CAJ | 학술논문

目的：本实验结合液氮深低温冻存技术与地塞米松预处理方法，研究其对猪角膜免疫原性影响情况及可能机制。 　　方法：选择新鲜猪角膜，以13 mm直径环钻制取角膜标本，依次放入四种冷冻保护液中冷平衡，“简化四步法”程序降温后放入液氮中保存，使用前40℃水浴复温。地塞米松作用组以同法制备植片，前三步冷平衡同前，最后分别放入含0．01，0．03，0．05 mg/mL地塞米松保护液中4℃孵育18h，再程序降温液氮冻存。 BALB/c小鼠50只，随机分为阳性对照组（新鲜组）、程序冻存组、地塞米松程序冻存组Ⅰ、地塞米松程序冻存组Ⅱ和地塞米松程序冻存组Ⅲ，每组10只。各组角膜植入到BALB/c小鼠背部皮下，术后14 d取出，做石蜡包埋， HE染色、CD25和FasL免疫组化染色，光镜观察。 　　结果：术后14 d剖取角膜植片，见植片与周围组织粘连，植片明显肿胀，半透明，色微黄。 HE染色结果：新鲜组角膜植片全层有大量淋巴细胞浸润；程序冻存组角膜浸润细胞较新鲜组减少，多位于内皮和上皮层分布；地塞米松作用各组细胞浸润数最少。免疫组化显示：新鲜组植片CD25＋和FasL＋浸润细胞数最多，与程序冻存组、各地塞米松作用组比较有明显统计学差异。程序冻存组CD25＋和FasL＋浸润细胞数多于各地塞米松作用组，比较有显著性差异。各地塞米松作用组CD25＋和FasL＋浸润细胞数差异比较无统计学意义。 　　结论：地塞米松作用组角膜免疫原性最低。各地塞米松作用组角膜免疫原性在0．01～0．05 mg/mL范围内无浓度依赖效应。
목적：본실험결합액담심저온동존기술여지새미송예처리방법，연구기대저각막면역원성영향정황급가능궤제。 　　방법：선택신선저각막，이13 mm직경배찬제취각막표본，의차방입사충냉동보호액중랭평형，“간화사보법”정서강온후방입액담중보존，사용전40℃수욕복온。지새미송작용조이동법제비식편，전삼보랭평형동전，최후분별방입함0．01，0．03，0．05 mg/mL지새미송보호액중4℃부육18h，재정서강온액담동존。 BALB/c소서50지，수궤분위양성대조조（신선조）、정서동존조、지새미송정서동존조Ⅰ、지새미송정서동존조Ⅱ화지새미송정서동존조Ⅲ，매조10지。각조각막식입도BALB/c소서배부피하，술후14 d취출，주석사포매， HE염색、CD25화FasL면역조화염색，광경관찰。 　　결과：술후14 d부취각막식편，견식편여주위조직점련，식편명현종창，반투명，색미황。 HE염색결과：신선조각막식편전층유대량림파세포침윤；정서동존조각막침윤세포교신선조감소，다위우내피화상피층분포；지새미송작용각조세포침윤수최소。면역조화현시：신선조식편CD25＋화FasL＋침윤세포수최다，여정서동존조、각지새미송작용조비교유명현통계학차이。정서동존조CD25＋화FasL＋침윤세포수다우각지새미송작용조，비교유현저성차이。각지새미송작용조CD25＋화FasL＋침윤세포수차이비교무통계학의의。 　　결론：지새미송작용조각막면역원성최저。각지새미송작용조각막면역원성재0．01～0．05 mg/mL범위내무농도의뢰효응。
AIM:To study the influences of dexamethasone on the immunogenicity of porcine cornea cryopreserved in liquid nitrogen and the involved mechanism. METHODS:Fresh porcine corneas were drilled down by 13mm-diameter trephine.Then the samples were put into four kinds of cryoprotectants in turn for cooling equilibrium with“simplified four-step” method and then stored in liquid nitrogen. All samples were thawed in water at 40℃before being used.The samples treated with dexamethasone were made and the preceding three cooling steps were same. But the samples were additionally incubated in 0.01, 0.03, 0.05mg/mL dexamethasone for 18h at 4℃before the last cooling step. Totally 50 BALB/c mice were randomly divided into 5 groups including 10 mice in each group: the control group;routine cooling group which were treated through four cooling steps; dexamethasone group I, dexamethasone group II and dexamethasone group III which were treated with 0.01, 0.03, 0.05mg/mL dexamethasone successively. After treatment, corneal samples were transplanted into the hypodermis on the back of mice.After 14d, the samples were taken out, imbedded in paraffin, and stained by HE and CD25/FasL immunohistochemistry.All the samples were investigated under light microscope. RESULTS:On the 14th day, all the samples were taken out.We found the samples which adhered to surrouding tissues swelled obviously and showed translucent and a bit yellow.Through HE staining, fresh corneal samples in the control group were infiltrated with large amount of lymphocytes throughout each layer; the amount of infiltrating cells in the routine cooling group was less than the control group in which most cells were distributed in endodermis and epitheliums. In the dexamethasone -treated groups, the amount of infiltrated cells were the least. Immunohistochemical results showed that compared with the the routine cooling group and dexamethasone-treated groups, the amount of CD25 and FasL in the control group was the most, the difference was significant.Compared with dexamethasone-treated groups, the amount of CD25 and FasL positive infiltrating cells in the routine cooling group were more.ALL the differences were significant.But there was no significant difference in the amount of CD25 and FasL positive infiltrating cells among each dexamethasone treated group. CONCLUSION: Corneas in dexamethasone -treated groups showed the lowest immunogenicity. Corneal immunogenicity was irrelevant with the concentration of dexamethasone at the range of 0.01-0.05mg/mL.