中华实验眼科杂志
中華實驗眼科雜誌
중화실험안과잡지
CHINESE JOURNAL OF EXPERIMENTAL OPHTHALMOLOGY
2015年
1期
16-20
,共5页
李红%吕建平%蔡善君%宿罡%武志鹏%宫鑫
李紅%呂建平%蔡善君%宿罡%武誌鵬%宮鑫
리홍%려건평%채선군%숙강%무지붕%궁흠
蓝光%光损伤%视网膜色素上皮%凋亡%线粒体%半胱氨酸天冬氨酸蛋白酶%蛋白激酶C%细胞培养
藍光%光損傷%視網膜色素上皮%凋亡%線粒體%半胱氨痠天鼕氨痠蛋白酶%蛋白激酶C%細胞培養
람광%광손상%시망막색소상피%조망%선립체%반광안산천동안산단백매%단백격매C%세포배양
Blue light%Light-induced injury%Retinal pigment epithelium%Apoptosis%Mitochondria%Cysteine-aspartic acid proteases%Protein kinase C%Cell culture
背景 研究已证实蓝光照射可导致视网膜色素上皮细胞(RPE)凋亡,但其机制目前尚不完全清楚. 目的 探讨线粒体凋亡通路是否参与蓝光照射诱导体外培养的人RPE细胞凋亡过程.方法 分离新鲜的供体视网膜,对人RPE细胞进行原代培养和传代,用角蛋白单克隆抗体行细胞鉴定.将体外培养的人RPE细胞分为无光照组、单纯光照组、光照+硝苯地平组、光照+钙磷酸结合蛋白C(calphostin C)组、光照+佛波酯(PMA)组.光照组细胞用(2 000±500) lx的蓝光照射人RPE细胞6h,然后继续培养24 h后终止.采用Western blot法比较两个组间RPE细胞中凋亡相关调控因子bax、bcl-2、bcl-xl的相对表达,以评价蓝光照射对RPE细胞凋亡的影响.光照+硝苯地平组、光照+calphostin C组、光照+PMA组细胞在蓝光照射前1h分别在培养基中加入相应药物,然后以(2 000±500) lx的蓝光照射人RPE细胞6h,并继续培养24 h,采用Westernblot法检测5个组细胞中caspase-9蛋白表达量的变化,观察钙通道和蛋白激酶C(PKC)通路对RPE细胞线粒体的影响.结果 培养的细胞生长良好,细胞质内充满色素颗粒,呈铺路石样排列,对角蛋白呈阳性反应.无光照组和单纯光照组均可见bax、bcl-2及bcl-xl蛋白条带,相对分子质量分别为23 000、26 000和30 000.与无光照组比较,单纯光照组bax、bcl-2和bcl-xl蛋白表达相对值(A)下降,差异均有统计学意义(t=-4.409,P=0.012;t=7.575,P=0.002;t=6.068,P=0.004).与无光照组比较,单纯光照组、光照+calphostin C组、光照+PMA组细胞中caspase-9蛋白表达均升高,差异均有统计学意义(P=0.005、0.002、0.000),而光照+硝苯地平组与无光照组比较差异无统计学意义(P=0.191).与单纯光照组比较,光照+PMA组caspase-9蛋白表达升高,差异有统计学意义(P=0.005);而光照+硝苯地平组及光照+calphostin C组caspase-9蛋白表达差异均无统计学意义(P=0.057、0.643). 结论 蓝光致体外培养的人RPE细胞凋亡,同时细胞中caspase-9表达增强,凋亡抑制基因bcl-2及bcl-xl表达下降,凋亡促进基因bax蛋白表达增强.线粒体凋亡通路参与蓝光照射致RPE细胞凋亡的过程;PKC通路可能参与了蓝光导致的人RPE细胞凋亡.
揹景 研究已證實藍光照射可導緻視網膜色素上皮細胞(RPE)凋亡,但其機製目前尚不完全清楚. 目的 探討線粒體凋亡通路是否參與藍光照射誘導體外培養的人RPE細胞凋亡過程.方法 分離新鮮的供體視網膜,對人RPE細胞進行原代培養和傳代,用角蛋白單剋隆抗體行細胞鑒定.將體外培養的人RPE細胞分為無光照組、單純光照組、光照+硝苯地平組、光照+鈣燐痠結閤蛋白C(calphostin C)組、光照+彿波酯(PMA)組.光照組細胞用(2 000±500) lx的藍光照射人RPE細胞6h,然後繼續培養24 h後終止.採用Western blot法比較兩箇組間RPE細胞中凋亡相關調控因子bax、bcl-2、bcl-xl的相對錶達,以評價藍光照射對RPE細胞凋亡的影響.光照+硝苯地平組、光照+calphostin C組、光照+PMA組細胞在藍光照射前1h分彆在培養基中加入相應藥物,然後以(2 000±500) lx的藍光照射人RPE細胞6h,併繼續培養24 h,採用Westernblot法檢測5箇組細胞中caspase-9蛋白錶達量的變化,觀察鈣通道和蛋白激酶C(PKC)通路對RPE細胞線粒體的影響.結果 培養的細胞生長良好,細胞質內充滿色素顆粒,呈鋪路石樣排列,對角蛋白呈暘性反應.無光照組和單純光照組均可見bax、bcl-2及bcl-xl蛋白條帶,相對分子質量分彆為23 000、26 000和30 000.與無光照組比較,單純光照組bax、bcl-2和bcl-xl蛋白錶達相對值(A)下降,差異均有統計學意義(t=-4.409,P=0.012;t=7.575,P=0.002;t=6.068,P=0.004).與無光照組比較,單純光照組、光照+calphostin C組、光照+PMA組細胞中caspase-9蛋白錶達均升高,差異均有統計學意義(P=0.005、0.002、0.000),而光照+硝苯地平組與無光照組比較差異無統計學意義(P=0.191).與單純光照組比較,光照+PMA組caspase-9蛋白錶達升高,差異有統計學意義(P=0.005);而光照+硝苯地平組及光照+calphostin C組caspase-9蛋白錶達差異均無統計學意義(P=0.057、0.643). 結論 藍光緻體外培養的人RPE細胞凋亡,同時細胞中caspase-9錶達增彊,凋亡抑製基因bcl-2及bcl-xl錶達下降,凋亡促進基因bax蛋白錶達增彊.線粒體凋亡通路參與藍光照射緻RPE細胞凋亡的過程;PKC通路可能參與瞭藍光導緻的人RPE細胞凋亡.
배경 연구이증실람광조사가도치시망막색소상피세포(RPE)조망,단기궤제목전상불완전청초. 목적 탐토선립체조망통로시부삼여람광조사유도체외배양적인RPE세포조망과정.방법 분리신선적공체시망막,대인RPE세포진행원대배양화전대,용각단백단극륭항체행세포감정.장체외배양적인RPE세포분위무광조조、단순광조조、광조+초분지평조、광조+개린산결합단백C(calphostin C)조、광조+불파지(PMA)조.광조조세포용(2 000±500) lx적람광조사인RPE세포6h,연후계속배양24 h후종지.채용Western blot법비교량개조간RPE세포중조망상관조공인자bax、bcl-2、bcl-xl적상대표체,이평개람광조사대RPE세포조망적영향.광조+초분지평조、광조+calphostin C조、광조+PMA조세포재람광조사전1h분별재배양기중가입상응약물,연후이(2 000±500) lx적람광조사인RPE세포6h,병계속배양24 h,채용Westernblot법검측5개조세포중caspase-9단백표체량적변화,관찰개통도화단백격매C(PKC)통로대RPE세포선립체적영향.결과 배양적세포생장량호,세포질내충만색소과립,정포로석양배렬,대각단백정양성반응.무광조조화단순광조조균가견bax、bcl-2급bcl-xl단백조대,상대분자질량분별위23 000、26 000화30 000.여무광조조비교,단순광조조bax、bcl-2화bcl-xl단백표체상대치(A)하강,차이균유통계학의의(t=-4.409,P=0.012;t=7.575,P=0.002;t=6.068,P=0.004).여무광조조비교,단순광조조、광조+calphostin C조、광조+PMA조세포중caspase-9단백표체균승고,차이균유통계학의의(P=0.005、0.002、0.000),이광조+초분지평조여무광조조비교차이무통계학의의(P=0.191).여단순광조조비교,광조+PMA조caspase-9단백표체승고,차이유통계학의의(P=0.005);이광조+초분지평조급광조+calphostin C조caspase-9단백표체차이균무통계학의의(P=0.057、0.643). 결론 람광치체외배양적인RPE세포조망,동시세포중caspase-9표체증강,조망억제기인bcl-2급bcl-xl표체하강,조망촉진기인bax단백표체증강.선립체조망통로삼여람광조사치RPE세포조망적과정;PKC통로가능삼여료람광도치적인RPE세포조망.
Background Studies determined that blue light exposure causes apoptosis of human retinal pigment epithelial (RPE) cells,but its mechanism is still below understood.Objective The aim of this study was to investigate whether or how mitochondrial apoptotic pathway is involved in blue-light induced apoptosis of human RPE cells in vitro.Methods Human RPE cells were isolated from fresh donor eyes and primarily cultured and passaged.The cells were identified with keratin antibody by immunochemistry.Then the cells were the non-light exposed group,simple light-exposed group,light-exposed+nifedipine group,light-exposed+calphostin C group and the light-exposed+phorbol myristate acetate (PMA) group.Human RPE cells in light-exposed group were consequently cultured for 24 hours following the exposure of (2 000±500)lx blue-light for 6 hours,and then the expression levels of bax,bcl-2,bcl-xl in the cells were detected by Western blot to evaluate the effect of blue light on the apoptosis.The cells in the light-exposed+nifedipine group,light-exposed+calphostin C group and the light-exposed+PMA group were treated with the corresponding drugs 1 hour prior to light irradiation and sequently received 6-hour light irradiation and 48-hour culture.The expression of caspase-9 protein in the cells were assayed with Western blot to assess the influence of Ca2+ channel and protein kinase C (PKC) pathway on mitochondria of RPE cells.Results Cultured cells grew well with visible pigment in cytoplasm.The cells showed the positive response for keratin and presented a cobblestone-like appearance.The expression bands of bax,bcl-2 and bcl-xl proteins were clearly visible at the molecular weight of 23 000,26 000 and 30 000 in both non-light exposed group and the simple light-exposed group,and the absorbance values of the cells to bax were elevated,while the absorbance values to bcl-2 and bcl-xl were declined in the simple light-exposed group compared with the non-light exposed group (t =-4.409,P =0.012 ;t =7.575,P =0.002 ; t =6.068,P =0.004).Compared with the non-light exposed group,the absorbance values of caspase-9 were significantly raised in the simple light-exposed group,light-exposed+calphostin C group and the lightexposed+PMA group (P=0.005,0.002,0.000),but no significant difference between the non-light exposed group and light-exposed+nifedipine group (P=0.191).Compared with the simple light-exposed group,the expression level was considerably higher in the light-exposed + PMA group (P =0.005) ; while that in the light-exposed + nifedipine group or light-exposed+calphostin C group was not significantly different (P=0.057,0.643).Conclusions Blue light exposure induces apoptosis of RPE cells by up-regulating the expressions of bax and caspase-9 proteins and down-regulating the expressions of bcl-2 and bcl-xl.The mitochondrial apoptosis pathway and PKC pathway participate in blue-light induced apoptosis of human RPE cells in vitro.
