中华眼科医学杂志(电子版)
中華眼科醫學雜誌(電子版)
중화안과의학잡지(전자판)
CHINESE JOURNAL OF OPHTHALMOLOGIC MEDICINE(ELECTRONIC EDITION)
2014年
6期
313-317
,共5页
神经干细胞%视网膜色素上皮细胞%基因表达%分化
神經榦細胞%視網膜色素上皮細胞%基因錶達%分化
신경간세포%시망막색소상피세포%기인표체%분화
Neural stem cell%Retinal pigment epithelium%Gene expression%Differentiation
目的:探讨神经干细胞( NSC)与成熟视网膜色素上皮细胞( RPE)间相关基因的表达差异。方法分离小鼠原代RPE并进行扩增培养。应用反转录-聚合酶链反应鉴定眼区发育相关基因在NSC和RPE的表达,分析相关差异。进一步应用荧光定量聚合酶链反应( PCR)对共同表达的基因进行定量分析,比较两者差异。测量指标的数据资料以均数±标准差( x珋±s)的形式表示;采用独立样本t检验的方法分别比较NSC和RPE间各基因表达量的差异。结果原代分离后的小鼠RPE细胞呈圆形生长,细胞内充满色素颗粒,一般48 h后,细胞可稳定贴壁生长,部分细胞呈双核或多核,并逐渐呈三角形或多边形状伸展。培养7 d左右,细胞生长接近融合状态,进行RPE细胞的特异蛋白免疫荧光染色,可见细胞角蛋白及S-100等显著表达。在眼区发育相关的主要转录因子中,NSC特异表达Sox2 mRNA,RPE细胞特异表达Chx10、Opn4及RPE65 mRNA,两者均表达nestin、Pax6、R、Mitf、Hes1、Zo-1及tublin mRNA。在NSC和RPE共同表达的相关基因中,Pax6、Mtif、Rx,Hes1及ZO-1在RPE的相对表达量都显著高于其在NSC的表达,差异有统计学意义( t=-16.751,-5.439,-8.109,-4.96,-9.172;P <0.05)。而nestin在两类细胞的表达比较,差异无统计学意义(t=-0.328,P >0.05)。结论在NSC向RPE诱导分化过程中,Chx10、Opn4及RPE65具有一定特异性,可作为RPE的鉴定蛋白。 NSC和RPE存在共同表达的基因特性,但表达水平存在较大差异,可以在诱导分化过程中进行调控来促进RPE的定向分化。
目的:探討神經榦細胞( NSC)與成熟視網膜色素上皮細胞( RPE)間相關基因的錶達差異。方法分離小鼠原代RPE併進行擴增培養。應用反轉錄-聚閤酶鏈反應鑒定眼區髮育相關基因在NSC和RPE的錶達,分析相關差異。進一步應用熒光定量聚閤酶鏈反應( PCR)對共同錶達的基因進行定量分析,比較兩者差異。測量指標的數據資料以均數±標準差( x珋±s)的形式錶示;採用獨立樣本t檢驗的方法分彆比較NSC和RPE間各基因錶達量的差異。結果原代分離後的小鼠RPE細胞呈圓形生長,細胞內充滿色素顆粒,一般48 h後,細胞可穩定貼壁生長,部分細胞呈雙覈或多覈,併逐漸呈三角形或多邊形狀伸展。培養7 d左右,細胞生長接近融閤狀態,進行RPE細胞的特異蛋白免疫熒光染色,可見細胞角蛋白及S-100等顯著錶達。在眼區髮育相關的主要轉錄因子中,NSC特異錶達Sox2 mRNA,RPE細胞特異錶達Chx10、Opn4及RPE65 mRNA,兩者均錶達nestin、Pax6、R、Mitf、Hes1、Zo-1及tublin mRNA。在NSC和RPE共同錶達的相關基因中,Pax6、Mtif、Rx,Hes1及ZO-1在RPE的相對錶達量都顯著高于其在NSC的錶達,差異有統計學意義( t=-16.751,-5.439,-8.109,-4.96,-9.172;P <0.05)。而nestin在兩類細胞的錶達比較,差異無統計學意義(t=-0.328,P >0.05)。結論在NSC嚮RPE誘導分化過程中,Chx10、Opn4及RPE65具有一定特異性,可作為RPE的鑒定蛋白。 NSC和RPE存在共同錶達的基因特性,但錶達水平存在較大差異,可以在誘導分化過程中進行調控來促進RPE的定嚮分化。
목적:탐토신경간세포( NSC)여성숙시망막색소상피세포( RPE)간상관기인적표체차이。방법분리소서원대RPE병진행확증배양。응용반전록-취합매련반응감정안구발육상관기인재NSC화RPE적표체,분석상관차이。진일보응용형광정량취합매련반응( PCR)대공동표체적기인진행정량분석,비교량자차이。측량지표적수거자료이균수±표준차( x류±s)적형식표시;채용독립양본t검험적방법분별비교NSC화RPE간각기인표체량적차이。결과원대분리후적소서RPE세포정원형생장,세포내충만색소과립,일반48 h후,세포가은정첩벽생장,부분세포정쌍핵혹다핵,병축점정삼각형혹다변형상신전。배양7 d좌우,세포생장접근융합상태,진행RPE세포적특이단백면역형광염색,가견세포각단백급S-100등현저표체。재안구발육상관적주요전록인자중,NSC특이표체Sox2 mRNA,RPE세포특이표체Chx10、Opn4급RPE65 mRNA,량자균표체nestin、Pax6、R、Mitf、Hes1、Zo-1급tublin mRNA。재NSC화RPE공동표체적상관기인중,Pax6、Mtif、Rx,Hes1급ZO-1재RPE적상대표체량도현저고우기재NSC적표체,차이유통계학의의( t=-16.751,-5.439,-8.109,-4.96,-9.172;P <0.05)。이nestin재량류세포적표체비교,차이무통계학의의(t=-0.328,P >0.05)。결론재NSC향RPE유도분화과정중,Chx10、Opn4급RPE65구유일정특이성,가작위RPE적감정단백。 NSC화RPE존재공동표체적기인특성,단표체수평존재교대차이,가이재유도분화과정중진행조공래촉진RPE적정향분화。
Objective To comparatively characterize gene expression in neural stem cells ( NSCs) and retinal pigment epithelial cells ( RPECs ) .Methods Primary RPECs were isolated from xxx mice andcultured in proliferation conditions.The mRNA abundance of key genes involved in eye development in NSCs and RPECs was quantified by quantitative reverse transcription-PCR, and the data were analyzed by independent samples t-test.Results Primary murine RPE cells had a round shape in the initial culture and were filled with pigment granules after separation.They became stably adherent 48 h after culture.Part of the cells had a dual-nucleus or multi-nucleus and gradually projected to become triangular or polygonal in shape. By day 7 of culture,cellular fusion occurred in approximately 90%of the cells.Immunofluorescence staining showed the presence of cytokeratin and S-100.NSC-related gene,SOX2,was highly expressed in NSCs while Chx10,Opn4 and RPE65,believed to play key roles in eye development in general and in RPE development in particular were expressed in RPECs.Nestin,Pax6,Rx,Mitf,Hes1,Zo-1 and tublin were expressed in both NSCs and RPECs,but the expression level was significantly higher in RPECs than in NSCs for all these genes (P<0.05) except for nestin (P>0.05).Conclusions Some genes are expressed in both NSCs and RPECs,but the expression level appears to be different significantly.Chx10,Opn4, RPE65 may play some roles in NSC to RPE differentiate into RPE.