CAJ | 학술논문

背景目前对角膜内皮移植载体的研究较多,但究竟何种载体更适合作为角膜内皮移植载体并无明确定论. 目的观察壳聚糖的细胞相容性及组织相容性,探讨该材料作为角膜内皮移植载体的可能性.方法 10只新西兰白兔用过量麻醉法处死后摘取新鲜眼球,用组织块培养法培养角膜内皮细胞(CECs),将壳聚糖膜片置于无菌48孔板中,每孔加入200 μl细胞悬液进行孵育,观察细胞的形态和密度.采用扫描和透射电子显微镜观察细胞表面和超微形态结构.用免疫荧光法检测壳聚糖膜片上培养的CECs中纤维连接蛋白(FN)、Ⅰ型胶原(Coll-Ⅰ)、连接黏附分子-1(ZO-1)的表达.健康新西兰白兔10只,左眼经前房植入壳聚糖膜片,右眼仅做前房穿刺作为对照组,术后裂隙灯显微镜下观察术眼眼前节炎症反应及角膜水肿情况.于术后1、4、8周分别测量术眼角膜厚度,术后2周用角膜内皮镜观察兔眼的CECs形态,测定CECs密度,术后1个月、3个月时摘除兔眼眼球,角膜组织行苏木精-伊红染色,观察角膜炎症反应情况.采用配对t检验对实验组和对照组间各测量指标的数据资料进行统计学分析. 结果 CECs在壳聚糖膜片孵育5d后形成完整的单层多角形细胞,形态规则,7d后达到90％融合,细胞40％呈六角形,细胞间连接紧密.扫描电子显微镜下可见兔CECs为近圆形或多边形,以桥粒紧密连接,细胞表面有微绒毛；透射电子显微镜下可见细胞膜表面的突起、伪足和微绒毛,细胞质内可见空泡和其上有核糖核蛋白体的扩张内质网,染色质丰富.免疫荧光检测显示,壳聚糖膜片上培养的CECs中FN、Coll-Ⅰ、ZO-1呈阳性表达.兔活体前房内植入含CECs的壳聚糖膜片后3d,裂隙灯显微镜下可见前房内渗出物和角膜水肿；术后14d左右,前房内渗出完全吸收,角膜恢复透明.术后1、4、8周,实验组与对照组间角膜厚度的差异均无统计学意义(t=1.377,P=0.265；t=1.795,P=0.165；t=0.390,P=0.760)；两组间CECs密度和六角形细胞率的差异均无统计学意义(P=0.365、0.062)；术后3个月时实验兔角膜组织病理学检查发现,壳聚糖膜片周围炎性细胞浸润消失,角膜内皮结构良好.结论壳聚糖载体膜片与体外培养的兔CECs生物相容性好,是CECs移植的潜在良好载体. 揹景目前對角膜內皮移植載體的研究較多,但究竟何種載體更適閤作為角膜內皮移植載體併無明確定論. 目的觀察殼聚糖的細胞相容性及組織相容性,探討該材料作為角膜內皮移植載體的可能性.方法 10隻新西蘭白兔用過量痳醉法處死後摘取新鮮眼毬,用組織塊培養法培養角膜內皮細胞(CECs),將殼聚糖膜片置于無菌48孔闆中,每孔加入200 μl細胞懸液進行孵育,觀察細胞的形態和密度.採用掃描和透射電子顯微鏡觀察細胞錶麵和超微形態結構.用免疫熒光法檢測殼聚糖膜片上培養的CECs中纖維連接蛋白(FN)、Ⅰ型膠原(Coll-Ⅰ)、連接黏附分子-1(ZO-1)的錶達.健康新西蘭白兔10隻,左眼經前房植入殼聚糖膜片,右眼僅做前房穿刺作為對照組,術後裂隙燈顯微鏡下觀察術眼眼前節炎癥反應及角膜水腫情況.于術後1、4、8週分彆測量術眼角膜厚度,術後2週用角膜內皮鏡觀察兔眼的CECs形態,測定CECs密度,術後1箇月、3箇月時摘除兔眼眼毬,角膜組織行囌木精-伊紅染色,觀察角膜炎癥反應情況.採用配對t檢驗對實驗組和對照組間各測量指標的數據資料進行統計學分析. 結果 CECs在殼聚糖膜片孵育5d後形成完整的單層多角形細胞,形態規則,7d後達到90％融閤,細胞40％呈六角形,細胞間連接緊密.掃描電子顯微鏡下可見兔CECs為近圓形或多邊形,以橋粒緊密連接,細胞錶麵有微絨毛；透射電子顯微鏡下可見細胞膜錶麵的突起、偽足和微絨毛,細胞質內可見空泡和其上有覈糖覈蛋白體的擴張內質網,染色質豐富.免疫熒光檢測顯示,殼聚糖膜片上培養的CECs中FN、Coll-Ⅰ、ZO-1呈暘性錶達.兔活體前房內植入含CECs的殼聚糖膜片後3d,裂隙燈顯微鏡下可見前房內滲齣物和角膜水腫；術後14d左右,前房內滲齣完全吸收,角膜恢複透明.術後1、4、8週,實驗組與對照組間角膜厚度的差異均無統計學意義(t=1.377,P=0.265；t=1.795,P=0.165；t=0.390,P=0.760)；兩組間CECs密度和六角形細胞率的差異均無統計學意義(P=0.365、0.062)；術後3箇月時實驗兔角膜組織病理學檢查髮現,殼聚糖膜片週圍炎性細胞浸潤消失,角膜內皮結構良好.結論殼聚糖載體膜片與體外培養的兔CECs生物相容性好,是CECs移植的潛在良好載體.
배경 목전대각막내피이식재체적연구교다,단구경하충재체경괄합작위각막내피이식재체병무명학정론. 목적 관찰각취당적세포상용성급조직상용성,탐토해재료작위각막내피이식재체적가능성.방법 10지신서란백토용과량마취법처사후적취신선안구,용조직괴배양법배양각막내피세포(CECs),장각취당막편치우무균48공판중,매공가입200 μl세포현액진행부육,관찰세포적형태화밀도.채용소묘화투사전자현미경관찰세포표면화초미형태결구.용면역형광법검측각취당막편상배양적CECs중섬유련접단백(FN)、Ⅰ형효원(Coll-Ⅰ)、련접점부분자-1(ZO-1)적표체.건강신서란백토10지,좌안경전방식입각취당막편,우안부주전방천자작위대조조,술후렬극등현미경하관찰술안안전절염증반응급각막수종정황.우술후1、4、8주분별측량술안각막후도,술후2주용각막내피경관찰토안적CECs형태,측정CECs밀도,술후1개월、3개월시적제토안안구,각막조직행소목정-이홍염색,관찰각막염증반응정황.채용배대t검험대실험조화대조조간각측량지표적수거자료진행통계학분석. 결과 CECs재각취당막편부육5d후형성완정적단층다각형세포,형태규칙,7d후체도90％융합,세포40％정륙각형,세포간련접긴밀.소묘전자현미경하가견토CECs위근원형혹다변형,이교립긴밀련접,세포표면유미융모；투사전자현미경하가견세포막표면적돌기、위족화미융모,세포질내가견공포화기상유핵당핵단백체적확장내질망,염색질봉부.면역형광검측현시,각취당막편상배양적CECs중FN、Coll-Ⅰ、ZO-1정양성표체.토활체전방내식입함CECs적각취당막편후3d,렬극등현미경하가견전방내삼출물화각막수종；술후14d좌우,전방내삼출완전흡수,각막회복투명.술후1、4、8주,실험조여대조조간각막후도적차이균무통계학의의(t=1.377,P=0.265；t=1.795,P=0.165；t=0.390,P=0.760)；량조간CECs밀도화륙각형세포솔적차이균무통계학의의(P=0.365、0.062)；술후3개월시실험토각막조직병이학검사발현,각취당막편주위염성세포침윤소실,각막내피결구량호.결론각취당재체막편여체외배양적토CECs생물상용성호,시CECs이식적잠재량호재체.
Background There are a lot of studies about the carrier of corneal endothelial transplantation,but the best carrier has not been defined.Objective This study was to investigate the biocompatibility of chitosan carrier with rabbit corneal endothelium in vivo.Methods Fresh eye-balls were obtained from 10 New Zealand white rabbits.Rabbit corneal endothelial cells (CECs) were isolated and cultured on chitosan carrier in vitro.The morphology and density of rabbits CECs were observed every day,and the expressions of fibronectin (FN),collagen-1 (Coil-I) and Zonula occludens 1 (ZO-1) were detected by immunoinfluorescence.The morphology and ultrastructure of CECs were observed under the scanning and transmission electron microscope.Chitosan carrier with CECs was implanted into the anterior chamber of the left eyes in ten healthy New Zealand white rabbits,and only paracentesis of anterior chamber was performed in the right eyes as controls.The inflammation of ocular anterior segment was examined under the slit lamp microscope,and corneal thickness was measured 1 week,4 and 8 weeks after operation.Corneal endothelium cell density and morphology were examined under the corneal endothelial microscope at postoperative 2 weeks.Corneal samples were collected for the regular histopathological examination to observe the inflammatory reaction at postoperative 1 month and 3 months.Paired t test was used for statistical analyses between the control group (left eyes) and the experimental group (right eyes).The use and care of the animals followed the Statement of ARVO.Results CECs formed an intact monolayer of cells with the uniform shape and size on the chitosan membrane after incubated for 5 days.The cells reached confluence of 90％ 7 days after cultured with the 40％ hexagon cells.Under the scanning electron nicroscope,rabbit CECs showed the round or polygon in the shape with the microvillus on the cell surface.The cells connected closely by desmosome.The processes,pseudopodiums and microvillus on the cellular surface,vacuole in the cytoplasm,expanded endoplasmic reticulum with ribosome and abundant chromatin were exhibited under the transmission electron microscope.The immunofluorescence examination revealed the positive expressions of FN,Coll-Ⅰ and ZO-1 in the CECs on the chitosan carrier.In the in vivo experiment,the exudation in the anterior chamber and corneal edema were seen under the slit lamp microscope 3 days after implantation of chitosan carrier with CECs.However,the inflammation was gone 14 days after operation.The differences of the corneal thickness were no significant between the experimental group and the control group 1 week and 4,8 weeks after operation (t =1.377,P=0.265;t =1.795,P=0.165 ; t =0.390,P =0.760).In addition,no significant differences were found in the CECs density and the hexagon cells rate between the two groups(P =0.365,0.062).The histopathological examination showed that the inflammatory cells around the chitosan membrane were disappeared 3 months after operation and showed a good corneal structure.Conclusions Chitosan carrier has a good biocompatibility with rabbit CECs and anterior chamber,and it may be a potentially good carrier for CECs transplantation.