黃雄高 魏雁濤 張少?zèng)_
玻璃體對(duì)視網(wǎng)膜色素上皮細(xì)胞收縮作用的研究△
黃雄高 魏雁濤 張少?zèng)_
玻璃體;視網(wǎng)膜色素上皮細(xì)胞;細(xì)胞收縮;細(xì)胞骨架蛋白
目的探討人玻璃體體外培養(yǎng)對(duì)人視網(wǎng)膜色素上皮細(xì)胞收縮性的影響。方法低傳代(3-5代)人視網(wǎng)膜色素上皮細(xì)胞分兩組體外培養(yǎng),一組用普通培養(yǎng)液(含體積分?jǐn)?shù)10%胎牛血清的DMEM/F12)培養(yǎng),一組用體積分?jǐn)?shù)25%玻璃體培養(yǎng),應(yīng)用I型膠原凝膠收縮實(shí)驗(yàn)檢測(cè)收縮力的變化,應(yīng)用免疫熒光技術(shù)檢測(cè)細(xì)胞骨架蛋白F-actin、α-SMA和vinculin的定位染色,應(yīng)用免疫印跡技術(shù)檢測(cè)α-SMA和vinculin蛋白的表達(dá)。結(jié)果與普通培養(yǎng)液培養(yǎng)相比,低傳代的人視網(wǎng)膜色素上皮細(xì)胞玻璃體中培養(yǎng)48 h,膠原凝膠面積為(22 127±2072)像素,明顯大于在普通培養(yǎng)液中的凝膠面積(16 084±1154)像素,細(xì)胞收縮能力顯著下降,兩組比較差異有統(tǒng)計(jì)學(xué)意義(t=4.412,P=0.012)。在普通培養(yǎng)液中,F(xiàn)-actin、vinculin主要分布于細(xì)胞周邊,vinculin形成的粘連斑粗大;而在玻璃體中培養(yǎng)后,F(xiàn)-actin重組,在細(xì)胞的一端形成扁平偽足結(jié)構(gòu),vinculin主要分布于細(xì)胞扁平偽足端和細(xì)胞中央,形成的粘連斑明顯變小。玻璃體培養(yǎng)后α-SMA蛋白表達(dá)顯著下降,與普通培養(yǎng)液相比差異有統(tǒng)計(jì)學(xué)意義(t=2.845,P=0.047);vinculin蛋白表達(dá)無(wú)顯著變化(t=0.198,P=0.852)。結(jié)論玻璃體培養(yǎng)可導(dǎo)致視網(wǎng)膜色素上皮細(xì)胞收縮性下降,這一作用可能與α-SMA蛋白表達(dá)下調(diào)及vinculin重新分布有關(guān)。
[眼科新進(jìn)展,2014,34(7):624-627]
增生性玻璃體視網(wǎng)膜病變的特征是沿著玻璃體和視網(wǎng)膜形成增生膜,增生膜的牽引導(dǎo)致視網(wǎng)膜脫離和視力喪失[1]。視網(wǎng)膜色素上皮(retinal pigment epithelium,RPE)細(xì)胞被證實(shí)經(jīng)歷了上皮間質(zhì)轉(zhuǎn)分化過(guò)程,并在增生膜的發(fā)展和收縮中起主要作用[2]。我們前期實(shí)驗(yàn)已證實(shí),玻璃體體外培養(yǎng)具有誘導(dǎo)RPE細(xì)胞間質(zhì)轉(zhuǎn)分化作用,并發(fā)現(xiàn)玻璃體培養(yǎng)引起的RPE細(xì)胞收縮性下降的現(xiàn)象與傳統(tǒng)觀點(diǎn)相悖[3]。鑒于此,我們進(jìn)行了深入研究,以期進(jìn)一步證實(shí)和探討可能的機(jī)制,現(xiàn)將結(jié)果報(bào)告如下。
1.1細(xì)胞培養(yǎng)健康人捐獻(xiàn)的眼球來(lái)源于中山大學(xué)中山眼科中心眼庫(kù),排除既往有眼病史者。分離視網(wǎng)膜獲取玻璃體,剪碎玻璃體纖維,通過(guò)0.22 μm濾器(美國(guó)Millipore公司)過(guò)濾收集玻璃體,等量分裝后-80 ℃冰箱保存。所用玻璃體體積分?jǐn)?shù)為25%,通過(guò)應(yīng)用3份體積的普通培養(yǎng)液[即含體積分?jǐn)?shù)10%胎牛血清(杭州四季青公司)的DMEM/F12(美國(guó)Gibco公司)]與1份體積的玻璃體混合配制而成[4]。按照Z(yǔ)heng等[5]報(bào)道的方法,通過(guò)胰蛋白酶消化分離,用普通培養(yǎng)液培養(yǎng)RPE細(xì)胞。低傳代細(xì)胞(3-5代)通過(guò)CK18免疫熒光染色鑒定[6-7]用于實(shí)驗(yàn)。RPE細(xì)胞分為兩組,分別應(yīng)用普通培養(yǎng)液(含體積分?jǐn)?shù)10%胎牛血清的DMEM/F12培養(yǎng)液)和體積分?jǐn)?shù)25%的玻璃體進(jìn)行培養(yǎng)。
1.2凝膠收縮將I型鼠尾膠原(美國(guó)Invitrogen公司)配制成濃度為2 mg·mL-1的中和膠原混合液加入24孔板,每孔0.5 mL。放于37 ℃、含體積分?jǐn)?shù)5%CO2細(xì)胞培養(yǎng)箱內(nèi)孵育60 min后凝固成膠原凝膠。胰蛋白酶消化RPE細(xì)胞后用無(wú)血清DMEM/F12培養(yǎng)液重懸計(jì)數(shù),將細(xì)胞密度為250×106L-1的RPE細(xì)胞接種于凝固的凝膠上(單層培養(yǎng))。2~8 h后細(xì)胞貼壁伸展,分組分別換為普通培養(yǎng)液或體積分?jǐn)?shù)25%玻璃體液,用無(wú)菌顯微鑷將凝膠與孔板壁分離使之懸浮,37 ℃、含體積分?jǐn)?shù)5%CO2細(xì)胞培養(yǎng)箱中孵育48 h后拍照,應(yīng)用Image-Pro Plus 6.0軟件分析膠原凝膠面積(以像素為單位),取3次獨(dú)立實(shí)驗(yàn)數(shù)據(jù)進(jìn)行統(tǒng)計(jì)學(xué)分析。
1.3免疫熒光分析將濃度為25×106L的RPE細(xì)胞懸液滴于消毒的載玻片上,待細(xì)胞貼壁后加入培養(yǎng)液制成細(xì)胞爬片。48 h后,細(xì)胞爬片用40 g·L-1多聚甲醛和冰冷丙酮固定,應(yīng)用兔抗α-SMA抗體(美國(guó)ABcam公司)和鼠抗人vinculin抗體(美國(guó)ABcam公司)作為一抗,用Dylight564羊抗兔IgG和Dylight488羊抗鼠IgG(杭州聯(lián)科公司)作為二抗完成免疫熒光檢測(cè)。用FITC-鬼筆環(huán)肽(美國(guó)Sigma公司)染色F-actin,用Dylight564羊抗鼠IgG作為二抗雙重染色檢測(cè)vinculin。用共聚焦顯微鏡(LSM 510 META,Zeiss公司)觀察α-SMA和vinculin蛋白定位表達(dá)的變化。
1.4免疫印跡分析兩組細(xì)胞去血清培養(yǎng)過(guò)夜后分別加入普通培養(yǎng)液或體積分?jǐn)?shù)25%玻璃體培養(yǎng)48 h,用RIPA和磷酸酶抑制劑(上海博彩公司)提取蛋白,BCA蛋白定量試劑盒(美國(guó)Thermo公司)微孔板法行蛋白定量。SDS-PAGE電泳分離蛋白,轉(zhuǎn)移至PVDF膜,50 g·L-1無(wú)脂牛奶封閉,孵育兔抗人α-SMA抗體(美國(guó)ABcam公司,150稀釋)、鼠抗人vinculin抗體(美國(guó)ABcam公司,11000稀釋)、兔抗人GAPDH抗體(杭州賢至生物科技有限公司,1500稀釋)一抗過(guò)夜。洗膜后,孵育共價(jià)結(jié)合的辣根過(guò)氧化物酶標(biāo)記的羊抗兔或鼠二抗(杭州聯(lián)科公司,15000稀釋),采用化學(xué)發(fā)光法檢測(cè)蛋白的表達(dá)。
2.1凝膠收縮膠原凝膠收縮實(shí)驗(yàn)結(jié)果顯示,兩種培養(yǎng)液培養(yǎng)48 h,體積分?jǐn)?shù)25%玻璃體培養(yǎng)組凝膠面積為(22 127±2072)像素,普通培養(yǎng)液培養(yǎng)組凝膠面積為(16 084±1154)像素。體積分?jǐn)?shù)25%玻璃體培養(yǎng)誘導(dǎo)凝膠收縮的程度顯著低于普通培養(yǎng)液誘導(dǎo)的凝膠收縮,兩組膠原凝膠面積比較差異具有統(tǒng)計(jì)學(xué)意義(t=4.412,P=0.012;見(jiàn)圖1)。
Figure 1 Images of collagen gel contraction.A:Gel contraction in normal medium after 48 hours;B:Gel contraction in volumn fraction 25% vitreous medium after 48 hours 膠原凝膠收縮像。A:普通培養(yǎng)液培養(yǎng)48 h凝膠收縮;B:體積分?jǐn)?shù)25%玻璃體培養(yǎng)48 h凝膠收縮
2.2免疫熒光分析結(jié)果免疫熒光染色結(jié)果顯示,RPE細(xì)胞在普通培養(yǎng)液中培養(yǎng)48 h后胞漿內(nèi)α-SMA的染色較強(qiáng),而細(xì)胞經(jīng)體積分?jǐn)?shù)25%玻璃體培養(yǎng)48 h后,胞漿內(nèi)α-SMA的染色減弱,明顯低于在普通培養(yǎng)液中的表達(dá)。F-actin骨架在普通培養(yǎng)液培養(yǎng)后主要分布于細(xì)胞的周邊部,形如細(xì)胞周邊的條帶;當(dāng)細(xì)胞在體積分?jǐn)?shù)25%玻璃體中培養(yǎng)48 h后,F(xiàn)-actin纖維在細(xì)胞的一端重排,形成扇形的扁平偽足,而在細(xì)胞另一端形成錐形拖尾。經(jīng)普通培養(yǎng)液培養(yǎng)48 h后,vinculin主要分布于細(xì)胞的邊緣,少部分位于細(xì)胞中央,形成的粘連斑粗大;經(jīng)體積分?jǐn)?shù)25%玻璃體培養(yǎng)48 h后,vinculin大部分從周邊向細(xì)胞F-actin富集的一端和細(xì)胞中央重新分布,vinculin形成的粘連斑也明顯變小(圖2)。
2.3免疫印跡分析結(jié)果免疫印跡結(jié)果顯示,玻璃體培養(yǎng)48 h,α-SMA蛋白的表達(dá)顯著下調(diào),vinculin蛋白表達(dá)變化不明顯(圖3A);兩組比較,α-SMA蛋白的表達(dá)差異具有統(tǒng)計(jì)學(xué)意義(t=2.845,P=0.047),vinculin蛋白表達(dá)差異無(wú)統(tǒng)計(jì)學(xué)意義(t=0.198,P=0.852,見(jiàn)圖3B)。
Figure 2 Immunofluorescence staining of α-SMA,F-actin and vinculin in two groups of culture medium after 48 hours.A,C,D,E in normal medium,B,F,G,H in 25% vitreous medium.A:α-SMA stained deeply in cytoplasm (×400);B:α-SMA stained weakly in cytoplasm (×400);C:F-actin was mainly distributed in circumferential bundles at the cell periphery (×1000);D:Vinculin was primarily distributed at the periphery of RPE cells,and formed large vinculin focal adhesions (×1000);E:Merged image of C and D,vinculin-formed focal adhesions were attached to the F-actin terminals (×1000);F:F-actin rearranged and formed lamellipodial projections at one edge of the cells and a cone-shaped tail at the other edge (×1000);G:Vinculin rearranged and smaller vinculin focal adhesions were primarily observed in the center of RPE cells and at the F-actin-accumulated edge (×1000);H:Merged image of F and G (×1000) 兩組培養(yǎng)液培養(yǎng)48 h,免疫熒光染色檢測(cè)α-SMA、F-actin及vinculin定位表達(dá)。A、C、D、E為在普通培養(yǎng)液中培養(yǎng),B、F、G、H為在體積分?jǐn)?shù)25%玻璃體中培養(yǎng)。A:α-SMA在胞漿染色較深(×400);B:α-SMA在胞漿染色較弱(×400);C:F-actin主要分布于細(xì)胞的周邊,呈條帶狀(×1000);D:vinculin主要分布于細(xì)胞周邊部,形成的粘連斑粗大(×1000);E:C和D合并圖像,vinculin形成的粘連斑位于F-actin兩端(×1000);F:F-actin重排,在細(xì)胞一端富集形成扁平偽足樣結(jié)構(gòu),另一端形成錐形拖尾(×1000);G:vinculin 重新分布,主要位于扁平偽足端和細(xì)胞中央部,粘連斑明顯變小(×1000);H:F和G合并圖像(×1000)
本研究結(jié)果表明,玻璃體培養(yǎng)減弱了RPE細(xì)胞介導(dǎo)的凝膠收縮,誘導(dǎo)細(xì)胞骨架F-actin重新排布,vinculin形成的粘連斑重新分布并由較大變?yōu)檩^小。在這個(gè)過(guò)程中,α-SMA蛋白表達(dá)顯著下調(diào),但vinculin蛋白表達(dá)無(wú)顯著變化。
傳統(tǒng)觀點(diǎn)認(rèn)為,在增生性玻璃體視網(wǎng)膜病變過(guò)程中,RPE細(xì)胞經(jīng)歷了成肌纖維細(xì)胞樣轉(zhuǎn)分化[5,7- 8],轉(zhuǎn)分化后RPE細(xì)胞的收縮性增強(qiáng)。我們的實(shí)驗(yàn)結(jié)果表明,玻璃體培養(yǎng)減弱了RPE細(xì)胞介導(dǎo)的凝膠收縮,這一結(jié)果與傳統(tǒng)觀點(diǎn)不一致。研究已證實(shí),在增生性玻璃體視網(wǎng)膜病變模型中RPE細(xì)胞發(fā)生間質(zhì)轉(zhuǎn)分化,α-SMA表達(dá)顯著增高[7],這一特征性的變化被認(rèn)為是上皮細(xì)胞成肌樣轉(zhuǎn)分化的標(biāo)志[7]。在視網(wǎng)膜前膜中,RPE細(xì)胞α-SMA的表達(dá)增加最終導(dǎo)致?tīng)坷砸暰W(wǎng)膜脫離[5]。這些充分證明RPE細(xì)胞中α-SMA表達(dá)增高是細(xì)胞收縮的動(dòng)力來(lái)源[5,7,9]。因此,我們分別應(yīng)用免疫熒光和免疫印跡技術(shù)檢測(cè)了兩種培養(yǎng)狀態(tài)下RPE細(xì)胞α-SMA的表達(dá)。結(jié)果證實(shí)玻璃體培養(yǎng)誘導(dǎo)RPE細(xì)胞α-SMA蛋白表達(dá)顯著下降,與我們以前通過(guò)RT-PCR檢測(cè)其mRNA表達(dá)結(jié)果一致[3]。有文獻(xiàn)報(bào)道,玻璃體培養(yǎng)的RPE細(xì)胞降低了成纖維細(xì)胞生長(zhǎng)因子(fibroblast growth factor,FGF)-2 mRNA和蛋白的表達(dá)[10],而FGF-2能誘導(dǎo)細(xì)胞α-SMA的表達(dá)[11]。是否玻璃體誘導(dǎo)的RPE細(xì)胞中FGF-2與α-SMA表達(dá)下降之間存在內(nèi)在聯(lián)系,尚需要進(jìn)一步研究。
Figure 3 Effects of two groups of culture medium on protein expression of α-SMA and vinculin.A:Protein expression of α-SMA and vinculin in two culture media after 48 hours;B:Comparison of α-SMA and vinculin protein expression 兩組培養(yǎng)液對(duì)α-SMA、vinculin蛋白表達(dá)的影響。A:兩組培養(yǎng)液培養(yǎng)48 h α-SMA、vinculin蛋白的表達(dá);B:α-SMA、vinculin蛋白表達(dá)的比較
本研究顯示,RPE細(xì)胞在普通培養(yǎng)液中培養(yǎng)48 h后,大部分F-actin分布于細(xì)胞的周邊,排列如帶狀,只有少部分跨過(guò)細(xì)胞漿。vinculin分布于細(xì)胞周邊與F-actin纖維末端相連。這種分布有利于保持正常的細(xì)胞形態(tài)和生理張力,使細(xì)胞處于相對(duì)靜止?fàn)顟B(tài)。細(xì)胞骨架是細(xì)胞轉(zhuǎn)分化形態(tài)和可塑性的結(jié)構(gòu)基礎(chǔ)。我們以前的研究已證實(shí),玻璃體誘導(dǎo)了RPE細(xì)胞間質(zhì)轉(zhuǎn)分化,主要表現(xiàn)為細(xì)胞形態(tài)改變,運(yùn)動(dòng)的可塑性增強(qiáng)[3]。這些表現(xiàn)是通過(guò)誘導(dǎo)細(xì)胞F-actin纖維絲的重組,在細(xì)胞的前進(jìn)緣形成扁平偽足來(lái)實(shí)現(xiàn)的[12],同時(shí)通過(guò)Rac1 GTPase/LIMK1/cofilin分子信號(hào)途徑來(lái)實(shí)現(xiàn)對(duì)細(xì)胞骨架重組和運(yùn)動(dòng)可塑性的調(diào)控[12-13]。文獻(xiàn)報(bào)道,vinculin被認(rèn)為是細(xì)胞機(jī)械動(dòng)力的調(diào)節(jié)子,并且vinculin作為一個(gè)機(jī)械偶聯(lián)子和F-actin黏附歸因于vinculin在脂質(zhì)膜形成大面積的粘連斑和定位分布于細(xì)胞與基質(zhì)、細(xì)胞與細(xì)胞黏附以及與其他蛋白黏附的多個(gè)蛋白位點(diǎn)[14]。作為細(xì)胞骨架蛋白之一的vinculin在細(xì)胞運(yùn)動(dòng)調(diào)節(jié)上具有雙重功能,具體的功能與所處的細(xì)胞外環(huán)境密切相關(guān)[15]。在二維平面基質(zhì),由vinculin所致的粘連斑和細(xì)胞骨架的穩(wěn)定性阻礙了細(xì)胞遷移;但在三維細(xì)胞基質(zhì),vinculin有助于細(xì)胞收縮力的產(chǎn)生,幫助細(xì)胞克服立體阻礙以增強(qiáng)細(xì)胞的侵襲[15]。在玻璃體誘導(dǎo)的RPE細(xì)胞間質(zhì)轉(zhuǎn)分化過(guò)程中,vinculin調(diào)整其分布位點(diǎn),從周邊向細(xì)胞前進(jìn)緣和中央移位。值得注意的是,由vinculin形成的粘連斑的面積較細(xì)胞在普通培養(yǎng)液中明顯變小。這說(shuō)明vinculin的再分布和粘連斑由大變小以及F-actin纖維絲的重排布構(gòu)成了玻璃體誘導(dǎo)的RPE細(xì)胞運(yùn)動(dòng)可塑性增強(qiáng)的結(jié)構(gòu)基礎(chǔ),導(dǎo)致細(xì)胞與細(xì)胞之間、細(xì)胞與基質(zhì)間的黏附力下降,促使細(xì)胞處于相互分散的不穩(wěn)定狀態(tài),從而降低了細(xì)胞的整體收縮力,可能也與細(xì)胞收縮力下降有關(guān)。另外vinculin蛋白的表達(dá)結(jié)果表明,vinculin呈組成性表達(dá)增強(qiáng),兩種培養(yǎng)液培養(yǎng)比較,其表達(dá)差異無(wú)統(tǒng)計(jì)學(xué)意義。
本研究的結(jié)果再次證明,玻璃體培養(yǎng)誘導(dǎo)的RPE細(xì)胞間質(zhì)轉(zhuǎn)分化屬于一種非成肌樣轉(zhuǎn)分化類(lèi)型,這一結(jié)果有利于我們深入理解玻璃體在增生性玻璃體視網(wǎng)膜病變中的作用。有關(guān)玻璃體誘導(dǎo)RPE細(xì)胞收縮力下降的分子信號(hào)機(jī)制尚需進(jìn)一步研究。
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date:Jan 6,2014
Supported by National Natural Science Foundation of China(No:81170866);Hainan Natural Science Foundation(No:814367)From theZhongshanOphthalmicCenter,SunYat-senUniversity(HUANG Xiong-Gao,WEI Yan-Tao,ZHANG Shao-Chong),Guangzhou510060,GuangdongProvince,China;HainanEyeHosipitalofZhongshanOphthalmicCenter,SunYat-senUniversity(HUANG Xiong-Gao),Haikou570311,HainanProvince,China
Study on contractility of retinal pigment epithelial cells induced by vitreous in vitro
HUANG Xiong-Gao,WEI Yan-Tao,ZHANG Shao-Chong
vitreous;retinal pigment epithelial cells;contractility
ObjectiveTo investigate the effects of vitreous culturedinvitroon contractility of human retinal pigment epithelial (RPE) cells.MethodsThe low-passage cultured RPE cells divided into two groups of treatment by volumn fraction 10% serum-containing Dulbecco minimum essential medium (DMEM)/F12 medium (normal medium) or the same medium supplemented with volumn fraction 25% human vitreous (25% vitreous medium).Cell contractility was tested with collagen gel contraction analysis.Staining location of F-actin,α-smooth muscle actin (α-SMA) and vinculin were detected by immunofluorescence.The expression levels of α-SMA and vinculin were detected by Western blotting.ResultsCompared to cells cultured in normal medium,low-passage number RPE cells cultured in 25% vitreous medium for 48 hours,the collagen gel area of (22 127±2072) pixels in 25% vitreous medium was significantly greater than the gel area of (16 084±1154) pixels in normal medium,contractility of the cells decreased remarkably(t=4.412,P=0.012).In normal medium cultured RPE cells,filamentous actin (F-actin) and vinculin-forming focal adhesions mainly at margin of cell,the focal adhesions were bigger.But in 25% vitreous treated cells,F-actin reorganized and formed pseudopodia at one side of cells,vinculin-forming focal adhesions redistributed mainly from margin to center and the side of actin polymerization,and became small.The expression level of α-SMA was down-regulated significantly (t=2.845,P=0.047),the expression level of vinculin was not significantly different between the two media (t=0.198,P=0.852 5).ConclusionVitreous decrease the contractility of RPE cells,this effect may be related to the down-regulated expression of α-SMA and the redistribution of vinculin.
黃雄高,男,1975年1月出生,湖北仙桃人,博士,副主任醫(yī)師,主要從事玻璃體視網(wǎng)膜疾病的基礎(chǔ)與臨床研究。聯(lián)系電話(huà):0898-68628561;E-mail:hxg_eye@163.com
AboutHUANGXiong-Gao:Male,born in January,1975.Medical doctor,associate chief physician.Tel:+86-898-68628561;E-mail:hxg_eye@163.com
2014-01-06
國(guó)家自然科學(xué)基金資助(編號(hào):81170866);海南省自然科學(xué)基金資助(編號(hào):814367)
510060 廣東省廣州市,中山大學(xué)中山眼科中心(黃雄高,魏雁濤,張少?zèng)_);570311 海南省??谑校猩窖劭浦行暮D涎劭漆t(yī)院(黃雄高)
張少?zèng)_,E-mail:zhshaochong@163.com
黃雄高,魏雁濤,張少?zèng)_.玻璃體對(duì)視網(wǎng)膜色素上皮細(xì)胞收縮作用的研究[J].眼科新進(jìn)展,2014,34(7):624?627.
10.13389/j.cnki.rao.2014.0171
【實(shí)驗(yàn)研究】
修回日期:2014-03-05
本文編輯:盛麗娜
Accepteddate:Mar 5,2014
Responsibleauthor:ZHANG Shao-Chong,E-mail:zhshaochong@163.com
[RecAdvOphthalmol,2014,34(7):624-627]