切换至 "中华医学电子期刊资源库"
高级检索
中华眼科医学杂志(电子版)

中华眼科医学杂志(电子版) ›› 2017, Vol. 07 ›› Issue (01) : 12 -17. doi: 10.3877/cma.j.issn.2095-2007.2017.01.003

所属专题： 文献；

论著

VEGF诱导对翼状胬肉成纤维细胞血管生成拟态影响的研究
陈俊杰1, 蓝育青2,†(), 吴共发3, 黄绮亭3, 曾宇婷3   
  1. 1. 511300　广东省　广州市增城区人民医院（中山大学孙逸仙纪念医院增城院区）眼科
    2. 510120　中山大学孙逸仙纪念医院眼科
    3. 511300　广东省　广州市增城区人民医院病理科
  • 收稿日期:2017-01-02 出版日期:2017-02-28
  • 通信作者: 蓝育青
  • 基金资助:
    广东省医学科研基金(A2016422); 广州市增城区人民医院优秀医学人才培育基金(2017－YX－01)

Study on fibroblasts in vasculogenic mimicry of pterygium induced by VEGF

Junjie Chen1, Yuqing Lan2,†(), Gongfa Wu3, Qiting Huang3, Yuting Zeng3   

  1. 1. Department of ophthalmology, Zengcheng District People′s Hospital of Guangzhou, Guangzhou 511300, China
    2. Department of ophthalmology, Sun Yat-Sen Memorial Hospital of Sun Yat-Sen University, Guangzhou 510120, China
    3. Department of Pathology, Zengcheng District People′s Hospital of Guangzhou, Guangzhou 511300, China
  • Received:2017-01-02 Published:2017-02-28
  • Corresponding author: Yuqing Lan
  • About author:
    Corresponding author: Lan Yuqing, Email:
全文 ( ) 下载PDF ( ) 导出引用
引用本文：

陈俊杰, 蓝育青, 吴共发, 黄绮亭, 曾宇婷. VEGF诱导对翼状胬肉成纤维细胞血管生成拟态影响的研究[J]. 中华眼科医学杂志(电子版), 2017, 07(01): 12-17.

Junjie Chen, Yuqing Lan, Gongfa Wu, Qiting Huang, Yuting Zeng. Study on fibroblasts in vasculogenic mimicry of pterygium induced by VEGF[J]. Chinese Journal of Ophthalmologic Medicine(Electronic Edition), 2017, 07(01): 12-17.

目的

探讨血管内皮生长因子(VEGF)诱导对人翼状胬肉成纤维细胞血管生成拟态(VM)的影响。

方法

收集在广州市增城区人民医院行翼状胬肉手术的6例新鲜的初发性人翼状胬肉标本(年龄41～58岁)。将人翼状胬肉新鲜组织剪碎后进行体外贴壁细胞常规培养，并采用波形蛋白(Vimentin)免疫细胞化学染色和广谱细胞角蛋白(CK)染色对人翼状胬肉成纤维细胞进行鉴定。分别设置对照组和不同浓度的VEGF(1 ng/ml、10 ng/ml及100 ng/ml)组，取生长良好的3～5代翼状胬肉成纤维细胞进行分组培养。采用细胞计数试剂盒－8法检测各组成纤维细胞的增殖能力。应用细胞三维培养技术和糖原染色法观察各组VM的进展情况，采用蛋白质印迹法检测各组细胞的基质金属蛋白酶(MMP)－2和低氧诱导因子－1α(HIF－1α)蛋白的表达。各组吸光度(OD值)、MMP－2及HIF－1α的描述均采用均数±标准差(±s)表示，对照组与不同浓度VEGF组的比较采用单向方差分析，当差异有统计学意义时，进一步两两比较。双变量相关性分析采用Spearman′s等级相关分析。

结果

人翼状胬肉成纤维细胞鉴定结果显示，细胞爬片经苏木精－伊红染色后，在显示镜下细胞呈长梭形、三角形、扇形或不规则状。细胞免疫化学染色结果显示，细胞的胞浆呈Vimentin阳性表达，广谱CK阴性表达。细胞计数试剂盒－8法检测细胞增殖结果显示，对照组、1 ng/ml组、10 ng/ml组及100 ng/ml组细胞的OD值分别为0.57±0.023、0.59±0.012、0.77±0.024及1.014±0.035。对照组和1 ng/ml组比较差异无统计学意义(t＝1.21，P>0.05)；10 ng/ml组和100 ng/ml组的OD值均明显高于对照组和1 ng/ml组(t＝14.78，12.34；P<0.05)；100 ng/ml组的OD值最高，与对照组、1 ng/ml组及10 ng/ml组比较，差异有统计学意义(t＝20.12，19.45，13.41；P<0.05)。细胞三维培养及糖染色显示，10 ng/ml组和100 ng/ml组均出现VM结构，而对照组和1 ng/ml组未发现VM结构。10 ng/ml组和100 ng/ml组VM结构的密度分别为4.40±1.14和12.40±2.07，差异有统计学意义(t＝－7.899，P<0.05)。对照组、1 ng/ml组、10 ng/ml组及100 ng/ml组细胞的MMP－2值分别为0.26±0.038、0.29±0.013、0.39±0.013及0.73±0.014；对照组、1 ng/ml组、10 ng/ml组及100 ng/ml组细胞的HIF－1α值分别为0.087±0.0086、0.087±0.0031、0.15±0.0016及0.37±0.026。对照组和1 ng/ml组中MMP－2和HIF－1α灰度值的差异均无统计学意义(t＝1.21，0.01；P>0.05)；10 ng/ml组中MMP－2和HIF－1α灰度值均显著高于对照组和1 ng/ml组(t＝7.92，6.85，17.64，17.82；P<0.05)；100 ng/ml组的中MMP－2和HIF－1α灰度值均显著高于其他3组(t＝28.48，26.45，18.72，25.31，24.78，15.45；P<0.05)。翼状胬肉成纤维细胞的血管生成拟态密度与MMP－2和HIF－1α表达呈显著正相关(r＝0.509，0.503；P<0.05)。

结论

VEGF能够增强人翼状胬肉成纤维细胞的增殖能力、促进VM出现及促使MMP－2和HIF－1α表达增高。提示VEGF因素可能是翼状胬肉出现VM的分子机制，并协同增强MMP－2和HIF－1α表达一起影响翼状胬肉的进展。

关键词: 翼状胬肉, 血管生成拟态, 血管内皮生长因子, 三维培养
Objective

To investigate the expression of vascular endothelial growth factor (VEGF) on human pterygium fibroblast angiogenesis induced by vasculogenic mimicry (VM) effect.

Methods

6 cases of primary pterygium were collected from Zengcheng District People′s Hospital of Guangzhou province (aged from 41 to 58). Cut fresh tissue of human pterygium, and in vitro adherent cell culture. The human pterygium fibroblasts were identified by immunocytochemical staining of Vimentin and cytokeratin. Groups were divided into control group and different concentrations of VEGF (1 ng/ml, 10 ng/ml and 100 ng/ml) group, take good growth of the 3 to 5 generations of pterygium fibroblasts were cultured. Cell counting kit-8 method was used to detect the proliferation ability of each component of the fibroblast cells, and the cell culture and PAS staining were used to observe the VM of each group, Western blotting was used to detect the expression of matrix metalloproteinase (MMP)-2 and hypoxia inducible factor -1α(HIF-1α) protein in the cells of each group of rats.Each absorbance(OD value), MMP-2, and HIF-1 alpha described by the mean and standard deviation (±s). Multiple groups were compared using one-way analysis of variance, when the difference was statistically significant, two further comparisons between the two. Bivariate correlation analysis using Spearman′s rank correlation analysis.

Results

Human pterygium fibroblasts identification results showed that the cell smear after hematoxylin eosin staining under the microscope in the display cell fusiform, triangle, fan-shaped or irregular shape. The results of immunocytochemical staining showed that the cytoplasm of the cells was positive for Vimentin, and the expression of CK was negative. The results of cell counting kit was used to detect cell proliferation showed that the OD value of control group and 1 ng/ml group, 10 ng/ml group and 100 ng/ml group cells were 0.57±0.023, 0.59±0.012, 0.77±0.024 and 1.014±0.035, differences in control group and 1 ng/ml group was not statistically significant (t=1.21, P>0.05). the OD value of 10 ng/ml group and 100 ng/ml group were higher than that of the control group and 1 ng/ml group (t=14.78, 12.34; P<0.05); the OD value of 100 ng/ml group is the highest, control group, 1 ng/ml and 10 ng/ml groups were significantly different (t=20.12, 19.45, 13.41; P<0.05). Three dimensional cell culture and PAS staining showed that 10 ng/ml group and the 100 ng/ml group had VM structure in the control group and the 1 ng/ml group, no VM appeared in the control group and group. 10 ng/ml group and the density of the 100 ng/ml were respectively 4.40±1.14 and 12.40±2.07, the difference was statistically significant (t=-7.899, P<0.05). The 1 ng/ml group, 10 ng/ml group and 100 ng/ml group cell MMP-2 values were 0.26±0.038, 0.29±0.013, 0.39±0.013 and 0.73±0.014 in the control group; and 1 ng/ml group, 10 ng/ml group and 100 ng/ml group cell HIF-1 values were 0.087±0.0086 and 0.087±0.0031, 0.15±0.0016 and 0.37±0.026 in control. The difference between MMP-2 and HIF-1 alpha gray value of 1 ng/ml group and control group had no statistical significance (t=1.21, 0.01; P>0.05); MMP-2 and HIF-1 alpha gray values were significantly higher than those in control group and 1 ng/ml group (t=7.92, 6.85, 17.64, 17.82; P<0.05); 100 ng/ml group of MMP-2 HIF-1 alpha and gray value were significantly higher than the other 3 groups (t=28.48, 26.45, 18.72, 25.31, 24.78, 15.45; P<0.05). Pterygium fibroblast cells in vasculogenic mimicry density and MMP-2 and HIF-1 expression was positively correlated, and closely correlated ( r=0.509, 0.503; P<0.05).

Conclusion

VEGF can promote cells proliferation and induce VM in human pterygium fibroblasts cells, also enhance the protein expression of MMP-2 and HIF-1α. VEGF may be the molecular mechanism of angiogenesis mimicry and synergistic enhancement MMP-2 and HIF-1α protein expression, thus influence the progress of pterygium.

Key words: Pterygium, Vasculogenic mimicry, Vascular endothelial growth factor, Three-dimensional culture
图1 免疫细胞化学染色法鉴定人翼状胬肉成纤维细胞染色图　A　图显示人翼状胬肉成纤维细胞呈Vimentin阳性表达，表现为细胞胞浆呈黄褐色；B图显示人翼状胬肉成纤维细胞不表达广谱CK，无黄褐色着色(×200)
图2 10 ng/ml组和100 ng/ml组的细胞三维培养图像　倒置显微镜下观察出现管道状、网格状结构(×200)
图3 对照组与不同浓度VEGF组的诱导对翼状胬肉成纤维细胞MMP－2和HIF－1α蛋白表达的影响
[1]
张梅,刘祖国,李永平, 等. 翼状胬肉上皮细胞p53蛋白的表达及其功能状态的研究[J]. 中华眼科杂志, 2002, 38(2)：115-116.
[2]
Weinstein O,Rosenthal G,Zirkin H, et al. Overexpression of p53 tumor suppressor gene in pterygia[J]. Eye, 2002, 16(5)：619-621.
[3]
Maniotis AJ,Folberg R,Hess A, et al. Vascular channel formation by human melanoma cells in vivo and in vitro: vasculogenic mimicry[J]. Am J Pathol, 1999, 155(3)：739-752.
[4]
Qiao L,Liang N,Zhang J, et al. Advanced research on vasculogenic mimicry in cancer[J]. J Cell Mol Med, 2015, 19(2)：315-326.
[5]
李永平,朱哲,张文忻. 翼状胬肉组织中血管拟态的初步研究[J]. 中华眼科杂志, 2007, 43(10)：872-875.
[6]
Matejíková J,Kucharská J,Pancza D, et al. The effect of antioxidant treatment and NOS inhibition on the incidence of ischemia－induced arrhythmias in the diabetic rat heart[J]. Physiological research, 2008, 57(Suppl. 2)：55-60.
[7]
张莉薇,席兴华. 翼状胬肉发病机制的分子生物学研究进展[J]. 国际眼科杂志, 2006, 6(6)：1404-1406.
[8]
Peng ML,Tsai YY,Tung JN, et al. Vascular endothelial growth factor gene polymorphism and protein expression in the pathogenesis of pterygium[J]. Bri J Ophthalmol, 2014, 98(4)：556-561.
[9]
Lee KW,Choi YH,Hwang SH, et al. Outdoor Air Pollution and Pterygium in Korea[J]. J Korean Med Sci, 2017, 32(1)：143-150.
[10]
Romano V,Steger B,Kovacova A, et al. Further evidence for heredity of pterygium[J]. Ophthalmic Genet, 2016, 37(4)：1-3.
[11]
Martins TGDS,Alves MR,Chammas R, et al. Mitomycin C in pterygium treatment[J]. Int J Ophthalmol, 2016, 9(3)：465-468.
[12]
陈永铃,崔颖,卢亚梅, 等. microRNAs的差异性表达在原发性及复发性翼状胬肉发病机制中的作用研究[J/CD]. 中华眼科医学杂志(电子版), 2016, 6(1)：26-32.
[13]
Jiménez B. Mechanistic insights on the inhibition of tumor angiogenesis[J]. J Mol Med, 2001, 78(12)：663-672.
[14]
刘阳,孙宪丽,李彬, 等. 翼状胬肉组织病理学研究及相关因子的检测[J]. 眼科, 2000, 9(6)：357-360.
[15]
陈俊杰,蓝育青,吴共发, 等. 血管生成拟态与翼状胬肉进行期及静止期的相关性研究[J]. 国际眼科杂志, 2015, 15(3)：414-417.
[16]
朱哲,李永平,张卉颖, 等. CD34在翼状胬肉中表达的意义[J]. 中华实验眼科杂志, 2006, 24(6)：569-572.
[17]
Yancopoulos GD,Davis S,Gale NW, et al. Vascular－specific growth factors and blood vessel formation[J]. Nature, 2000, 407(6801)：242-248.
[18]
李海燕,刘克兰,袁志刚, 等. 翼状胬肉中Ki－67和VEGF的表达[J]. 中国实用眼科杂志, 2006, 24(9)：946-948.
[19]
李明渊,唐仁泓. HIF－1与VEGF在翼状胬肉中的表达及意义[J]. 中华实验眼科杂志, 2009, 27(3)：214-217.
[20]
黄廷球,莫菊彩,王自海, 等. VEGF在翼状胬肉发病与复发中的作用[J]. 放射免疫学杂志, 2010, 23(6)：666-667.
[21]
张雷,于晓艳,丛璐. VEGF在翼状胬肉组织中的表达[J]. 中国实验诊断学, 2006, 10(12)：1464-1465.
[22]
朱芳,李振宇,任精华, 等. VEGF与肿瘤血管生成拟态关系的研究(英文)[J]. 中德临床肿瘤学杂志, 2009, 14(11)：20-24.
[23]
宋颖,杨文蕾,张琳. VEGF、SDF－1、Ki－67、PCNA及Survivin与翼状胬肉的关系分析[J]. 国际眼科杂志, 2015(12)：2120-2122.
[24]
Mei J,Gao Y,Zhang L, et al. VEGF－siRNA silencing induces apoptosis, inhibits proliferation and suppresses vasculogenic mimicry in osteosarcoma in vitro[J]. Exp Oncol, 2008, 30(1)：29-34.
[25]
王俊艳,王立,孙涛, 等. VEGF－α诱导对卵巢癌血管生成拟态形成及侵袭、迁移能力的影响[J]. 中国组织化学与细胞化学杂志, 2011, 20(1)：48-52.
[26]
肖丽,姜达. 乳腺癌血管生成拟态及其与基质金属蛋白酶－2表达的相关性研究[J]. 河北医药, 2015, 37(14)：2085-2089.
[27]
李丕嵩,李建一,张文海, 等. 血管生成拟态在恶性肿瘤的研究进展[J]. 国际肿瘤学杂志, 2014, 41(11)：824-827.
[28]
Zhang J,Qiao L,Liang N, et al. Vasculogenic mimicry and tumor metastasis[J]. Journal of BUON, 2016, 21(3)：533-541.
[29]
Zhu W,Sun W,Zhang JT, et al. Norcantharidin enhances TIMP－2 anti－vasculogenic mimicry activity for human gallbladder cancers through downregulating MMP－2 and MT1－MMP[J]. Int J Oncol, 2014, 46(2)：627-640.
[30]
Li S,Meng W,Guan Z, et al. The hypoxia－related signaling pathways of vasculogenic mimicry in tumor treatment[J]. Biomedicine & pharmacotherapy, 2016, 80(80)：127-135.
[31]
Kirschmann DA,Seftor EA,Hardy KM, et al. Molecular Pathways: Vasculogenic Mimicry in Tumor Cells: Diagnostic and Therapeutic Implications[J]. Clinical Cancer Research, 2012, 18(10)：2726-2732.
[32]
张百红. 缺氧导致肿瘤发生的机制[J]. 国际肿瘤学杂志, 2012, 39(2)：108-110.
[33]
奚再兴,张贺,何小姣, 等. 缺氧与胶质瘤细胞血管生成拟态现象的体外研究[J]. 潍坊医学院学报, 2012, 34(3)：180-182.
[34]
张劲涛,林凯金,贾永峰, 等. 缺氧诱导因子－1α对口腔鳞状细胞癌血管生成拟态相关基因影响的实验研究[J]. 中华临床医师杂志(电子版), 2012, 6(19)：5814-5820.
[35]
Zhou TJ,Huang XH,Gong L, et al. Vasculogenic mimicry and hypoxia－inducible factor－1α expression in cervical squamous cell carcinoma[J]. Genet Mol Res, 2016, 15(1)：7396.
[36]
Kütscher C,Lampert FM,Kunze M, et al. Overexpression of hypoxia－inducible factor－1 alpha improves vasculogenesis－related functions of endothelial progenitor cells[J]. Microvascular Research, 2016, 105(105)：85-92.
[37]
胡佳丽,高玉. 低氧诱导因子－1α在视网膜母细胞瘤发生发展中作用的研究进展[J]. 国际眼科杂志, 2016, 16(8)：1477-1479.
[38]
Huang LE,Gu J,Schau M, et al. Regulation of hypoxia－inducible factor 1alpha is mediated by an O2－dependent degradation domain via the ubiquitin－proteasome pathway[J]. Proc Nat Acad Sci USA, 1998, 95(14)：7987-7992.
[39]
Qiao L,Liang N,Zhang J, et al. Advanced research on vasculogenic mimicry in cancer[J]. J Cell Mol Med, 2015, 19(2)：315-326.
[40]
李明渊,唐仁泓. HIF－1与VEGF在翼状胬肉中的表达及意义[J]. 中华实验眼科杂志, 2009, 27(3)：214-217.
[41]
王建文,郑建秋,罗丹. 基质金属蛋白酶－2、－9在眼部翼状胬肉组织中的表达及意义[J]. 哈尔滨医科大学学报, 2015, 49(5)：412-415.
[42]
张丹娜,杨桂芳,刘钊臣. Bcl－2和MMP－9在翼状胬肉中的表达及意义[J]. 国际眼科杂志, 2012, 12(9)：1729-1730.
[43]
曾军,姜德咏,刘湘平, 等. 基质金属蛋白酶在翼状胬肉的表达[J]. 眼科学报, 2004, 20(4)：242-245.
[1] 张梦璐, 邢泽宇, 王昕, 刘嘉琦, 刘刚, 王翔. 血管内皮生长因子在炎性乳腺癌中的应用[J]. 中华乳腺病杂志(电子版), 2021, 15(05): 302-306.
[2] 史旭锋, 魏冬, 余小平, 周军利. 血管内皮生长因子介导的血管新生促进创面愈合的研究进展[J]. 中华损伤与修复杂志(电子版), 2021, 16(06): 536-538.
[3] 王博, 白子锐, 李坚. 近红外二区新型血管内皮生长因子受体靶向探针在结直肠癌小鼠模型中的应用[J]. 中华普通外科学文献(电子版), 2023, 17(03): 173-177.
[4] 芦丹, 杨硕, 刘旭. VEGF、HMGB1、hs-CRP/Alb在AECOPD伴呼吸衰竭中的变化及预后分析[J]. 中华肺部疾病杂志(电子版), 2023, 16(04): 532-534.
[5] 王玉芹, 刘冠群, 曲福君. IDH1和VEGF对非小细胞肺癌的诊断意义[J]. 中华肺部疾病杂志(电子版), 2022, 15(06): 847-849.
[6] 何继强, 任安伟, 王飞. 周围型肺癌CT特征与PCNA、VEGF及Bcl-2表达水平的相关性[J]. 中华肺部疾病杂志(电子版), 2022, 15(04): 527-529.
[7] 林红卫, 李王平, 金发光. PM2.5激活HIF-1α-NF-κB/VEGF通路对肺损伤的影响[J]. 中华肺部疾病杂志(电子版), 2022, 15(03): 316-322.
[8] 翁晓芹, 秦嘉阳, 张扬, 崔进, 沈红. HIF-1α/VEGF-VEGFR2/Nrp-1表达对NSCLC患者Treg增殖的影响[J]. 中华肺部疾病杂志(电子版), 2021, 14(06): 717-722.
[9] 龙慧玲, 林蜜, 邵婷. 三维球体间充质干细胞培养技术的研究进展及其应用[J]. 中华细胞与干细胞杂志(电子版), 2023, 13(04): 229-234.
[10] 崔宏宇, 杨一佺, 郭黎霞, 吕爱国, 张志宏, 张新, 杨艳萍, 申然, 连丽英, 曹志刚, 王立芳, 胡建华, 范肃洁. 改良Ahmed青光眼引流阀植入术治疗闭角期新生血管性青光眼疗效的临床研究[J]. 中华眼科医学杂志(电子版), 2023, 13(02): 76-81.
[11] 吴培培, 陈秀丽, 冯程程, 徐海峰. 玻璃体腔注射雷珠单抗联合地塞米松治疗视网膜中央静脉阻塞继发黄斑水肿的临床研究[J]. 中华眼科医学杂志(电子版), 2022, 12(01): 24-30.
[12] 吴晓翔, 杨波, 李景漩, 张凤玲, 郭桂辉, 郑少培. 脐动脉超声检查联合NLR、sFlt-1/PLGF对妊娠高血压综合征患者不良妊娠结局的预测价值[J]. 中华临床医师杂志(电子版), 2023, 17(03): 266-271.
[13] 李辉, 李海霞, 王秀香. 血必净注射液辅助治疗原发性肝癌介入术后综合征的临床研究[J]. 中华介入放射学电子杂志, 2022, 10(01): 27-31.
[14] 颜凡辉, 赵明俐, 李颖, 郭方明, 詹景冬, 赵英杰, 王阳, 张艳芬, 赵笑梅. 急性冠脉综合征患者冠脉血管病变程度与血清TNF-α、VEGF水平相关性研究[J]. 中华诊断学电子杂志, 2023, 11(03): 158-164.
[15] 徐高丽, 张建兴, 周健, 沈文俊, 谷志远, 徐国超. 静压力下缺氧诱导因子-1α信号通路对髁突软骨细胞增殖与凋亡的调控作用[J]. 中华老年病研究电子杂志, 2021, 08(03): 24-29.
阅读次数
全文


摘要

版权所有 中华医学会 中华医学电子音像出版社有限责任公司  京ICP备14006079号-1  网络出版服务许可证：(署)网出证(京)字第075号