摘要
目的建立器官培养保存角膜的方法。方法按照已有配方配制器官培养保存液和运输液。将24只新鲜猪角膜随机分成A、B、C三组,悬浮于上述保存液中,DIN瓶密闭保存于37℃恒温孵箱。分别将A、B、C三组角膜放入保存液中保存4、11、18d,再转到含5%葡聚糖T500的运输液中脱水3d。保存后的每个角膜都从中央平分成两半,其中一半常规测厚度、做台盼蓝-茜素红染色观察内皮细胞形态、计算内皮细胞数量和台盼蓝阳性细胞数;另一半撕取后弹力膜,4%多聚甲醛固定后TUNEL法测定内皮细胞凋亡。采取上述实验方法,分别测定并比较保存前和保存后第7、14、21d时猪角膜的水肿程度、内皮细胞数量及丢失率、台盼蓝阳性率、内皮细胞凋亡情况。结果三组猪角膜均透明。随保存时间的延长而出现内皮细胞形态欠规则、细胞丢失率升高、台盼蓝阳性细胞数增多、TUNEL阳性细胞数量增加。结论实验室建立了器官培养保存猪角膜的方法。按此配方配制的保存液可维持猪角膜活性达3周。
Objective To develop a method of corneal organ culture preservation. Methods Prepare the preservation medium and transport medium according to the formula.24 fresh Porcine corneas were divided into three groups by random,and they were correspondingly floated in preservation medium in DIN bottles which were put in 37℃ calorstat. Every 8 corneas were preserved in preservation medium for 4,11 ,18 days respectively. They were shifted to transport medium A with 5 % daxtran 500 and kept for 3 days before use. After preservation, every cornea was cut into two pieces,which a half piece was measured thickness, stained with trypan bluealizarin bordeaux, observed the appearance and shape of CECs, counted CECs and trypan blue positive CECs,and the other piece was ripped away the posterior elastic membrane to detect the apoptosis CECs after fixed by 4% paraformaldehyde by TUNEL technique.By the way above-mentioned, porcine corneas＂ thickness ,the lost rate of CEC, trypan blue positive CECs and condition of apoptosis were determined and compared respectively before preservation and after 7,14,21 days＂ preservation. Results The procine corneas which were preserved for 3 weeks kept transparent. The longer the corneas were preserved, the shape of endothelial cell not more regular, and the lost rate of endothelial cell was higher,and the number of typan-blue positive ECs was increased, and the number of TUNEL positive cell was raised. Conclusion The method we built for corneal preservation was feasible. The preservation medium can keep the activity of porcine cornea for 3 weeks.
出处
《安徽医学》
2009年第8期846-849,共4页
Anhui Medical Journal
基金
安徽省卫生厅临床医学重点学科技术进步专项课题(2004z027)
关键词
角膜
器官培养
猪
Cornea
Organ culture
Porcine
