中华实验眼科杂志
中華實驗眼科雜誌
중화실험안과잡지
CHINESE JOURNAL OF EXPERIMENTAL OPHTHALMOLOGY
2013年
1期
8-12
,共5页
毛羽翔%林少芬%曾美珍%田景毅%唐仕波
毛羽翔%林少芬%曾美珍%田景毅%唐仕波
모우상%림소분%증미진%전경의%당사파
视网膜血管内皮细胞%细胞培养%免疫组织化学%细胞鉴定
視網膜血管內皮細胞%細胞培養%免疫組織化學%細胞鑒定
시망막혈관내피세포%세포배양%면역조직화학%세포감정
Retinal microvascular endothelial cell%Cell culture%Immunohistochemistry%Cell identification
背景 优化人视网膜血管内皮细胞的培养和鉴定方法在视网膜血管性疾病的研究中有重要作用,以往研究者已成功培养了人视网膜血管内皮细胞,但进一步简化其方法并达到获取量大而纯的细胞是基础研究和临床研究的基础. 目的 探索一种更为简便快速、收获量大且纯度高的视网膜血管内皮细胞的改良培养方法,并对目标细胞的抗原表达特点进行分析,比较眼科新生血管性疾病研究中常用的两种血管内皮细胞的标志性蛋白表达情况.方法 利用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Ⅰ用二步消化法获取人视网膜血管内皮细胞,在传统培养方法中采用含质量分数10％胎牛血清的人血管内皮细胞培养液的基础上,添加内皮细胞生长因子-β(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)包被以促进培养细胞的贴壁.观察收获的目标细胞的形态特征,采用活体显微镜进行形态学观察、常规组织病理学观察目标细胞的生长状态,免疫组织化学法检测Ⅷ因子、CD31、CD34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血管内皮细胞,原代培养的细胞72 h贴壁,第9～10天细胞达到融合状态,呈铺路石样.常规组织学观察显示,细胞核呈鲜亮蓝色,细胞质呈淡红色.培养的细胞对Ⅷ因子、CD31、CD34相关抗原表达呈阳性反应.结果显示培养的血管内皮细胞均有CD31、CD34同时表达,但其阳性染色程度低于Ⅷ因子相关抗原阳性反应,人脐静脉血管内皮细胞(UVECs)与人血管内皮细胞相同. 结论 在胰蛋白酶、胶原蛋白酶消化法的基础上用含10％优质胎牛血清的人内皮细胞培养基中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血管内皮细胞的目的.
揹景 優化人視網膜血管內皮細胞的培養和鑒定方法在視網膜血管性疾病的研究中有重要作用,以往研究者已成功培養瞭人視網膜血管內皮細胞,但進一步簡化其方法併達到穫取量大而純的細胞是基礎研究和臨床研究的基礎. 目的 探索一種更為簡便快速、收穫量大且純度高的視網膜血管內皮細胞的改良培養方法,併對目標細胞的抗原錶達特點進行分析,比較眼科新生血管性疾病研究中常用的兩種血管內皮細胞的標誌性蛋白錶達情況.方法 利用正常人角膜移植供體眼毬分離視網膜組織,採用質量分數2％胰蛋白酶和質量分數0.133％膠原酶Ⅰ用二步消化法穫取人視網膜血管內皮細胞,在傳統培養方法中採用含質量分數10％胎牛血清的人血管內皮細胞培養液的基礎上,添加內皮細胞生長因子-β(β-ECGF)和肝素鈉,對分離的細胞進行體外培養,培養皿用纖維黏連蛋白(FN)包被以促進培養細胞的貼壁.觀察收穫的目標細胞的形態特徵,採用活體顯微鏡進行形態學觀察、常規組織病理學觀察目標細胞的生長狀態,免疫組織化學法檢測Ⅷ因子、CD31、CD34在細胞中的錶達以鑒定目標細胞. 結果 應用胰蛋白酶、膠原酶二步消化法可成功穫取人視網膜血管內皮細胞,原代培養的細胞72 h貼壁,第9～10天細胞達到融閤狀態,呈鋪路石樣.常規組織學觀察顯示,細胞覈呈鮮亮藍色,細胞質呈淡紅色.培養的細胞對Ⅷ因子、CD31、CD34相關抗原錶達呈暘性反應.結果顯示培養的血管內皮細胞均有CD31、CD34同時錶達,但其暘性染色程度低于Ⅷ因子相關抗原暘性反應,人臍靜脈血管內皮細胞(UVECs)與人血管內皮細胞相同. 結論 在胰蛋白酶、膠原蛋白酶消化法的基礎上用含10％優質胎牛血清的人內皮細胞培養基中添加生長因子和肝素鈉,併用FN包被培養皿進行體外培養,可達到快速、大量分離和純化人視網膜血管內皮細胞的目的.
배경 우화인시망막혈관내피세포적배양화감정방법재시망막혈관성질병적연구중유중요작용,이왕연구자이성공배양료인시망막혈관내피세포,단진일보간화기방법병체도획취량대이순적세포시기출연구화림상연구적기출. 목적 탐색일충경위간편쾌속、수획량대차순도고적시망막혈관내피세포적개량배양방법,병대목표세포적항원표체특점진행분석,비교안과신생혈관성질병연구중상용적량충혈관내피세포적표지성단백표체정황.방법 이용정상인각막이식공체안구분리시망막조직,채용질량분수2％이단백매화질량분수0.133％효원매Ⅰ용이보소화법획취인시망막혈관내피세포,재전통배양방법중채용함질량분수10％태우혈청적인혈관내피세포배양액적기출상,첨가내피세포생장인자-β(β-ECGF)화간소납,대분리적세포진행체외배양,배양명용섬유점련단백(FN)포피이촉진배양세포적첩벽.관찰수획적목표세포적형태특정,채용활체현미경진행형태학관찰、상규조직병이학관찰목표세포적생장상태,면역조직화학법검측Ⅷ인자、CD31、CD34재세포중적표체이감정목표세포. 결과 응용이단백매、효원매이보소화법가성공획취인시망막혈관내피세포,원대배양적세포72 h첩벽,제9～10천세포체도융합상태,정포로석양.상규조직학관찰현시,세포핵정선량람색,세포질정담홍색.배양적세포대Ⅷ인자、CD31、CD34상관항원표체정양성반응.결과현시배양적혈관내피세포균유CD31、CD34동시표체,단기양성염색정도저우Ⅷ인자상관항원양성반응,인제정맥혈관내피세포(UVECs)여인혈관내피세포상동. 결론 재이단백매、효원단백매소화법적기출상용함10％우질태우혈청적인내피세포배양기중첨가생장인자화간소납,병용FN포피배양명진행체외배양,가체도쾌속、대량분리화순화인시망막혈관내피세포적목적.
Background To optimize the culture method of human retinal microvascular endothelial cells is very important for the study of retinal angiogenesis disease.Human retinal microvascular endothelial cells have been successfully cultured in previous studies,but further improvement of the culture method to harvest higher yields and purity cells is still needed.Objective This study was to design a modified method to isolate and purify human retinal microvascular endothelial cells much easily and quickly,and to compare the expression of specific markers of vascular endothelial cells,factor Ⅷ and CD31/CD34 in the cells.Methods The use of human donor eyeballs was approved by the Ethic Commission of Zhongshan Ophthalmic Center of Sun Yat-sen University.The retina tissue from healthy donor was isolated and digested by the two-step digestion method with 2％ trypsin and 0.133％ collagenase Ⅳ.Human retinal microvascular endothelial cells were collected and plated in 60 mm dishes coated by 0.1％ fibronectin and cultured in endothelial cell-specialized medium supplemented with 10％ fetal bovine serum,0.3 mg/L β-endothelial cell growth factor (ECGF) and 100 ng/L sodium heparin.During the culturing,the growth situation of the cells was monitored by morphological observation,and immunohistochemical staining was performed to probe vascular endothelial cell-specific membrane protein CD31,CD34 and factor Ⅷ for identification of the cell purity.Results Human retinal microvascular endothelial cells were isolated successfully from the retina by the twostep digestion method.The primary cultured cells adhered to well 72 hours later and achieved confluence with the typical cobblestone appearance 9 to 10 days after cultured.The cells exhibited the blue nuclei and reddish cytoplasm by regular haematoxylin and eosin stain and showed a strong positive response for CD31,CD34 and factor Ⅷ by immunohistochemistry.The positive dye of CD31 and CD34 was lower than Ⅷ factor in both endothelial cells.Conclusions Modified culture method of human retinal microvascular endothelial cells can improve cell culture result and purify target cells.
