氨来呫诺对大鼠骨髓间充质干细胞成骨分化的影响
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摘要
目的探讨氨来呫诺调控大鼠骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)成骨分化的作用,为其在抗骨质疏松治疗等方面的应用提供理论依据。方法体外分离、培养SD大鼠BMSCs。(1)通过CCK8法检测不同浓度氨来呫诺对BMSCs增殖的影响;(2)设置对照组和氨来呫诺(25μmol/L)处理组,利用成骨诱导培养基进行诱导,诱导后第10天进行碱性磷酸酶(alkaline phosphatase,ALP)染色检测;诱导后第21天进行茜素红染色;(3)实时荧光定量PCR检测诱导后第3、7、14天成骨分化相关基因ALP、Runt相关转录因子2(runt-related transcription factor 2,RUNX2)、骨桥蛋白(osteopontin,OPN)的表达水平。结果 (1)CCK8检测结果显示25μmol/L氨来呫诺对BMSCs增殖没有明显影响,50μmol/L氨来呫诺显著抑制BMSCs的增殖,故在后续实验中均采用25μmol/L氨来呫诺作为效应浓度。(2)干预后第10天,氨来呫诺处理组较对照组的ALP染色阳性面积明显增多;干预后第21天,氨来呫诺处理组较对照组的钙结节形成密集,钙结节数目明显增多。(3)干预后第7天,氨来呫诺处理组较对照组中ALP的表达显著提高;第14天时,氨来呫诺处理组较对照组中RUNX2、OPN的表达显著提高。结论氨来呫诺可能通过促进BMSCs成骨分化相关基因表达,进而促进BMSCs细胞中ALP的表达和钙结节形成,促进BMSCs成骨分化。
Objective To investigate the effect of amlexanox on osteogenic differentiation of rat bonemarrow mesenchymal stem cells(BMSCs), and provide theoretical basis for anti-osteoporosis treatment.Methods The SD rat BMSCs were isolated and cultured in vitro. The effect of different concentrations ofamlexanox on the proliferation of BMSCs was detected by CCK8 assay. The cells were divided into control groupand amlexanox(25 μmol/L) treatment group, and they were induced by osteogenic induction medium. Alkalinephosphatase(ALP) staining test was performed on the 10 thday and alizarin red staining on the 21 stday. Theexpression levels of ALP, runt-related transcription factor 2(RUNX2) and osteopontin(OPN) m RNA on the day3, 7 and 14 were detected by real-time quantitative reverse transcription polymerase chain reaction(RT-q PCR).Results CCK8 test results showed that 25 μmol/L amlexanox had no significant effect on the proliferation ofBMSCs, but 50 μmol/L amlexanox did, so the 25 μmol/L amlexanox was used for the effect concentration. Thepositive staining area in amlexanox group was significantly increased at 10 thday after treatment and theformation of calcium nodules in amlexanox group was increased at the 21 stday compared to the control group.The m RNA expression levels of ALP were significantly promoted at the 7 thday, and the expression levels ofRUNX2 and OPN were simultaneously promoted at the 14 thday. Conclusion Amlexanox could promote theosteogenic differentiation of BMSCs by promoting the expression of osteogenic differentiation related genes of BMSCs.
Objective To investigate the effect of amlexanox on osteogenic differentiation of rat bonemarrow mesenchymal stem cells(BMSCs), and provide theoretical basis for anti-osteoporosis treatment.Methods The SD rat BMSCs were isolated and cultured in vitro. The effect of different concentrations ofamlexanox on the proliferation of BMSCs was detected by CCK8 assay. The cells were divided into control groupand amlexanox(25 μmol/L) treatment group, and they were induced by osteogenic induction medium. Alkalinephosphatase(ALP) staining test was performed on the 10 thday and alizarin red staining on the 21 stday. Theexpression levels of ALP, runt-related transcription factor 2(RUNX2) and osteopontin(OPN) m RNA on the day3, 7 and 14 were detected by real-time quantitative reverse transcription polymerase chain reaction(RT-q PCR).Results CCK8 test results showed that 25 μmol/L amlexanox had no significant effect on the proliferation ofBMSCs, but 50 μmol/L amlexanox did, so the 25 μmol/L amlexanox was used for the effect concentration. Thepositive staining area in amlexanox group was significantly increased at 10 thday after treatment and theformation of calcium nodules in amlexanox group was increased at the 21 stday compared to the control group.The m RNA expression levels of ALP were significantly promoted at the 7 thday, and the expression levels ofRUNX2 and OPN were simultaneously promoted at the 14 thday. Conclusion Amlexanox could promote theosteogenic differentiation of BMSCs by promoting the expression of osteogenic differentiation related genes of BMSCs.
引文
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[2]Jiang Y,Jahagirdar BN,Reinhardt RL,et al.Pluripotency of mes-enchymal stem cells derived from adult marrow[J].Nature,2002,418(6893):41-49.
[3]Justesen J,Stenderup K,Ebbesen EN,et al.Adipocyte tissue vol-ume in bone marrow is increased with aging and in patients withosteoporosis[J].Biogerontology,2001,2(3):165-171.
[4]Verma S,Rajaratnam JH,Denton J,et al.Adipocytic proportionof bone marrow is inversely related to bone formation in osteopo-rosis[J].J Clin Pathol,2002,55(9):693-698.
[5]Antebi B,Pelled G,Gazit D.Stem cell therapy for osteoporosis[J].Curr Osteoporos Rep,2014,12(1):41-47.
[6]Phetfong J,Sanvoranart T,Nartprayut K,et al.Osteoporosis:thecurrent status of mesenchymal stem cell-based therapy[J].CellMol Biol Lett,2016,21:12.
[7]Abbasi F,Raoof M,Khatami R,et al.Effectiveness of Amlexanoxand Adcortyl for the treatment of recurrent aphthous ulcers[J].JClin Exp Dent,2016,8(4):e368-e372.
[8]Bell J.Amlexanox for the treatment of recurrent aphthous ulcers[J].Clin Drug Investig,2005,25(9):555-566.
[9]Zhang Y,Guan H,Li J,et al.Amlexanox suppresses osteoclasto-genesis and prevents ovariectomy-induced bone loss[J].Sci Rep,2015,5:13575.
[10]Beyer Nardi N,da Silva Meirelles L.Mesenchymal stem cells:isolation,in vitro expansion and characterization[J].Handb ExpPharmacol,2006,(174):249-282.
[11]Sreejit P,Dilip KB,Verma RS.Generation of mesenchymal stemcell lines from murine bone marrow[J].Cell Tissue Res,2012,350(1):55-68.
[12]Deng P,Chen QM,Hong C,et al.Histone methyltransferases anddemethylases:regulators in balancing osteogenic and adipogenicdifferentiation of mesenchymal stem cells[J].Int J Oral Sci,2015,7(4):197-204.
[13]Wang C,Meng H,Wang X,et al.Differentiation of bone marrowmesenchymal stem cells in osteoblasts and adipocytes and its rolein treatment of osteoporosis[J].Med Sci Monit,2016,22:226-233.
[14]Reilly SM,Ahmadian M,Zamarron BF,et al.A subcutaneousadipose tissue-liver signalling axis controls hepatic gluconeo-genesis[J].Nat Commun,2015,6:6047.
[15]Reilly SM,Chiang SH,Decker SJ,et al.An inhibitor of the proteinkinases TBK1 and IKK-?improves obesity-related metabolic dys-functions in mice[J].Nat Med,2013,19(3):313-321.
[16]Bhattarai T,Bhattacharya K,Chaudhuri P,et al.Correlation ofcommon biochemical markers for bone turnover,serum calcium,and alkaline phosphatase in post-menopausal women[J].Malays JMed Sci,2014,21(1):58-61.
[17]Miyamoto S,Miyamoto Y,Shibata Y,et al.In situ quasi-static anddynamic nanoindentation tests on calcified nodules formed by os-teoblasts:Implication of glucocorticoids responsible for osteoblastcalcification[J].Acta Biomater,2015,12:216-226.
[18]Weng JJ,Su Y.Nuclear matrix-targeting of the osteogenic factorRunx2 is essential for its recognition and activation of the alkalinephosphatase gene[J].Biochim Biophys Acta,2013,1830(3):2839-2852.
[19]Mc Kee MD,Nanci A.Osteopontin at mineralized tissue interfacesin bone,teeth,and osseointegrated implants:ultrastructural distri-bution and implications for mineralized tissue formation,turnover,and repair[J].Microsc Res Tech,1996,33(2):141-164.