Deltex-1对骨髓间充质干细胞增殖及其向平滑肌细胞分化的影响
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摘要
目的探讨Deltex-1对骨髓间充质干细胞(bone marrow mesenchymal stem cells,bMSCs)增殖及其向平滑肌细胞(smooth muscle cells,SMCs)分化的影响。方法分离培养大鼠骨髓bMSCs,采用流式细胞仪检测鉴定。通过Deltex-1过表达腺病毒载体p Ad/Deltex-1感染bMSCs后,采用CCK-8(cell count kit-8)法检测Deltex-1过表达对bMSCs增殖的影响;将未感染、经Deltex-1病毒感染及经空病毒感染的bMSCs与SMCs共培养,采用免疫荧光细胞化学、RT-PCR及Western blot分别检测其bMSCs中SMCs标志物平滑肌肌球蛋白重链(smooth muscle myosin heavy chain,SM-MHC)及Notch信号通路相关因子Notch-1的表达,单纯培养的bMSCs、SMCs的相同检测作正常对照。结果成功分离、培养大鼠bMSCs。CCK-8检测证实:与对照比较,经Deltex-1病毒感染的bMSCs生长减慢,48 h开始显得更为明显(P<0.05,P<0.01);免疫荧光细胞化学、RT-PCR及Western blot检测显示:与对照比较,经Deltex-1病毒感染的bMSCs与SMCs共培养后显著表达SMCs的标志物SM-MHC,较弱表达Notch信号通路相关因子Notch-1(P<0.01)。结论 Deltex-1过表达可抑制bMSCs的增殖,并促进其向SMCs分化。
Objective To investigate the effect of Deltex-1 overexpression on the proliferation of bone marrow mesenchymal stem cells( bMSCs) and their differentiation into smooth muscle cells( SMCs) in vitro.Methods Rat bMSCs were isolated,cultured and identified. The effect of Deltex-1 overexpression on the proliferation of bMSCs infected with the adenovirus vector p Ad/Deltex-1 was observed using cell count kit-8( CCK-8). In a co-culture system with SMCs,bMSCs infected with p Ad/Deltex-1 or an empty viral vector or with no treatment were examined for the expression of smooth muscle myosin heavy chain( SM-MHC,a marker of SMCs) using immunofluorescence cytochemistry staining,RT-PCR and Western blotting,with bMSCs and SMCs without treatment in separate cultures as normal controls. Results Compared with the control cells,bMSCs infected by the adenovirus vector p Ad/Deltex-1 exhibited significantly lowered growth rate,which was especially obvious at 48 h after the infection( P < 0. 05, 0. 01). The results of immunofluorescence cytochemistry,real-time PCR and Western blotting all showed that bMSCs infected with the Deltex-1 vector and co-cultured with SMCs expressed a significantly increased level of SM-MHC with a lowed expression of Notch-1( P < 0. 01). Conclusion Deltex-1 overexpression causes suppression of bMSC proliferation and promotes the differentiation of bMSCs into smooth muscle cells in vitro.
Objective To investigate the effect of Deltex-1 overexpression on the proliferation of bone marrow mesenchymal stem cells( bMSCs) and their differentiation into smooth muscle cells( SMCs) in vitro.Methods Rat bMSCs were isolated,cultured and identified. The effect of Deltex-1 overexpression on the proliferation of bMSCs infected with the adenovirus vector p Ad/Deltex-1 was observed using cell count kit-8( CCK-8). In a co-culture system with SMCs,bMSCs infected with p Ad/Deltex-1 or an empty viral vector or with no treatment were examined for the expression of smooth muscle myosin heavy chain( SM-MHC,a marker of SMCs) using immunofluorescence cytochemistry staining,RT-PCR and Western blotting,with bMSCs and SMCs without treatment in separate cultures as normal controls. Results Compared with the control cells,bMSCs infected by the adenovirus vector p Ad/Deltex-1 exhibited significantly lowered growth rate,which was especially obvious at 48 h after the infection( P < 0. 05, 0. 01). The results of immunofluorescence cytochemistry,real-time PCR and Western blotting all showed that bMSCs infected with the Deltex-1 vector and co-cultured with SMCs expressed a significantly increased level of SM-MHC with a lowed expression of Notch-1( P < 0. 01). Conclusion Deltex-1 overexpression causes suppression of bMSC proliferation and promotes the differentiation of bMSCs into smooth muscle cells in vitro.
引文
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[2]YIOU R,HOGREL J Y,LOCHE C M,et al.Periurethral skeletal myofibre implantation in patients with urinary incontinence and intrinsic sphincter deficiency:a phase I clinical trial[J].BJU Int,2013,111(7):1105-1116.DOI:10.1111/j.1464-410X.2012.11682.x.
[3]WILLIAMS J K,DEAN A,BADLANI G,et al.Regenerative Medicine Therapies for Stress Urinary Incontinence[J].J Urol,2016,196(6):1619-1626.DOI:10.1016/j.juro.2016.05.136.
[4]SHI B,LONG X,ZHAO R,et al.Featured Article:Transplantation of mesenchymal stem cells carrying the human receptor activity-modifying protein 1 gene improves cardiac function and inhibits neointimal proliferation in the carotid angioplasty and myocardial infarction rabbit model[J].Exp Biol Med,2014,239(3):356-365.DOI:10.1177/1535370213517619.
[5]YUAN J,HUANG G,XIAO Z,et al.Overexpression ofβ-NGF promotes differentiation of bone marrow mesenchymal stem cells into neurons through regulation of AKT and MAPK pathway[J].Mol Cell Biochem,2013,383(1/2):201-211.DOI:10.1007/s11010-013-1768-6.
[6]CAO J,WEI Y,LIAN J,et al.Notch signaling pathway promotes osteogenic differentiation of mesenchymal stem cells by enhancing BMP9/Smad signaling[J].Int J Mol Med,2017,40(2):378-388.DOI:10.3892/ijmm.2017.3037.
[7]CHENG Y C,HUANG Y C,YEH T H,et al.Deltex1 is inhibited by the Notch-Hairy/E(Spl)signaling pathway and induces neuronal and glial differentiation[J].Neural Dev,2015,10:28.DOI:10.1186/s13064-015-0055-5.
[8]ZHANG P,YANG Y,NOLO R,et al.Regulation of NOTCH signaling by reciprocal inhibition of HES1 and Deltex 1 and its role in osteosarcoma invasiveness[J].Oncogene,2010,29(20):2916-2926.DOI:10.1038/onc.2010.62.
[9]王荥,王延洲,徐惠成.deltex-1腺病毒载体的构建及其在大鼠b MSCs中的表达[J].第三军医大学学报,2011,33(15):1555-1558.Wang Y,Wang Y Z,Xu H C.Expression of deltex-1 in rat bone marrow mesenchymal stem cells by adenovirus vector[J].J Third Mil Med Univ,2011,33(15):1555-1558.
[10]YANG D Y,SHEU M L,SU H L,et al.Dual regeneration of muscle and nerve by intravenous administration of human amniotic fluid-derived mesenchymal stem cells regulated by stromal cell-derived factor-1alpha in a sciatic nerve injury model[J].J Neurosurg,2012,116(6):1357-1367.DOI:10.3171/2012.2.JNS111360.
[11]GURRIAR N-RODR GUEZ U,SANTOS-ZAS I,GONZ LEZ-S NCHEZ J,et al.Action of obestatin in skeletal muscle repair:stem cell expansion,muscle growth,and microenvironment remodeling[J].Mol Ther,2015,23(6):1003-1021.DOI:10.1038/mt.2015.40.
[12]WANG Z,WEN Y,LI Y H,et al.Smooth muscle precursor cells derived from human pluripotent stem cells for treatment of stress urinary incontinence[J].Stem Cells Dev,2016,25(6):453-461.DOI:10.1089/scd.2015.0343.
[13]CAO J,WEI Y,LIAN J,et al.Notch signaling pathway promotes osteogenic differentiation of mesenchymal stem cells by enhancing BMP9/Smad signaling[J].Int J Mol Med,2017,40(2):378-388.DOI:10.3892/ijmm.2017.3037.
[14]HSIAO H W,LIU W H,WANG C J,et al.Deltexl is a target of the transcription factor NFAT that promotes T cell anergy[J].Immunity,2009,31(1):72-83.DOI:10.1016/j.immuni.2009.04.017.
[15]FOX V,GOKHALE P J,WALSH J R,et al.Cell-cell signaling through NOTCH regulates human embryonic stem cell proliferation[J].Stem cells,2008,26(3):715-723.DOI:10.1634/stemcells.2007-0368.
[16]LIN C H,LILLY B.Endothelial cells direct mesenchymal stem cells toward a smooth muscle cell fate[J].Stem Cells Dev,2014,23(21):2581-2590.DOI:10.1089/scd.2014.0163.
[17]CIRIA M,GARC A N A,ONTORIA-OVIEDO I,et al.Mesenchymal Stem Cell Migration and Proliferation Are Mediated by Hypoxia-Inducible Factor-1alpha Upstream of Notch and SUMO Pathways[J].Stem Cells Dev,2017,26(13):973-985.DOI:10.1089/scd.2016.0331.
[18]SANCHO R,CREMONA C A,Behrens A.Stem cell and progenitor fate in the mammalian intestine:Notch and lateral inhibition in homeostasis and disease[J].EMBO Rep,2015,16(5):571-581.DOI:10.15252/embr.201540188.