巨噬细胞对血管内皮细胞增殖、Hoxb2、血管内皮生长因子受体和整合素ανβ3表达的影响

南方医科大学学报 ›› 2005, Vol. 25 ›› Issue (02): 148-151.

巨噬细胞对血管内皮细胞增殖、Hoxb2、血管内皮生长因子受体和整合素ανβ3表达的影响

刘亮¹, 刘旭盛², 张晓启³, 明佳², 徐辉⁴, 程天民⁴

1. 南方医科大学南方医院烧伤科, 广东, 广州, 510515;
2. 第三军医大学西南医院烧伤研究所, 重庆, 400038;
3. 解放军234医院烧伤科, 辽宁, 朝阳, 122000;
4. 第三军医大学复合伤研究所, 重庆, 400038

出版日期:2005-02-20 发布日期:2005-02-20
基金资助:
收稿日期:2004-10-8。
基金项目:国家重点基础发展规划项目(G1999054205)
作者简介:刘亮(1971- ),男,2003年毕业于第三军医大学,医学硕士,主治医师、讲师,电话:020-61641114-87250,E-mail:liuliang9@yahoo.com.cn
通讯作者:刘旭盛,023-68752865,E-mail:liuxsp@cta.cq.cn

Effects of macrophages on the biological behaviors and VEGF receptor mRNA, Hoxb2 mRNA, and integrin ανβ3 expressions of vascular endothelial cells

LIU Liang¹, LIU Xu-sheng², ZHANG Xiao-qi³, MING Jia², XU Hui⁴, CHENG Tian-min⁴

1. 南方医科大学南方医院烧伤科, 广东, 广州, 510515;
2. 第三军医大学西南医院烧伤研究所, 重庆, 400038;
3. 解放军234医院烧伤科, 辽宁, 朝阳, 122000;
4. 第三军医大学复合伤研究所, 重庆, 400038

Online:2005-02-20 Published:2005-02-20

摘要/Abstract

摘要： 目的建立人巨噬细胞系U937与人脐静脉血管内皮细胞系ECV-304体外共培养模型,以刀豆蛋白A(ConA)作为U937激活剂,研究巨噬细胞调节血管生成的机制。方法实验分4组:ECV-304、ConA+ECV-304、U937+ECV-304和ConA+U937+ECV-304。将ECV-304细胞接种,待60%融合时按照不同的分组进行共培养48h后,流式细胞仪检测细胞周期的变化;采用3H-TdR掺入试验检测内皮细胞DNA合成变化;RT-PCR技术检测内皮细胞血管内皮生长因子(VEGF)受体KDR和同源盒(homebox)Hoxb2基因mRNA表达水平的变化;免疫荧光在流式细胞仪上检测整合素受体ανβ3表达的变化。结果 ConA激活的U937细胞可使内皮细胞S期、DNA合成明显增加(P<0.01);ConA刺激的U937细胞使内皮细胞VEGF受体KDR(0.879±0.003)、Hoxb2基因mRNA的表达水平(0.947±0.003)和整合素受体ανβ3的表达水平(10.26±1.73)明显上调(P<0.01)。结论 ConA活化的巨噬细胞可通过影响内皮细胞的细胞周期、DNA合成、VEGF受体KDR、Hoxb2及整合素受体ανβ3的表达来促进内皮细胞的增殖、迁移及与基质的黏附,从而调节血管的生成。

Abstract: Objective To explore the mechanism by which macrophages regulate angiogenesis by co-culturing human umbilical vein endothelial cells(ECV-304) with human macrophage cells(U937) stimulated by concanavalin A(ConA). Methods Monolayer ECV-304 cells growing to 60% confluence were co-cultured with 1×105/ml U937 cells in the presence or absence of ConA(ConA+U937+ECV-304 and U937+ECV-304 groups, respectively), with non-treated and ConA-treated ECV-304 cells serving as the control groups(ECV-304 and ConA+ECV-304 groups, respectively). Forty-eight h later, U937 cells were removed from the cell co-culture for examining changes in DNA synthesis of ECV-304 cells with 3H-TdR incorporation assay and for cell cycle analysis with flow cytometry. RT-PCR was employed to assess the influence of macrophages stimulated by ConA on the expression of the target genes. With immunofluorescent method, the changes in the expression of integrin receptor ανβ3 of ECV-304 were determined. Results A significant increase in S-phase ECV-304 cells with enhanced DNA synthesis was observed after co-culture of the cells with ConA-stimulated U937 cells(P<0.01), which also resulted in significant up-regulation of the expressions of KDR mRNA(0.879±0.003), Hoxb2 mRNA(0.947±0.003) and integrin receptor ανβ3(10.26±1.73). Conclusion Macrophages can accelerate the proliferation, migration and adhesion of the vascular endothelial cells to the basilar membrane matrix by affecting their cell cycle, DNA synthesis, expression of KDR mRNA, Hoxb2 mRNA and integrin ανβ3, so as to modulate the angiogenetic process of the latter cells.

中图分类号:

R329.28

刘亮¹, 刘旭盛², 张晓启³, 明佳², 徐辉⁴, 程天民⁴. 巨噬细胞对血管内皮细胞增殖、Hoxb2、血管内皮生长因子受体和整合素ανβ3表达的影响[J]. 南方医科大学学报, 2005, 25(02): 148-151.

LIU Liang¹, LIU Xu-sheng², ZHANG Xiao-qi³, MING Jia², XU Hui⁴, CHENG Tian-min⁴. Effects of macrophages on the biological behaviors and VEGF receptor mRNA, Hoxb2 mRNA, and integrin ανβ3 expressions of vascular endothelial cells[J]. Journal of Southern Medical University, 2005, 25(02): 148-151.

参考文献

[1] 付小兵,王德文.现代创伤修复学[M].北京:人民军医出版社,1999 91-3
[2] Pettet G, Chaplain MA, McElwain DL, et al. On the role of angio genesis in wound healing [J]. Proc R Soc Lond B Biol Sci, 1996, 263(1376): 1487-93.
[3] Domenico F, Marie FP, Clemente C, et al. Relationship between DNA methylation and gene expression of the HOXB gene cluster in small cell lung cancers[J]. FEBS Lett, 1996, 380(8): 103-7.
[4] Mark W, Lingen DD. Role ofleukocytes and endothelial cells in the development of angiogenesis in inflammation and wound healing [J]. Arch Pathol Lab Med, 2001, 125(1): 67-71.
[5] Alisa EK, Peter J, Polverini S, et al. Induction ofneovascularization by activated human monocytes [J]. J Leukoc Biol, 1986, 39(2): 233-8.
[6] Clark EA, Brugge JS. Integrins and signal transduction pathways: the road taken [J]. Science, 1995,268(5208): 233-9.
[7] Horton MA. The αvβ3 integrin "ventronectin receptor" [J]. Int J Biochem Cell Biol, 1997, 29(5): 721-5.
[8] Clark RA, Tonnesen MG, Gailit J, et al. Transient function expression of αvβ3 on vascular cells during wound repair [J]. Am J Pathol, 1996, 148(5): 1407-21.
[9] Sepp NT, Li LJ, Lee KH, et al. Basic fibroblast growth factor increases expression of the αvβ3 complex on human microvessel endothelial cells[J]. J Invest Dermatol, 1994, 103(3): 295-9.
[10] Sheu JR, Yen MH, Kan YC, et al. Inhibition of angiogenesis in vitro and in vivo: Compparison of the relative activities of triflavin, an Arg-Gly-Asp-containing peptide snd snti-αvβ3 integrin monoclonal antibody[J]. Boichem Biophys Acta, 1997, 1336(3): 445-54.
[11] Mcginnis W, Levine MS, Hafen E, et al. A conserved DNA sequence in homeotic genes of the Drosophilia Antennapedia and bithorax complexes[J]. Nature, 1984, 308(29): 428-9.
[12] Akam M. Hox genes and the evolution of diverse body plans [J]. Biol Sci, 1995, 349(1329): 313-9.
[13] 张晓启,刘旭盛,刘亮同源盒基因B2反义寡核苷酸对内皮细胞的影响[J].中华烧伤杂志,2001,17(6):.348-50.Zhang XQ, Liu XS, Liu L. The influence of Hoxb2 cells-sense oligodeoxynucleotides on the proliferation and expression of human umbilical vein endothelial cells [J]. Chin J Burns, 2001, 17(6):348-50.
[14] 张晓启,刘旭盛.B2同源盒基因反义寡核苷酸对原代脐静脉内皮细胞增殖及细胞周期的影响[J].中华创伤杂志,2002,18(1):47-8.Zhang XQ, Liu XS. The influence of Hoxb2 cells-sense oligo deoxynucleotides on the proliferation and cell cycle of human umbilical vein endothelial cells [J]. Chin J Traumatol, 2002, 18(1):47-8.