CAJ | 학술논문

目的比较体外不同培养条件对视网膜及脑神经干细胞(NSCs)视紫红质的纯化能力及不同诱导条件对NSCs的诱导分化能力.方法取胚兔视网膜及大脑皮质制备单细胞悬液,分别在5种不同培养基中进行体外培养并纯化.选取无血清条件下传3代的NSCs,分别在2种培养基中诱导8～10 d.采用免疫荧光法及流式细胞仪检测所获得细胞的神经干细胞和视网膜神经上皮细胞抗原的表达.结果免疫荧光法显示无血清培养条件下2种组织来源的NSCs均部分表达巢蛋白.流式细胞术显示2种诱导方式均部分细胞表达巢蛋白,较诱导前明显降低,而视紫红质及Thy1.1的表达均较诱导前明显增高.经5%胎牛血清(FBS)诱导的细胞表达视紫红质较联合应用全反式视黄酸(ATRA)时高,差异均有统计学意义(χ2=30.59,6.76;P<0.01).但Thy1.1表达低于后者,差异也有统计学意义(χ2=6.19,22.92;P=0.01).结论无血清培养基添加N2、EGF、bFGF、LIF 4种因子可以获得最佳纯化效果.2种诱导培养基都能够诱导NSCs的分化,视网膜NSCs分化为视网膜神经上皮细胞的能力高于大脑皮质NSCs.
목적 비교체외불동배양조건대시망막급뇌신경간세포(NSCs)시자홍질적순화능력급불동유도조건대NSCs적유도분화능력.방법 취배토시망막급대뇌피질제비단세포현액,분별재5충불동배양기중진행체외배양병순화.선취무혈청조건하전3대적NSCs,분별재2충배양기중유도8～10 d.채용면역형광법급류식세포의검측소획득세포적신경간세포화시망막신경상피세포항원적표체.결과 면역형광법현시무혈청배양조건하2충조직래원적NSCs균부분표체소단백.류식세포술현시2충유도방식균부분세포표체소단백,교유도전명현강저,이시자홍질급Thy1.1적표체균교유도전명현증고.경5%태우혈청(FBS)유도적세포표체시자홍질교연합응용전반식시황산(ATRA)시고,차이균유통계학의의(χ2=30.59,6.76;P<0.01).단Thy1.1표체저우후자,차이야유통계학의의(χ2=6.19,22.92;P=0.01).결론 무혈청배양기첨가N2、EGF、bFGF、LIF 4충인자가이획득최가순화효과.2충유도배양기도능구유도NSCs적분화,시망막NSCs분화위시망막신경상피세포적능력고우대뇌피질NSCs.
Objective To isolate neural stem cells (NSCs) from rabbit retina and brain,and induce differentiation of those NSCs using different culture media.Methods Single-cell suspensions of retina and cerebral cortex were prepared from rabbit embryo,cultured in 5 types of different media to isolate the NSCs by continual passages.After 3 passages,NSCs were induced to differentiation in 2 types of different media for 8 to 10 days.NSCs and induced-retinal ceils were examined by immunofluorescence and flow cytometry for the expression pattern of some specific antigens.Results Immunofluorescence showed that NSCs from retina and brain,cultured in serum-free media,both expressed Nestin partially.Flow cytometry showed that Nestin positive cells were significantly decreased while the Rhodopsin and Thy1.1 positive cells were increased after induction.Compared with the combined induction of all-trans retinoid acid (ATRA) and serum,5 % FBS (fetal bovine serum) led to higher expression of Rhodopsin (P<0.01),but lower expression of Thy1.1 (P=0.01).Conclusion Serum-free media with N2,EGF,bFGF,LIF is the best for NSCs purification.Both induciton media can induce NSCs to differentiate.Retina NSCs have higher potentials to differentiate into retinal neuro-epithelial cells than brain NSCs.