长效、中效、短效3种激素影响miR-672-5p在大鼠成骨细胞的差异表达
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摘要
背景:前期研究中发现,甲泼尼龙干预后miR-672-5p在大鼠成骨细胞的表达上调,提示miR-672-5p可能在激素性股骨头坏死中发挥作用。目的:分析长效、中效、短效3种激素影响miR-672-5p在大鼠成骨细胞的差异表达。方法:SD大鼠原代成骨细胞体外培养,取第3代大鼠成骨细胞加入DMEM高糖培养基,并分别加入终浓度为1×10~(-5) mol/L的地塞米松(长效)、甲泼尼龙(中效)及氢化可的松(短效),并设空白对照组;4组分别细胞总RNA提取,反转录反应,定量PCR反应;算出2~(-ΔΔCT)值评估miR-672-5p的表达量。结果与结论:(1)与空白组相比,3组激素的miR-672-5p在大鼠成骨细胞的表达均显示出上调,其中甲泼尼龙组为2.35倍,提示表达最高;甲泼尼龙组miR-672-5p表达显著高于氢化可的松组和地塞米松组(均P <0.05),但氢化可的松组与地塞米松组比较差异无显著性意义;(2)结果说明,中效激素的miR-672-5p在大鼠成骨细胞的表达量最大,提示甲泼尼龙在激素性股骨头坏死中可能起重要角色。
BACKGROUND:Preliminary study has found that miR-672-5p can up-regulate osteoblast expression in rats, suggesting that miR-672-5p may participate in osteonecrosis of the femoral head.OBJECTIVE:To investigate the differential expression of miR-672-5p in osteoblasts in rats after intervention by long-acting, intermediateacting and short-acting hormones.METHODS:Primary osteoblasts of Sprague-Dawley rats were cultured in vitro. The third generation rat osteoblasts were added into DMEM high glucose medium, and then 1×10~(-5) mol/L methylprednisolone, hydrocortisone and dexamethasone were added, respectively. The cells cultured with normal DMEM were used as blank control group. Total RNA extraction was performed for reverse transcription reaction and quantitative PCR. The 2~(-ΔΔCT) value was calculated to evaluate miR-672-5p expression.RESULTS AND CONCLUSION:Compared with the blank control group, the expression level of miR-672-5p in the other three groups was up-regulated, and that in the methylprednisolone group was 2.35 times, showing the highest level. The expression level of miR-672-5p in the methylprednisolone group was significantly higher than that in the hydrocortisone and dexamethasone groups(both P < 0.05). There was no significant difference between hydrocortisone and dexamethasone groups. These results indicate that the expression of miR-672-5p in rat osteoblasts by intermediate-acting hormone is the highest, which suggests that methylprednisolone may play an important role in steroid-associated osteonecrosis of the femoral head.
BACKGROUND:Preliminary study has found that miR-672-5p can up-regulate osteoblast expression in rats, suggesting that miR-672-5p may participate in osteonecrosis of the femoral head.OBJECTIVE:To investigate the differential expression of miR-672-5p in osteoblasts in rats after intervention by long-acting, intermediateacting and short-acting hormones.METHODS:Primary osteoblasts of Sprague-Dawley rats were cultured in vitro. The third generation rat osteoblasts were added into DMEM high glucose medium, and then 1×10~(-5) mol/L methylprednisolone, hydrocortisone and dexamethasone were added, respectively. The cells cultured with normal DMEM were used as blank control group. Total RNA extraction was performed for reverse transcription reaction and quantitative PCR. The 2~(-ΔΔCT) value was calculated to evaluate miR-672-5p expression.RESULTS AND CONCLUSION:Compared with the blank control group, the expression level of miR-672-5p in the other three groups was up-regulated, and that in the methylprednisolone group was 2.35 times, showing the highest level. The expression level of miR-672-5p in the methylprednisolone group was significantly higher than that in the hydrocortisone and dexamethasone groups(both P < 0.05). There was no significant difference between hydrocortisone and dexamethasone groups. These results indicate that the expression of miR-672-5p in rat osteoblasts by intermediate-acting hormone is the highest, which suggests that methylprednisolone may play an important role in steroid-associated osteonecrosis of the femoral head.
引文
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[9]Wang C,Meng H,Wang Y,Analysis of early stage osteonecrosis of the human femoral head and the mechanism of femoral head collapse.Int J Biol Sci.2018;14(2):156-164.
[10]Gangji V,DeMaertelaer V,Hauzeur JP.Autologous bone marrow cell implantation in the treatment of non-traumatic osteonecrosis of the femoral head:five year followup of a prospective controlled study.Bone.2011;49:1005-1009.
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[13]Chen C,Qu Z,Yin X,et al.Efficacy of umbilical cord-derived mesenchymal stem cell-based therapy for osteonecrosis of the femoral head:A three-year follow-up study.Molecular Medicine Reports.2016;14(5):4209.
[14]Song H,Tao L,Wang F,et al.Effect of bone mesenchymal stem cells transplantation on the micro-environment of early osteonecrosis of the femoral head.Int J Clin Exp Pathol.2015;8(11):14528-14534.
[15]Oshima K,Nampei A,Matsuda M,et al.Adiponectin increases bone mass by suppressing osteoclast and activating osteoblast.Biochem Biophys Res Commun.2005;331(2):520-526.
[16]Shinoda Y,Yamaguchi M,Ogata N,et al.Regulation of Bone Formation by Adiponectin Through Autocrine/Paracrine and Endocrine Pathways.J Cellular Biochemistry.2006;99:196-208.
[17]黄俊,尹宗生.脂联素与大鼠激素性股骨头坏死的修复[J].中国组织工程研究与临床康复,2010,14(15):2680-2683.
[18]Ko JY,Wang FS,Wang CJ,et al.Increased Dickkopf-1expression accelerates bone cell apoptosis in femoral head osteonecrosis.Bone.2010;46(3):584-591.
[19]Ueda S,Ichiseki T,Yoshitomi Y,et al.Osteocytic cell necrosis is caused by a combination of glucocorticoid-induced Dickkopf-1 and hypoxia.Med Mol Morphol.2015;48(2):69-75.
[20]王镜山.进展型股骨头坏死患者血清抗酒石酸酸性磷酸酶5b及Ⅱ型胶原C-末端肽的表达及意义[J].广东医学,2015,36(23):3690-3692.
[21]Tan X,Cai D,Wu Y,et al.Comparative analysis of serum proteomes:discovery of proteins associated with osteonecrosis of the femoral head.Transl Res.2006;148(3):114-119.
[22]Wu RW,Wang FS,Ko JY,et al.Comparative serum proteome expression of osteonecrosis of the femoral head in adults.Bone.2008;43(3):561-566.
[23]成学琴,鲍华英,张爱华,等.儿童原发性肾病综合征不同种类糖皮质激素治疗的临床研究[J].临床儿科杂志,2013,31(2):159-161.
[24]邓勇,欧书强,邱自辉,等.国内不同剂量甲泼尼龙治疗重型手足口病疗效Meta分析[J].中国病毒病杂志,2014,4(1):53-57.
[25]王斌.系统性红斑狼疮患者促肾上腺皮质激素的变化及不同剂型糖皮质激素治疗对其的影响[D].合肥:安徽医科大学,2012.
[26]倪丹红.某医院糖皮质激素类药物的用药现状、合理性及安全性分析评价[D].长沙:中南大学,2013.
[27]赵籥陶,黄慈波.糖皮质激素的合理使用[J].临床药物治疗杂志,2010,25(1):23-28.
[28]Powell C,Chang C,Naguwa SM,et al.Steroid induced osteonecrosis:An analysis of steroid dosing risk.Autoimmun Rev.2010;9(11):721-743.
[29]Zhang NF,Li ZR,Wei HY,et al.Steroid-induced osteonecrosis:the number of lesions is related to the dosage.J Bone Joint Surg Br.2008;90(9):1239-1243.
[30]汪轩,张琳,邢婧,等.激素剂量对激素性股骨头坏死模型建立的影响及系统评价[J].浙江中医药大学学报,2016,40(1):19-25.
[31]张永乐,王义生,张世清,等.大剂量甲强龙对兔髋关节周围骨组织BMP-2表达的影响[J].郑州大学学报(医学版),2012,47(3):358-361.
[32]Zhao Z,Xue Y,Hong D,et al.Polymorphisms in the Glucocorticoid Receptor Gene and Associations with Glucocorticoid-Induced Avascular Osteonecrosis of the Femoral Head.Genet Test Mol Biomarkers.2017;21(5):322-327.
[33]Riccio I,Pota E,Marcarelli M,et al.Osteonecrosis as a complication in pediatric patients with acute lymphoblastic leukemia.Pediatr Med Chir.2016;38(3):118.
[34]Tse SM,Mok CC.Time trend and risk factors of avascular bone necrosis in patients with systemic lupus erythematosus.Lupus.2017;26(7):715-722.
[35]Padhye B,Dalla-Pozza L,Little D,et al.Incidence and outcome of osteonecrosis in children and adolescents after intensive therapy for acute lymphoblastic leukemia(ALL).Cancer Med.2016;5(5):960-7.
[36]杨波,杜春晓,李义凯,等.硬膜外腔注射不同浓度泼尼松对大鼠血糖、血脂及股骨头形态学的影响[J].广东医学,2012,33(2):174-176.
[37]张文信,刘美芬,叶青合,等.地塞米松对兔骨髓间充质干细胞凋亡及细胞周期的影响[J].广东医学,2012,33(2):170-172.
[38]肖春生,林娜,林诗富,等.不同糖皮质激素诱导鸡股骨头坏死的实验研究[J].中国骨伤,2010,23(3):184-187.
[39]王兴山.循环microRNA与激素性股骨头坏死相关性的初步研究[D].北京:北京协和医学院,2012.
[40]Xu J,Fan G,Chen S,et al.Methylprednisolone inhibition of TNF-alpha expression and NF-kB activation after spinal cord injury in rats.Brain Res Mol Brain Res.1998;59(2):135-142.
[41]Glass DA 2nd,Bialek P,Ahn JD,et al.Canonical Wnt signaling in differentiated osteoblasts controls osteoclast differentiation.Developmental Cell.2005;8(5):751-764.
[42]Olkku A,Mahonen A.Calreticulin mediated glucocorticoid receptor export is involved in beta-catenin translocation and Wnt signalling inhibition in human osteoblastic cells.Bone.2009;44(4):555-565.
[43]Smith E,Coetzee GA,Frenkel B.Glucocorticoids inhibit cell cycle progression in differentiating osteoblasts via glycogen synthase kinase-3beta.J Biol Chem.2002;277(20):18191-18197.
[2]Weinstein RS.Glucocorticoid-induced osteonecrosis.Endocrine.2012;41(2):183-90.
[3]Zalavras C,Shah S,Birnbaum MJ,et al.Role of apoptosis in glucocorticoid-induced osteoporosis and osteonecrosis.Crit Rev Eukaryot Gene Expr.2003;13(2-4):221-235.
[4]李鹏飞,孙楠,樊粤光,等.激素干预对大鼠成骨细胞mi RNA表达的影响[J].广东医学,2016,37(9):1261-1264.
[5]Li P,Sun N,Zeng J,et al.Differential expression of mi R-672-5p and mi R-146a-5p in osteoblasts in rats after steroid intervention.Gene.2016;591(1):69-73.
[6]Lu J,Getz G,Miska EA,et al.MicroRNA expression profiles classify human cancers.Nature.2005;435(7043):834-838.
[7]李鹏飞,孙楠,樊粤光,等.袁氏生脉成骨片对激素干预后大鼠成骨细胞的差异表达mi RNA的实验研究[J].中华中医药学刊,2017,35(1):91-94+260.
[8]Li P,Sun N,Zeng J,et al.Differential expression of mi R-672-5p and mi R-146a-5p in osteoblasts in rats after steroid intervention.Gene.2016;591(1):69-73.
[9]Wang C,Meng H,Wang Y,Analysis of early stage osteonecrosis of the human femoral head and the mechanism of femoral head collapse.Int J Biol Sci.2018;14(2):156-164.
[10]Gangji V,DeMaertelaer V,Hauzeur JP.Autologous bone marrow cell implantation in the treatment of non-traumatic osteonecrosis of the femoral head:five year followup of a prospective controlled study.Bone.2011;49:1005-1009.
[11]王宇,贾全章.组织工程化骨治疗股骨头坏死的动物实验研究现状[J].中国矫形外科杂志,2011,19(9):740-742.
[12]Kawate K,Yajima H,Ohgushi H,et al.Artificial tissue-engineered approach for the treatment of steroid-induced osteonecrosis of the femoral head:transplantation of autologous mesenchymal stem cells cultured with beta-tricalcium phosphate ceramics and free vascularized fibula.Organs.2006;30(12):960-962.
[13]Chen C,Qu Z,Yin X,et al.Efficacy of umbilical cord-derived mesenchymal stem cell-based therapy for osteonecrosis of the femoral head:A three-year follow-up study.Molecular Medicine Reports.2016;14(5):4209.
[14]Song H,Tao L,Wang F,et al.Effect of bone mesenchymal stem cells transplantation on the micro-environment of early osteonecrosis of the femoral head.Int J Clin Exp Pathol.2015;8(11):14528-14534.
[15]Oshima K,Nampei A,Matsuda M,et al.Adiponectin increases bone mass by suppressing osteoclast and activating osteoblast.Biochem Biophys Res Commun.2005;331(2):520-526.
[16]Shinoda Y,Yamaguchi M,Ogata N,et al.Regulation of Bone Formation by Adiponectin Through Autocrine/Paracrine and Endocrine Pathways.J Cellular Biochemistry.2006;99:196-208.
[17]黄俊,尹宗生.脂联素与大鼠激素性股骨头坏死的修复[J].中国组织工程研究与临床康复,2010,14(15):2680-2683.
[18]Ko JY,Wang FS,Wang CJ,et al.Increased Dickkopf-1expression accelerates bone cell apoptosis in femoral head osteonecrosis.Bone.2010;46(3):584-591.
[19]Ueda S,Ichiseki T,Yoshitomi Y,et al.Osteocytic cell necrosis is caused by a combination of glucocorticoid-induced Dickkopf-1 and hypoxia.Med Mol Morphol.2015;48(2):69-75.
[20]王镜山.进展型股骨头坏死患者血清抗酒石酸酸性磷酸酶5b及Ⅱ型胶原C-末端肽的表达及意义[J].广东医学,2015,36(23):3690-3692.
[21]Tan X,Cai D,Wu Y,et al.Comparative analysis of serum proteomes:discovery of proteins associated with osteonecrosis of the femoral head.Transl Res.2006;148(3):114-119.
[22]Wu RW,Wang FS,Ko JY,et al.Comparative serum proteome expression of osteonecrosis of the femoral head in adults.Bone.2008;43(3):561-566.
[23]成学琴,鲍华英,张爱华,等.儿童原发性肾病综合征不同种类糖皮质激素治疗的临床研究[J].临床儿科杂志,2013,31(2):159-161.
[24]邓勇,欧书强,邱自辉,等.国内不同剂量甲泼尼龙治疗重型手足口病疗效Meta分析[J].中国病毒病杂志,2014,4(1):53-57.
[25]王斌.系统性红斑狼疮患者促肾上腺皮质激素的变化及不同剂型糖皮质激素治疗对其的影响[D].合肥:安徽医科大学,2012.
[26]倪丹红.某医院糖皮质激素类药物的用药现状、合理性及安全性分析评价[D].长沙:中南大学,2013.
[27]赵籥陶,黄慈波.糖皮质激素的合理使用[J].临床药物治疗杂志,2010,25(1):23-28.
[28]Powell C,Chang C,Naguwa SM,et al.Steroid induced osteonecrosis:An analysis of steroid dosing risk.Autoimmun Rev.2010;9(11):721-743.
[29]Zhang NF,Li ZR,Wei HY,et al.Steroid-induced osteonecrosis:the number of lesions is related to the dosage.J Bone Joint Surg Br.2008;90(9):1239-1243.
[30]汪轩,张琳,邢婧,等.激素剂量对激素性股骨头坏死模型建立的影响及系统评价[J].浙江中医药大学学报,2016,40(1):19-25.
[31]张永乐,王义生,张世清,等.大剂量甲强龙对兔髋关节周围骨组织BMP-2表达的影响[J].郑州大学学报(医学版),2012,47(3):358-361.
[32]Zhao Z,Xue Y,Hong D,et al.Polymorphisms in the Glucocorticoid Receptor Gene and Associations with Glucocorticoid-Induced Avascular Osteonecrosis of the Femoral Head.Genet Test Mol Biomarkers.2017;21(5):322-327.
[33]Riccio I,Pota E,Marcarelli M,et al.Osteonecrosis as a complication in pediatric patients with acute lymphoblastic leukemia.Pediatr Med Chir.2016;38(3):118.
[34]Tse SM,Mok CC.Time trend and risk factors of avascular bone necrosis in patients with systemic lupus erythematosus.Lupus.2017;26(7):715-722.
[35]Padhye B,Dalla-Pozza L,Little D,et al.Incidence and outcome of osteonecrosis in children and adolescents after intensive therapy for acute lymphoblastic leukemia(ALL).Cancer Med.2016;5(5):960-7.
[36]杨波,杜春晓,李义凯,等.硬膜外腔注射不同浓度泼尼松对大鼠血糖、血脂及股骨头形态学的影响[J].广东医学,2012,33(2):174-176.
[37]张文信,刘美芬,叶青合,等.地塞米松对兔骨髓间充质干细胞凋亡及细胞周期的影响[J].广东医学,2012,33(2):170-172.
[38]肖春生,林娜,林诗富,等.不同糖皮质激素诱导鸡股骨头坏死的实验研究[J].中国骨伤,2010,23(3):184-187.
[39]王兴山.循环microRNA与激素性股骨头坏死相关性的初步研究[D].北京:北京协和医学院,2012.
[40]Xu J,Fan G,Chen S,et al.Methylprednisolone inhibition of TNF-alpha expression and NF-kB activation after spinal cord injury in rats.Brain Res Mol Brain Res.1998;59(2):135-142.
[41]Glass DA 2nd,Bialek P,Ahn JD,et al.Canonical Wnt signaling in differentiated osteoblasts controls osteoclast differentiation.Developmental Cell.2005;8(5):751-764.
[42]Olkku A,Mahonen A.Calreticulin mediated glucocorticoid receptor export is involved in beta-catenin translocation and Wnt signalling inhibition in human osteoblastic cells.Bone.2009;44(4):555-565.
[43]Smith E,Coetzee GA,Frenkel B.Glucocorticoids inhibit cell cycle progression in differentiating osteoblasts via glycogen synthase kinase-3beta.J Biol Chem.2002;277(20):18191-18197.