小胶质细胞的嘌呤受体在脉络膜新生血管小鼠热损伤模型中的表达

目的　研究小胶质细胞的嘌呤受体（P2）在脉络膜新生血管（CNV）形成中的作用及其抑制剂磷酸吡哆醛-6-偶氮（苯-2，4-二磺酸）四钠盐水合物（PPADS）对小胶质细胞的功能和CNV形成的影响。方法　选取6~8周龄C57BL/6J小鼠25只，随机分为正常对照组（5只）和模型组（20只），模型组小鼠均制作激光诱导CNV模型，模型组小鼠在激光后第1、4、7、14天处死并取材视网膜和脉络膜组织。选取CX3CR1^GFP/+基因敲除小鼠15只，制作激光诱导CNV模型后随机分为3组，第1组和第2组分别于激光后在玻璃体内立即注射2 μL PPADS（PPADS注射组）或磷酸盐缓冲液（PBS对照组），第3组在激光后每天一次PPADS局部滴眼（PPADS眼药组），共3 d。CX3CR1^GFP/+小鼠于激光后第4天麻醉后行视网膜自发荧光和荧光素眼底血管造影（FFA）检查后处死取材视网膜。各组小鼠取材后分别进行免疫荧光化学染色和实时荧光定量PCR检测P2受体（P2X4、P2X7、P2Y2和P2Y12）的表达。结果　激光诱导CNV形成后，局部激活的小胶质细胞可表达P2受体（P2X4、P2X7、P2Y2和P2Y12）。与正常对照组相比，P2X4、P2X7、P2Y2、P2Y12 mRNA表达量在激光损伤后第1天即可增加，第4天达峰值（t=6.05、2.95、3.67、5.98,均为 P<0.01）；随后渐下降，至第14天时，仅有极少量激活的小胶质细胞表达目标受体。PPADS抑制P2受体后，与PBS对照组P2X4、P2X7、P2Y2和P2Y12受体mRNA的含量相比，PPADS注射组（t=5.54、9.82、3.86、7.91,均为P<0.01）和PPADS眼药组（t=3.24、5.89、6.75、4.97,均为P<0.01）均明显下降。与PBS对照组相比，PPADS眼药组与PPADS注射组的小胶质细胞的自发荧光强度均明显减弱（均为P<0.01），与PBS对照组相比，PPADS注射组和PPADS眼药组CNV渗漏荧光强度均明显减少（均为P<0.01）。但在PPADS注射组和PPADS眼药组之间，CNV荧光渗漏强度差异无统计学意义（P=0.864）。结论　激光诱导CNV形成后，局部激活的小胶质细胞可表达P2受体，P2受体抑制剂PPADS可显著影响小胶质细胞的功能，并抑制CNV的形成。

Expression of purine receptors of microglia cells in choroidal neovascularization mouse model

Objective　To investigate the role of P2 receptors of microglia in choroidal neovascularization (CNV) and the effect of its inhibitor PPADS on microglial function and the formation of CNV.Methods　A total of 25 C57BL/6J mice aged from 6 to 8 weeks were selected, including 5 mice in the normal control group and 20 mice in model groups randomly. All mice in model groups were made into laser-induced CNV models. The mice in model groups were sacrificed at the 1st, 4th, 7th and 14th days after laser treatment, and the retinal and choroid tissues were collected. A total of 15 CX3CR1^GFP/+ knockout mice were selected and made into laser-induced CNV models, then randomly divided into three groups. The first group and the second group were injected with 2 μL PPADS (PPADS injection group) or phosphate buffer solution (PBS control group) immediately after laser treatment, and the third group was treated with PPADS eye drops once a day (PPADS eye drops group) for a total of 3 days. On the 4th day after laser treatment, all of CX3CR1^GFP/+ mice underwent retinal autofluorescence and fundus fluorescein angiography (FFA) examination, then the retinal and choroid tissues were collected. The expression of P2 receptors (P2X4, P2X7, P2Y2 and P2Y12) were detected by immunofluorescence chemical staining and quantitative real-time PCR.Results　P2 receptors (P2X4, P2X7, P2Y2, and P2Y12) were expressed in locally activated microglia after laser treatment. Compared with normal control group, the mRNA expression levels of P2X4, P2X7, P2Y2 and P2Y12 increased on the 1st day after laser injury, and reached the peak on the 4th day (t=6.05,2.95,3.67,5.98, all P<0.01). By day 14, only a very small number of activated microglia expressed the target receptors. After inhibition by PPADS, compared with the PBS control group, the mRNA expression levels of P2X4, P2X7, P2Y2 and P2Y12 were significantly decreased in the PPADS injection group (t=5.54,9.82,3.86,7.91, all P<0.01) and the PPADS eye drops group (t=3.24,5.89,6.75,4.97, all P<0.01). The spontaneous fluorescence intensity of microglia in the PPADS eye drops group and the PPADS injection group was significantly decreased when compared with PBS control group (both P<0.01). The fluorescence intensity of CNV leakage in the PPADS injection group and the PPADS eye drops group was significantly decreasedcompared with the PBS control group (both P<0.01). However, there was no statistically significant difference in CNV fluorescence leakage intensity between the PPADS injection group and the PPADS eye drops group (P=0.864).Conclusion　After laser induced CNV, locally activated microglia can express P2 receptors. The P2 receptor inhibitor, PPADS, can significantly affect the function of microglia and inhibit the formation of CNV.