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
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谢亚佳, 邓真, 范德生. 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[J]. 上海中医药杂志, 2020,54(2):85-90.
XIE Yajia, DENG Zhen, FAN Desheng. Effect and mechanism of curcumin on osteogenic differentiation of bone marrow mesenchymal stem cells in high glucose environment [J]. Shanghai Journal of Traditional Chinese Medicine, 2020,54(2):85-90.
谢亚佳, 邓真, 范德生. 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[J]. 上海中医药杂志, 2020,54(2):85-90. DOI: 10.16305/j.1007-1334.2020.02.020.
XIE Yajia, DENG Zhen, FAN Desheng. Effect and mechanism of curcumin on osteogenic differentiation of bone marrow mesenchymal stem cells in high glucose environment [J]. Shanghai Journal of Traditional Chinese Medicine, 2020,54(2):85-90. DOI: 10.16305/j.1007-1334.2020.02.020.
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-κB p65核转位;Western blot检测NF-κB p65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100 mL α-MEM培养基中加入2 mmol/L谷氨酰胺、10 mmol/L β-甘油磷酸钠、10 nmol/L地塞米松、50 mg/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OCN mRNA表达。 结果:接种培养7 d,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1 d、3 d时增殖能力无显著性差异(P>0.05);5 d时与正常组比较,高糖组细胞增殖能力显著降低(P<0.05);1 d、3 d、5 d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-κB p65活化入核增多,姜黄素组则抑制高糖浓度下细胞NF-κB p65核转位,Western blot显示姜黄素可抑制NF-κB p65核蛋白表达。 各组细胞成骨诱导14 d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14 d后,与正常组比较,高糖组成骨分化相关基因Runx2、OCN mRNA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、OCN mRNA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-κB p65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. MethodsRat 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/L β-glycerophosphate, 10 nmol/L dexamethasone and 50 mg/L ascorbic acid were added into every 100 ml α-MEM medium. Alizarin red staining was used to detect the formation of calcium nodules. The expression of Runx2 and OCN mRNA 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 expression of NF - κ B p65. After 14 days of osteogenesis induction, alizarin red staining showed a large number of mineralized nodules in the normal glucose concentration group, and only a small number of mineralized nodules in the high glucose concentration group. Curcumin treatment could significantly promote the formation of calcium nodules in the high glucose concentration group. The results of PCR showed that the expression of Runx2 and OCN mRNA in high glucose concentration group was significantly lower than that in normal glucose concentration group (P<0.05); The expression of Runx2 and OCN mRNA in osteogenic differentiation of BMSCs was significantly promoted in the high glucose + curcumin group (P<0.05). Conclusion:Curcumin could promote osteogenic differentiation of bone marrow mesenchymal stem cells in high glucose environment. The mechanism may be related to the inhibition of activation of NF-κB p65 in high glucose concentration.
姜黄素糖尿病骨质疏松高糖骨髓间充质干细胞成骨分化
curcumindiabetic osteoporosishigh glucoseBMSCsosteogenic differentiation
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