中华眼底病杂志
中華眼底病雜誌
중화안저병잡지
CHINESE JOURNAL OF OCULAR FUNDUS DISEASES
2014年
4期
395-398
,共4页
宋伟涛%张雪咏%熊思齐%蒋剑%文丹%喻一心%夏晓波
宋偉濤%張雪詠%熊思齊%蔣劍%文丹%喻一心%夏曉波
송위도%장설영%웅사제%장검%문단%유일심%하효파
小神经胶质细胞%视网膜神经节细胞%干细胞%受体,Notch%动物实验
小神經膠質細胞%視網膜神經節細胞%榦細胞%受體,Notch%動物實驗
소신경효질세포%시망막신경절세포%간세포%수체,Notch%동물실험
Microglia%Retinal ganglion cells%Stem cells%Receptors,notch%Animal experimentation
目的 观察Notch信号通路抑制剂对鼠Müller细胞来源的视网膜干细胞的调控作用.方法 Sprague Dawley大鼠20只,鼠龄10～20 d.显微镜下分离视网膜,采用反复不完全胰蛋白酶消化法传代培养.取传代3次的细胞用于实验.Müller细胞诱导去分化,荧光显微镜下观察细胞增生状态,免疫荧光细胞化学染色,逆转录-聚合酶链反应(RT-PCR)、蛋白免疫印迹法(Western blot)检测视网膜干细胞特异性表达产物巢蛋白(Nestin)、Ki-67的表达;5-乙炔基2'脱氧尿嘧啶核苷(Edu)染色检测细胞核阳性率.视网膜干细胞随机分为Notch信号通路γ分泌酶抑制剂(GSI)干预组(GSI组)和对照组,采用免疫荧光染色方法统计分析神经节细胞的比例变化.结果 荧光显微镜观察结果显示,去分化培养细胞增生聚集成球.免疫荧光细胞化学染色结果显示,Nestin、Ki-67表达率分别为(92.94±6.48)％、(85.96±6.04)％.细胞核Edu阳性率为(82.80±6.65)％.RT-PCR、Western blot检测结果显示,神经球中Nestin、Ki-67 mRNA、蛋白均呈高表达,纯化后的Müller细胞中无表达.GSI组、对照组神经节细胞阳性率分别为(16.98±2.87)％、(11.17±0.71)％.两组细胞阳性率比较,差异有统计学意义(t=3.210,P=0.002).结论 Notch信号通路是视网膜干细胞向神经节细胞分化过程中重要的调控基因.
目的 觀察Notch信號通路抑製劑對鼠Müller細胞來源的視網膜榦細胞的調控作用.方法 Sprague Dawley大鼠20隻,鼠齡10～20 d.顯微鏡下分離視網膜,採用反複不完全胰蛋白酶消化法傳代培養.取傳代3次的細胞用于實驗.Müller細胞誘導去分化,熒光顯微鏡下觀察細胞增生狀態,免疫熒光細胞化學染色,逆轉錄-聚閤酶鏈反應(RT-PCR)、蛋白免疫印跡法(Western blot)檢測視網膜榦細胞特異性錶達產物巢蛋白(Nestin)、Ki-67的錶達;5-乙炔基2'脫氧尿嘧啶覈苷(Edu)染色檢測細胞覈暘性率.視網膜榦細胞隨機分為Notch信號通路γ分泌酶抑製劑(GSI)榦預組(GSI組)和對照組,採用免疫熒光染色方法統計分析神經節細胞的比例變化.結果 熒光顯微鏡觀察結果顯示,去分化培養細胞增生聚集成毬.免疫熒光細胞化學染色結果顯示,Nestin、Ki-67錶達率分彆為(92.94±6.48)％、(85.96±6.04)％.細胞覈Edu暘性率為(82.80±6.65)％.RT-PCR、Western blot檢測結果顯示,神經毬中Nestin、Ki-67 mRNA、蛋白均呈高錶達,純化後的Müller細胞中無錶達.GSI組、對照組神經節細胞暘性率分彆為(16.98±2.87)％、(11.17±0.71)％.兩組細胞暘性率比較,差異有統計學意義(t=3.210,P=0.002).結論 Notch信號通路是視網膜榦細胞嚮神經節細胞分化過程中重要的調控基因.
목적 관찰Notch신호통로억제제대서Müller세포래원적시망막간세포적조공작용.방법 Sprague Dawley대서20지,서령10～20 d.현미경하분리시망막,채용반복불완전이단백매소화법전대배양.취전대3차적세포용우실험.Müller세포유도거분화,형광현미경하관찰세포증생상태,면역형광세포화학염색,역전록-취합매련반응(RT-PCR)、단백면역인적법(Western blot)검측시망막간세포특이성표체산물소단백(Nestin)、Ki-67적표체;5-을결기2'탈양뇨밀정핵감(Edu)염색검측세포핵양성솔.시망막간세포수궤분위Notch신호통로γ분비매억제제(GSI)간예조(GSI조)화대조조,채용면역형광염색방법통계분석신경절세포적비례변화.결과 형광현미경관찰결과현시,거분화배양세포증생취집성구.면역형광세포화학염색결과현시,Nestin、Ki-67표체솔분별위(92.94±6.48)％、(85.96±6.04)％.세포핵Edu양성솔위(82.80±6.65)％.RT-PCR、Western blot검측결과현시,신경구중Nestin、Ki-67 mRNA、단백균정고표체,순화후적Müller세포중무표체.GSI조、대조조신경절세포양성솔분별위(16.98±2.87)％、(11.17±0.71)％.량조세포양성솔비교,차이유통계학의의(t=3.210,P=0.002).결론 Notch신호통로시시망막간세포향신경절세포분화과정중중요적조공기인.
Objective To observe the role of Notch signaling pathway inhibitor in differentiationprocess of stem cells derived from retinal Müller cells into the ganglion cell.Methods Retinas of Sprague Dawley rat at postnatal 10-20 days were dissociated from eye balls.The third passage of Müller cells was used in this experiment,which cultured by repeated incomplete pancreatic enzyme digestion method.The retinal Müller cells were induced in the serum-free dedifferentiation medium.The cell proliferation state was observed under an inverted microscope.The expression of the specific markers Nestin and Ki-67 of retinal stem cells was measured by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot.The positive rate of nucleus was detected by Edu.The retinal stem cells was divided into Gamma secretase inhibtor-Ⅰ (GSI) group and control group,the rate of ganglion cells was counted by using immunofluorescence staining.Results The cell proliferation had gathered to form a sphere.Immunofluorescence staining showed that the expressions of Nestin and Ki-67 were (92.94 ± 6.48％) and (85.96±6.04％) respectively.Edu positive rate of nucleus was (82.80±6.65)％.RT-PCR and Western blot further confirmed the high expression of Nestin and Ki-67 in the cell spheres but not in the Müller cells.The positive rate of ganglion cells were (16.98±2.87)％ and (11.17±0.71)％ in GSI group and control group respectively,with the significant difference (t =3.210,P =0.002).Conclusion Notch signaling pathway is an important regulatory gene in stem cells differentiated into retinal ganglion cell.
