中国组织工程研究
中國組織工程研究
중국조직공정연구
Journal of Clinical Rehabilitative Tissue Engineering Research
2014年
1期
94-99
,共6页
许中中%余晓菲%杜连心%李晶%王丽娅
許中中%餘曉菲%杜連心%李晶%王麗婭
허중중%여효비%두련심%리정%왕려아
干细胞%培养%角膜上皮干细胞%活体激光扫描角膜共焦显微镜%免疫荧光染色%定位%国家自然科学基金
榦細胞%培養%角膜上皮榦細胞%活體激光掃描角膜共焦顯微鏡%免疫熒光染色%定位%國傢自然科學基金
간세포%배양%각막상피간세포%활체격광소묘각막공초현미경%면역형광염색%정위%국가자연과학기금
stem cells%epithelium,corneal%microscopy,confocal%fluoroimmunoassay
背景：眼表存在两种形式的上皮干细胞即角膜上皮干细胞和结膜上皮干细胞，角膜上皮干细胞在角膜上皮细胞更新和角膜透明的维持方面起着重要作用。<br> 目的：采用活体激光扫描角膜共焦显微镜和免疫荧光染色技术相结合的方法，从活体和体外层面上对角膜上皮干细胞进行定位研究。<br> 方法：收集2009年9月至2012年9月来河南省眼科研究所就诊的单侧角膜缘干细胞缺乏患者，使用活体激光扫描角膜共焦显微镜检查患者双眼，健侧眼为对照。扫描方位依次为中央角膜及上、下、左、右方的角膜缘，记录扫描图像并分析。眼球材料来自于河南省眼库，切取角膜中央和角膜缘组织，组织包埋剂包被、冰冻切片，切片厚度5-7μm；免疫荧光染色技术检测p63、ABCG2、K3和Connexin 43在角膜中央及角膜缘上皮层的表达。<br> 结果与结论：共有24例患者确诊为单侧角膜缘干细胞缺乏，活体激光扫描角膜共焦显微镜下患侧眼角膜病变区可见结膜细胞及杯状细胞；角膜缘区域Vogt栅栏状结构消失，色素细胞消失，取而代之的是大量纤维瘢痕化组织。免疫荧光染色示表达ABCG2和p63的细胞主要在角膜缘上皮基底层，尤其在近结膜侧的角膜缘及角膜缘中间部表达相对较高，而中央角膜上皮层细胞不表达；K3及Connexin43在角膜缘上皮基底细胞层不表达，中央角膜上皮全层表达。通过活体激光扫描角膜共焦显微镜观察及干细胞标记物检测显示角膜上皮干细胞主要存在于角膜缘外2/3区域的Vogt栅栏基底部及钉突结构中。
揹景：眼錶存在兩種形式的上皮榦細胞即角膜上皮榦細胞和結膜上皮榦細胞，角膜上皮榦細胞在角膜上皮細胞更新和角膜透明的維持方麵起著重要作用。<br> 目的：採用活體激光掃描角膜共焦顯微鏡和免疫熒光染色技術相結閤的方法，從活體和體外層麵上對角膜上皮榦細胞進行定位研究。<br> 方法：收集2009年9月至2012年9月來河南省眼科研究所就診的單側角膜緣榦細胞缺乏患者，使用活體激光掃描角膜共焦顯微鏡檢查患者雙眼，健側眼為對照。掃描方位依次為中央角膜及上、下、左、右方的角膜緣，記錄掃描圖像併分析。眼毬材料來自于河南省眼庫，切取角膜中央和角膜緣組織，組織包埋劑包被、冰凍切片，切片厚度5-7μm；免疫熒光染色技術檢測p63、ABCG2、K3和Connexin 43在角膜中央及角膜緣上皮層的錶達。<br> 結果與結論：共有24例患者確診為單側角膜緣榦細胞缺乏，活體激光掃描角膜共焦顯微鏡下患側眼角膜病變區可見結膜細胞及杯狀細胞；角膜緣區域Vogt柵欄狀結構消失，色素細胞消失，取而代之的是大量纖維瘢痕化組織。免疫熒光染色示錶達ABCG2和p63的細胞主要在角膜緣上皮基底層，尤其在近結膜側的角膜緣及角膜緣中間部錶達相對較高，而中央角膜上皮層細胞不錶達；K3及Connexin43在角膜緣上皮基底細胞層不錶達，中央角膜上皮全層錶達。通過活體激光掃描角膜共焦顯微鏡觀察及榦細胞標記物檢測顯示角膜上皮榦細胞主要存在于角膜緣外2/3區域的Vogt柵欄基底部及釘突結構中。
배경：안표존재량충형식적상피간세포즉각막상피간세포화결막상피간세포，각막상피간세포재각막상피세포경신화각막투명적유지방면기착중요작용。<br> 목적：채용활체격광소묘각막공초현미경화면역형광염색기술상결합적방법，종활체화체외층면상대각막상피간세포진행정위연구。<br> 방법：수집2009년9월지2012년9월래하남성안과연구소취진적단측각막연간세포결핍환자，사용활체격광소묘각막공초현미경검사환자쌍안，건측안위대조。소묘방위의차위중앙각막급상、하、좌、우방적각막연，기록소묘도상병분석。안구재료래자우하남성안고，절취각막중앙화각막연조직，조직포매제포피、빙동절편，절편후도5-7μm；면역형광염색기술검측p63、ABCG2、K3화Connexin 43재각막중앙급각막연상피층적표체。<br> 결과여결론：공유24례환자학진위단측각막연간세포결핍，활체격광소묘각막공초현미경하환측안각막병변구가견결막세포급배상세포；각막연구역Vogt책란상결구소실，색소세포소실，취이대지적시대량섬유반흔화조직。면역형광염색시표체ABCG2화p63적세포주요재각막연상피기저층，우기재근결막측적각막연급각막연중간부표체상대교고，이중앙각막상피층세포불표체；K3급Connexin43재각막연상피기저세포층불표체，중앙각막상피전층표체。통과활체격광소묘각막공초현미경관찰급간세포표기물검측현시각막상피간세포주요존재우각막연외2/3구역적Vogt책란기저부급정돌결구중。
BACKGROUND:There are two types of epithelial stem cells in the ocular surface tissue:corneal epithelial stem cells and conjunctival epithelial stem cells. The corneal epithelial stem cells play an important role in renewal of corneal epithelial cells and maintenance of corneal transparency. <br> OBJECTIVE:To study the location of corneal epithelial stem cells using laser in vivo confocal microscopy and immunofluorescent staining. <br> METHODS:Patients with unilateral limbal stem celldeficiency who went to Henan Eye Institute from September 2009 to September 2012 were enrol ed in this study. Bilateral eyes were scanned by laser in vivo confocal microscopy, and the healthy eye was imaged as a control. The central cornea and limbus were scanned and images were recorded for statistical analysis. The eye bal s were obtained from Henan Eye Bank, China. Central cornea and limbus were dissected and embedded in the OCT compound for frozen section and the proper thickness of the section was 5-7μm. Immunofluorescent staining was used to detect the expression of p63, ABCG2, K3 and Connexin 43 in the epithelial layers of central cornea and limbus. <br> RESULTS AND CONCLUSION:Twenty-four patients diagnosed with unilateral limbal stem celldeficiency were recruited. Under confocal microscopy, in the affected eyes, the typical morphology of conjunctival epithelial cells and goblet cells was detected instead of corneal epithelial cells;in the limbus, a great amount of fiber scarring tissue was detected instead of Vogt palisade, rete pegs and pigment cells. Immunofluorescent staining showed the expression of p63, ABCG2 was mainly in the basal layer of limbal epithelium, especial y in the outer and middle parts, but the expression of p63 and ABCG2 was not detected in the epithelial celllayers of central cornea. K3 and Connexin43 were not expressed in suprabasal layers of limbal epithelium, but in central cornea, they were expressed highly in the whole epithelial celllayers. Laser in vivo confocal microscopy and immunofluorescent staining showed the corneal epithelial stem cells were located in the basal layer of outer and middle limbal epithelium, mainly in Vogt palisade and rete pegs.
