刺五加苷诱导大鼠间充质干细胞成骨分化的作用
|
摘要
目的探讨刺五加苷诱导大鼠间充质干细胞(BMSCs)成骨分化的作用。方法提取SD大鼠的BMSCs,培养3代后采用流式细胞术鉴定表面抗原CD45、CD29、CD90的表达;在经典成骨培养液中加入不同浓度的刺五加苷而分为9组:A组(1×10~(–4)mol/L),B组(1×10~(–5)mol/L),C组(1×10~(–6)mol/L),D组(1×10–7mol/L),E组(1×10~(–8)mol/L),F组(1×10~(–9)mol/L),G组(1×10~(–10)mol/L),H组(经典成骨组),I组(阴性对照组)。采用细胞计数试剂盒(CCK-8)检测细胞增殖能力,反转录定量PCR(RT-qPCR)检测骨形成蛋白(BMP)表达情况,筛选出刺五加苷的最佳诱导浓度。以最佳诱导浓度培养12d后,RT-qPCR检测成骨分化相关基因Runt相关转录因子(RUNX)、成骨细胞特异性转录因子(OSX)、骨涎蛋白(BSP)、骨钙素(OCN)的mRNA表达水平,Western blotting检测Notch跨膜受体蛋白1(Notch1)、毛发增强分裂蛋白(Hes1)的蛋白表达水平;第21天时行矿化钙结节茜素红染色。结果第3代BMSCs表面抗原符合干细胞鉴定标准。CCK-8检测结果显示,A、B两组在培养120h后明显抑制了BMSCs的增殖,后期培养中将舍弃A、B两组;而RTqPCR结果显示,添加有刺五加苷的C～G组中,E组BMP表达量最高(4.91±0.46),因此选择1×10~(–8)mol/L作为刺五加苷的最佳诱导浓度进行后续实验。RT-qPCR检测结果显示,E组OSX表达量(30.72±1.96)明显高于I组(1.02±0.27)和H组(9.99±0.59,P<0.05),BSP表达量(8.15±0.47)高于I组(1.09±0.31)和H组(6.03±0.8,P<0.05),OCN表达量(5.91±0.68)高于I组(1.18±2.91)和H组(3.05±0.53,P<0.05),RUNX表达量(1.99±0.09)虽高于I组(1.02±0.19),却低于H组(2.51±0.06,P<0.05)。Westernblotting检测结果显示,E组成骨诱导后Notch1表达量(4608±103)较I组(2638±308)高,却低于H组(5218±182,P<0.05),Hes1表达量(8885±17)较I组(6241±461)增高,却低于H组(12 289±629,P<0.05)。茜素红染色结果显示刺五加苷浓度为10–8mol/L时矿化钙结节多于经典成骨组,提示该浓度刺五加苷成骨诱导效果较经典成骨组更佳。结论刺五加苷能协同地塞米松促进BMSCs的成骨分化,其作用可能与Notch通路有关。
Objective To investigate the effect of acanthopanax on inducing osteogenesis of rat bone marrow stem cells(BMSCs). Methods BMSCs were extracted from SD rats, and the surface antigens CD45, CD29 and CD90 were identified by flow cytometry at the third generation. Different concentrations of acanthopanax were added to the classical osteogenic medium to make it being 9 groups: A(1×10~(–4) mol/L), B(1×10~(–5) mol/L), C(1×10~(–6) mol/L), D(1×10~(–7) mol/L), E(1×10~(–8) mol/L), F(1×10~(–9) mol/L),G(1×10~(–10) mol/L), H(classical osteogenic group), and I(negative control group). The cell counting kit CCK-8 was used to detect cell proliferation, RT-qPCR was performed to detect the mRNA expression of bone morphogenetic protein(BMP), and then the optimal concentration of acanthopanax was selected and used to the later experiments. On the 12 th day of culturing with optimal concentration, RT-qPCR was performed to detect osteogenic differentiation-related gene expression: RUNX, OSX, BSP and OCN.Western blotting was used to detect the levels of transmembrane receptor protein 1(Notch1) and hairy enhancer of split 1(Hes1).On the 21 th day of culturing, the mineralized calcium nodules were stained with alizarin red. Results The surface antigens of the third generation BMSCs were consistent with the stem cell identification criteria. CCK-8 results indicated that the proliferation of BMSCs was inhibited in group A and group B 120 h after cultivation, so the two groups were discarded in the later culture. RTqPCR results showed that among groups C-G with acanthopanax, the expression of BMP in group E(1×10~(–8) mol/L) was the highest(4.91±0.46), so 1×10~(–8) mol/L was selected as the optimal concentration of acanthopanax to finish the later experiments.The results of RT-qPCR showed that the expression of OSX was significantly higher in group E(30.72±1.96) than in group I(1.02±0.27) and group H(9.99±0.59, P<0.05); the expression of BSP(8.15±0.47) was higher than in group I(1.09±0.31)and group H(6.03±0.8, P<0.05); and the expression of OCN(5.91±0.68) was higher than in group I(1.18±2.91) and group H(3.05±0.53, P<0.05). However, the expression of RUNX was higher in group E(1.99±0.09) than in group I(1.02±0.19, P<0.05),but was lower than in group H(2.51±0.06, P<0.05). Western blotting suggested that the Notch1 in group E(4608±103) was higher than in group I(2638±308), but lower than in group H(5218±182, P<0.05); Hes1 expression in group E(8885±17) was higher than in group I( 6241±461), but lower than in H group(12289±629, P<0.05). The alizarin red staining indicated that the number of mineralized calcium nodules was higher in group E than in group H, suggesting that the osteogenic effect in group E(with acanthopanax concentration of 10~(–8) mol/L) is better than in group H. Conclusion Acanthopanax may cooperate with dexamethasone to promote the osteogenesis of BMSCs, which may be related to the Notch signaling pathway.
Objective To investigate the effect of acanthopanax on inducing osteogenesis of rat bone marrow stem cells(BMSCs). Methods BMSCs were extracted from SD rats, and the surface antigens CD45, CD29 and CD90 were identified by flow cytometry at the third generation. Different concentrations of acanthopanax were added to the classical osteogenic medium to make it being 9 groups: A(1×10~(–4) mol/L), B(1×10~(–5) mol/L), C(1×10~(–6) mol/L), D(1×10~(–7) mol/L), E(1×10~(–8) mol/L), F(1×10~(–9) mol/L),G(1×10~(–10) mol/L), H(classical osteogenic group), and I(negative control group). The cell counting kit CCK-8 was used to detect cell proliferation, RT-qPCR was performed to detect the mRNA expression of bone morphogenetic protein(BMP), and then the optimal concentration of acanthopanax was selected and used to the later experiments. On the 12 th day of culturing with optimal concentration, RT-qPCR was performed to detect osteogenic differentiation-related gene expression: RUNX, OSX, BSP and OCN.Western blotting was used to detect the levels of transmembrane receptor protein 1(Notch1) and hairy enhancer of split 1(Hes1).On the 21 th day of culturing, the mineralized calcium nodules were stained with alizarin red. Results The surface antigens of the third generation BMSCs were consistent with the stem cell identification criteria. CCK-8 results indicated that the proliferation of BMSCs was inhibited in group A and group B 120 h after cultivation, so the two groups were discarded in the later culture. RTqPCR results showed that among groups C-G with acanthopanax, the expression of BMP in group E(1×10~(–8) mol/L) was the highest(4.91±0.46), so 1×10~(–8) mol/L was selected as the optimal concentration of acanthopanax to finish the later experiments.The results of RT-qPCR showed that the expression of OSX was significantly higher in group E(30.72±1.96) than in group I(1.02±0.27) and group H(9.99±0.59, P<0.05); the expression of BSP(8.15±0.47) was higher than in group I(1.09±0.31)and group H(6.03±0.8, P<0.05); and the expression of OCN(5.91±0.68) was higher than in group I(1.18±2.91) and group H(3.05±0.53, P<0.05). However, the expression of RUNX was higher in group E(1.99±0.09) than in group I(1.02±0.19, P<0.05),but was lower than in group H(2.51±0.06, P<0.05). Western blotting suggested that the Notch1 in group E(4608±103) was higher than in group I(2638±308), but lower than in group H(5218±182, P<0.05); Hes1 expression in group E(8885±17) was higher than in group I( 6241±461), but lower than in H group(12289±629, P<0.05). The alizarin red staining indicated that the number of mineralized calcium nodules was higher in group E than in group H, suggesting that the osteogenic effect in group E(with acanthopanax concentration of 10~(–8) mol/L) is better than in group H. Conclusion Acanthopanax may cooperate with dexamethasone to promote the osteogenesis of BMSCs, which may be related to the Notch signaling pathway.
引文
[1] Guo X, Guo SN, Lv FM. Progress in TCM treatment of postmenopausal osteoporosis[J]. Xinjiang Tradit Chin Med,2018, 36(2):141-144.[郭玄,郭世宁,吕发明.绝经后骨质疏松症的中医治疗进展[J].新疆中医药,2018, 36(2):141-144.]
[2] Liu YY, Wu T. Drug regulation of osteogenic differentiation of bone marrow stromal cells and the treatment of osteoporosis[J].Chin J Clin Rehabil, 2005, 9(26):184-185.[刘钰瑜,吴铁.药物调控骨髓基质细胞成骨分化与骨质疏松症治疗的方向[J].中国临床康复,2005, 9(26):184-185.]
[3] Zhao WH, Shen GY, Ren H, et al. Research progress in the regulation of osteoporosis-related signaling pathways by the active monomer component of rhizoma drynariae[J]. Chin J Osleoporos, 2017, 23(1):122-129, 140.[招文华,沈耿杨,任辉,等.骨碎补活性单体成分调控骨质疏松症相关信号通路的研究进展[J].中国骨质疏松杂志,2017, 23(1):122-129,140.]
[4] Fang N. The extract of Eucommia ulmoides Oliv and its monomeric quercetin and(+)-syringarea inhibited the adipogenic differentiation of BMSCs[D]. Nanchang:Nanchang University, 2014.[方宁.杜仲叶提取物及其单体槲皮素、(+)-丁香脂素抑制BMSCs成脂分化研究[D].南昌:南昌大学,2014.]
[5] Borrelli F, Ernst E. Alternative and complementary therapies for the menopause[J]. Maturitas, 2010, 66(4):333-343.
[6] Liu J, Zhang Z, Guo Q, et al. Syringin prevents bone loss in ovariectomized mice via TRAF6 mediated inhibition of NF-κB and stimulation of PI3K/AKT[J]. Phytomedicine, 2018, 42:43-50.
[7] Wharton KA, Yedvobnick B, Finnerty VG, et al. Opa:a novelfamily of transcribed repeats shared by the Notch locus and other developmentally regulated loci in D. melanogaster[J]. Cell,1985, 40(1):55-62.
[8] Yu GY, Zheng GZ, Chang B, et al. Naringin stimulates osteogenic differentiation of rat bone marrow stromal cells via activation of the notch signaling pathway[J]. Stem Cells Int, 2016, 2016:7130653.
[9] Lee WJ, Hah YS, Ock SA, et al. Cell source-dependent in vivo immunosuppressive properties of mesenchymal stem cells derived from the bone marrow and synovial fluid of minipigs[J].Exp Cell Res, 2015, 333(2):273-288.
[10] Pittenger MF, Mackay AM, Beck SC, et al. Multilineage potential of adult human mesenchymal stem cells[J]. Science, 1999,284(5411):143-147.
[11] Sinha KM, Zhou X. Genetic and molecular control of osterix in skeletal formation[J].J Cell Biochem, 2013, 114(5):975-984.
[12] Fisher LW, Fedarko NS. Six genes expressed in bones and teeth encode the current members of the SIBLING family of proteins[J]. Connect Tissue Res, 2003, 44(Suppl 1):33-40.
[13] Bouleftour W, Bouet G, Granito RN, et al. Blocking the expression of both bone sialoprotein(BSP)and osteopontin(OPN)impairs the anabolic action of PTH in mouse calvaria bone[J].J Cell Physiol, 2015, 230(3):568-577.
[14] Ducy P, Desbois C, Boyce B, et al. Increased bone formation in osteocalcin-deficient mice[J]. Nature, 1996, 382(6590):448-452.
[15] Komori T, Yagi H, Nomura S, et al. Targeted disruption of Cbfal results in a complete lack of bone formation owing to maturational arrest of osteoblasts[J]. Cell, 1997, 89(5):755-764.
[16] Komori T. Roles of Runx2 in skeletal development[j]. Adv Exp Med Biol, 2017, 962:83-93.
[17] Liu W, Toyosawa S, Furuichi T, et al. Overexpression of Cbfal in osteoblasts inhibits osteoblast maturation and causes osteopenia with multiple fractures[J].J Cell Biol, 2001, 155(1):157-166.
[18] Maruyama Z, Yoshida CA, Furuichi T, et al. Runx2 determines bone maturity and turnover rate in postnatal bone development and is involved in bone loss in estrogen deficiency[J]. Dev Dyn,2007, 236(7):1876-1890.
[19] Ma H, Wu Y, Zhang H. Notch signaling in bone formation and related skeletal diseases[J]. Chin J Med Genet, 2015, 32(2):274-279.
[20] Ramasamy SK, Kusumbe AP, Wang L, et al. Endothelial Notch activity promotes angiogenesis and osteogenesis in bone[J].Nature, 2014, 507(7492):376-380.
[21] Tezuka K, Yasuda M, Watanabe N, et al. Stimulation of osteoblastic cell differentiation by Notch[J].J Bone Miner Res,2002, 17(2):231-239.
[22] Lee JS, Thomas DM, Gutierrez G, et al. HES1 cooperates with pRb to activate RUNX2-dependent transcription[J]. J Bone Miner Res, 2006, 21(6):921-933.
[23] Li T, Ferns K, Yan ZQ, et al. Acanthopanax senticosus:photochemistry and anticancer potential[J]. Am J Chin Med,2016, 44(8):1543-1558.
[2] Liu YY, Wu T. Drug regulation of osteogenic differentiation of bone marrow stromal cells and the treatment of osteoporosis[J].Chin J Clin Rehabil, 2005, 9(26):184-185.[刘钰瑜,吴铁.药物调控骨髓基质细胞成骨分化与骨质疏松症治疗的方向[J].中国临床康复,2005, 9(26):184-185.]
[3] Zhao WH, Shen GY, Ren H, et al. Research progress in the regulation of osteoporosis-related signaling pathways by the active monomer component of rhizoma drynariae[J]. Chin J Osleoporos, 2017, 23(1):122-129, 140.[招文华,沈耿杨,任辉,等.骨碎补活性单体成分调控骨质疏松症相关信号通路的研究进展[J].中国骨质疏松杂志,2017, 23(1):122-129,140.]
[4] Fang N. The extract of Eucommia ulmoides Oliv and its monomeric quercetin and(+)-syringarea inhibited the adipogenic differentiation of BMSCs[D]. Nanchang:Nanchang University, 2014.[方宁.杜仲叶提取物及其单体槲皮素、(+)-丁香脂素抑制BMSCs成脂分化研究[D].南昌:南昌大学,2014.]
[5] Borrelli F, Ernst E. Alternative and complementary therapies for the menopause[J]. Maturitas, 2010, 66(4):333-343.
[6] Liu J, Zhang Z, Guo Q, et al. Syringin prevents bone loss in ovariectomized mice via TRAF6 mediated inhibition of NF-κB and stimulation of PI3K/AKT[J]. Phytomedicine, 2018, 42:43-50.
[7] Wharton KA, Yedvobnick B, Finnerty VG, et al. Opa:a novelfamily of transcribed repeats shared by the Notch locus and other developmentally regulated loci in D. melanogaster[J]. Cell,1985, 40(1):55-62.
[8] Yu GY, Zheng GZ, Chang B, et al. Naringin stimulates osteogenic differentiation of rat bone marrow stromal cells via activation of the notch signaling pathway[J]. Stem Cells Int, 2016, 2016:7130653.
[9] Lee WJ, Hah YS, Ock SA, et al. Cell source-dependent in vivo immunosuppressive properties of mesenchymal stem cells derived from the bone marrow and synovial fluid of minipigs[J].Exp Cell Res, 2015, 333(2):273-288.
[10] Pittenger MF, Mackay AM, Beck SC, et al. Multilineage potential of adult human mesenchymal stem cells[J]. Science, 1999,284(5411):143-147.
[11] Sinha KM, Zhou X. Genetic and molecular control of osterix in skeletal formation[J].J Cell Biochem, 2013, 114(5):975-984.
[12] Fisher LW, Fedarko NS. Six genes expressed in bones and teeth encode the current members of the SIBLING family of proteins[J]. Connect Tissue Res, 2003, 44(Suppl 1):33-40.
[13] Bouleftour W, Bouet G, Granito RN, et al. Blocking the expression of both bone sialoprotein(BSP)and osteopontin(OPN)impairs the anabolic action of PTH in mouse calvaria bone[J].J Cell Physiol, 2015, 230(3):568-577.
[14] Ducy P, Desbois C, Boyce B, et al. Increased bone formation in osteocalcin-deficient mice[J]. Nature, 1996, 382(6590):448-452.
[15] Komori T, Yagi H, Nomura S, et al. Targeted disruption of Cbfal results in a complete lack of bone formation owing to maturational arrest of osteoblasts[J]. Cell, 1997, 89(5):755-764.
[16] Komori T. Roles of Runx2 in skeletal development[j]. Adv Exp Med Biol, 2017, 962:83-93.
[17] Liu W, Toyosawa S, Furuichi T, et al. Overexpression of Cbfal in osteoblasts inhibits osteoblast maturation and causes osteopenia with multiple fractures[J].J Cell Biol, 2001, 155(1):157-166.
[18] Maruyama Z, Yoshida CA, Furuichi T, et al. Runx2 determines bone maturity and turnover rate in postnatal bone development and is involved in bone loss in estrogen deficiency[J]. Dev Dyn,2007, 236(7):1876-1890.
[19] Ma H, Wu Y, Zhang H. Notch signaling in bone formation and related skeletal diseases[J]. Chin J Med Genet, 2015, 32(2):274-279.
[20] Ramasamy SK, Kusumbe AP, Wang L, et al. Endothelial Notch activity promotes angiogenesis and osteogenesis in bone[J].Nature, 2014, 507(7492):376-380.
[21] Tezuka K, Yasuda M, Watanabe N, et al. Stimulation of osteoblastic cell differentiation by Notch[J].J Bone Miner Res,2002, 17(2):231-239.
[22] Lee JS, Thomas DM, Gutierrez G, et al. HES1 cooperates with pRb to activate RUNX2-dependent transcription[J]. J Bone Miner Res, 2006, 21(6):921-933.
[23] Li T, Ferns K, Yan ZQ, et al. Acanthopanax senticosus:photochemistry and anticancer potential[J]. Am J Chin Med,2016, 44(8):1543-1558.