Hey1表达对BMP-9诱导下C3H10T1/2细胞的成骨分化及增殖影响
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摘要
目的探讨Notch信号通路重要靶点Hey1表达水平改变对BMP-9诱导C3H10T1/2细胞成骨分化与增殖的影响。方法构建过表达Hey1慢病毒LV-Hey1、抑制Hey1表达慢病毒LV-sh Hey1,分别感染C3H10T1/2细胞干预Hey1表达水平,以LV-Blank(空质粒)感染C3H10T1/2细胞作为对照;以荧光显微镜对慢病毒感染效果、实时荧光定量PCR以及Western blot对Hey1表达水平进行验证,筛选不同Hey1表达水平的稳定细胞系。用含BMP-9的条件培养基诱导不同Hey1表达水平的C3H10T1/2细胞(分别为BMP-9+C3H10T1/2组、BMP-9+C3H10T1/2-Hey1组、BMP-9+C3H10T1/2-sh Hey1组),以正常培养基培养的细胞作为对照(C3H10T1/2组、C3H10T1/2-Blank组)。培养后48 h,实时荧光定量PCR及Western blot测定成骨分化相关转录因子Runx2、骨桥蛋白、骨钙素m RNA及蛋白表达水平;4、5、6、7 d行MTT检测及4、5、10 d行流式细胞仪测定细胞增殖能力;4、7 d时ELISA测定细胞ALP表达水平并行染色观察。结果成功建立不同Hey1表达水平稳定细胞系。成骨方面,各时间点与BMP-9+C3H10T1/2组比较,BMP-9诱导下Hey1过表达的BMP-9+C3H10T1/2-Hey1组细胞Runx2、骨桥蛋白、骨钙素m RNA及蛋白表达水平以及成骨分化标志物ALP含量均显著增加(P<0.05),抑制Hey1表达的BMP-9+C3H10T1/2-sh Hey1组细胞以上指标均显著降低(P<0.05)。对细胞增殖活力影响方面,与BMP-9+C3H10T1/2组比较,BMP-9+C3H10T1/2-Hey1组MTT检测吸光度(A)值及细胞G2+S期百分比均提高(P<0.05);而抑制Hey1表达BMP-9+C3H10T1/2-sh Hey1组以上指标均降低(P<0.05)。结论 Hey1表达是BMP-9诱导C3H10T1/2细胞成骨分化重要环节,同时影响细胞早期增殖。
Objective To investigate the effect of Notch signaling pathway important target Hey1 expression on the differentiation and proliferation of C3H10T1/2 cells induced by bone morphogenetic protein 9(BMP-9). Methods Hey1 lentivirus and Hey1 short hairpin RNA lentivirus were constructed and used to infect C3H10T1/2 cells to change the expression level of Hey1 in C3H10T1/2 cells. C3H10T1/2 cells infected with LV-Blank(empty plasmid) as control. The Hey1 expression levels of different groups were detected by fluorescence microscope, realtime fluorescence quantitative PCR, and Western blot. The C3H10T1/2 cells with different Hey1 expression level were induced by BMP-9 conditioned medium(BMP-9+C3H10T1/2 group, BMP-9+C3H10T1/2-Hey1 group, and BMP-9+C3H10T1/2-sh Hey1 group); the cells of control groups(C3H10T1/2 group and C3H10T1/2-Blank group) were cultured with normal medium. The m RNA and protein expression levels of osteogenesis related transcription factors(Runx2, osteopontin, and osteocalcin) were detected at 48 hours by real-time fluorescence quantitative PCR and Western blot assay. The cells proliferation and cycles were detected by MTT assay at 4, 5, 6, and 7 days and flow cytometry at 4, 5, and 10 days. The alkaline phosphatase(ALP) activity was analyzed by ELISA and observed by ALP staining at 4 and 7 days. Results C3H10T1/2 cell lines with different Hey1 expression levels were successfully established. In osteogenesis compared with BMP-9+C3H10T1/2 group, overexpression of Hey1 enhanced the m RNA and protein expressions of transcription factors(Runx2, osteopontin, and osteocalcin), and the expression of osteogenic differentiation marker(ALP)(P<0.05); however, inhibition of Hey1 expression significantly decreased the above indexes(P<0.05). In cell proliferation activity compared with BMP-9+C3H10T1/2 group, overexpression of Hey1 increased absorbance(A) value in MTT assay and pecentage of G2+S cells in cytometry assay, but inhibition of Hey1 expression significantly decreased the indexes(P<0.05). Conclusion Expression of Hey1 is the important link in the osteogenic differentiation process of C3H10T1/2 cells induced by BMP-9, and plays an important role in the regulation of early cell proliferation.
Objective To investigate the effect of Notch signaling pathway important target Hey1 expression on the differentiation and proliferation of C3H10T1/2 cells induced by bone morphogenetic protein 9(BMP-9). Methods Hey1 lentivirus and Hey1 short hairpin RNA lentivirus were constructed and used to infect C3H10T1/2 cells to change the expression level of Hey1 in C3H10T1/2 cells. C3H10T1/2 cells infected with LV-Blank(empty plasmid) as control. The Hey1 expression levels of different groups were detected by fluorescence microscope, realtime fluorescence quantitative PCR, and Western blot. The C3H10T1/2 cells with different Hey1 expression level were induced by BMP-9 conditioned medium(BMP-9+C3H10T1/2 group, BMP-9+C3H10T1/2-Hey1 group, and BMP-9+C3H10T1/2-sh Hey1 group); the cells of control groups(C3H10T1/2 group and C3H10T1/2-Blank group) were cultured with normal medium. The m RNA and protein expression levels of osteogenesis related transcription factors(Runx2, osteopontin, and osteocalcin) were detected at 48 hours by real-time fluorescence quantitative PCR and Western blot assay. The cells proliferation and cycles were detected by MTT assay at 4, 5, 6, and 7 days and flow cytometry at 4, 5, and 10 days. The alkaline phosphatase(ALP) activity was analyzed by ELISA and observed by ALP staining at 4 and 7 days. Results C3H10T1/2 cell lines with different Hey1 expression levels were successfully established. In osteogenesis compared with BMP-9+C3H10T1/2 group, overexpression of Hey1 enhanced the m RNA and protein expressions of transcription factors(Runx2, osteopontin, and osteocalcin), and the expression of osteogenic differentiation marker(ALP)(P<0.05); however, inhibition of Hey1 expression significantly decreased the above indexes(P<0.05). In cell proliferation activity compared with BMP-9+C3H10T1/2 group, overexpression of Hey1 increased absorbance(A) value in MTT assay and pecentage of G2+S cells in cytometry assay, but inhibition of Hey1 expression significantly decreased the indexes(P<0.05). Conclusion Expression of Hey1 is the important link in the osteogenic differentiation process of C3H10T1/2 cells induced by BMP-9, and plays an important role in the regulation of early cell proliferation.
引文
1 Selvamurugan N,Kwok S,Vasilov A,et al.Effects of BMP-2 and pulsed electromagnetic field(PEMF)on rat primary osteoblastic cell proliferation and gene expression.J Orthop Res,2007,25(9):1213-1220.
2唐大刚,王淼,张艳亮,等.Heyl基因参与调控BMP9诱导的C3H10T1/2细胞成骨分化.中国生物工程杂志,2015,35(6):14-20.
3 Sekiya I,Larson BL,Vuoristo JT,et al.Comparison of effect of BMP-2,-4,and-6 on in vitro cartilage formation of human adult stem cells from bone marrow stroma.Cell Tissue Res,2005,320(2):269-276.
4 Qian X,Zhang C,Chen G,et al.Effects of BMP-2 and FGF2 on the osteogenesis of bone marrow-derived mesenchymal stem cells in hindlimb-unloaded rats.Cell Biochem Biophys,2014,70(2):1127-1136.
5 Zhang X,Guo WG,Cui H,et al.In vitro and in vivo enhancement of osteogenic capacity in a synthetic BMP-2 derived peptide-coated mineralized collagen composite.J Tissue Eng Regen Med,2013.[Epub ahead of print]
6 Wegman F,Bijenhof A,Schuijff L,et al.Osteogenic differentiation as a result of BMP-2 plasmid DNA based gene therapy in vitro and in vivo.Eur Cell Mater,2011,21:230-242.
7 Zhang Y,Madhu V,Dighe AS,et al.Osteogenic response of human adipose-derived stem cells to BMP-6,VEGF,and combined VEGF plus BMP-6 in vitro.Growth Factors,2012,30(5):333-343.
8 Cheng H,Jiang W,Phillips FM,et al.Osteogenic activity of the fourteen types of human bone morphogenetic proteins(BMPs).J Bone Joint Surg(Am),2003,85-A(8):1544-1552.
9 Sharff KA,Song WX,Luo X,et al.Heyl basic helix-loop-helix protein plays an important role in mediating BMP9-induced osteogenic differentiation of mesenchymal progenitor cells.J Biol Chem,2009,284(1):649-659.
10 Lavery K,Hawley S,Swain P,et al.New insights into BMP-7 mediated osteoblastic differentiation of primary human mesenchymal stem cells.Bone,2009,45(1):27-41.
11 Ugarte F,Ryser M,Thieme S,et al.Notch signaling enhances osteogenic differentiation while inhibiting adipogenesis in primary human bone marrow stromal cells.Exp Hematol,2009,37(7):867-875.
12 Weber D,Wiese C,Gessler M.Hey bHLH transcription factors.Curr Top Dev Biol,2014,110:285-315.
13张晓艳,杨伦韵,谢佳瑛,等.携带siRbpj重组腺病毒载体的构建及其对BMP9诱导骨髓间充质干细胞成骨分化作用影响.重庆医科大学学报,2014,39(5):601-606.
14 Moellering RE,Cornejo M,Davis TN,et al.Direct inhibition of the NOTCH transcription factor complex.Nature,2009,462(7270):182-188.
15 Tezuka K,Yasuda M,Watanabe N,et al.Stimulation of osteoblastic cell differentiation by Notch.J Bone Miner Res,2002,17(2):231-239.
16 McLarren KW,Lo R,Grbavec D,et al.The mammalian basic helix loop helix protein HES-1 binds to and modulates the transactivating function of the runt-related factor Cbfal.J Biol Chem,2000,275(1):530-538.
17 Suh JH,Lee HW,Lee JW,et al.Hesl stimulates transcriptional activity of Runx2 by increasing protein stabilization during osteoblast differentiation.Biochem Biophys Res Commun,2008,367(1):97-102.
18 Lin GL,Hankenson KD.Integration of BMP,Wnt,and notch signaling pathways in osteoblast differentiation.J Cell Biochem,2011,112(12):3491-3501.
19 Nobta M,Tsukazaki T,Shibata Y,et al.Critical regulation of bone morphogenetic protein-induced osteoblastic differentiation by Deltal/Jagged 1-activated Notchl signaling.J Biol Chem,2005,280(16):15842-15848.
20 Tezuka K,Yasuda M,Watanabe N,et al.Stimulation of osteoblastic cell differentiation by Notch.J Bone Miner Res,2002,17(2):231-239.
21赵艳芳,宋涛,刘跃亮,等.RUNX2对BMP9诱导的间充质干细胞C3H10T1/2成骨分化的影响.中国生物工程杂志,2013,33(2):21-26.
22 Sharff KA,Song WX,Luo X,et al.Heyl basic helix-loop-helix protein plays an important role in mediating BMP9-induced osteogenic differentiation of mesenchymal progenitor cells.J Biol Chem,2009,284(1):649-659.
23 Salie R,Kneissel M,Vukevic M,et al.Ubiquitous overexpression of Heyl transcription factor leads to osteopenia and chondrocyte hypertrophy in bone.Bone,2010,46(3):680-694.
2唐大刚,王淼,张艳亮,等.Heyl基因参与调控BMP9诱导的C3H10T1/2细胞成骨分化.中国生物工程杂志,2015,35(6):14-20.
3 Sekiya I,Larson BL,Vuoristo JT,et al.Comparison of effect of BMP-2,-4,and-6 on in vitro cartilage formation of human adult stem cells from bone marrow stroma.Cell Tissue Res,2005,320(2):269-276.
4 Qian X,Zhang C,Chen G,et al.Effects of BMP-2 and FGF2 on the osteogenesis of bone marrow-derived mesenchymal stem cells in hindlimb-unloaded rats.Cell Biochem Biophys,2014,70(2):1127-1136.
5 Zhang X,Guo WG,Cui H,et al.In vitro and in vivo enhancement of osteogenic capacity in a synthetic BMP-2 derived peptide-coated mineralized collagen composite.J Tissue Eng Regen Med,2013.[Epub ahead of print]
6 Wegman F,Bijenhof A,Schuijff L,et al.Osteogenic differentiation as a result of BMP-2 plasmid DNA based gene therapy in vitro and in vivo.Eur Cell Mater,2011,21:230-242.
7 Zhang Y,Madhu V,Dighe AS,et al.Osteogenic response of human adipose-derived stem cells to BMP-6,VEGF,and combined VEGF plus BMP-6 in vitro.Growth Factors,2012,30(5):333-343.
8 Cheng H,Jiang W,Phillips FM,et al.Osteogenic activity of the fourteen types of human bone morphogenetic proteins(BMPs).J Bone Joint Surg(Am),2003,85-A(8):1544-1552.
9 Sharff KA,Song WX,Luo X,et al.Heyl basic helix-loop-helix protein plays an important role in mediating BMP9-induced osteogenic differentiation of mesenchymal progenitor cells.J Biol Chem,2009,284(1):649-659.
10 Lavery K,Hawley S,Swain P,et al.New insights into BMP-7 mediated osteoblastic differentiation of primary human mesenchymal stem cells.Bone,2009,45(1):27-41.
11 Ugarte F,Ryser M,Thieme S,et al.Notch signaling enhances osteogenic differentiation while inhibiting adipogenesis in primary human bone marrow stromal cells.Exp Hematol,2009,37(7):867-875.
12 Weber D,Wiese C,Gessler M.Hey bHLH transcription factors.Curr Top Dev Biol,2014,110:285-315.
13张晓艳,杨伦韵,谢佳瑛,等.携带siRbpj重组腺病毒载体的构建及其对BMP9诱导骨髓间充质干细胞成骨分化作用影响.重庆医科大学学报,2014,39(5):601-606.
14 Moellering RE,Cornejo M,Davis TN,et al.Direct inhibition of the NOTCH transcription factor complex.Nature,2009,462(7270):182-188.
15 Tezuka K,Yasuda M,Watanabe N,et al.Stimulation of osteoblastic cell differentiation by Notch.J Bone Miner Res,2002,17(2):231-239.
16 McLarren KW,Lo R,Grbavec D,et al.The mammalian basic helix loop helix protein HES-1 binds to and modulates the transactivating function of the runt-related factor Cbfal.J Biol Chem,2000,275(1):530-538.
17 Suh JH,Lee HW,Lee JW,et al.Hesl stimulates transcriptional activity of Runx2 by increasing protein stabilization during osteoblast differentiation.Biochem Biophys Res Commun,2008,367(1):97-102.
18 Lin GL,Hankenson KD.Integration of BMP,Wnt,and notch signaling pathways in osteoblast differentiation.J Cell Biochem,2011,112(12):3491-3501.
19 Nobta M,Tsukazaki T,Shibata Y,et al.Critical regulation of bone morphogenetic protein-induced osteoblastic differentiation by Deltal/Jagged 1-activated Notchl signaling.J Biol Chem,2005,280(16):15842-15848.
20 Tezuka K,Yasuda M,Watanabe N,et al.Stimulation of osteoblastic cell differentiation by Notch.J Bone Miner Res,2002,17(2):231-239.
21赵艳芳,宋涛,刘跃亮,等.RUNX2对BMP9诱导的间充质干细胞C3H10T1/2成骨分化的影响.中国生物工程杂志,2013,33(2):21-26.
22 Sharff KA,Song WX,Luo X,et al.Heyl basic helix-loop-helix protein plays an important role in mediating BMP9-induced osteogenic differentiation of mesenchymal progenitor cells.J Biol Chem,2009,284(1):649-659.
23 Salie R,Kneissel M,Vukevic M,et al.Ubiquitous overexpression of Heyl transcription factor leads to osteopenia and chondrocyte hypertrophy in bone.Bone,2010,46(3):680-694.