中华实验眼科杂志
中華實驗眼科雜誌
중화실험안과잡지
Chinese Journal of Experimental Ophthalmology
2015年
9期
774-780
,共7页
殷秋菊%吴一湘%于莉%刘勋%杨春波%李筱荣
慇鞦菊%吳一湘%于莉%劉勛%楊春波%李篠榮
은추국%오일상%우리%류훈%양춘파%리소영
姜黄素%人%胚样小体/药物作用%干细胞%细胞分化/药物作用%基因表达/药物作用%眼色素上皮%Wnt蛋白/代谢
薑黃素%人%胚樣小體/藥物作用%榦細胞%細胞分化/藥物作用%基因錶達/藥物作用%眼色素上皮%Wnt蛋白/代謝
강황소%인%배양소체/약물작용%간세포%세포분화/약물작용%기인표체/약물작용%안색소상피%Wnt단백/대사
Curcumin%Humans%Embryoid bodies/drug effects%Stem cells%Cell differentiation/drug effects%Gene expression/drug effect%Pigment epithelium,Eye%Wnt proteins/metabolism
背景 来源于多能干细胞的视网膜色素上皮(RPE)细胞移植治疗年龄相关性黄斑变性(AMD)和视网膜色素变性(RP)是近年研究热点,但RPE体外分化诱导效率低下、成本高等一直是难以克服的障碍.研究表明,姜黄素(curcumin)可促进人胚胎干细胞(ESCs)的定向诱导分化,但其对人ESCs向RPE样细胞的定向分化的作用机制尚不清楚. 目的 研究体外培养的人ESCs向视网膜色素上皮(RPE)样细胞的诱导分化过程,探讨curcumin对人ESCs向RPE细胞定向诱导效率的影响及其机制. 方法 将人ESCs株进行体外培养,将处于对数生长期的ESCs传代至基质膜(Matrigel)包被的6孔板中,以mTeSRTM1培养基培养至过渡融合状态后更换为含质量分数87％ KnockOutTM DMEM、质量分数10％血清替代物(SR)、质量分数1％非必需氨基酸和质量分数1％谷氨酰胺及青链霉素双抗的分化诱导体系,同时加入终浓度为1 μmol/L的curcumin处理24 h,对照组培养基中未加入curcumin.分别于诱导培养3周及5周时提取细胞RNA及蛋白,采用荧光定量逆转录PCR(RT-PCR)法检测诱导RPE(iRPE)细胞中干细胞标志物、RPE相关标志物及Wnt/β-catenin信号通路相关因子mRNA的相对表达水平;采用Western blot法及免疫荧光染色检测人ESCs、iRPE细胞及人RPE细胞中相关标志物蛋白的表达水平;通过细胞吞噬实验检测iRPE细胞的吞噬功能. 结果 Curcumin组诱导后3周时即可见iRPE细胞色素化,随着时间延长色素化程度更高,而对照组细胞在诱导后5周时开始出现细胞色素化.RT-PCR结果显示,curcumin诱导后3周及5周,curcumin组iRPE细胞中ESCs标志物NANOG mRNA的相对表达水平明显低于对照组,差异均有统计学意义(t=13.086,P=0.022;t=34.186,P=0.004),而RPE标志物Pax6、RX、CRALBP及RPE65 mRNA的相对表达量明显高于对照组,差异均有统计学意义(均P＜0.01).Western blot检测显示,CRALBP、RPE65和MITF蛋白在iRPE细胞中表达,表达强度与人RPE细胞相近,而人ESCs不表达上述蛋白.免疫荧光染色显示,iRPE细胞中Pax6、MITF和ZO-1蛋白表达阳性,分别定位于细胞质和细胞膜,可见curcumin组得到的细胞为iRPE细胞,纯化效率达到100％,体外细胞吞噬实验显示,iRPE细胞的细胞质内呈现的聚苯乙烯荧光微球接近阳性对照组,而阴性对照组iRPE细胞中未见到聚苯乙烯荧光微球.诱导培养后第3周和第5周时,curcumin组细胞中Wnt信号通路下游靶因子Lef1、MYC和TCF7,Wnt受体FZD3及Wnt配体Wnt2B和Wnt7B的mRNA相对表达水平明显高于对照组,差异均有统计学意义(均P＜0.01). 结论 Curcumin能提高人ESCs向RPE样细胞的定向诱导效率,其主要机制可能是通过促进Wnt/β-catenin信号通路的活性,从而促进iRPE细胞的分化和成熟.
揹景 來源于多能榦細胞的視網膜色素上皮(RPE)細胞移植治療年齡相關性黃斑變性(AMD)和視網膜色素變性(RP)是近年研究熱點,但RPE體外分化誘導效率低下、成本高等一直是難以剋服的障礙.研究錶明,薑黃素(curcumin)可促進人胚胎榦細胞(ESCs)的定嚮誘導分化,但其對人ESCs嚮RPE樣細胞的定嚮分化的作用機製尚不清楚. 目的 研究體外培養的人ESCs嚮視網膜色素上皮(RPE)樣細胞的誘導分化過程,探討curcumin對人ESCs嚮RPE細胞定嚮誘導效率的影響及其機製. 方法 將人ESCs株進行體外培養,將處于對數生長期的ESCs傳代至基質膜(Matrigel)包被的6孔闆中,以mTeSRTM1培養基培養至過渡融閤狀態後更換為含質量分數87％ KnockOutTM DMEM、質量分數10％血清替代物(SR)、質量分數1％非必需氨基痠和質量分數1％穀氨酰胺及青鏈黴素雙抗的分化誘導體繫,同時加入終濃度為1 μmol/L的curcumin處理24 h,對照組培養基中未加入curcumin.分彆于誘導培養3週及5週時提取細胞RNA及蛋白,採用熒光定量逆轉錄PCR(RT-PCR)法檢測誘導RPE(iRPE)細胞中榦細胞標誌物、RPE相關標誌物及Wnt/β-catenin信號通路相關因子mRNA的相對錶達水平;採用Western blot法及免疫熒光染色檢測人ESCs、iRPE細胞及人RPE細胞中相關標誌物蛋白的錶達水平;通過細胞吞噬實驗檢測iRPE細胞的吞噬功能. 結果 Curcumin組誘導後3週時即可見iRPE細胞色素化,隨著時間延長色素化程度更高,而對照組細胞在誘導後5週時開始齣現細胞色素化.RT-PCR結果顯示,curcumin誘導後3週及5週,curcumin組iRPE細胞中ESCs標誌物NANOG mRNA的相對錶達水平明顯低于對照組,差異均有統計學意義(t=13.086,P=0.022;t=34.186,P=0.004),而RPE標誌物Pax6、RX、CRALBP及RPE65 mRNA的相對錶達量明顯高于對照組,差異均有統計學意義(均P＜0.01).Western blot檢測顯示,CRALBP、RPE65和MITF蛋白在iRPE細胞中錶達,錶達彊度與人RPE細胞相近,而人ESCs不錶達上述蛋白.免疫熒光染色顯示,iRPE細胞中Pax6、MITF和ZO-1蛋白錶達暘性,分彆定位于細胞質和細胞膜,可見curcumin組得到的細胞為iRPE細胞,純化效率達到100％,體外細胞吞噬實驗顯示,iRPE細胞的細胞質內呈現的聚苯乙烯熒光微毬接近暘性對照組,而陰性對照組iRPE細胞中未見到聚苯乙烯熒光微毬.誘導培養後第3週和第5週時,curcumin組細胞中Wnt信號通路下遊靶因子Lef1、MYC和TCF7,Wnt受體FZD3及Wnt配體Wnt2B和Wnt7B的mRNA相對錶達水平明顯高于對照組,差異均有統計學意義(均P＜0.01). 結論 Curcumin能提高人ESCs嚮RPE樣細胞的定嚮誘導效率,其主要機製可能是通過促進Wnt/β-catenin信號通路的活性,從而促進iRPE細胞的分化和成熟.
배경 래원우다능간세포적시망막색소상피(RPE)세포이식치료년령상관성황반변성(AMD)화시망막색소변성(RP)시근년연구열점,단RPE체외분화유도효솔저하、성본고등일직시난이극복적장애.연구표명,강황소(curcumin)가촉진인배태간세포(ESCs)적정향유도분화,단기대인ESCs향RPE양세포적정향분화적작용궤제상불청초. 목적 연구체외배양적인ESCs향시망막색소상피(RPE)양세포적유도분화과정,탐토curcumin대인ESCs향RPE세포정향유도효솔적영향급기궤제. 방법 장인ESCs주진행체외배양,장처우대수생장기적ESCs전대지기질막(Matrigel)포피적6공판중,이mTeSRTM1배양기배양지과도융합상태후경환위함질량분수87％ KnockOutTM DMEM、질량분수10％혈청체대물(SR)、질량분수1％비필수안기산화질량분수1％곡안선알급청련매소쌍항적분화유도체계,동시가입종농도위1 μmol/L적curcumin처리24 h,대조조배양기중미가입curcumin.분별우유도배양3주급5주시제취세포RNA급단백,채용형광정량역전록PCR(RT-PCR)법검측유도RPE(iRPE)세포중간세포표지물、RPE상관표지물급Wnt/β-catenin신호통로상관인자mRNA적상대표체수평;채용Western blot법급면역형광염색검측인ESCs、iRPE세포급인RPE세포중상관표지물단백적표체수평;통과세포탄서실험검측iRPE세포적탄서공능. 결과 Curcumin조유도후3주시즉가견iRPE세포색소화,수착시간연장색소화정도경고,이대조조세포재유도후5주시개시출현세포색소화.RT-PCR결과현시,curcumin유도후3주급5주,curcumin조iRPE세포중ESCs표지물NANOG mRNA적상대표체수평명현저우대조조,차이균유통계학의의(t=13.086,P=0.022;t=34.186,P=0.004),이RPE표지물Pax6、RX、CRALBP급RPE65 mRNA적상대표체량명현고우대조조,차이균유통계학의의(균P＜0.01).Western blot검측현시,CRALBP、RPE65화MITF단백재iRPE세포중표체,표체강도여인RPE세포상근,이인ESCs불표체상술단백.면역형광염색현시,iRPE세포중Pax6、MITF화ZO-1단백표체양성,분별정위우세포질화세포막,가견curcumin조득도적세포위iRPE세포,순화효솔체도100％,체외세포탄서실험현시,iRPE세포적세포질내정현적취분을희형광미구접근양성대조조,이음성대조조iRPE세포중미견도취분을희형광미구.유도배양후제3주화제5주시,curcumin조세포중Wnt신호통로하유파인자Lef1、MYC화TCF7,Wnt수체FZD3급Wnt배체Wnt2B화Wnt7B적mRNA상대표체수평명현고우대조조,차이균유통계학의의(균P＜0.01). 결론 Curcumin능제고인ESCs향RPE양세포적정향유도효솔,기주요궤제가능시통과촉진Wnt/β-catenin신호통로적활성,종이촉진iRPE세포적분화화성숙.
Background Pluripotent stem cell-derived retinal pigment epithelial (RPE) cells holds great promise for the treatment of age-related macular degeneration (AMD) and retinitis pigmentosa (RP),but the poor induction efficiency and the according high cost of RPE differentiation hindere its clinical applications.Curcumin is proved to have a promoting effect on the induced differentiation of embryonic stem cells (ESCs).However,the mechanism of curcumin on differentiation of human ESCs into RPE-like cells remains unclear.Objective This study aimed to explore the underlying molecular mechanism of curcumin on directed differentiation of human ESCs into RPE-like cells.Methods Human ESCs strains were cultured in the Matrigel-coated 6-well plate with mTeSRTM 1 medium until over-confluence,and basic fibroblast growth factor was withdrawn there after to induce automatic differentiation.Curcumin at the final concentration 1 μmol/L was added in the first day of differentiation for 24 hours,and the cells without curcumin in the medium served as the control group.Total RNA and protein were extracted at 3 weeks and 5 weeks after induction.RT-PCR,Western blot and immunofluorescence were performed to examine the expressions of the biomarks of stem cells and RPE cells as well as Wnt/β-catenin signaling pathway components.The endocytosis of polystyrene microsphere by induced RPE (iRPE) cells was investigated to verify their function of phagocytosis which features RPE cells.Results Pigmented cells were found from 3 weeks through 5 weeks after induction in the curcumin group,but only less pigmented cells were seen in the fifth week after induction in the control group.In the third and fifth week after induction,the relative expression levels of NANOG mRNA in the iRPE cells were significantly lower than those in the control group (t =13.086,P =0.022;t =34.186,P =0.004),and the relative expression levels of Pax6,RX,CRALBP and RPE65 mRNA were higher in the curcumin group than those of the control group (all at P＜0.01).Western blot assay showed that the expressing bands for CRALBP,RPE65 and MITF enhanced in iRPE cells with a similar appearance in human RPE cells.However,these expressions were all absent in human ESCs.Immunofluorescence staining showed the positive expressions of Pax6,MITF and ZO-1 in cytoplasm of iRPE cells in the curcumin group with a purified efficacy 100％.The fluorescence dye-doped polystyrene microspheres in cytoplasm were obvious in the iRPE cells like positive controls,but the polystyrene microsphere was absent in the negative controls.From 3 weeks through 5 weeks after induced,the relative expression levels of Lef1,MYC and TCF7 mRNA (the dwnstream target genes of Wnt signaling pathway),FZD3 mRNA (Wnt receptor),Wnt2B mRNA (Wnt ligand) and Wnt7B mRNA were significantly reduced in the curcumin group compared with the control group (all at P＜0.01).Conclusions Curcumin promotes the differentiation of human ESCs into RPE-like cells by stimulating the activation of Wnt signaling pathway,and therefore accelerate the differentiation and mature of iRPE cells.
