CAJ | 학술논문

背景角膜盲是中国主要的致盲眼病之一.随着组织工程技术的进步和发展,组织工程移植膜的构建将为角膜病的治疗开辟新的途径,而选择和培养理想的种子细胞是工程化角膜的基础. 目的采用胰蛋白酶消化法体外分离培养角膜内皮细胞(CECs)并进行常规培养,观察并鉴定细胞体外生长的生物学特性,为实验研究和工程化角膜的构建提供种子细胞. 方法 3月龄新西兰白兔12只,摘除新鲜眼球,光学显微镜下将兔角膜带有内皮细胞的后弹力层面完整分离,胰蛋白酶消化、纯化后得到CECs,进行体外培养.倒置显微镜下观察细胞的形态变化;锥虫蓝染色法计算培养细胞的存活率并绘制细胞生长曲线;采用CM-Dil染色法进行细胞标记传代;采用苏木精-伊红染色法观察培养细胞的形态,并用免疫组织化学染色法观察神经元烯醇化酶(NSE)抗体的表达,对细胞进行细胞表型鉴定;扫描电子显微镜下观察培养细胞的表面结构;采用免疫荧光染色法观察细胞质紧密连接蛋白1(ZO-1)的表达.结果揭取带有内皮细胞的后弹力层联合酶消化法可快速获得大量纯化的CECs,且快速贴壁生长并增生,体外培养5～7 d即融合形成单层,呈铺路石样排列,培养细胞的存活率为95％;CM-Dil染色显示培养的CECs呈现红色环状的荧光颗粒,标记率达90％以上,连续传代后仍显示红色荧光.苏木精-伊红染色显示培养细胞呈多角形,核圆,细胞质中NSE呈棕黄色染色.扫描电子显微镜下可见细胞表面有丰富的微绒毛,细胞间足突相互连接,细胞体积大而扁平;荧光显微镜下可见细胞间ZO-1呈绿色荧光. 结论通过完整分离角膜后弹力层联合胰蛋白酶消化法可体外培养、纯化和传代兔CECs,传2～3代的细胞具有CECs的生物学特性,有望为组织工程化角膜内皮移植膜的构建提供优质的种子细胞来源.
배경 각막맹시중국주요적치맹안병지일.수착조직공정기술적진보화발전,조직공정이식막적구건장위각막병적치료개벽신적도경,이선택화배양이상적충자세포시공정화각막적기출. 목적 채용이단백매소화법체외분리배양각막내피세포(CECs)병진행상규배양,관찰병감정세포체외생장적생물학특성,위실험연구화공정화각막적구건제공충자세포. 방법 3월령신서란백토12지,적제신선안구,광학현미경하장토각막대유내피세포적후탄력층면완정분리,이단백매소화、순화후득도CECs,진행체외배양.도치현미경하관찰세포적형태변화;추충람염색법계산배양세포적존활솔병회제세포생장곡선;채용CM-Dil염색법진행세포표기전대;채용소목정-이홍염색법관찰배양세포적형태,병용면역조직화학염색법관찰신경원희순화매(NSE)항체적표체,대세포진행세포표형감정;소묘전자현미경하관찰배양세포적표면결구;채용면역형광염색법관찰세포질긴밀련접단백1(ZO-1)적표체.결과 게취대유내피세포적후탄력층연합매소화법가쾌속획득대량순화적CECs,차쾌속첩벽생장병증생,체외배양5～7 d즉융합형성단층,정포로석양배렬,배양세포적존활솔위95％;CM-Dil염색현시배양적CECs정현홍색배상적형광과립,표기솔체90％이상,련속전대후잉현시홍색형광.소목정-이홍염색현시배양세포정다각형,핵원,세포질중NSE정종황색염색.소묘전자현미경하가견세포표면유봉부적미융모,세포간족돌상호련접,세포체적대이편평;형광현미경하가견세포간ZO-1정록색형광. 결론 통과완정분리각막후탄력층연합이단백매소화법가체외배양、순화화전대토CECs,전2～3대적세포구유CECs적생물학특성,유망위조직공정화각막내피이식막적구건제공우질적충자세포래원.
Background Corneal blindness is one of the major blinding eye diseases in China.With the development and progress of tissue engineering technology,tissue-engineered corneas offers a new approach to the treatment of corneal diseases.To select and cultivate ideal seed cells is a foundation of construction of tissueengineered corneas.Objective This study was to evaluate the efficiency of stripe off the Descemet membrane with endothelium plus enzymic digestion in the isolation of corneal endothelial cells and analyze the bionomics of rabbit corneal endothelial cells (CECs) in vitro.Methods Descemet membrane was stripped from fresh cornea of New Zealand rabbit under the dissection microscope.Descemet membrane with endothelium was incubated in trypsin and EDTA solution at 37 ℃ and then purified for CECs subculture in vitro.The morphology of the cultured cells was observed under the inverted microscope and marked by CM-Dil dye solution.Then the shape of the cells was observed by hematoxylin and eosin staining and the cells were identified for the expression of neuron specific enolase (NSE) using immunochemistry.The viability of the cells were evaluated by trypan blue staining.The surface structure of the cells were examined under the scanning electron microscope.Intercellular zonula occludens-1 (ZO-1) was identified by immunofluorecsence staining.Results A large number of purified CECs were obtained from Descemet membrane with endothelium through enzymic digestion.Cultured cells grew well and formed monolayer 5-7 days later with the cobblestone stone-like arrangement.The survival rate of the cells was 95％.CECs presented with the red annular fluorescence for CM-Dil with the labeling rate ＞90％.NSE was positively expressed in the cytoplasm.Polygon CECs were seen by hematoxylin and eosin staining and showed the brown staining.Abundant microvilli on the cellular surface and interconnected foot process were seen under the scanning electron microscope.ZO-1 showed the green fluorescence.Conclusions The method of striping off the corneal Descemet membrane with endothelium plus enzymic digestion can obtain abundant CECs.Cultured cells have good biological properties.This study may offer a feasible application in the engineering of corneal transplant membrane.