中华眼底病杂志
中華眼底病雜誌
중화안저병잡지
CHINESE JOURNAL OF OCULAR FUNDUS DISEASES
2015年
1期
57-61
,共5页
吴一湘%杨春波%于敏%李筱荣
吳一湘%楊春波%于敏%李篠榮
오일상%양춘파%우민%리소영
苯丙氨酸/药物作用%胚胎干细胞%细胞分化%视网膜色素上皮
苯丙氨痠/藥物作用%胚胎榦細胞%細胞分化%視網膜色素上皮
분병안산/약물작용%배태간세포%세포분화%시망막색소상피
Phenylalanine/drug effects%Embryonic stem cells%Cell differentiation%Retinal pigment epithelium
目的 观察苯丙氨酸对胚胎干细胞(ESC)向视网膜色素上皮(RPE)细胞定向分化诱导效率的影响.方法 H1人ESC(hESC)分为对照组和实验组,以mTeSR培养基常规培养.细胞克隆过度融合后对照组以撤除碱性成纤维细胞生长因子、含10％ knockout血清替代物的培养基诱导hESC自主分化至第7周.实验组从第3周开始在诱导培养基中添加0.2 mmol/L苯丙氨酸培养至第7周.观察两组细胞形态学变化;实时荧光定量聚合酶链反应(RT-PCR)检测人类配对盒基因6(Pax6)、小眼球相关转录因子(MITF)、RPE65、酪氨酸酶(tyrosinase)、Wnt配体(Wnt3a)、淋巴样增强因子-1(Lef1)、转录因子7(Tcf7)基因mRNA的表达;蛋白免疫印迹法(Western blot)检测MITF、RPE65蛋白的表达;流式细胞技术(FCM)检测RPE65阳性细胞比例.Sprague Dawley大鼠12只,随机分为对照组和治疗组;单眼内路法视网膜下腔注射,对照组注射2 μl 1倍磷酸盐缓冲液,治疗组注射2 μl纯化的hESC-RPE.治疗后6周行视网膜电图(ERG)检查,观察暗适应3.0 a-b波振幅.结果 诱导分化第7周,实验组出现大量肉眼可见的色素团块,明显多于对照组.色素化区域内可见多边形蜂窝状排列的单层细胞,胞质充满色素颗粒.RT-PCR检测结果显示,实验组Pax6、MITF、RPE65、tyrosinase(t=39.44、26.14、31.28、77.24)、Wnt3a、Lef1、Tcf7(t=46.11、27.69、17.42)mRNA表达量高于对照组,差异有统计学意义(P＜0.01).Western blot检测结果显示,实验组MITF、RPE65蛋白表达量高于对照组.FCM检测结果显示,实验组、对照组RPE65阳性比例分为(18.91±1.76)％、(0.88±0.09)％;实验组RPE65阳性比较明显高于对照组,差异有统计学意义(P＜0.01).ERG检查结果显示,治疗组大鼠暗适应3.0 a-b波振幅较对照组显著提高,差异有统计学意义(t=7.543,P＜0.01).结论 苯丙氨酸显著提高hESC向RPE定向诱导效率.hESC诱导来源的RPE细胞视网膜下移植能改善视网膜变性模型动物视功能.
目的 觀察苯丙氨痠對胚胎榦細胞(ESC)嚮視網膜色素上皮(RPE)細胞定嚮分化誘導效率的影響.方法 H1人ESC(hESC)分為對照組和實驗組,以mTeSR培養基常規培養.細胞剋隆過度融閤後對照組以撤除堿性成纖維細胞生長因子、含10％ knockout血清替代物的培養基誘導hESC自主分化至第7週.實驗組從第3週開始在誘導培養基中添加0.2 mmol/L苯丙氨痠培養至第7週.觀察兩組細胞形態學變化;實時熒光定量聚閤酶鏈反應(RT-PCR)檢測人類配對盒基因6(Pax6)、小眼毬相關轉錄因子(MITF)、RPE65、酪氨痠酶(tyrosinase)、Wnt配體(Wnt3a)、淋巴樣增彊因子-1(Lef1)、轉錄因子7(Tcf7)基因mRNA的錶達;蛋白免疫印跡法(Western blot)檢測MITF、RPE65蛋白的錶達;流式細胞技術(FCM)檢測RPE65暘性細胞比例.Sprague Dawley大鼠12隻,隨機分為對照組和治療組;單眼內路法視網膜下腔註射,對照組註射2 μl 1倍燐痠鹽緩遲液,治療組註射2 μl純化的hESC-RPE.治療後6週行視網膜電圖(ERG)檢查,觀察暗適應3.0 a-b波振幅.結果 誘導分化第7週,實驗組齣現大量肉眼可見的色素糰塊,明顯多于對照組.色素化區域內可見多邊形蜂窩狀排列的單層細胞,胞質充滿色素顆粒.RT-PCR檢測結果顯示,實驗組Pax6、MITF、RPE65、tyrosinase(t=39.44、26.14、31.28、77.24)、Wnt3a、Lef1、Tcf7(t=46.11、27.69、17.42)mRNA錶達量高于對照組,差異有統計學意義(P＜0.01).Western blot檢測結果顯示,實驗組MITF、RPE65蛋白錶達量高于對照組.FCM檢測結果顯示,實驗組、對照組RPE65暘性比例分為(18.91±1.76)％、(0.88±0.09)％;實驗組RPE65暘性比較明顯高于對照組,差異有統計學意義(P＜0.01).ERG檢查結果顯示,治療組大鼠暗適應3.0 a-b波振幅較對照組顯著提高,差異有統計學意義(t=7.543,P＜0.01).結論 苯丙氨痠顯著提高hESC嚮RPE定嚮誘導效率.hESC誘導來源的RPE細胞視網膜下移植能改善視網膜變性模型動物視功能.
목적 관찰분병안산대배태간세포(ESC)향시망막색소상피(RPE)세포정향분화유도효솔적영향.방법 H1인ESC(hESC)분위대조조화실험조,이mTeSR배양기상규배양.세포극륭과도융합후대조조이철제감성성섬유세포생장인자、함10％ knockout혈청체대물적배양기유도hESC자주분화지제7주.실험조종제3주개시재유도배양기중첨가0.2 mmol/L분병안산배양지제7주.관찰량조세포형태학변화;실시형광정량취합매련반응(RT-PCR)검측인류배대합기인6(Pax6)、소안구상관전록인자(MITF)、RPE65、락안산매(tyrosinase)、Wnt배체(Wnt3a)、림파양증강인자-1(Lef1)、전록인자7(Tcf7)기인mRNA적표체;단백면역인적법(Western blot)검측MITF、RPE65단백적표체;류식세포기술(FCM)검측RPE65양성세포비례.Sprague Dawley대서12지,수궤분위대조조화치료조;단안내로법시망막하강주사,대조조주사2 μl 1배린산염완충액,치료조주사2 μl순화적hESC-RPE.치료후6주행시망막전도(ERG)검사,관찰암괄응3.0 a-b파진폭.결과 유도분화제7주,실험조출현대량육안가견적색소단괴,명현다우대조조.색소화구역내가견다변형봉와상배렬적단층세포,포질충만색소과립.RT-PCR검측결과현시,실험조Pax6、MITF、RPE65、tyrosinase(t=39.44、26.14、31.28、77.24)、Wnt3a、Lef1、Tcf7(t=46.11、27.69、17.42)mRNA표체량고우대조조,차이유통계학의의(P＜0.01).Western blot검측결과현시,실험조MITF、RPE65단백표체량고우대조조.FCM검측결과현시,실험조、대조조RPE65양성비례분위(18.91±1.76)％、(0.88±0.09)％;실험조RPE65양성비교명현고우대조조,차이유통계학의의(P＜0.01).ERG검사결과현시,치료조대서암괄응3.0 a-b파진폭교대조조현저제고,차이유통계학의의(t=7.543,P＜0.01).결론 분병안산현저제고hESC향RPE정향유도효솔.hESC유도래원적RPE세포시망막하이식능개선시망막변성모형동물시공능.
Objective To investigate the impact of L-Phenylalanine on the efficiency of retinal pigment epithelial (RPE) cell derivation from human embryonic stem cells (hESCs) and explore the underlying mechanisms.Methods H1 hESCs were routinely cultured with mTeSR medium and divided into control and experimental groups.When cells reached over-confluence,spontaneous differentiation was triggered using 10％ KSR differentiation medium without bFGF.L-Phenylalanine (0.2 mmol/L) was supplemented in the experimental group from the 3rd week.The expression of RPE markers and Wnt signaling components in the two groups was detected by Real time-RCR,Western blot and Flow cytometry analyses.Purified hESC-RPE cells and PBS were injected into the subretinal space of sodium iodine-induced retinal degeneration rats separately.Retinal function was assessed by ERG 6 weeks after the transplantation.Results On the 7th week,much more pigment cell clumps appeared in the experimental group compared to the control group.Within these areas there were monolayer hexagonal RPE cells full of pigment granules.The experimental group showed significantly higher expression of Pax6,MITF,Tyrosinase,RPE65,Wnt3a,Lef1 and Tcf7 genes than the control group (P＜0.01).Higher expression level of MITF and RPE65 proteins and higher percentage of RPE65 (+) cells (P＜0.01) were detected in the experimental group.6 weeks after sub-retinal transplantation of hESC-RPE cells,the amplitudes of a-b wave in the transplanted eyes were significantly higher than those in the control eyes (P＜0.01) at the stimulus intensity of 3.0 cd · s/m2.Conclusions L-Phenylalanine effectively promoted the differentiation of embryonic stem cells into retinal pigment epithelial cells,and its impacts on the Wnt/β-catenin signaling pathway may partially explain the underlying mechanisms.Subretinal transplantation of hESC-RPE remarkably improved the retinal functions of retinal degenerative animal models.
