙牙髓干细胞增殖和成骨分化的影响  

Effect of emodin at different concentrations on proliferation and osteogenic differentiation of stem cells from human exfoliated deciduous teeth

作　　者：岳海云 徐广振 毕迎春 YUE Hai-yun;XU Guang-zhen;BI Ying-chun(Department of Basic Medical Sciences,Department of Neurosurgery,Department of Stomatology,the 960th Hospital of the PLA,Jinan 250031,China)

机构地区：[1]解放军第九六〇医院基础医学实验室,济南250031 [2]解放军第九六〇医院神经外科,济南250031 [3]解放军第九六〇医院口腔科,济南250031

出　　处：《口腔颌面修复学杂志》2024年第6期409-415,共7页Chinese Journal of Prosthodontics

基　　金：解放军第九六〇医院院长基金面上项目(项目编号:2021MS03,2023MS04)。

摘　　要：目的:研究大黄素对人乳牙牙髓干细胞的增殖和成骨分化的影响。方法:分离人乳牙牙髓干细胞,镜下观察细胞形态,成骨诱导后经茜素红染色和碱性磷酸酶染色鉴定其成骨分化能力,流式细胞术检测细胞表面标记物鉴定其干细胞源性。不同浓度的大黄素预处理人乳牙牙髓干细胞,通过CCK-8法检测细胞的增殖能力,茜素红染色、碱性磷酸酶染色检测细胞成骨分化能力,通过RT-qPCR检测碱性磷酸酶、RUNX2和骨钙素的mRNA表达,Western blot检测成骨相关蛋白RUNX2的蛋白表达。结果:①CCK-8结果显示,大黄素在浓度为0.1-10μmol/L时可以明显促进人乳牙牙髓干细胞的增殖,而浓度为100μmol/L则显著抑制了细胞的增殖;②碱性磷酸酶染色和茜素红染色显示,随着大黄素浓度的升高,碱性磷酸酶染色和茜素红染色逐渐加深,10μmol/L大黄素处理组碱性磷酸酶染色最深,矿化结节数量也最多;③RT-qPCR结果显示,大黄素促进碱性磷酸酶、RUNX2和骨钙素mRNA的表达量(P<0.05)。④Western blot结果显示,成骨相关蛋白RUNX2的表达随着大黄素的处理浓度增加而增加。结论:大黄素在1-10μmol/L范围内对人乳牙牙髓干细胞的增殖和成骨分化具有促进作用,且浓度为10μmol/L时效果最明显。Objective:In order to explore the impact of emodin on the proliferation and osteogenic differentiation of stem cells from human exfoliated deciduous teeth(SHED).Methods:The morphology of the cells was observed under a microscope.Following osteogenic induction,the osteogenic differentiation capacity of the stem cells was determined by alizarin red staining and alkaline phosphatase staining.SHEDs were exposed to varying concentrations of emodin.The proliferation capacity of the cells was determined using the CCK-8 method.Alizarin red staining and alkaline phosphatase staining were utilized to determine the osteogenic differentiation capacity of the cells.The mRNA expressions of alkaline phosphatase,RUNX2,and osteocalcin were detected by RT-qPCR.Western blot was employed to ascertain the protein expression of RUNX2.Results:①The CCK-8 results demonstrated that,at concentrations of 0.1-10μmol/L,emodin greatly increased the proliferation of SHEDs,whereas at concentrations of 100μmol/L,emodin significantly reduced the proliferation of SHEDs.②The alkaline phosphatase staining and alizarin red staining showed that with the increase of emodin concentration,the alkaline phosphatase staining and alizarin red staining gradually deepened,and the 10μmol/L emodin treatment group showed the deepest alkaline phosphatase staining and the largest number of mineralized nodules.③RT-qPCR results showed that emodin promoted the expression of early osteogenic differentiation related genes alkaline phosphatase and RUNX2 and late osteogenic differentiation related gene osteocalcin mRNA(P<0.05).④Western blot results showed that the expression of RUNX2 was increased with the increase of emodin concentration in human primary tooth pulp stem cells.Conclusion:Emodin can promote the proliferation and osteogenic differentiation of SHEDs in the range of 1-10μmol/L,and the effect is most obvious at the concentration of 10μmol/L.

关 键 词：乳牙牙髓干细胞 成骨分化 大黄素 牙髓干细胞 

分 类 号：R780.2[医药卫生—口腔医学]