中华眼底病杂志
中華眼底病雜誌
중화안저병잡지
CHINESE JOURNAL OF OCULAR FUNDUS DISEASES
2014年
4期
399-403
,共5页
王菁%蔡萌%李静%田蓉%林少芬%田景毅%李佳%俞德超%罗燕
王菁%蔡萌%李靜%田蓉%林少芬%田景毅%李佳%俞德超%囉燕
왕정%채맹%리정%전용%림소분%전경의%리가%유덕초%라연
视网膜色素上皮%受体,补体3b/拮抗剂和抑制剂%补体激活%动物实验
視網膜色素上皮%受體,補體3b/拮抗劑和抑製劑%補體激活%動物實驗
시망막색소상피%수체,보체3b/길항제화억제제%보체격활%동물실험
Retinal pigment epithelium%Receptors,complement 3b/antagonists & inhibitors%Complement activation%Animal experimentation
目的 观察补体受体1(CR1)对补体激活的氧化应激状态下人视网膜色素上皮(hRPE)单层细胞屏障的保护作用.方法 原代培养3～5代hRPE细胞,建立稳定的hRPE单层细胞屏障模型.500 μmol/L叔丁基过氧化氢和10％正常人血清处理hRPE细胞,建立hRPE单层细胞屏障补体激活的氧化损伤模型.将补体激活的氧化应激状态下hRPE单层细胞屏障分为模型组和CR1治疗组.模型组加入1 μl的磷酸盐缓冲液,CR1治疗组加入1 μl的CR1溶液使其终浓度为1μg/ml,继续培养4h.以正常hRPE单层细胞屏障作为正常对照组,检测模型组和CR1治疗组细胞的跨表皮细胞膜电阻(TER)值.酶联免疫吸附试验检测模型组和CR1治疗组血管内皮生长因子(VEGF)、趋化因子配体2(CCL2)、C3a、C5a、膜攻击复合物(MAC)的蛋白量.结果 hRPE单层细胞屏障于接种后3周稳定形成.模型组、CR1治疗组补体激活的氧化损伤hRPE细胞TER值分别为正常hRPE细胞的54.01％、63.48％.模型组与CR1治疗组补体激活的氧化损伤hRPE细胞TER值比较,差异有统计学意义(t=21.60,P＜0.05).CR1治疗组VEGF、CCL2蛋白量分别较模型组下降了11.48％、23.47％;两组VEGF、CCL2蛋白量比较,差异均有统计学意义(t=3.26、2.43,P＜0.05).CR1治疗组C3a、C5a、MAC蛋白量较模型组分别下降了24.00％、27.87％、22.44％;两组C3a、C5a、MAC蛋白量比较,差异均有统计学意义(t=9.86、2.63、6.94,P＜0.05).结论 CR1对补体激活的氧化应激状态下hRPE单层细胞屏障具有保护作用,抑制补体激活、下调CCL2和VEGF表达可能是其机制.
目的 觀察補體受體1(CR1)對補體激活的氧化應激狀態下人視網膜色素上皮(hRPE)單層細胞屏障的保護作用.方法 原代培養3～5代hRPE細胞,建立穩定的hRPE單層細胞屏障模型.500 μmol/L叔丁基過氧化氫和10％正常人血清處理hRPE細胞,建立hRPE單層細胞屏障補體激活的氧化損傷模型.將補體激活的氧化應激狀態下hRPE單層細胞屏障分為模型組和CR1治療組.模型組加入1 μl的燐痠鹽緩遲液,CR1治療組加入1 μl的CR1溶液使其終濃度為1μg/ml,繼續培養4h.以正常hRPE單層細胞屏障作為正常對照組,檢測模型組和CR1治療組細胞的跨錶皮細胞膜電阻(TER)值.酶聯免疫吸附試驗檢測模型組和CR1治療組血管內皮生長因子(VEGF)、趨化因子配體2(CCL2)、C3a、C5a、膜攻擊複閤物(MAC)的蛋白量.結果 hRPE單層細胞屏障于接種後3週穩定形成.模型組、CR1治療組補體激活的氧化損傷hRPE細胞TER值分彆為正常hRPE細胞的54.01％、63.48％.模型組與CR1治療組補體激活的氧化損傷hRPE細胞TER值比較,差異有統計學意義(t=21.60,P＜0.05).CR1治療組VEGF、CCL2蛋白量分彆較模型組下降瞭11.48％、23.47％;兩組VEGF、CCL2蛋白量比較,差異均有統計學意義(t=3.26、2.43,P＜0.05).CR1治療組C3a、C5a、MAC蛋白量較模型組分彆下降瞭24.00％、27.87％、22.44％;兩組C3a、C5a、MAC蛋白量比較,差異均有統計學意義(t=9.86、2.63、6.94,P＜0.05).結論 CR1對補體激活的氧化應激狀態下hRPE單層細胞屏障具有保護作用,抑製補體激活、下調CCL2和VEGF錶達可能是其機製.
목적 관찰보체수체1(CR1)대보체격활적양화응격상태하인시망막색소상피(hRPE)단층세포병장적보호작용.방법 원대배양3～5대hRPE세포,건립은정적hRPE단층세포병장모형.500 μmol/L숙정기과양화경화10％정상인혈청처리hRPE세포,건립hRPE단층세포병장보체격활적양화손상모형.장보체격활적양화응격상태하hRPE단층세포병장분위모형조화CR1치료조.모형조가입1 μl적린산염완충액,CR1치료조가입1 μl적CR1용액사기종농도위1μg/ml,계속배양4h.이정상hRPE단층세포병장작위정상대조조,검측모형조화CR1치료조세포적과표피세포막전조(TER)치.매련면역흡부시험검측모형조화CR1치료조혈관내피생장인자(VEGF)、추화인자배체2(CCL2)、C3a、C5a、막공격복합물(MAC)적단백량.결과 hRPE단층세포병장우접충후3주은정형성.모형조、CR1치료조보체격활적양화손상hRPE세포TER치분별위정상hRPE세포적54.01％、63.48％.모형조여CR1치료조보체격활적양화손상hRPE세포TER치비교,차이유통계학의의(t=21.60,P＜0.05).CR1치료조VEGF、CCL2단백량분별교모형조하강료11.48％、23.47％;량조VEGF、CCL2단백량비교,차이균유통계학의의(t=3.26、2.43,P＜0.05).CR1치료조C3a、C5a、MAC단백량교모형조분별하강료24.00％、27.87％、22.44％;량조C3a、C5a、MAC단백량비교,차이균유통계학의의(t=9.86、2.63、6.94,P＜0.05).결론 CR1대보체격활적양화응격상태하hRPE단층세포병장구유보호작용,억제보체격활、하조CCL2화VEGF표체가능시기궤제.
Objective To observe the effect of complement receptor 1 (CR1) on barrier of cultured human retinal epithelial cells (hRPE) under complement-activated oxidative stress.Methods The third to fifth passage of hRPE cultured on Transwell insert were used to establish a stable hRPE monolayer barrier.The hRPE monolayer barrier was exposed to 500 μmol/L ten-butyl hydroperoxide and 10％ normal human serum to establish the hRPE monolayer barrier model of complement-activated oxidative stress in vitro.hRPE monolayer barriers under complement-activated oxidative stress were divided into two groups including model group and CR1 treatment (1 μg/ml) group.Model group and CR1 treatment group were treated with 1 μl phosphate buffer solution (PBS) or CR1 for 4 hours.Normal hRPE monolayer barrier were used as control in transepithelial resistance (TER) measurement experiment.TER was measured to evaluate the barrier function of hRPE.The hRPE-secreted vascular endothelial growth factor (VEGF) and chemokine (C-C Motif) Ligand 2 (CCL2),together with complement bioactive fragments (C3a,C5a) and membrane-attack complex (MAC) in the supernatant were detected by enzyme-linked immune sorbent assay.Results Stable hRPE monolayer barrier was established 3 weeks after hRPE seeded on Transwell insert.Complement-activated oxidative stress resulted in a sharp decrease of TER to 54.51％ compared with normal hRPE barrier.CR1 treatment could significantly improve TER of barrier under complement-activated oxidative stress to 63.48％ compared with normal hRPE barrier(t =21.60,P＜0.05).Compared with model group,CR1 treatment could significantly decrease the concentration of VEGF and CCL2 by 11.48％ and 23.47 ％ secreted by hRPE under complement-activated oxidative stress (t =3.26,2.43; P＜0.05).Compared with model group,CR1 treatment could also decreased the concentration of C3a,C5a and MAC by 24.00 ％,27.87 ％,22.44 ％.The difference were statistically significant (t =9.86,2.63,6.94 ; P＜0.05).Conclusions CR1 could protect the barrier function of hRPE cells against complement-activated oxidative stress.The underlying mechanism may involve inhibiting complement activation and downregulating the expression of VEGF and CCL2.
