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ICAM-1基因转染对小鼠间充质干细胞成脂分化功能的影响 被引量:3

Effects of ICAM-1 gene transfection on the adipogenic differentiation of MSCs
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摘要 目的探讨细胞间黏附分子1(ICAM-1)基因转染对小鼠间充质干细胞(MSC)向脂肪细胞分化的影响。方法构建含小鼠ICAM-1基因全长DNA片段的MIGRl-ICAM-1重组逆转录病毒质粒,连同空质粒MIGRl及包装质粒ECOS分别转染T293细胞,收集相关病毒上清并感染小鼠MSC细胞系C3H10T1/2细胞,荧光倒置显微镜下观察及real.timePCR法和流式细胞术检测转染效率,将获得稳定过表达ICAM-1的C3H10T1/2细胞(C3H10T1/2-ICAM-1细胞)和转入空质粒的C3H10T1/2细胞(C3H10T1/2-MIGRl细胞)向脂肪细胞诱导分化,以原位油红O染色和real—timePCR检测成脂分化能力及其关键转录因子C/EBPoL和PPARy mRNA的表达水平。结果成功构建MIGRl-ICAM一1逆转录病毒载体;感染C3H10T1/2细胞获得稳定过表达ICAM-1的C3H10T1/2-ICAM-1细胞和空载体对照C3H10T1/2一MIGRl细胞。成脂细胞诱导分化结果显示,无论是在自分化还是诱导分化组中,C3H10T1/2-ICAM-1细胞与转染空载体的细胞相比脂滴变小,其脂肪细胞数量分别为[(3.2±0.5)/孔、(12.2±3.8)/孔],显著少于转染空载体的细胞[(11.2±0.4)/孔、(51.3±2.8)/孔](P〈0.05);C3H10T1/2一ICAM一1细胞自分化组和诱导分化组C/EBPoLmRNA表达水平分别为1.2±0.7和2.9±0.9);PPAR-,/mRNA表达水平分别为1557.6±70.2和7547.0±442.2,均较转染空载体细胞的5.8±0.5和23.0±2.3:2453.04-215.6和9856.3±542.2下调(P〈0.05)。结论细胞表面ICAM一1过表达可抑制小鼠MSC的成脂分化。 Objective To explore the effects of ICAM-1 gene transfection on the differentiation of MSCs to adipocytes. Methods The recombinant retroviral expression plasmid MIGRI-ICAM-1 containing full length of mouse ICAM-1 gene was constructed. The constructed plasmid MIGRI-ICAM-1, empty plasmid MIGR1 and packaging plasmid ECOS were transfected into T293 cell lines and then the superuatant generated from T293 cells were used to infect mouse MSCs cell line C3H10T 1/2. The transfective efficiency was determined by inverted fluorescence microscope, real-time PCR and flow cytometry. Furthermore, ICAM-1 overexpressing MSCs (C3H10T 1/2-ICAM-1 ) and empty vector transfection MSCs (C3H10T 1/2-MIGR1 ) were cultured in medium with or without induction reagents, Oil-red-O staining was used to detect the lipid accumulation, and the expression of transcriptional factors C/EBPct and PPARy, which were key factors in the differentiation of MSCs to adipocytes, were tested by real-time-PCR. Results The recombinant retrovirus vector containing mouse ICAM-1 gene was successful constructed. After transfeetion into MSCs cell line C3H10T 1/2, the overexpression ICAM-1 MSCs cell line (C3H10T 1/2-ICAM-1 ) and control cell line (C3H10T 1/2-MIGR1 ) were obtained. Furthermore, these two cell lines were treated without or with adipocytic induction reagents, C3H10T 1/2-ICAM-1 showed significantly lower mRNA expression level for C/EBPa [(1.2±0. 7), (2.9 ±0.9)1 and PPARy [(1557.6 ±70.2),(7547.0 ±442.2)] when compared with C3H10T 1/2- MIGR1 E(5.8±0.5),(23.0±2.3) and (2453.0±215.6),(9856.3 ±542.2) I (P 〈 0.05 ). Moreover, little lipid droplet and decreased quantity of adipocytes were detected in C3 H 10T 1/2-ICAM-1 [ ( 3.2 ± 0.5 )/well, ( 12.2 ±3.8 )/well than that in C3 H10T 1/2-MIGR1 [ ( 11.2 ± 0.4)/well, (51.3 ±2.8)/well] (P 〈0.05). Conclusion Overexpression of ICAM-1 in MSCs can inhibit its adipocytie differentiation.
出处 《中华血液学杂志》 CAS CSCD 北大核心 2013年第5期435-439,共5页 Chinese Journal of Hematology
基金 国家自然科学基金(31070996、31171084) 国家自然科学基金青年基金(81101342) 天津市自然科学基金(12JCYBJC17100)
关键词 基因 ICAM-1 间充质干细胞 基因转染 细胞分化 Gene, ICAM-1 Mesenchymal stem cell Gene transfection Cellular differentia-tion
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参考文献11

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同被引文献23

  • 1Farbod Rastegar,Deana Shenaq,Eric R Wagner,Stephanie H Kim,Russell R Reid,Hue H Luu,Rex C Haydon.Mesenchymal stem cells: Molecular characteristics and clinical applications[J].World Journal of Stem Cells,2010,2(4):67-80. 被引量:35
  • 2Buschmann K,Koch L,Braach N,et al.CXCLl-triggered Interaction of LFA1 and ICAM1 Control Glucose-Induced leukocyte recruitment during Inflammation in vivo.Mediators Inflamm,doi:10.1155/2012/739176.
  • 3Ren G,Zhao X,Zhang L,et al.Inflammatory cytokine-induced intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 in mesenchymal stem cells are critical for immunosuppression.J Immunol,2010; 184(5):2321-2328.
  • 4Zhu H,Guo ZK,Jiang XX,et al.A protocol for isolation and culture of mesenchymal stem cells from mouse compact bone.Nat Protoc,2010;5(3):550-560.
  • 5Nombela-Arrieta C,Ritz J,Silberstein LE.The elusive nature and function of mesenchymal stem ceils.Nat Rev Mol Cell Biol,2011 ;12(2):126-131.
  • 6Bost F,Aouadi M,Caron L,et al.The role of MAPKs in adipocyte differentiation and obesity.Biochimie.2005 ; 87 (1):51-56.
  • 7Lee JS,Park JH,Kwon IK,Lim JY.Retinoic acid inhibits BMP4-induced C3H10T 1/2 stem cell commitment to adipocyte via downregulating Smad/P38MAPK signaling.Biochem Biophys Res Commun.2011 ;409(3):550-555.
  • 8Hu X, Wei L, Taylor TM, et al. Hypoxic preconditioning enhances bone marrow mesenchymal stem cell migration via Kv2.1 channel and FAK activation. Am J Physiol, 2011 ,301 (2) :362 -372.
  • 9Buschmann K, Koch L, Braach N, et al. CXCL1-Triggered Interac- tion of LFA1 and ICAM1 Control Glucose-Induced Leukocyte Re- cruitment during Infammation In Vivo. Mediators Inflamm, 2012, 2012: 739176.
  • 10Ren G, Zhao X, Zhang L, et al. Inflammatory cytokine-induced in- tercellular adhesion molecule-1 and vascular cell adhesion molecule-1 in mesenchymal stem cells are critical for immunosuppression. J Im- munol, 2010, 184(5) : 2321 -2328.

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