低氧诱导趋化因子受体表达对视网膜前体细胞迁移能力的影响

Hypoxia-induced changes of retinal progenitor cells migration by chemotaxis factor 4

背景体外研究表明，趋化性细胞因子受体4（CXCR4）及其配体基质细胞衍生因子-1（SDF．1）在诱导视网膜前体细胞（RPCs）定向迁移的过程中可能起重要作用。RPCs表达CXCR4升高能增强干细胞的趋化活性，从而提高移植细胞的定向迁移能力。目的探讨RPCs在低氧条件下CXCR4受体的表达。方法分离孕龄17d的NIH小鼠的胚胎视网膜细胞并制备成含5×10^6～10×10^6个／L细胞的悬液，将细胞接种到25cm2培养瓶中，用全神经球贴壁培养法进行培养。RPCs在正常O2（体积分数16％O2）和低O2（体积分数10％O2）环境中培养12h和24h后，用逆转录聚合酶链反应（RT—PCR）法检测CXCR4和缺氧诱导因子-1（HIF-1）mRNA的表达；流式细胞仪（FACS）检测RPCs中CXCR4阳性细胞的百分比；Boyden小室实验观察30μg／L的SDF-1对RPCs的趋化效应。结果10％O2培养12h和24h后，RPCs中CXCR4mRNA的表达量（CXCR4mRNA／B—actinmRNA）分别为0．28±0．07和0．48±0．17，比正常氧培养组的0．16±0．02升高了1．75倍和3．00倍，10％O2培养12h和24h后RPCs中HIF-1mRNA表达量（HIF—1mRNA／B—actinmRNA）分别为0．18±0．07和0．38±0．13，比正常氧培养组的0．06±0．01升高了3．00倍和6．30倍，差异有统计学意义（P〈0．01）。Boyden小室实验表明，10％O2培养12h和24h后SDF-1对RPCs的趋化效应由正常氧的13．00％分别上升到36．00％和46．00％。FACS检测表明，10％O2诱导12h和24h后，RPCs中CXCR4阳性细胞率由正常氧浓度的9．01％分别上升到26．90％和46．10％，差异均有统计学意义（P〈0．01）。结论RPCs在低氧条件下CXCR4受体表达增加，同时对SDF-1的趋化能力增强。HIF-1的表达增加是CXCR4表达增高的可能机制。

Background In vitro study showed that chemotaxis consist of chemotaxis factor 4 （CXCR4） and stromal cells derived factor-1 （ SDF-1 ） and may play a role in the orientation and migration of retinal progenitor cells （RPCs） toward lesion. Overexpression of CXCR4 in RPCs can enhance the chemotaxis activity. Objective This work was to explore the feasibility and underlying mechanism of up-regulation of CXCR4 on RPCs induced by hypoxia. Methods RPCs were retained in an incubator with normal O2volume （ 16% ） or hypoxia condition （ 10% O2 ） for 12 hours and 24 hours respectively. Flow cytometer cell analysis screening （FACS） was conduced to measure the proportion of CXCR4-expressing cells, and CXCR4, HIF-1 mRNA were analyzed by reverse transcription-polymerse chain reaction （ RT-PCR）. The chemotical effect of 30 mg/L SDF-1 to RPCs cultured under the hypoxia condition was assessed using Boyden chamber. Results The expression level of CXCR4 （CXCR4 mRNA/β-actin mRNA） in RPCs cultured by 10% O2 for 12 and 24 hours were 0. 28±0.07and 0.48±0.17 and increased by 1.75 and 3.00 fold more than that of 16% O2 culture group （0. 16±0.02） （ P〈0. 01 ）. The expression level of HIF-1 mRNA （ HIF-1mRNA/β-actin mRNA） in RPCs cultured by 10% O2for 12 and 24 hours were 0. 18±0. 07and 0. 38±0. 13 and increased by 3. 00 and 6.30 fold more than that of 16% O2 culture group （0.06±0.01） （P〈0. 01 ）. The chemotical effect of 30 βg/L SDF-1 to RPCs increased from 13.00% in 16% O2 culture group to 36.00% and 46.00% in the cells cultured by 10% O2for 12 and 24 hours. FACS revealed that the proportion of CXCR4＋ cells in hypoxia-exposure for 12 and 24 hours were 26.90% and 46. 10% ,respectively,but that in 16% O2 culture group was 9. 10% ,showing a statistically significant difference （P 〈 0.01 ）. Conclusions RPCs induced by hypoxia can enhance the expression of CXCR4 in RPE cells and the chemotaxia to SDF-1. The overexpression of HIF-1 in RPCs may be involved in the up-regulation of CXCR4 expression.