中华实验眼科杂志
中華實驗眼科雜誌
중화실험안과잡지
CHINESE JOURNAL OF EXPERIMENTAL OPHTHALMOLOGY
2011年
2期
113-117
,共5页
孙涛%曹晖%许迅%顾青%许琳%朱弼珺
孫濤%曹暉%許迅%顧青%許琳%硃弼珺
손도%조휘%허신%고청%허림%주필군
胰岛素样生长因子结合蛋白相关蛋白-1%血管内皮细胞生长因子%视网膜%血管内皮细胞%血管新生
胰島素樣生長因子結閤蛋白相關蛋白-1%血管內皮細胞生長因子%視網膜%血管內皮細胞%血管新生
이도소양생장인자결합단백상관단백-1%혈관내피세포생장인자%시망막%혈관내피세포%혈관신생
Insulin-like growth factor binding protein-related protein 1%Vascular endothelial growth factor%Retina%Vascular endothelial cell%Angiogenesis
背景 寻求有效的血管内皮生长因子(VEGF)抑制剂是治疗和预防新生血管性眼病的关键.胰岛索样生长因子结合蛋白相关蛋白-1(IGFBP-rP1)是一种新发现的抑血管新生因子,推测其在眼内有抑制VEGF的作用.目的 探讨IGFBP-rP1对VEGF体外诱导视网膜新生血管形成的抑制作用及其机制.方法使用含质量分数10%FBS的DMEM对猕猴视网膜/脉络膜血管内皮细胞株(RF/6A)进行扩增培养,利用免疫荧光细胞化学染色法观察RF/6A细胞表达IGFBP-rP1的情况.RF/6A细胞血清饥饿法培养24h后分为对照组、10 mg/L VEGF组,50、100、200 ms/L IGFBP-rP1+10 mg/L VEGF组进行干预,分别利用MTS比色法、Transwell实验和流式细胞术比较IGFBP-rP1(0、50、100、200 mg/L)联合VEGF(10 ms/L)作用后,RF/6A细胞在增生、移行和凋亡等生物学行为方面的变化.结果 RF/6A细胞用不同质量浓度的IGFBP-rP1培养后细胞质呈FITC激发后的绿色荧光,细胞核呈PI激发后的红色荧光,而对照组细胞仅见细胞核的红色荧光.10 ms/L VEGF组RF/6A细胞的A490值、移行细胞数与对照组相比明显增加,差异均有统计学意义(t=-15.191,P=0.000;t=-21.274,P=0.000),细胞凋亡率明显下降,差异有统计学意义(t=10.228,P=0.000).与10 mg/L VEGF组相比,IGFBP-rP1(50、100、200 mg/L)+10 mg/L VEGF组RF/6A细胞的A490值、移行细胞数明显下降(均P<0.05).50、100、200 mg/L IGFBP-rP1+10 ms/L VEGF组RF/6A细胞的细胞凋亡率分别提高了(1.26±0.04)%、(1.50±0.07)%和(1.93±0.27)%,各组的总体差异有统计学意义(F=274.273,P=0.000).结论 IGFBP-rP1作为一种内源性因子,通过促细胞凋亡机制抑制VEGF诱导的视网膜血管的生成.
揹景 尋求有效的血管內皮生長因子(VEGF)抑製劑是治療和預防新生血管性眼病的關鍵.胰島索樣生長因子結閤蛋白相關蛋白-1(IGFBP-rP1)是一種新髮現的抑血管新生因子,推測其在眼內有抑製VEGF的作用.目的 探討IGFBP-rP1對VEGF體外誘導視網膜新生血管形成的抑製作用及其機製.方法使用含質量分數10%FBS的DMEM對獼猴視網膜/脈絡膜血管內皮細胞株(RF/6A)進行擴增培養,利用免疫熒光細胞化學染色法觀察RF/6A細胞錶達IGFBP-rP1的情況.RF/6A細胞血清饑餓法培養24h後分為對照組、10 mg/L VEGF組,50、100、200 ms/L IGFBP-rP1+10 mg/L VEGF組進行榦預,分彆利用MTS比色法、Transwell實驗和流式細胞術比較IGFBP-rP1(0、50、100、200 mg/L)聯閤VEGF(10 ms/L)作用後,RF/6A細胞在增生、移行和凋亡等生物學行為方麵的變化.結果 RF/6A細胞用不同質量濃度的IGFBP-rP1培養後細胞質呈FITC激髮後的綠色熒光,細胞覈呈PI激髮後的紅色熒光,而對照組細胞僅見細胞覈的紅色熒光.10 ms/L VEGF組RF/6A細胞的A490值、移行細胞數與對照組相比明顯增加,差異均有統計學意義(t=-15.191,P=0.000;t=-21.274,P=0.000),細胞凋亡率明顯下降,差異有統計學意義(t=10.228,P=0.000).與10 mg/L VEGF組相比,IGFBP-rP1(50、100、200 mg/L)+10 mg/L VEGF組RF/6A細胞的A490值、移行細胞數明顯下降(均P<0.05).50、100、200 mg/L IGFBP-rP1+10 ms/L VEGF組RF/6A細胞的細胞凋亡率分彆提高瞭(1.26±0.04)%、(1.50±0.07)%和(1.93±0.27)%,各組的總體差異有統計學意義(F=274.273,P=0.000).結論 IGFBP-rP1作為一種內源性因子,通過促細胞凋亡機製抑製VEGF誘導的視網膜血管的生成.
배경 심구유효적혈관내피생장인자(VEGF)억제제시치료화예방신생혈관성안병적관건.이도색양생장인자결합단백상관단백-1(IGFBP-rP1)시일충신발현적억혈관신생인자,추측기재안내유억제VEGF적작용.목적 탐토IGFBP-rP1대VEGF체외유도시망막신생혈관형성적억제작용급기궤제.방법사용함질량분수10%FBS적DMEM대미후시망막/맥락막혈관내피세포주(RF/6A)진행확증배양,이용면역형광세포화학염색법관찰RF/6A세포표체IGFBP-rP1적정황.RF/6A세포혈청기아법배양24h후분위대조조、10 mg/L VEGF조,50、100、200 ms/L IGFBP-rP1+10 mg/L VEGF조진행간예,분별이용MTS비색법、Transwell실험화류식세포술비교IGFBP-rP1(0、50、100、200 mg/L)연합VEGF(10 ms/L)작용후,RF/6A세포재증생、이행화조망등생물학행위방면적변화.결과 RF/6A세포용불동질량농도적IGFBP-rP1배양후세포질정FITC격발후적록색형광,세포핵정PI격발후적홍색형광,이대조조세포부견세포핵적홍색형광.10 ms/L VEGF조RF/6A세포적A490치、이행세포수여대조조상비명현증가,차이균유통계학의의(t=-15.191,P=0.000;t=-21.274,P=0.000),세포조망솔명현하강,차이유통계학의의(t=10.228,P=0.000).여10 mg/L VEGF조상비,IGFBP-rP1(50、100、200 mg/L)+10 mg/L VEGF조RF/6A세포적A490치、이행세포수명현하강(균P<0.05).50、100、200 mg/L IGFBP-rP1+10 ms/L VEGF조RF/6A세포적세포조망솔분별제고료(1.26±0.04)%、(1.50±0.07)%화(1.93±0.27)%,각조적총체차이유통계학의의(F=274.273,P=0.000).결론 IGFBP-rP1작위일충내원성인자,통과촉세포조망궤제억제VEGF유도적시망막혈관적생성.
Background Antagonists against vascular endothelial growth factor (VECF) play key roles in treating and preventing neovascular ophthalmopathy. As a novel anti-angiogenic factor, insulin-like growth factor binding protein-related protein 1 (IGFBP-rP1) might be an antagonist against VEGF in eye. Objective This study was to explore the inhibitory effect of IGFBP-rP1, a novel anti-angiogenic factor, on VEGF-induced retinal angiogenesis in vitro. Methods The retina-choroid endothelial cell line ( RF/6A ) was cultured in DMEM containing 10% fetal bovine serum. Culture cells were divided into control group(free-serum culture group) ,10mg/L VEGF culture group and different concentrations of IGFBP-rP1 (50,100,200 mg/L) +10 mg/L VEGF group. The expression of IGFBP-rP1 in the cells was detected by immunofluorescence assay. The proliferation and migration of RF/6A cells were evaluated using MTS colorimetric assay and the chemotactic motility assay, respectively. Flow cytometry was used to detect the apoptosis of RF/6A cells. Results The immunofluorescence assay RF/6A cells showed the green fluorescence in cytoplasm and red fluorescence in nuclei after cells were exposed to any concentration of IGFBP-rP1 ,but only red fluorescence was seen in nuclei in control cells. After stimulation of 10 mg/L VEGF,the proliferation value (A490) was elevated and the numbers of cell migration were increased in comparison with control group (t = -15. 191, P = 0. 000; t = -21. 274, P = 0. 000 ) , but the cellular apoptosis rate was lower than the control group (t - 10. 228, P = 0. 000 ) . After treated with various concentrations of IGFBP-rP +10% VEGF, the proliferation and migration of RF/6A cells were significantly decreased in comparison with only 10% VEGF group (F = 534. 158,P = 0. 000;F = 2742. 323,P = 0.000,respectively) ,and the inhibitory effects were gradually enhanced with the increase of IGFBP-rP1 levels (P<0. 05). The apoptosis rate of RF/6A cells in 50,100 and 200 mg/L + 10 mg/L VEGF groups increased by ( 1. 26±0. 04)% ,( 1. 50±0. 07)% and ( 1. 93±0. 27)% respectively,showing significant differences among different groups ( F = 274. 273, P = 0. 000). Conclusion IGFBP-rP1 inhibits the proliferation and activity of retina and choroid endothelial cells induced by VEGF at a concentration-independent manner. It appears to be as a novel endogenous inhibitory factor in retinal angiogenesis.
