【作者】 胡柯；

【导师】 赵敏；

【作者基本信息】 重庆医科大学 ， 眼科学， 2005， 硕士

【摘要】 目的:1. 研究羊膜的实用制备工艺,包括清洗方法、抗生素液和贴载物的选择及切割工具的使用,针对目前几种羊膜制备方法的利弊,探索一种较规范实用的制备程序。2. 通过观察比较新鲜羊膜(FAM)、甘油保存羊膜(GPAM)、酒精保存羊膜(APAM)、真空干燥羊膜(VXAM)移植治疗兔角膜碱烧伤的临床效果及羊膜在眼表的转归,探讨不同保存方法羊膜移植的作用以及羊膜在移植中基底膜和生物活性成分的效应和作用机制。 方法:1. 制备工艺的研究:⑴取健康剖宫产产妇胎盘,剥离羊膜,分别用两种方法清洗表面:生理盐水冲洗+注射器局部加压冲洗法和生理盐水冲洗+无菌纱布轻擦洗法去除羊膜表面血迹,随机抽取 2 种羊膜作组织病理及超微结构检查,比较羊膜上皮情况。⑵将洗净羊膜分别置于含 1000 U/ml 庆大霉素+2.5μg /ml 两性霉素 B 和 2000 U/ml 庆大霉素+2.5μg /ml 两性霉素 B 的抗生素液中浸泡 20min,4h 后抽样作细菌、真菌及支原体培养。⑶将羊膜分别平铺于手术粘贴巾、1um 孔径硝酸纤维素滤膜、普通中速滤纸及优质无纺布(灭菌手术帽)上,观察在生理盐水中及甘油保存液中的贴附情况及贴载物物理性状改变。⑷分别用三种自制羊膜环切刀对羊膜进行切割,比较切割效果。制成新鲜羊膜、甘油保存羊膜、酒精保存羊膜及真空干燥羊膜备用。2. 建立新西兰大白兔角膜碱烧伤模型,将 60 只新西兰大白兔随机分为新鲜羊膜移植组、甘油保存羊膜移植组、酒精保存羊膜移植组、真空干燥羊膜重庆医科大学硕士研究生学位论文 3移植组及碱烧伤对照组,每组 12 只,术后在裂隙灯显微镜下观察羊膜植片变化、角膜混浊水肿及新生血管情况并作荧光素染色;各组分别于术后 2w、1m、2m、3m 取标本作组织病理切片(HE)和透射电镜(TEM),观察组织病理和超微结构的改变和羊膜植片的转归。结果:1. 制备工艺:⑴HE 提示用生理盐水冲洗+注射器局部冲洗法羊膜上皮损伤情况明显轻于用冲洗+纱布擦洗后羊膜,前者上皮结构基本完整,后者上皮部份区域脱失。TEM 提示经两种方法处理的羊膜上皮均有程度不同改变。⑵经两种浓度抗生素液处理后的羊膜的细菌、真菌和支原体培养结果均为阴性。⑶硝酸纤维素滤膜对羊膜的贴附性最好,滤膜无形变;手术粘贴巾羊膜贴附性虽好,但易发生卷曲变形,剪切后更加明显;中速滤纸虽也可贴附,但复水后不能起到支撑作用,并发生纤维脱落,附着于羊膜上,不利于手术;优质无纺布复水后不变形,但贴附较差,羊膜易游离需用缝线缝合固定,不能被羊膜环切刀切割。⑷设计的三种羊膜环切刀,均能将羊膜连同贴载物完整切下。以旋转冲压式羊膜环切刀操作最为方便省力,易消毒。2. 移植术后开始观察至 90 天,FAMG、GPAMG 和 VXAMG 效果都显著优于 APAMG,后者与 CG 间无显著差异。羊膜移植后植片 18 天左右开始溶解脱落,至 1 月左右全部溶解,在各种组织切片及电镜标本中均未能见到羊膜组织。在抑制新生血管方面新鲜羊膜移植组显著优于甘油保存移植组及真空干燥羊膜移植组(P﹤0.05)。角膜混浊程度与荧光素染色方面新鲜羊膜、甘油羊膜和真空干燥羊膜移植组间无显著性差异(P﹥0.05)。新鲜羊膜移植组效果最好,甘油保存羊膜移植组及真空干燥羊膜组间无明显差异。 结论:1. 制备工艺:⑴羊膜上皮脆弱,取材及清洗过程中易受损伤,在操作中应动作轻柔,对表面血迹用生理盐水直接冲洗尽量不擦洗以保护上皮,减少损伤。⑵含 1000 U/ml 庆大霉素+2.5μg /ml 两性霉更多还原

【Abstract】 Purpose 1.Search the methods of cleaning, selection of carriersand manufacture of the cutter. Aim at advantages and disadvantages ofseveral kinds of facture amnion ,to raise a normative method of factureamnion. 2.By observing and comparing the effects on curing rabbitcorneal alkali burn on using FAM,PAM, VXAM and VPAM, by observingthe ending of amnion on rabbit’s ocular surface. Discuss the efficiency onthe different ways to keeping the amnion and find the functions ofbasement membrane and organism activity material. Method 1.To research the technics of preparing amnion.⑴Toobtain placenta from health pregnant woman at the time Caesareansection ,peel off the amnion, cleaning the surface of the amnion in twoways: saline and injector irrigating; saline irrigating and use carbasus toscrub the bloodstain on the amnion. Collect amnions in two treating waysto observe HE and TEM, to compare epithelia of amnion.⑵Put thecleaning amnions into 1000 U/ml cidomycin and 2.5μg /ml amphotericin B;2000 U/ml cidomycin and 2.5μg /ml amphotericin B for 20 min individual,4 hours later colture the amnions for bacteria, fungi and mycoplasm.⑶Tilethe amnion on operation paste scarf, 1um colloxylin coat, normal filterpaper and operating cap individual,observe the sticking stat in saline andglycerin preservation solution.⑷cutting amnion to patch by three kinds ofself-made trepans. observing and comparing the differences during wholeoperation. Then made them into FAM, PAM, VXAM and VPAM. 2. 60New Zealand rabbits were used to make corneal alkali burn models anddivided into FAM,PAM, VXAM, VPAM and CG, every group have 12rabbits. After operation, we observe the change of amnion transplant,corneal opacity, CNV and corneal fluorescein staining by slit lampmicroscope. 2 w, 1m,2m and 3m after operation, we did ultrastructuremorphological and histology observation. Results 1. On the technics of preparing amnion ⑴HE show that thedamage swashed by injector on amniotic epithelial are lighter than wipedby gauze. The former amniotic epithelial are close to integrity, the latter aredecoherence in some area. TEM show that there are damages on two kindsof amniotic epithelial.⑵The result of bacteria, fungi and mycoplasmcultivation are all negative. ⑶Colloxylin paper is the best choices onstickiness and physical capability; operation affix paper have goodstickiness . After cutting, it is easy to metamorphose. Filter paper can’tsupport the amnion after immerged in saline. It’s fibre will fall off.Operating cap have good physical capability, but bad stickiness. It need tobe sutured with amnion.⑷All three kinds of trepans can cut amnion withcarrier effectively. The stamping trepan is the most convenient andlaborsaving, antisepsis is dependable. 2. Observe three months,FAMG,GPAMG and VXAMG have better curative effect than APAMG.APAMG and CG have not distinct difference. The amnion graft begin todissolve and desquamation after 18 days, as far as 1 month it disappearedcompletely. Amnion tissue didn’t found under LM and EM. FAMG havebetter anti-CNV than GPAMG and VXAMG(P﹤0.05).FAMG,GPAMGand VXAMG have not distinct difference in corneal opacity andfluorescein staining(P﹥0.05). FAMG have the best curative effect.GPAMG and VXAMG have not distinct difference. Conclusions:1. On the technics of preparing amnion ⑴Because ofthe fragile of amniotic epithelia, it is easy to damage during gaining andcleaning, scrubing the amnions directely will damage the amniotic epithelia,so the action should be light and soft. Use the saline and injector flushingcan cleaning the bloodstain, protecting the epithelia, decreasing thedamages.⑵Lower concentration of 1000 U/ml cidomycin and 2.5μg /mlamphotericin B can assure effects of aseptic.⑶Colloxylin paper is the bestchoice as the carrier for it physical ability and stickiness.⑷The stampingtrepan can cut amnion expediently and normatively. It is easy to produce inlarge number. Simplify the operation.2.On transplantation ⑴ Aftertransplantation, VXAM、GPAM and FAM can accelerate corneal epithelialhealing, lighten inflam更多还原