趋化因子受体4拮抗剂对实验性角膜新生血管的抑制作用及其机制
추화인자수체4길항제대실험성각막신생혈관적억제작용급기궤제
Inhibition of experimental corneal neovascularization by chemokine receptor 4 antagonist
  • 万方数据
    中华实验眼科杂志 중화실험안과잡지
    CHINESE JOURNAL OF EXPERIMENTAL OPHTHALMOLOGY
    2012年 10期 877-881 ,共5页

    蔡琴华%刘高勤%夏春林%陆培荣

    채금화%류고근%하춘림%륙배영

    趋化因子受体4%拮抗剂新生血管%碱烧伤%趋化因子 趨化因子受體4%拮抗劑新生血管%堿燒傷%趨化因子
    추화인자수체4%길항제신생혈관%감소상%추화인자
    Chemokine receptor 4%Antagonist neovascularization%Alkali burn%Chemokine
    背景 基质细胞源性细胞因子1 α/趋化因子受体4(SDF-1α/CXCR4)信号途径是机体内介导多种细胞趋化、黏附以及增生的关键分子,能通过促进血管内皮祖细胞向肿瘤组织的迁移而促进肿瘤新生血管的发生,同时也参与新生血管性眼病的病理过程. 目的 探讨阻断CXCR4信号通路对实验性角膜新生血管(CNV)发生发展的抑制作用及机制.方法 收集8周龄BALB/c小鼠80只,将浸有1 mol/L NaOH的滤纸贴附在左眼角膜中央40 s以诱导小鼠CNV,按随机数字表法将动物分为透明质酸钠组(质量分数0.2％透明质酸钠点眼)及CXCR4拮抗剂组(0.2％透明质酸钠配制的CXCR4拮抗剂点眼),自碱烧伤当日分别用相应的药物点眼共14d,于第14天裂隙灯下观察两组小鼠的CNV,然后制备角膜悬液,用流式细胞技术检测角膜悬液中CD31的表达值；制备角膜组织切片,以逆转录PCR(RT-PCR)和Western blot法检测CXCR4 mRNA和蛋白在小鼠角膜组织中的表达,以免疫组织化学法检测角膜组织内CD31阳性标记的血管内皮细胞.以ELISA法检测角膜组织裂解液内血管内皮生长因子(VEGF)的表达.使用CXCR4拮抗剂干预小鼠腹腔来源的巨噬细胞,检测粒细胞巨噬细胞刺激因子(GM-CSF)刺激后培养上清液中VEGF的表达.结果 小鼠角膜碱烧伤后2周,裂隙灯下可见CNV达高峰,与透明质酸钠组比较,CXCR4拮抗剂组CNV明显减少；角膜免疫组织化学检测显示,CXCR4拮抗剂组小鼠角膜中CD31阳性染色强度弱于透明质酸钠组,CD31阳性细胞数量明显减少；进一步流式细胞技术检测发现,CXCR4拮抗剂组CD31阳性表达率为(9.50±2.34)％,明显低于透明质酸钠组的(17.50±3.16)％,差异有统计学意义(t=-7.312,P＜0.05);RT-PCR和Western blot法检测表明,碱烧伤后2、4、7d小鼠角膜组织中CXCR4 mRNA和蛋白的表达均明显高于正常小鼠角膜组织,组间比较差异均有统计学意义(P＜0.01；P＜0.05).ELISA检测结果显示,CXCR4拮抗剂点眼后4d、7d角膜组织内VEGF表达明显低于透明质酸钠组,差异均有统计学意义(t=10.927、5.151,P＜0.05).体外实验发现,细胞培养后12、24和48 h,GM-CSF+CXCR4拮抗剂组上清液中VEGF的表达水平明显低于GM-CSF组,差异均有统计学意义(P＜0.05). 结论 CXCR4拮抗剂能通过下调VEGF的表达抑制实验性CNV的形成.
    배경 기질세포원성세포인자1 α/추화인자수체4(SDF-1α/CXCR4)신호도경시궤체내개도다충세포추화、점부이급증생적관건분자,능통과촉진혈관내피조세포향종류조직적천이이촉진종류신생혈관적발생,동시야삼여신생혈관성안병적병리과정. 목적 탐토조단CXCR4신호통로대실험성각막신생혈관(CNV)발생발전적억제작용급궤제.방법 수집8주령BALB/c소서80지,장침유1 mol/L NaOH적려지첩부재좌안각막중앙40 s이유도소서CNV,안수궤수자표법장동물분위투명질산납조(질량분수0.2％투명질산납점안)급CXCR4길항제조(0.2％투명질산납배제적CXCR4길항제점안),자감소상당일분별용상응적약물점안공14d,우제14천렬극등하관찰량조소서적CNV,연후제비각막현액,용류식세포기술검측각막현액중CD31적표체치；제비각막조직절편,이역전록PCR(RT-PCR)화Western blot법검측CXCR4 mRNA화단백재소서각막조직중적표체,이면역조직화학법검측각막조직내CD31양성표기적혈관내피세포.이ELISA법검측각막조직렬해액내혈관내피생장인자(VEGF)적표체.사용CXCR4길항제간예소서복강래원적거서세포,검측립세포거서세포자격인자(GM-CSF)자격후배양상청액중VEGF적표체.결과 소서각막감소상후2주,렬극등하가견CNV체고봉,여투명질산납조비교,CXCR4길항제조CNV명현감소；각막면역조직화학검측현시,CXCR4길항제조소서각막중CD31양성염색강도약우투명질산납조,CD31양성세포수량명현감소；진일보류식세포기술검측발현,CXCR4길항제조CD31양성표체솔위(9.50±2.34)％,명현저우투명질산납조적(17.50±3.16)％,차이유통계학의의(t=-7.312,P＜0.05);RT-PCR화Western blot법검측표명,감소상후2、4、7d소서각막조직중CXCR4 mRNA화단백적표체균명현고우정상소서각막조직,조간비교차이균유통계학의의(P＜0.01；P＜0.05).ELISA검측결과현시,CXCR4길항제점안후4d、7d각막조직내VEGF표체명현저우투명질산납조,차이균유통계학의의(t=10.927、5.151,P＜0.05).체외실험발현,세포배양후12、24화48 h,GM-CSF+CXCR4길항제조상청액중VEGF적표체수평명현저우GM-CSF조,차이균유통계학의의(P＜0.05). 결론 CXCR4길항제능통과하조VEGF적표체억제실험성CNV적형성.
    Background Stromal-derived factor 1α /chemokine receptor 4(SDF-1α/CXCR4) axis is one of the important signals which mediates several different activities such as chemotaxis,adhesion,proliferation and survival resulting in recruitment to sites of immune and inflammatory reactions.Considerable evidence suggests that CXCR4/SDF-1α axis is involved in tumor angiogenesis and plays a key role in the development of ocular neovascularization.Objective The purpose of this study was to explore the effect of CXCR4 antagonist on the development of cxperimental corneal neovascularization(CNV).Methods CNV model was established in the left eye of 8-weekold clean BALB/c mouse by putting the filter with 1 mol/L NaOH at the central cornea for 40 seconds.The animals were randomizcd into hyaluronate group and CXCR4 antagonist group,and the edydrops was topically administered respectively on the day of modeling 4 times per day for 14 days.CNV was examined under the slit lamp at the fourteenth day,and then the corneal suspension and section were made.Expressions of CXCR4 mRNA and protein in corneas were detected using RT-PCR and Western blot.The CD31 level in cornea was assayed by flowcytometry and immunochemistry.The expression of VEGF in burned corneas and suspension from mouse peritoneal macrophages stimulated with CXCR4 antagonist in vitro was detected by ELISA.The use of the animal followed Ragulations for the Administration of Affairs Concerning Experimental Animals by State Science and Technology Commission.Results Two weeks after corneal alkali burn,the growth of CNV peaked under the slit lamp.Compared with hyaluronate group,CNV was obviously decreased in the CXCR4 antagonist group.Immunochemistry showed that intensity of positive staining for CD31 in cornea in the CXCR4 antagonist group was weaker than the hyaluronate group.Flowcytometry clarified that CD31 positive cells rate was 9.50％ ±2.34％ in the CXCR4 antagonist group and 17.50％ ±3.16％ in the hyaluronate group,showing a significant difference between them (t=-7.312,P＜0.05).In 2,4,7 days after cornea alkali burn,the expressions of CXCR4 mRNA and protein were significantly enhanced in burn corneas compared with normal corneas(P＜0.01 ;P＜0.05).ELISA showed that the VEGF expression level in corneal tissue and supernatant of mouse peritoneal macrophages in vitro were significantly lower in the CXCR4 antagonist group than that of hyaluronate group(t =10.927,5.151,P＜0.05).The expression level of VEGF in corneal suspension was lower in the GM-CSH+CXCR4 antogonist group than that in the GM-CSH group (P＜0.05).Conclusions CXCR4 antagonist can reduce experimental CNV by down-regulating VEGF expression in cornea.
    원문보기 원문다운로드 사이트로이동
저자의 최근 논문