背景 应用羊膜移植术治疗角膜溃疡的方法在临床上已广泛开展,但目前国内有关将羊膜作为药物载体治疗感染性眼表疾病的研究较少见. 目的 探讨载药羊膜在治疗眼表感染性疾病中的缓释性能并评价其疗效.方法 利用体外抑菌实验比较羊膜浸泡不同质量浓度(5、20、30 g/L)的左氧氟沙星不同时间(5、15、30、60 min)后平均抑菌环直径的改变.利用角膜基质层接种法将浊度为0.7麦氏单位的金黄色葡萄球菌菌悬液0.1ml自角膜近中央处注入角膜基质层,形成直径4.0～6.0 mm的混浊区,建立20只家兔单眼感染性角膜溃疡模型,采用随机数字表法将动物模型随机分为单纯羊膜移植联合点药组和载药羊膜移植组,每组各10只,两组家兔造模后24 h分别行单纯羊膜移植术或载药羊膜移植术.单纯羊膜移植联合点药组术后用质量分数0.5％左氧氟沙星滴眼液点眼,每日3次,分别于点眼后0.5、1.0、2.0、3.5、5.5h抽取房水0.1ml,载药羊膜移植组于术后1、3、7、10、14、21d抽取房水0.1ml,用高效液相色谱分析法检测各时间点兔眼房水中左氧氟沙星的质量浓度.各组大鼠在裂隙灯显微镜下观察角膜病变情况及角膜溃疡的面积变化,按照改进的McNeill角膜病变程度分级法对角膜病变进行评分,并于术后4周行兔眼角膜组织病理学检查.结果 左氧氟沙星体外抑菌实验表明,平均抑菌环直径随药物质量浓度的增加而增大,相同质量浓度左氧氟沙星浸泡15 min的羊膜与浸泡5 min相比抑菌环直径增大,差异均有统计学意义(P＜0.01),但与浸泡30 min、60 min的羊膜相比差异均无统计学意义(P＞0.05).单纯羊膜移植联合点药组术后房水中左氧氟沙星质量浓度随时间的延长而逐渐降低,0.5h为(0.873 ±0.264) mg/L,5.5h为(0.106±0.027) mg/L;载药羊膜移植组术后1、3、7d房水中左氧氟沙星质量浓度仍高于单纯羊膜移植联合点药组点药后0.5h,差异均有统计学意义(均P=0.00).载药羊膜移植组家兔用药后1、2、4周角膜病变评分分别为1.7±0.6、1.3±0.5和0.2±0.4,明显低于单纯羊膜移植联合点药组的2.2±0.8、2.0±0.6和1.5±0.8,差异均有统计学意义(F分组=9.49,P=0.01；F时间=22.96,P=0.01).术后1周载药羊膜移植组角膜溃疡面积明显小于单纯羊膜移植联合点药组,差异有统计学意义[(1.6±0.6)mm2 vs.(3.2±0.8)mm2,t=3.98,P=0.00].组织病理学检查显示,单纯羊膜移植联合点药组术后4周角膜各层排列不规则,出现1～2层新生鳞状上皮,基质层结构紊乱,可见较多炎性细胞浸润及残留血管腔；载药羊膜移植组术后4周角膜有4～5层新生鳞状上皮,排列整齐,仅见少量炎性细胞浸润及残留血管腔.结论 左氧氟沙星载药羊膜移植至角膜后具有一定的药物缓释性能,可以提高房水中的有效药物质量浓度,对金黄色葡萄球菌感染性角膜溃疡的疗效优于点眼的用药方式.
揹景 應用羊膜移植術治療角膜潰瘍的方法在臨床上已廣汎開展,但目前國內有關將羊膜作為藥物載體治療感染性眼錶疾病的研究較少見. 目的 探討載藥羊膜在治療眼錶感染性疾病中的緩釋性能併評價其療效.方法 利用體外抑菌實驗比較羊膜浸泡不同質量濃度(5、20、30 g/L)的左氧氟沙星不同時間(5、15、30、60 min)後平均抑菌環直徑的改變.利用角膜基質層接種法將濁度為0.7麥氏單位的金黃色葡萄毬菌菌懸液0.1ml自角膜近中央處註入角膜基質層,形成直徑4.0～6.0 mm的混濁區,建立20隻傢兔單眼感染性角膜潰瘍模型,採用隨機數字錶法將動物模型隨機分為單純羊膜移植聯閤點藥組和載藥羊膜移植組,每組各10隻,兩組傢兔造模後24 h分彆行單純羊膜移植術或載藥羊膜移植術.單純羊膜移植聯閤點藥組術後用質量分數0.5％左氧氟沙星滴眼液點眼,每日3次,分彆于點眼後0.5、1.0、2.0、3.5、5.5h抽取房水0.1ml,載藥羊膜移植組于術後1、3、7、10、14、21d抽取房水0.1ml,用高效液相色譜分析法檢測各時間點兔眼房水中左氧氟沙星的質量濃度.各組大鼠在裂隙燈顯微鏡下觀察角膜病變情況及角膜潰瘍的麵積變化,按照改進的McNeill角膜病變程度分級法對角膜病變進行評分,併于術後4週行兔眼角膜組織病理學檢查.結果 左氧氟沙星體外抑菌實驗錶明,平均抑菌環直徑隨藥物質量濃度的增加而增大,相同質量濃度左氧氟沙星浸泡15 min的羊膜與浸泡5 min相比抑菌環直徑增大,差異均有統計學意義(P＜0.01),但與浸泡30 min、60 min的羊膜相比差異均無統計學意義(P＞0.05).單純羊膜移植聯閤點藥組術後房水中左氧氟沙星質量濃度隨時間的延長而逐漸降低,0.5h為(0.873 ±0.264) mg/L,5.5h為(0.106±0.027) mg/L;載藥羊膜移植組術後1、3、7d房水中左氧氟沙星質量濃度仍高于單純羊膜移植聯閤點藥組點藥後0.5h,差異均有統計學意義(均P=0.00).載藥羊膜移植組傢兔用藥後1、2、4週角膜病變評分分彆為1.7±0.6、1.3±0.5和0.2±0.4,明顯低于單純羊膜移植聯閤點藥組的2.2±0.8、2.0±0.6和1.5±0.8,差異均有統計學意義(F分組=9.49,P=0.01；F時間=22.96,P=0.01).術後1週載藥羊膜移植組角膜潰瘍麵積明顯小于單純羊膜移植聯閤點藥組,差異有統計學意義[(1.6±0.6)mm2 vs.(3.2±0.8)mm2,t=3.98,P=0.00].組織病理學檢查顯示,單純羊膜移植聯閤點藥組術後4週角膜各層排列不規則,齣現1～2層新生鱗狀上皮,基質層結構紊亂,可見較多炎性細胞浸潤及殘留血管腔；載藥羊膜移植組術後4週角膜有4～5層新生鱗狀上皮,排列整齊,僅見少量炎性細胞浸潤及殘留血管腔.結論 左氧氟沙星載藥羊膜移植至角膜後具有一定的藥物緩釋性能,可以提高房水中的有效藥物質量濃度,對金黃色葡萄毬菌感染性角膜潰瘍的療效優于點眼的用藥方式.
배경 응용양막이식술치료각막궤양적방법재림상상이엄범개전,단목전국내유관장양막작위약물재체치료감염성안표질병적연구교소견. 목적 탐토재약양막재치료안표감염성질병중적완석성능병평개기료효.방법 이용체외억균실험비교양막침포불동질량농도(5、20、30 g/L)적좌양불사성불동시간(5、15、30、60 min)후평균억균배직경적개변.이용각막기질층접충법장탁도위0.7맥씨단위적금황색포도구균균현액0.1ml자각막근중앙처주입각막기질층,형성직경4.0～6.0 mm적혼탁구,건립20지가토단안감염성각막궤양모형,채용수궤수자표법장동물모형수궤분위단순양막이식연합점약조화재약양막이식조,매조각10지,량조가토조모후24 h분별행단순양막이식술혹재약양막이식술.단순양막이식연합점약조술후용질량분수0.5％좌양불사성적안액점안,매일3차,분별우점안후0.5、1.0、2.0、3.5、5.5h추취방수0.1ml,재약양막이식조우술후1、3、7、10、14、21d추취방수0.1ml,용고효액상색보분석법검측각시간점토안방수중좌양불사성적질량농도.각조대서재렬극등현미경하관찰각막병변정황급각막궤양적면적변화,안조개진적McNeill각막병변정도분급법대각막병변진행평분,병우술후4주행토안각막조직병이학검사.결과 좌양불사성체외억균실험표명,평균억균배직경수약물질량농도적증가이증대,상동질량농도좌양불사성침포15 min적양막여침포5 min상비억균배직경증대,차이균유통계학의의(P＜0.01),단여침포30 min、60 min적양막상비차이균무통계학의의(P＞0.05).단순양막이식연합점약조술후방수중좌양불사성질량농도수시간적연장이축점강저,0.5h위(0.873 ±0.264) mg/L,5.5h위(0.106±0.027) mg/L;재약양막이식조술후1、3、7d방수중좌양불사성질량농도잉고우단순양막이식연합점약조점약후0.5h,차이균유통계학의의(균P=0.00).재약양막이식조가토용약후1、2、4주각막병변평분분별위1.7±0.6、1.3±0.5화0.2±0.4,명현저우단순양막이식연합점약조적2.2±0.8、2.0±0.6화1.5±0.8,차이균유통계학의의(F분조=9.49,P=0.01；F시간=22.96,P=0.01).술후1주재약양막이식조각막궤양면적명현소우단순양막이식연합점약조,차이유통계학의의[(1.6±0.6)mm2 vs.(3.2±0.8)mm2,t=3.98,P=0.00].조직병이학검사현시,단순양막이식연합점약조술후4주각막각층배렬불규칙,출현1～2층신생린상상피,기질층결구문란,가견교다염성세포침윤급잔류혈관강；재약양막이식조술후4주각막유4～5층신생린상상피,배렬정제,부견소량염성세포침윤급잔류혈관강.결론 좌양불사성재약양막이식지각막후구유일정적약물완석성능,가이제고방수중적유효약물질량농도,대금황색포도구균감염성각막궤양적료효우우점안적용약방식.
Background Treatment of corneal ulceration by transplanting drug-loaded amniotic membrane has been used widespreadly abroad,however,seldom study is found in China up to now.Objective This study was to explore the sustained release property and the efficacy of the drug-loaded amniotic membrane.Methods The bacteriostatic area of amniotic membrane fragments immersed with different concentrations (5,20,30 g/L) of levofloxacin for different time points (5,15,30,60 minutes) was evaluated by in vitro test.Bacterial corneal ulceration models were established in 20 rabbits by injected 0.7 MCF staphylococcus aureus suspension (0.1 ml) into the central corneal stroma to form the cloudy area of 4.0-6.0 mm.Then the rabbits were randomized into two groups.Regular amniotic membrane transplantation was performed laterally and 0.5％ levofloxacin drops was topically administered after operation in the amnion+levofloxacin drops group,and drug-loading amnion transplantation was used in the drug-loading amnion group.Aqueous humor of 0.1 ml was collected in 30 minutes,1 hour,2,3.5,5.5 hours after levofloxacin was administered and 1 day,3,7,10,14,21 days after operation for the detect of levofloxacin level with high-performance liquid chromatography.The corneal symptom was scored based on McNeill's criteria in 1 week,2 weeks and 4 weeks and the ulceration area was assessed under the slit lamp in the first week.The pathological examination was carried out in the fourth week after surgery.Results The mean bacteriostatic area was bigger with the increase of levofloxacin concentration,and bacteriostatic area in amnion immersed for 15 minutes was bigger than that of 5 minutes (P＜0.01).The levofloxacin concentration of aqueous humor after transplantation was decreased by extending the time,and that in 30 minutes and 5.5 hours after operation was (0.873±0.264) mg/L and (0.106±0.027) mg/L,respectively,in the amnion+levofloxacin drops group,and that in day 1,3,7 after surgery in the drug-loading amnion group was higher than at 30 minutes in the amnion+levofloxacin drops group,showing all significant differences (all P =0.00).In the first,second and fourth week after operation,the corneal symptom score was 1.7±0.6,1.3±0.5,0.2±0.4 in the drug-loading amnion group and 2.2±0.8,2.0±0.6,1.5±0.8 in the amnion+ levofloxacin drops group,with the significant differences among the different groups and time points (Fgroup =9.49,P =0.01 ;Ftime =22.96,P=0.01).The corneal ulceration area was (1.6±0.6) mm2 in the drug-loading amnion group and (3.2±0.8) mm2 in the amnion+levofloxacin drops group 1 week after operation,showing a significant difference between them (t =3.98,P =0.00).Histopathological revealed that the various layers of cornea tissue appeared irregular arrangement in the amnion + levofloxacin drops group 4 weeks after operation with 1-2 layers of new squamous epithelium.Disorder hypothallus structure,more inflammatory cells and residual vascular cavity were visible.However,new squamous epithelium of 4-5 layers was seen in the drug-loading amnion group,and inflammatory cells and residual vascular cavity were less than the amnion+levofloxacin drops group 4 weeks after operation.Conclusions Levofloxacin-loaded amniotic membrane can sustained release levofloxacin and maintain an effective drug concentration in aqueous humor,which improves the treating efficacy for staphylococcus aureus corneal ulceration.
