中华实验眼科杂志
中華實驗眼科雜誌
중화실험안과잡지
CHINESE JOURNAL OF EXPERIMENTAL OPHTHALMOLOGY
2011年
10期
868-871
,共4页
视网膜前体细胞/移植%趋化性细胞因子受体4/基质细胞衍生因子-1%低氧
視網膜前體細胞/移植%趨化性細胞因子受體4/基質細胞衍生因子-1%低氧
시망막전체세포/이식%추화성세포인자수체4/기질세포연생인자-1%저양
Retinal progenitor cells/transplantation%Chemotaxis factor receptor/stromal cells derived factor-1%Hypoxia
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5×106～10×106个/L细胞的悬液,将细胞接种到25 cm2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24 h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30 μg/L的SDF-1对RPCs的趋化效应.结果 10％ O2培养12h和24 h后,RPCs中CXCR4 mRNA的表达量(CXCR4 mRNA/β-actin mRNA)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24 h后RPCs中HIF-1 mRNA表达量(HIF-1 mRNA/β-actin mRNA)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24 h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24 h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.H1F-1的表达增加是CXCR4表达增高的可能机制.
揹景 體外研究錶明,趨化性細胞因子受體4(CXCR4)及其配體基質細胞衍生因子-1(SDF-1)在誘導視網膜前體細胞(RPCs)定嚮遷移的過程中可能起重要作用.RPCs錶達CXCR4升高能增彊榦細胞的趨化活性,從而提高移植細胞的定嚮遷移能力.目的 探討RPCs在低氧條件下CXCR4受體的錶達.方法 分離孕齡17d的NIH小鼠的胚胎視網膜細胞併製備成含5×106～10×106箇/L細胞的懸液,將細胞接種到25 cm2培養瓶中,用全神經毬貼壁培養法進行培養.RPCs在正常O2(體積分數16％O2)和低O2(體積分數10％O2)環境中培養12h和24 h後,用逆轉錄聚閤酶鏈反應(RT-PCR)法檢測CXCR4和缺氧誘導因子-1(HIF-1)mRNA的錶達；流式細胞儀(FACS)檢測RPCs中CXCR4暘性細胞的百分比；Boyden小室實驗觀察30 μg/L的SDF-1對RPCs的趨化效應.結果 10％ O2培養12h和24 h後,RPCs中CXCR4 mRNA的錶達量(CXCR4 mRNA/β-actin mRNA)分彆為0.28±0.07和0.48+0.17,比正常氧培養組的0.16+0.02升高瞭1.75倍和3.00倍,10％ O2培養12h和24 h後RPCs中HIF-1 mRNA錶達量(HIF-1 mRNA/β-actin mRNA)分彆為0.18±0.07和0.38±0.13,比正常氧培養組的0.06±0.01升高瞭3.00倍和6.30倍,差異有統計學意義(P＜0.01).Boyden小室實驗錶明,10％O2培養12h和24 h後SDF-1對RPCs的趨化效應由正常氧的13.00％分彆上升到36.00％和46.00％.FACS檢測錶明,10％ O2誘導12h和24 h後,RPCs中CXCR4暘性細胞率由正常氧濃度的9.01％分彆上升到26.90％和46.10％,差異均有統計學意義(P＜0.01).結論 RPCs在低氧條件下CXCR4受體錶達增加,同時對SDF-1的趨化能力增彊.H1F-1的錶達增加是CXCR4錶達增高的可能機製.
배경 체외연구표명,추화성세포인자수체4(CXCR4)급기배체기질세포연생인자-1(SDF-1)재유도시망막전체세포(RPCs)정향천이적과정중가능기중요작용.RPCs표체CXCR4승고능증강간세포적추화활성,종이제고이식세포적정향천이능력.목적 탐토RPCs재저양조건하CXCR4수체적표체.방법 분리잉령17d적NIH소서적배태시망막세포병제비성함5×106～10×106개/L세포적현액,장세포접충도25 cm2배양병중,용전신경구첩벽배양법진행배양.RPCs재정상O2(체적분수16％O2)화저O2(체적분수10％O2)배경중배양12h화24 h후,용역전록취합매련반응(RT-PCR)법검측CXCR4화결양유도인자-1(HIF-1)mRNA적표체；류식세포의(FACS)검측RPCs중CXCR4양성세포적백분비；Boyden소실실험관찰30 μg/L적SDF-1대RPCs적추화효응.결과 10％ O2배양12h화24 h후,RPCs중CXCR4 mRNA적표체량(CXCR4 mRNA/β-actin mRNA)분별위0.28±0.07화0.48+0.17,비정상양배양조적0.16+0.02승고료1.75배화3.00배,10％ O2배양12h화24 h후RPCs중HIF-1 mRNA표체량(HIF-1 mRNA/β-actin mRNA)분별위0.18±0.07화0.38±0.13,비정상양배양조적0.06±0.01승고료3.00배화6.30배,차이유통계학의의(P＜0.01).Boyden소실실험표명,10％O2배양12h화24 h후SDF-1대RPCs적추화효응유정상양적13.00％분별상승도36.00％화46.00％.FACS검측표명,10％ O2유도12h화24 h후,RPCs중CXCR4양성세포솔유정상양농도적9.01％분별상승도26.90％화46.10％,차이균유통계학의의(P＜0.01).결론 RPCs재저양조건하CXCR4수체표체증가,동시대SDF-1적추화능력증강.H1F-1적표체증가시CXCR4표체증고적가능궤제.
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)inRPCs 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-1 mRNA/β-actin mRNA)in RPCs cultured by 10％ O2 for 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 μg/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 H1F-1 in RPCs may be involved in the up-regulation of CXCR4 expression.
