中华实验眼科杂志
中華實驗眼科雜誌
중화실험안과잡지
CHINESE JOURNAL OF EXPERIMENTAL OPHTHALMOLOGY
2013年
11期
1031-1036
,共6页
陈惠康%张济明%李龙标%钱益勇%刘高勤%罗宝根%费梅
陳惠康%張濟明%李龍標%錢益勇%劉高勤%囉寶根%費梅
진혜강%장제명%리룡표%전익용%류고근%라보근%비매
热休克蛋白/B6%中和性抗体%脉络膜血管内皮细胞%新生血管%迁移%凋亡
熱休剋蛋白/B6%中和性抗體%脈絡膜血管內皮細胞%新生血管%遷移%凋亡
열휴극단백/B6%중화성항체%맥락막혈관내피세포%신생혈관%천이%조망
Heat shock protein/B6%Neutralizing antibody%Choroidal vascular endothelial cell%Neovascularization%Migration%Apoptosis
背景 血管内皮细胞的增生和迁移是血管发生的主要环节.新近的研究发现,热休克蛋白B6(HspB6)可促进多种与血管新生相关因子的分泌,导致病理状态下新生血管的发生,研究HspB6中和性抗体对人脉络膜血管内皮细胞增生、迁移和管腔形成能力的影响对于脉络膜新生血管相关疾病的靶向治疗具有重要意义. 目的 探讨HspB6中和性抗体对人脉络膜血管内皮细胞管腔形成能力的影响及其机制.方法 对人脉络膜血管内皮细胞株进行培养和传代,取对数生长期细胞用于实验.应用逆转录PCR(RT-PCR)和流式细胞术检测HspB6 mRNA及其蛋白在人脉络膜血管内皮细胞中的表达.将基质胶铺于96孔板中,凝固后加入细胞悬液,细胞密度为2×104个/孔,将不同质量浓度(100 μg/L、500 μg/L)HspB6中和性抗体分别加入培养孔,未加入HspB6中和性抗体的细胞作为对照组(0μg/L HspB6中和性抗体组),每组设3个复孔.细胞孵育12h后,采用体外三维成型法检测各组完整管腔形成的数量,以评价HspB6中和性抗体对人脉络膜血管内皮细胞的管腔形成能力.96孔板培养人脉络膜血管内皮细胞,培养液中加入不同质量浓度(0、50、100、500 μg/L)HspB6中和性抗体孵育24 h,然后采用细胞计数试剂盒-8(CCK-8)检测细胞的增生值.采用Transwell小室法检测HspB6中和性抗体作用24 h后各组穿过小室膜的“贴壁”细胞数目,以评估人脉络膜内皮细胞的增生和迁移情况.收集HspB6中和性抗体干预后的人脉络膜血管内皮细胞,流式细胞术检测HspB6中和性抗体作用后各组人脉络膜血管内皮细胞的凋亡率. 结果 人脉络膜血管内皮细胞在基因和蛋白水平均可表达HspB6分子.0、100、500 μg/L HspB6中和性抗体组管腔形成数目分别为(67.25±5.75)、(60.39±6.41)和(39.76±10.73)个/视野,总体比较差异有统计学意义(F=10.210,P=0.012),其中500 μg/L HspB6中和性抗体组管腔形成数目明显少于0 μg/L HspB6中和性抗体组,差异有统计学意义(P=0.005).随着HspB6中和性抗体质量浓度的增加和作用时间的延长,其对细胞增生和迁移的抑制率均明显增加,差异均有统计学意义(F质量浓度=7.485,P=0.002;F时间=16.684,P=0.001).Transwell小室法检测显示,0、50、100、500 μg/L HspB6中和性抗体组细胞迁移数目分别为(14.0±2.5)、(11.1±0.8)、(6.6±0.1)、(6.7±0.2)个,其中100 μg/L、500 μg/L HspB6中和性抗体干预24 h后细胞迁移数目较0μg/L HspB6中和性抗体组明显减少,差异均有统计学意义(均P=0.000).流式细胞术检测结果发现,HspB6中和性抗体组人脉络膜血管内皮细胞的凋亡率为(22.73±2.53)％,对照组为(13.33±2.08)％,差异有统计学意义(t=4.967,P=0.008). 结论 HspB6中和性抗体对脉络膜血管内皮细胞的管腔形成有抑制作用,可能通过抑制血管内皮细胞的增生、迁移以及促进细胞的凋亡等途径发挥效应.
揹景 血管內皮細胞的增生和遷移是血管髮生的主要環節.新近的研究髮現,熱休剋蛋白B6(HspB6)可促進多種與血管新生相關因子的分泌,導緻病理狀態下新生血管的髮生,研究HspB6中和性抗體對人脈絡膜血管內皮細胞增生、遷移和管腔形成能力的影響對于脈絡膜新生血管相關疾病的靶嚮治療具有重要意義. 目的 探討HspB6中和性抗體對人脈絡膜血管內皮細胞管腔形成能力的影響及其機製.方法 對人脈絡膜血管內皮細胞株進行培養和傳代,取對數生長期細胞用于實驗.應用逆轉錄PCR(RT-PCR)和流式細胞術檢測HspB6 mRNA及其蛋白在人脈絡膜血管內皮細胞中的錶達.將基質膠鋪于96孔闆中,凝固後加入細胞懸液,細胞密度為2×104箇/孔,將不同質量濃度(100 μg/L、500 μg/L)HspB6中和性抗體分彆加入培養孔,未加入HspB6中和性抗體的細胞作為對照組(0μg/L HspB6中和性抗體組),每組設3箇複孔.細胞孵育12h後,採用體外三維成型法檢測各組完整管腔形成的數量,以評價HspB6中和性抗體對人脈絡膜血管內皮細胞的管腔形成能力.96孔闆培養人脈絡膜血管內皮細胞,培養液中加入不同質量濃度(0、50、100、500 μg/L)HspB6中和性抗體孵育24 h,然後採用細胞計數試劑盒-8(CCK-8)檢測細胞的增生值.採用Transwell小室法檢測HspB6中和性抗體作用24 h後各組穿過小室膜的“貼壁”細胞數目,以評估人脈絡膜內皮細胞的增生和遷移情況.收集HspB6中和性抗體榦預後的人脈絡膜血管內皮細胞,流式細胞術檢測HspB6中和性抗體作用後各組人脈絡膜血管內皮細胞的凋亡率. 結果 人脈絡膜血管內皮細胞在基因和蛋白水平均可錶達HspB6分子.0、100、500 μg/L HspB6中和性抗體組管腔形成數目分彆為(67.25±5.75)、(60.39±6.41)和(39.76±10.73)箇/視野,總體比較差異有統計學意義(F=10.210,P=0.012),其中500 μg/L HspB6中和性抗體組管腔形成數目明顯少于0 μg/L HspB6中和性抗體組,差異有統計學意義(P=0.005).隨著HspB6中和性抗體質量濃度的增加和作用時間的延長,其對細胞增生和遷移的抑製率均明顯增加,差異均有統計學意義(F質量濃度=7.485,P=0.002;F時間=16.684,P=0.001).Transwell小室法檢測顯示,0、50、100、500 μg/L HspB6中和性抗體組細胞遷移數目分彆為(14.0±2.5)、(11.1±0.8)、(6.6±0.1)、(6.7±0.2)箇,其中100 μg/L、500 μg/L HspB6中和性抗體榦預24 h後細胞遷移數目較0μg/L HspB6中和性抗體組明顯減少,差異均有統計學意義(均P=0.000).流式細胞術檢測結果髮現,HspB6中和性抗體組人脈絡膜血管內皮細胞的凋亡率為(22.73±2.53)％,對照組為(13.33±2.08)％,差異有統計學意義(t=4.967,P=0.008). 結論 HspB6中和性抗體對脈絡膜血管內皮細胞的管腔形成有抑製作用,可能通過抑製血管內皮細胞的增生、遷移以及促進細胞的凋亡等途徑髮揮效應.
배경 혈관내피세포적증생화천이시혈관발생적주요배절.신근적연구발현,열휴극단백B6(HspB6)가촉진다충여혈관신생상관인자적분비,도치병리상태하신생혈관적발생,연구HspB6중화성항체대인맥락막혈관내피세포증생、천이화관강형성능력적영향대우맥락막신생혈관상관질병적파향치료구유중요의의. 목적 탐토HspB6중화성항체대인맥락막혈관내피세포관강형성능력적영향급기궤제.방법 대인맥락막혈관내피세포주진행배양화전대,취대수생장기세포용우실험.응용역전록PCR(RT-PCR)화류식세포술검측HspB6 mRNA급기단백재인맥락막혈관내피세포중적표체.장기질효포우96공판중,응고후가입세포현액,세포밀도위2×104개/공,장불동질량농도(100 μg/L、500 μg/L)HspB6중화성항체분별가입배양공,미가입HspB6중화성항체적세포작위대조조(0μg/L HspB6중화성항체조),매조설3개복공.세포부육12h후,채용체외삼유성형법검측각조완정관강형성적수량,이평개HspB6중화성항체대인맥락막혈관내피세포적관강형성능력.96공판배양인맥락막혈관내피세포,배양액중가입불동질량농도(0、50、100、500 μg/L)HspB6중화성항체부육24 h,연후채용세포계수시제합-8(CCK-8)검측세포적증생치.채용Transwell소실법검측HspB6중화성항체작용24 h후각조천과소실막적“첩벽”세포수목,이평고인맥락막내피세포적증생화천이정황.수집HspB6중화성항체간예후적인맥락막혈관내피세포,류식세포술검측HspB6중화성항체작용후각조인맥락막혈관내피세포적조망솔. 결과 인맥락막혈관내피세포재기인화단백수평균가표체HspB6분자.0、100、500 μg/L HspB6중화성항체조관강형성수목분별위(67.25±5.75)、(60.39±6.41)화(39.76±10.73)개/시야,총체비교차이유통계학의의(F=10.210,P=0.012),기중500 μg/L HspB6중화성항체조관강형성수목명현소우0 μg/L HspB6중화성항체조,차이유통계학의의(P=0.005).수착HspB6중화성항체질량농도적증가화작용시간적연장,기대세포증생화천이적억제솔균명현증가,차이균유통계학의의(F질량농도=7.485,P=0.002;F시간=16.684,P=0.001).Transwell소실법검측현시,0、50、100、500 μg/L HspB6중화성항체조세포천이수목분별위(14.0±2.5)、(11.1±0.8)、(6.6±0.1)、(6.7±0.2)개,기중100 μg/L、500 μg/L HspB6중화성항체간예24 h후세포천이수목교0μg/L HspB6중화성항체조명현감소,차이균유통계학의의(균P=0.000).류식세포술검측결과발현,HspB6중화성항체조인맥락막혈관내피세포적조망솔위(22.73±2.53)％,대조조위(13.33±2.08)％,차이유통계학의의(t=4.967,P=0.008). 결론 HspB6중화성항체대맥락막혈관내피세포적관강형성유억제작용,가능통과억제혈관내피세포적증생、천이이급촉진세포적조망등도경발휘효응.
Background The proliferation and migration of vascular endothelial cells is a primary link during angiogenesis.Studies showed that heat shock protein B6 (HspB6) promotes the secretion of multiple angiogenesis-related factors and therefore leads to neovascularization.Understanding the effects of neutralizing HspB6 antibody on the biological behavior of human choroidal vascular endothelial cells has an important significance in the target treatment of choroidal neovacularization diseases.Objective This study was to address the role and mechanism of neutralizing HspB6 antibody in tube formation of human choroidal vascular endothelial cells.Methods Human choroidal vascular endothelial cell line was normally cultured and harvested for total RNA extraction.Expressions of HspB6 mRNA and protein in human choroidal vascular endothelial cells were detected by reverse transcription PCR (RT-PCR) and flow cytometry (FCM).The cells were seeded on 96-well plate covered with matrigel at the density of 2×104/hole.Then the neutralizing HspB6 antibody at the concentration of 100 μg/Land 500 μg/L was added into the medium respectively,and the control cells were set without the addition of HspB6 antibody.The number of capillary tubes was calculated 12 hours after culture by three-dimensional matrigel assay.In addition,0,50,100,500 μg/L of neutralizing HspB6 antibody were added into the cell medium separately for 24hours,cell counting kit-8 (CCK-8) method was employed to assay the inhibitory rate(IR) of the cells.Transwell test was used to count the cell number across chamber membrane for the evaluation of migration ability of the cells.The apoptosis of the cells was assayed by FCM.Results Both HspB6 mRNA and protein were expressed on human choroidal vascular endothelial cells.The number of capillary tube formation of human choroidal vascular endothelial cells was (67.25±5.75),(60.39±6.41) and (39.76±10.73) /field in the 0,100 and 500 μg/L neutralizing HspB6 antibody groups,with significant difference among them (F =10.210,P =0.012),and the tube number was significantly less in the 500 μg/L neutralizing HspB6 antibody group compared with 0 μg/L neutralizing HspB6 group (P =0.005).The IR of neutralizing HspB6 antibody to the cellular proliferation and migration was enhanced with the increases of concentration and time lapse(Fconcentration =7.485,P =0.002 ; Ftime =16.684,P =0.001).The number of the cells through Transwell chamber membrane was 14.0 ± 2.5,11.1 ± 0.8,6.6 ± 0.1,6.7 ± 0.2 in the 0,50,100,500 μg/L neutralizing HspB6 antibody group respectively,and that in the 100 μg/L and 500 μg/L neutralizing HspB6 antibody group was lessened in comparison with the 0 μg/L neutralizing HspB6 antibody group(both at P=0.000).The apoptosis rate of the cells was (22.73 ± 2.53)％ in the neutralizing HspB6 antibody group,which was significantly lower than (13.33±2.08) ％ of the control group (t=4.967,P=0.008).Conclusions Neutralizing HspB6 antibody inhibits capillary tube formation of human choroidal endothelial cells in vitro in dose-and timedependent manner,probably through suppressing the proliferation and migration and promoting the apoptosis of choroidal endothelial cells.
