CAJ | 학술논문

背景人视网膜胶质细胞在视网膜增生性疾病的研究中有重要作用,以往研究者已成功地培养了人视网膜胶质细胞,但方法学有待进一步改进以达到细胞收获量更大的目的. 目的建立快速、收获量大且纯度高的视网膜胶质细胞的培养方法,对目标细胞的抗原表达特点进行分析. 方法取正常人角膜移植供体眼球分离视网膜组织,采用质量分数2％胰蛋白酶和质量分数0.133％胶原酶Ⅵ用二步法消化获取人视网膜胶质细胞,用含质量分数10％胎牛血清的人内皮细胞培养液,其中添加内皮细胞生长因子(β-ECGF)和肝素钠,对分离的细胞进行体外培养,培养皿用纤维黏连蛋白(FN)包被以促进人视网膜胶质细胞贴壁.观察收获的目标细胞的形态特征,采用活体显微镜下形态学观察、常规组织学观察法观察目标细胞的生长,同时采用免疫组织化学法检测胶质纤维酸性蛋白(GFAP)、波形蛋白(Vimentin)、神经元特异性烯醇化酶(NSE)、S-100、CD34、Ⅷ因子在细胞中的表达以鉴定目标细胞. 结果应用胰蛋白酶、胶原酶二步消化法可成功获取人视网膜胶质细胞,原代培养的细胞72 h贴壁,第9～10天细胞达到融合状态呈花瓣状；常规组织学观察显示细胞核呈鲜亮蓝色,细胞质(盘膜)呈淡红色,培养细胞GFAP、Vimentin呈强阳性表达,NSE、S100、CD34、Ⅷ因子相关抗原表达呈阴性反应. 结论应用胰蛋白酶、胶原蛋白酶消化法以及利用10％胎牛血清的人内皮细胞培养基,添加生长因子和肝素钠,并用FN包被培养皿进行体外培养可达到快速、大量分离和纯化人视网膜胶质细胞的目的,鉴定结果提示培养的目标细胞为人视网膜胶质细胞,其形态与以往报道的有所不同,具体特点尚需进一步研究.
배경 인시망막효질세포재시망막증생성질병적연구중유중요작용,이왕연구자이성공지배양료인시망막효질세포,단방법학유대진일보개진이체도세포수획량경대적목적. 목적 건립쾌속、수획량대차순도고적시망막효질세포적배양방법,대목표세포적항원표체특점진행분석. 방법 취정상인각막이식공체안구분리시망막조직,채용질량분수2％이단백매화질량분수0.133％효원매Ⅵ용이보법소화획취인시망막효질세포,용함질량분수10％태우혈청적인내피세포배양액,기중첨가내피세포생장인자(β-ECGF)화간소납,대분리적세포진행체외배양,배양명용섬유점련단백(FN)포피이촉진인시망막효질세포첩벽.관찰수획적목표세포적형태특정,채용활체현미경하형태학관찰、상규조직학관찰법관찰목표세포적생장,동시채용면역조직화학법검측효질섬유산성단백(GFAP)、파형단백(Vimentin)、신경원특이성희순화매(NSE)、S-100、CD34、Ⅷ인자재세포중적표체이감정목표세포. 결과 응용이단백매、효원매이보소화법가성공획취인시망막효질세포,원대배양적세포72 h첩벽,제9～10천세포체도융합상태정화판상；상규조직학관찰현시세포핵정선량람색,세포질(반막)정담홍색,배양세포GFAP、Vimentin정강양성표체,NSE、S100、CD34、Ⅷ인자상관항원표체정음성반응. 결론 응용이단백매、효원단백매소화법이급이용10％태우혈청적인내피세포배양기,첨가생장인자화간소납,병용FN포피배양명진행체외배양가체도쾌속、대량분리화순화인시망막효질세포적목적,감정결과제시배양적목표세포위인시망막효질세포,기형태여이왕보도적유소불동,구체특점상수진일보연구.
Background Human retinal gliocytes play an important role in proliferative diseases,which are the basis of in vitro studies.Researchers have cultured human retinal gliocytes in the past.In our study,we found that the cells we cultured presented a unique shape different from those by other researchers. Objective This study was to design to produce a new culture and purification method for retinal gliocyte in vitro. Methods Retinal tissue was isolated from human eyeballs and digested using the two-step digestion method (2％ pancreatin and 0.133％collagenase Ⅵ) to harvest the retinal glio cytes.The cells were collected and cultured in endothelial cell-targeted nutrient culture containing 10％ fetal calf serum and supplemented with β-endothelial cell growth factor (ECGF) and sodium heparin,and the culture dishes were coated with fibronectin(FN) to promote the attachment of retinal gliocyte.During the culturing process,the gliocytes were identified by the observation of morphological characteristic and regular histological examination.The identification of the cells also was performed by immunochemistry targeting glial fibrillary acidic protein (GFAP),Vimentin,neuron specific enolase ( NSE ),S-100,CD34,and Ⅷ factor. Results Retinal gliocytes were isolated successfully from the human retina by the two-step digestion method.Primary cultured cells attached after 72 hours and achieved confluency between day 9 and 10 that were aligned petaliform in shape.Regular histological examination after H&E staining showed blue cell nuclei and light red cytoplasm.The target cells presented with strong responses for GFAP and Vimentin and no response for NSE,S-100,CD34 and Ⅷ factor.Conclusions Large amount of purified human retinal gliocytes can be obtained by two-step digestion and cultured in endothelial cells-targeted culture medium supplemented with β-ECGF and sodium heparin in plates coated with FN.The cultured cells expressed markers for retinal gliocytes.However,specific features of these cells remain to be further elucidated.