姜黄素促进高糖环境下骨髓间充质干细胞成骨分化的作用及机制研究  

Effect and mechanism of curcumin on osteogenic differentiation of bone marrow mesenchymal stem cells in high glucose environment

作　　者：谢亚佳 邓真[3] 范德生[4] XIE Yajia;DENG Zhen;FAN Desheng(Department of Oral Medicine,Shanghai Stomatological Hospital,Fudan University,Shanghai 200001,China;Oral Biomedical Engineering Laboratory,Shanghai Stomatological Hospital,Fudan University,Shanghai 200001,China;Department of Traditional Chinese Osteopathy and traumatology,Baoshan Branch,Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine,Shanghai 201900,China;Department of Pathology,Baoshan Branch,Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine,Shanghai 201900,China)

机构地区：[1]复旦大学附属口腔医院(筹),上海市口腔病防治院口腔内科,上海200001 [2]复旦大学附属口腔医院(筹)口腔生物医学工程实验室,上海200001 [3]上海中医药大学附属曙光医院宝山分院中医骨伤科,上海201900 [4]上海中医药大学附属曙光医院宝山分院病理科,上海201900

出　　处：《上海中医药杂志》2020年第2期85-90,共6页Shanghai Journal of Traditional Chinese Medicine

基　　金：上海市自然科学基金项目(19ZR1438100);上海市宝山区科委科技创新专项资金项目(18-E-9);上海中医药大学附属曙光医院宝山分院国自然培育项目(GZRPYJJ-201804);上海市口腔病防治院院级课题(SSDC-2016-09).

摘　　要：目的研究姜黄素对高糖环境下骨髓间充质干细胞(BMSCs)成骨分化的影响及可能机制。方法原代分离培养大鼠骨髓间充质干细胞,分为正常组、高糖组和高糖+姜黄素组。CCK-8检测细胞增殖;免疫荧光染色检测细胞NF-κBp65核转位;Westernblot检测NF-κBp65核蛋白表达。成骨能力检测分为正常糖浓度成骨诱导组、高糖浓度成骨诱导组、高糖+姜黄素成骨诱导组,其中成骨诱导培养为每100mLα-MEM培养基中加入2mmol/L谷氨酰胺、10mmol/Lβ-甘油磷酸钠、10nmol/L地塞米松、50mg/L抗坏血酸。茜素红染色检测细胞钙结节形成,荧光定量PCR检测成骨相关基因Runx2和OCNmRNA表达。结果接种培养7d,倒置显微镜下可见骨髓间充质干细胞集落,呈克隆性生长;传代培养以后细胞伸展为长梭形。CCK-8结果表明,与正常组比较,高糖组细胞1d、3d时增殖能力无显著性差异(P>0.05);5d时与正常组比较,高糖组细胞增殖能力显著降低(P<0.05);1d、3d、5d时姜黄素组与高糖组比较细胞增殖能力差异均无统计学意义(P>0.05)。与正常组比较,高糖组细胞NF-κBp65活化入核增多,姜黄素组则抑制高糖浓度下细胞NF-κBp65核转位,Westernblot显示姜黄素可抑制NF-κBp65核蛋白表达。各组细胞成骨诱导14d后茜素红染色,正常组可见大量矿化结节,高糖组仅见少量矿化结节,而姜黄素处理可以明显促进高糖浓度下钙结节形成。PCR结果显示,各组细胞成骨诱导14d后,与正常组比较,高糖组成骨分化相关基因Runx2、OCNmRNA表达显著降低,差异有统计学意义(P<0.05);高糖+姜黄素组则可以促进高糖环境下骨髓间充质干细胞成骨分化相关基因Runx2、OCNmRNA表达,差异有统计学意义(P<0.05)。结论姜黄素能促进高糖环境下骨髓间充质干细胞成骨分化,其机制可能与抑制高糖浓度下细胞NF-κBp65活化相关。Objective To study the effect of curcumin on osteogenic differentiation of bone marrow mesenchymal stem cells(BMSCs)in high glucose environment and its possible mechanism.Methods Rat bone marrow mesenchymal stem cells were isolated and cultured in vitro.The cells were divided into normal glucose concentration group,high glucose concentration group and high glucose+curcumin group.CCK-8 was used to detect the cell proliferation activity;The nuclear translocation of NF-kappa B p65 was detected by immunofluorescence staining and the expression of NF-kappa B p65 was detected by Western blot.The osteogenic ability was divided into three groups:normal glucose concentration osteogenic induction group,high glucose concentration osteogenic induction group and high glucose+curcumin osteogenic induction group,in which 2 mmol/L glutamine,10 mmol/Lβ-glycerophosphate,10 nmol/L dexamethasone and 50 mg/L ascorbic acid were added into every 100 mlα-MEM medium.Alizarin red staining was used to detect the formation of calcium nodules.The expression of Runx2 and OCN mR NA was detected by fluorescence quantitative PCR.Results After 7 days of inoculation and culture,BMSCs were found to grow clonally under inverted microscope,and after subculture,the cells extended into long fusiform.CCK-8 results showed that there was no significant difference in cell proliferation activity between the high glucose group and the normal group on day 1 and day 3(P>0.05);The cell proliferation activity in the high glucose group was significantly lower than that in the normal group on day 5(P<0.05);There was no significant difference in cell proliferation activity between the curcumin group and the high glucose group on day 1,day 3 and day 5(P>0.05).Compared with the normal glucose concentration group,the activation of NF-κB p65 increased in the high glucose concentration group,while the curcumin group inhibited the nuclear translocation of NF-κB p65 in the high glucose concentration.Western blot showed that curcumin could inhibit the nucleoprotein exp

关 键 词：姜黄素 糖尿病骨质疏松 高糖 骨髓间充质干细胞 成骨分化 

分 类 号：R285.5[医药卫生—中药学]