中华实验眼科杂志
中華實驗眼科雜誌
중화실험안과잡지
CHINESE JOURNAL OF EXPERIMENTAL OPHTHALMOLOGY
2011年
2期
107-112
,共6页
角膜内皮细胞%细胞培养%细胞鉴定%组织工程%Na+-K+ATP酶
角膜內皮細胞%細胞培養%細胞鑒定%組織工程%Na+-K+ATP酶
각막내피세포%세포배양%세포감정%조직공정%Na+-K+ATP매
Corneal endothelial cell%Cellular culture%Cellular identification%Tissue engineering%Na+-K+ ATPase
背景 如何选择高质量、生物学特性更接近在体生理状态的角膜内皮种子细胞是组织工程角膜研究的基础.角膜内皮细胞(CECs)的培养、鉴定及生物学特性检测是优选角膜内皮种子细胞的瓶颈问题.目的 建立兔CECs原代培养及鉴定的方法,检测传代后细胞的生物学特性.方法从30只新西兰大白兔的角膜组织完整撕除后弹力层及CECs层,采用胰蛋白酶消化法进行原代培养,观察培养细胞的生长状态.分别从形态学、基因水平和蛋白水平鉴定细胞:茜素红染色法观察细胞形态,并与新鲜角膜组织的内皮细胞进行对比;逆转录聚合酶链反应(RT-PCR)法检测Ⅳα2型胶原(COL4A2)、血管内皮生长因子受体2(FLK1)、Na+-K+ATP酶α1亚单位(ATP1A1)、水通道蛋白1(AQP1)、电压依丛性阴离子通道(VDACs)等相对特异性基因;免疫细胞化学法观察神经元特异性烯醇化酶(NSE)、Na+-K+ATP酶及紧密连接蛋白ZO-1的表达.采用MTT比色法测定传代细胞的增生活性变化,采用超微量ATP酶试剂盒及免疫荧光法分别测定传代细胞Na+-K+ATP酶活性及其表达量的变化.结果 原代培养的兔CECs多在24h内贴壁,2～3d达融合,形态呈六边形.随着传代代数的增加,细胞形态逐渐发生改变,第2代、第3代细胞可见空泡样变.原代培养的细胞茜素红染色可见清晰的细胞轮廓,与在体的CECs形态相似.RT-PCR法检测可见COL4A2、FLKI、ATPIA1、AQPI、VDACs等基因在培养细胞中表达.免疫荧光染色结果显示NSE、Na+-K+ATP酶、ZO-1在培养细胞中呈阳性表达.MTT检测结果显示,传代后细胞的增生活性逐渐下降,尤其第2代、第3代细胞下降明显.定量检测结果显示随着传代代数的增加,兔CECs的Na+-K+ATP酶活性逐渐降低,各代细胞的Na+-K+ATP酶活性总体比较的差异有统计学意义(F=77.174,P=0.000).结论 成功用酶消化法建立了体外培养兔CECs的方法,并从多个层次建立了纯化细胞的鉴定方法.研究结果证实经传代后的CECs的增生活性和功能均有所下降,因此在进行相关的研究时应选用前2代的细胞.
揹景 如何選擇高質量、生物學特性更接近在體生理狀態的角膜內皮種子細胞是組織工程角膜研究的基礎.角膜內皮細胞(CECs)的培養、鑒定及生物學特性檢測是優選角膜內皮種子細胞的瓶頸問題.目的 建立兔CECs原代培養及鑒定的方法,檢測傳代後細胞的生物學特性.方法從30隻新西蘭大白兔的角膜組織完整撕除後彈力層及CECs層,採用胰蛋白酶消化法進行原代培養,觀察培養細胞的生長狀態.分彆從形態學、基因水平和蛋白水平鑒定細胞:茜素紅染色法觀察細胞形態,併與新鮮角膜組織的內皮細胞進行對比;逆轉錄聚閤酶鏈反應(RT-PCR)法檢測Ⅳα2型膠原(COL4A2)、血管內皮生長因子受體2(FLK1)、Na+-K+ATP酶α1亞單位(ATP1A1)、水通道蛋白1(AQP1)、電壓依叢性陰離子通道(VDACs)等相對特異性基因;免疫細胞化學法觀察神經元特異性烯醇化酶(NSE)、Na+-K+ATP酶及緊密連接蛋白ZO-1的錶達.採用MTT比色法測定傳代細胞的增生活性變化,採用超微量ATP酶試劑盒及免疫熒光法分彆測定傳代細胞Na+-K+ATP酶活性及其錶達量的變化.結果 原代培養的兔CECs多在24h內貼壁,2～3d達融閤,形態呈六邊形.隨著傳代代數的增加,細胞形態逐漸髮生改變,第2代、第3代細胞可見空泡樣變.原代培養的細胞茜素紅染色可見清晰的細胞輪廓,與在體的CECs形態相似.RT-PCR法檢測可見COL4A2、FLKI、ATPIA1、AQPI、VDACs等基因在培養細胞中錶達.免疫熒光染色結果顯示NSE、Na+-K+ATP酶、ZO-1在培養細胞中呈暘性錶達.MTT檢測結果顯示,傳代後細胞的增生活性逐漸下降,尤其第2代、第3代細胞下降明顯.定量檢測結果顯示隨著傳代代數的增加,兔CECs的Na+-K+ATP酶活性逐漸降低,各代細胞的Na+-K+ATP酶活性總體比較的差異有統計學意義(F=77.174,P=0.000).結論 成功用酶消化法建立瞭體外培養兔CECs的方法,併從多箇層次建立瞭純化細胞的鑒定方法.研究結果證實經傳代後的CECs的增生活性和功能均有所下降,因此在進行相關的研究時應選用前2代的細胞.
배경 여하선택고질량、생물학특성경접근재체생리상태적각막내피충자세포시조직공정각막연구적기출.각막내피세포(CECs)적배양、감정급생물학특성검측시우선각막내피충자세포적병경문제.목적 건립토CECs원대배양급감정적방법,검측전대후세포적생물학특성.방법종30지신서란대백토적각막조직완정시제후탄력층급CECs층,채용이단백매소화법진행원대배양,관찰배양세포적생장상태.분별종형태학、기인수평화단백수평감정세포:천소홍염색법관찰세포형태,병여신선각막조직적내피세포진행대비;역전록취합매련반응(RT-PCR)법검측Ⅳα2형효원(COL4A2)、혈관내피생장인자수체2(FLK1)、Na+-K+ATP매α1아단위(ATP1A1)、수통도단백1(AQP1)、전압의총성음리자통도(VDACs)등상대특이성기인;면역세포화학법관찰신경원특이성희순화매(NSE)、Na+-K+ATP매급긴밀련접단백ZO-1적표체.채용MTT비색법측정전대세포적증생활성변화,채용초미량ATP매시제합급면역형광법분별측정전대세포Na+-K+ATP매활성급기표체량적변화.결과 원대배양적토CECs다재24h내첩벽,2～3d체융합,형태정륙변형.수착전대대수적증가,세포형태축점발생개변,제2대、제3대세포가견공포양변.원대배양적세포천소홍염색가견청석적세포륜곽,여재체적CECs형태상사.RT-PCR법검측가견COL4A2、FLKI、ATPIA1、AQPI、VDACs등기인재배양세포중표체.면역형광염색결과현시NSE、Na+-K+ATP매、ZO-1재배양세포중정양성표체.MTT검측결과현시,전대후세포적증생활성축점하강,우기제2대、제3대세포하강명현.정량검측결과현시수착전대대수적증가,토CECs적Na+-K+ATP매활성축점강저,각대세포적Na+-K+ATP매활성총체비교적차이유통계학의의(F=77.174,P=0.000).결론 성공용매소화법건립료체외배양토CECs적방법,병종다개층차건립료순화세포적감정방법.연구결과증실경전대후적CECs적증생활성화공능균유소하강,인차재진행상관적연구시응선용전2대적세포.
Background How to harvest the purified corneal endothelial seed cells is very important for the corneal tissue engineering technology. Herein,to establish a good culture method and effective identification method of corneal endothelial cells ( CECs) is the key. Objective Present study wag to establish the cultivating and identifying approach of the rabbit CECs and detect the biological characteristics of passaged cells. Methods Rabbits CECs were isolated from Descemet's membrane peeled off completely in 30 New Zealand white rabbits and then digested with 0. 25% trypsin-0. 02% EDTA and primarily cultured in CECs medium containing 15% fetal bovine serum. The growth situate and cellular morphology of rabbit CECs were observed under the inverted phase-contrast microscope following the alizarin red staining. Rabbit CECs were identified by cellular morphology as well as in gene and protein level, including the detection of expressions of collagen type IV α2(COL4A2) ,vascular endothelial growth factor receptor 2 ( FLK1 ) , Na+-K+ ATPase alpha 1 subunit ( ATP1 A1 ) , aquaporin 1 ( AQP1 ) , voltage-dependent anion channels ( VDACs) by reverse transcription-polymerase chain reaction ( RT-PCR ). The expression and distribution of neurone specific enolase ( NSE) , Na+-K+ ATPase, zonula occludens-1 ( ZO-1 ) were also detected by immunocytochemistry under the fluorescence microscope. The proliferation activity of the passage cells was dynamically observed by MTT assay,and Na+-K+ ATPase activity of different generations cells was detected by ATPase kit. Results Majority of the primarily cultured cells adhered in 24 hours, infused in 2-3 days with the hexagon shape in appearance. The morphology of the cells was very varied with passage. Alizarin red staining showed a well- defined and well-lined cellular morphology similar to the corneal cells in vivo. Target genes of C0L4A2, FLK1, ATPIAI ,AQPI and VDACs were positively expressed in the cells. However,the expression of CK12 was absent in the cells. NSE, Na+-K+ ATPase, ZO-1 positive cells were respectively observed under the laser confocal scanning microscopy. MTT results showed a gradually low growth curve with the passage. Quantitative results also revealed that the Na+-K+ ATPase activity was gradually declined in different generations of cells ( F = 77. 174, P = 0. 000 ). Conclusion The enzyme digestion is a better approach of isolating and culturing comeal endothelial cells. Cultured cells can be identified by cells staining, gene expression and protein level. Earlier generation of CECs are ideal seed cells for cornea tissue engineering.
