壳聚糖膜与兔角膜内皮细胞生物相容性的研究

Biocompatibility of chitosan carrier with rabbit corneal endothelium

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.

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.