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

Inhibition of experimental corneal neovascularization by chemokine receptor 4 antagonist

背景基质细胞源性细胞因子1α/趋化因子受体4（SDF-1αCXCR4）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1mol／LNaOH的滤纸贴附在左眼角膜中央40S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CXCR4mRNA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CXCR4mRNA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48h，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。

Background Stromal-derived factor 1 α/chemokine receptor 4（SDF-lcUCXCR4） 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 experimental corneal neovascularization（CNV）. Methods CNV model was established in the left eye of 8-week- old clean BALB/c mouse by putting the filter with 1 mol/L NaOH at the central cornea for 40 seconds. The animals were randomized 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 immunoehemistry. 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. Flowcytomctry clarified that CD31 positive ceils 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.