中华实验眼科杂志
中華實驗眼科雜誌
중화실험안과잡지
CHINESE JOURNAL OF EXPERIMENTAL OPHTHALMOLOGY
2011年
10期
900-906
,共7页
杜连心%余晓菲%许中中%张红敏%杜晓峰%王丽娅
杜連心%餘曉菲%許中中%張紅敏%杜曉峰%王麗婭
두련심%여효비%허중중%장홍민%두효봉%왕려아
角膜/角膜缘%干细胞%激光扫描共焦显微镜%细胞培养%羊膜载体
角膜/角膜緣%榦細胞%激光掃描共焦顯微鏡%細胞培養%羊膜載體
각막/각막연%간세포%격광소묘공초현미경%세포배양%양막재체
Cornea/limbus%stem cells%Laser scanning confocal microscopy%Cell culture%Markers
背景 目前严重的致盲性眼表疾病的治疗方法主要是自体或异体角膜缘干细胞移植,但存在着供体材料来源有限、术后免疫排斥等问题.体外培养角膜缘干细胞用于成为研究热点,优化其培养方法是提高培养效率的前提.目的 以共焦显微镜作为指导进行取材,对人角膜缘干细胞进行体外培养和鉴定,为构建人角膜缘干细胞植片奠定基础.方法 应用激光扫描共焦显微镜对10例10眼白内障术前检查的患者行全角膜及角膜缘分区扫描,记录角膜及角膜缘上皮层、前弹力层图像,对所有检查资料进行研究分析.依据共焦显微镜对角膜缘上皮层的结构分析,对眼库提供的正常供体角膜缘组织进行取材,实验共分6批取组织进行培养,共接种103张组织块.用组织块培养法、以羊膜为载体进行体外培养,分别在培养的第5天、第10天利用免疫荧光技术检测p63蛋白、细胞角蛋白19(CK19)和角蛋白3(K3)及involucrin蛋白的表达对培养细胞的表型进行鉴定.结果 共焦显微镜扫描显示,角膜缘上皮层细胞聚集成细长条索,呈栅栏状外观,其间可见大量高反光颗粒状物质；在基底部扫描时观察到“细胞岛”样现象；角膜缘部位斜切面观察可见上皮层下有排列整齐的乳头状突起.接种的103块组织块中出膜生长74块,平均出膜率为(68.62±16.94)％,平均出膜时间为(5.83±2.04)d,第3批和第6批组织块的出膜率明显高于第2批和第4批,差异均有统计学意义(P＜0.05),第4批和第6批组织块的出膜时间明显长于第1、2、3、5批,差异有统计学意义(P＜0.05).在培养的第5天和第10天,角膜缘干细胞表达p63蛋白的阳性细胞比例分别为4.05％和36.52％,CK19阳性细胞比例分别为26.07％和40.55％,K3的阳性细胞比例分别为57.88％和40.81％,表达involucrin蛋白的阳性细胞比例分别为64.66％和59.19％.结论 在共焦显微镜引导下选取角膜缘组织进行角膜缘干细胞培养结果可靠,羊膜作为载体可以使体外培养的人角膜缘干细胞良好生长.
揹景 目前嚴重的緻盲性眼錶疾病的治療方法主要是自體或異體角膜緣榦細胞移植,但存在著供體材料來源有限、術後免疫排斥等問題.體外培養角膜緣榦細胞用于成為研究熱點,優化其培養方法是提高培養效率的前提.目的 以共焦顯微鏡作為指導進行取材,對人角膜緣榦細胞進行體外培養和鑒定,為構建人角膜緣榦細胞植片奠定基礎.方法 應用激光掃描共焦顯微鏡對10例10眼白內障術前檢查的患者行全角膜及角膜緣分區掃描,記錄角膜及角膜緣上皮層、前彈力層圖像,對所有檢查資料進行研究分析.依據共焦顯微鏡對角膜緣上皮層的結構分析,對眼庫提供的正常供體角膜緣組織進行取材,實驗共分6批取組織進行培養,共接種103張組織塊.用組織塊培養法、以羊膜為載體進行體外培養,分彆在培養的第5天、第10天利用免疫熒光技術檢測p63蛋白、細胞角蛋白19(CK19)和角蛋白3(K3)及involucrin蛋白的錶達對培養細胞的錶型進行鑒定.結果 共焦顯微鏡掃描顯示,角膜緣上皮層細胞聚集成細長條索,呈柵欄狀外觀,其間可見大量高反光顆粒狀物質；在基底部掃描時觀察到“細胞島”樣現象；角膜緣部位斜切麵觀察可見上皮層下有排列整齊的乳頭狀突起.接種的103塊組織塊中齣膜生長74塊,平均齣膜率為(68.62±16.94)％,平均齣膜時間為(5.83±2.04)d,第3批和第6批組織塊的齣膜率明顯高于第2批和第4批,差異均有統計學意義(P＜0.05),第4批和第6批組織塊的齣膜時間明顯長于第1、2、3、5批,差異有統計學意義(P＜0.05).在培養的第5天和第10天,角膜緣榦細胞錶達p63蛋白的暘性細胞比例分彆為4.05％和36.52％,CK19暘性細胞比例分彆為26.07％和40.55％,K3的暘性細胞比例分彆為57.88％和40.81％,錶達involucrin蛋白的暘性細胞比例分彆為64.66％和59.19％.結論 在共焦顯微鏡引導下選取角膜緣組織進行角膜緣榦細胞培養結果可靠,羊膜作為載體可以使體外培養的人角膜緣榦細胞良好生長.
배경 목전엄중적치맹성안표질병적치료방법주요시자체혹이체각막연간세포이식,단존재착공체재료래원유한、술후면역배척등문제.체외배양각막연간세포용우성위연구열점,우화기배양방법시제고배양효솔적전제.목적 이공초현미경작위지도진행취재,대인각막연간세포진행체외배양화감정,위구건인각막연간세포식편전정기출.방법 응용격광소묘공초현미경대10례10안백내장술전검사적환자행전각막급각막연분구소묘,기록각막급각막연상피층、전탄력층도상,대소유검사자료진행연구분석.의거공초현미경대각막연상피층적결구분석,대안고제공적정상공체각막연조직진행취재,실험공분6비취조직진행배양,공접충103장조직괴.용조직괴배양법、이양막위재체진행체외배양,분별재배양적제5천、제10천이용면역형광기술검측p63단백、세포각단백19(CK19)화각단백3(K3)급involucrin단백적표체대배양세포적표형진행감정.결과 공초현미경소묘현시,각막연상피층세포취집성세장조색,정책란상외관,기간가견대량고반광과립상물질；재기저부소묘시관찰도“세포도”양현상；각막연부위사절면관찰가견상피층하유배렬정제적유두상돌기.접충적103괴조직괴중출막생장74괴,평균출막솔위(68.62±16.94)％,평균출막시간위(5.83±2.04)d,제3비화제6비조직괴적출막솔명현고우제2비화제4비,차이균유통계학의의(P＜0.05),제4비화제6비조직괴적출막시간명현장우제1、2、3、5비,차이유통계학의의(P＜0.05).재배양적제5천화제10천,각막연간세포표체p63단백적양성세포비례분별위4.05％화36.52％,CK19양성세포비례분별위26.07％화40.55％,K3적양성세포비례분별위57.88％화40.81％,표체involucrin단백적양성세포비례분별위64.66％화59.19％.결론 재공초현미경인도하선취각막연조직진행각막연간세포배양결과가고,양막작위재체가이사체외배양적인각막연간세포량호생장.
Background Human limbal allograft transplantation or limbal autograft transplantation are the primary approaches to the severe corneal-blindness,but their application in clinic were limited because of the defects of donor material.With the development of tissue engineering technology,transplantation of in vitro cultured limbal epithelial stem cells is being an advanced management.Objective The aim of this work was to expand human limbal epithelial stem cells ex vivo under the guidance of confocal microscope and to lay the foundation for fabricating ex vivo cultured cell sheets.Methods Ten eyes of ten patients were examined with the Heidelberg Retina Tomography Ⅲ Rostock Cornea Module(HRT3-RCM)to elucidate the structure of the human corneoscleral limbus and to correlate limbal epithelial dimensions.According to the analysis of the images of limbal epithelia,the limbal tissues provided by Eye Bank of Henan Eye Institute were cut into suitable explants.Then,this study was conducted to expand limbal epithelial stem cells ex vivo on denuded amniotic membrane.The phenotypes of primary cultured cells were evaluated by morphology and immunofluorescent staining with antibodies for limbal epithelial stem cell markers (p63,cytokeratinl9)and differentiation markers(keratin 3,involucrin).This experimental procedure was approved by the Ethic Committee of Henan Provincial People's Hospital.The written informed consent was obtained from subjects before initiation of any examination.Results The palisade morphology of human limbus was imaged clearly on the laser scanning in vivo confocal microscopy and many hyperreflective cells were observed in palisade basal cells.The cell-island phenomenon was seen in the basement membrane under the laser scanning in vivo confocal microscopy.The oblique sections of limbus showed many papilla-like epithelial columns below the superficial limbal epithelia.Throughout the experiment duration,the epithelial cells grew well with the migration rates from limbal tissue (68.62± 16.94)％ and the migration time(5.83 ±2.04)days,which depended on the tissue freshness.Compared with the second and forth batch of tissue,the migration rates of the third and sixth batch of tissues were significantly higher(P＜0.05),and the migration time was evidently longer in the forth and sixth batch of tissue compared with the first,second,third and fifth batch(P＜0.05).The positively expressing rates in the cultured corneal stem cells were 4.05％ and 36.52％ for p63,26.07％ and 40.55％ for CK19,57.88％ and 40.81％ for K3,64.66％ and 59.19％ for involucrin.Conclusion Human limbal epithelial stem cells can be successfully and purposefully obtained from the limbal tissue based on the guidance confocal miscroscope.The cultured corneal stem cells can grow well on the denuded amniotic membrane
