CN109771370B - 一种眼内手术用前房灌注液及其用途 - Google Patents
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Abstract
本发明公开了一种眼内手术用前房灌注液及其用途,在平衡眼内压的作用下,还含有一定量的L‑丙氨酰‑L‑谷氨酰胺。白内障囊外摘除术、人工晶状体植入、玻璃体切除术等眼内手术均需较长时间的液体灌注,而常用眼科灌注液为平衡盐溶液,缺乏氧以及营养物质,对角膜内皮细胞的影响不容忽视。本发明提供的眼内手术用前房灌注液应用于术中灌注,既对缺血缺氧有保护作用又能提供营养物质,作用于角膜内皮细胞时,可以减少角膜内皮细胞凋亡的数量,减轻术后角膜肿胀,达到更好的手术效果。
Description
技术领域
本发明属于医药领域,具体涉及一种眼内手术用前房灌注液及其用途。
背景技术
随着显微镜技术的发展,内眼手术日趋成熟,数量逐年上升,挽救了很多失明患者。在眼内手术中,为平衡眼内压,眼内灌注是必不可少的。白内障囊外摘除术、人工晶状体植入、玻璃体切除术等均需较长时间的液体灌注。常规眼内灌注液为眼科平衡盐溶液,该溶液缺乏氧且不含营养物质,在较长手术过程中,与角膜内皮细胞长时间接触,容易使得细胞失去活性甚至发生凋亡,而角膜移植术后患者的视力恢复与角膜内皮细胞的活性有很大关系。谷氨酰胺(Glutamine,Gln)是体内含量最丰富的游离氨基酸,约占人体游离氨基酸总量的60%,还是许多生物合成途径的氮源。危重病人术后通过肠外营养补充谷氨酰胺可以增加血清中热休克蛋白含量,减少感染发生率,提高抗氧化能力。然而,Gln在加热灭菌后性质不稳定,并且在水溶液中溶解度低。L-丙氨酰-L-谷氨酰胺(L-alanyl-L-glutamine,Ala-Glu),又名丙谷二肽,属于氨基酸类的化学药剂,在体内可以释放谷氨酰胺,目前主要应用于手术后的创伤的肠外和肠内营养补充、组织器官缺血再灌注损伤的保护,还没有在眼内手术中作为前房灌注液应用的报道。
发明内容
本发明的目的在于克服现有技术的不足之处,提供了一种眼内手术用前房灌注液及其用途,在平衡眼内压的作用下,还含有一定量的L-丙氨酰-L-谷氨酰胺,解决了谷氨酰胺性质不稳定、溶解度低的缺点;该眼内手术用前房灌注液既对缺血缺氧有保护作用又能提供营养物质,应用于术中灌注,以该灌注液作用于角膜内皮细胞时,可以减少角膜内皮细胞凋亡的数量,减轻术后角膜肿胀,达到更好的手术效果。
本发明解决其技术问题所采用的技术方案之一是:
一种眼内手术用前房灌注液,为含有0.217~1.090mg/mL的L-丙氨酰-L-谷氨酰胺的眼科平衡盐溶液。
一实施例中:为含有1.085~1.087mg/mL的L-丙氨酰-L-谷氨酰胺的眼科平衡盐溶液。
一实施例中:所述眼科平衡盐溶液含有5.95~6.00mg/mL的氯化钠,0.29~0.30mg/mL的氯化钾,0.19~0.20mg/mL的氯化钙,3.05~3.15mg/mL的乳酸钠。
本发明解决其技术问题所采用的技术方案之二是:
一种上述的眼内手术用前房灌注液在制备术中用前房灌注制剂中的用途。
本技术方案与背景技术相比,它具有如下优点:
本发明的眼内手术用前房灌注液,在平衡眼内压的作用下,还含有一定量的L-丙氨酰-L-谷氨酰胺,L-丙氨酰-L-谷氨酰胺在加热灭菌后性质稳定,在水中溶解度高,L-丙氨酰-L-谷氨酰胺在体内分解为谷氨酰胺和丙氨酸,是谷胱甘肽的前体,可以增加谷胱甘肽的含量,而谷胱甘肽能有效清除手术过程中产生的有毒害作用的自由基,显著提高细胞抗凋亡能力。本发明的眼内手术用前房灌注液应用于术中前房灌注,可以对角膜内皮细胞起营养作用,并在手术应激的情况下保护细胞,提高细胞抗氧化、抗凋亡能力,减轻术后角膜肿胀,达到更好的手术效果。
附图说明
下面结合附图和实施例对本发明作进一步说明。
图1为TUNEL法检测体外实验组和体外对照组处理1h和2h后角膜内皮细胞凋亡照片,图中A:体外正常实验组;B:对照组术后1h;C:对照组术后2h;D:实验组术后1h;E:实验组术后2h。
图2为免疫荧光法检测体外实验组和体外对照组处理1h和2h后角膜内皮细胞骨架F-actin照片,图中A:体外正常实验组;B:对照组术后1h;C:对照组术后2h;D:实验组术后1h;E:实验组术后2h。
图3为体内实验组和体内对照组处理1h后角膜内皮细胞的活体共聚焦照片,图中A:正常组;B:对照组术后1h;C:实验组术后1h。
具体实施方式
下面通过实施例具体说明本发明的内容:
实施例1眼内手术用前房灌注液的制备:
本实施例的眼内手术用前房灌注液每1mL含有如下成分:
氯化钠 5.95~6.00mg
氯化钾 0.29~0.30mg
氯化钙 0.19~0.20mg
乳酸钠 3.05~3.15mg
L-丙氨酰-L-谷氨酰胺 0.217~1.086mg。
实施例2灌注液处理角膜实验及前房灌注实验
一、实验对象
健康清洁级6~8周龄ICR小鼠30只,体重20~25g,雌雄不限。
二、实验试剂
L-丙氨酰-L-谷氨酰胺、TUNEL法凋亡试剂盒、F-actin。
三、实验仪器
手术显微镜、正置多光子激光共聚焦系统Olympus FV1000MPE-B、扫描电子显微镜Jeo1JSM-6390LV、海德堡共焦激光角膜显微镜。
四、实验方法
1.分离角膜
颈椎脱臼法处死小鼠,摘除眼球。生理盐水冲洗后,将小鼠角膜沿角膜缘剪下,除去虹膜、睫状体等。分离角膜的操作均在手术显微镜下进行,防止对角膜的机械损伤。
2.处理角膜
2.1体外实验组
将角膜迅速移入装有实施例1所得灌注液的容器中,此时计为0时,于1h和2h后取出角膜,4%多聚甲醛4℃固定过夜,通过TUNEL法检测细胞凋亡,免疫荧光染色法F-actin染色查看角膜内皮细胞骨架。
2.2体外对照组
按以上步骤分离角膜,将角膜迅速移入常规灌注液(为眼科平衡盐溶液,即不添加L-丙氨酰-L-谷氨酰胺,其余组分及含量与实施例1的眼内手术用前房灌注液相同)中,此时计为0时,于1h和2h后取出角膜,4%多聚甲醛4℃固定过夜,通过TUNEL法检测细胞凋亡,免疫荧光染色法F-actin染色查看角膜内皮细胞骨架。
2.3体外正常组
按以上步骤分离角膜,不经过任何处理,4%多聚甲醛4℃固定过夜,通过TUNEL法检测细胞凋亡,免疫荧光染色法F-actin染色查看角膜内皮细胞骨架。
3.前房灌注
通过1%戊巴比妥5mL/kg将小鼠麻醉,在室温下,复方托吡卡胺滴眼液散瞳,开睑器开睑,在角膜缘4点方位,用32G针头以15°角距角膜缘2mm处穿刺入前房,置入34G的金属灌注针头并固定,避免接触角膜内皮层及虹膜表面,流量为每分钟10滴。
3.1体内实验组
迅速将实施例1所得灌注液与灌注针头相连,以稳定流速进行前房灌注,此时计为0时,于1h后停止灌注,立即采用活体共聚焦拍摄角膜内皮细胞照片。
3.2体内对照组
迅速将常规灌注液与灌注针头相连,以稳定流速进行前房灌注,此时计为0时,于1h后停止灌注,立即采用活体共聚焦拍摄角膜内皮细胞照片。
3.3体内正常组
不经过任何灌注处理,采用活体共聚焦拍摄角膜内皮细胞照片。
4.TUNEL法检测细胞凋亡
将上述步骤得到的角膜放置在1.5mL离心管内,内皮细胞面朝上,1XPBS冲洗后,用冷丙酮固定3min;1XPBS冲洗后,用1%TD-Buffer室温透膜10min,重复3次;1XPBS冲洗后,与Equilibration Buffer 4℃孵育1h,再与试剂盒提供配制的rTDT溶液4℃避光孵育4h;与2XSSC溶液4℃避光孵育40min终止反应,1XPBS冲洗后DAPI封片。
5.F-actin染色观察细胞形态
将上述步骤得到的角膜放置在1.5mL离心管内,内皮细胞面朝上,1XPBS冲洗后,用冷丙酮固定3min;1XPBS冲洗后用1%TD-Buffer透膜10min,重复3次;1XPBS冲洗后与F-actin染色液室温孵育90min;1XPBS冲洗后DAPI封片。
五、实验结果
1.TUNEL法检测细胞凋亡
如图1,镜下观察凋亡角膜内皮细胞的细胞核被染成绿色,未凋亡内皮细胞的细胞核呈蓝色。1h时,实验组和对照组的凋亡细胞数量稀少,随着观察时间的延长,实验组和对照组在2h的凋亡细胞数量逐渐增多,实验组的凋亡细胞数量显著少于对照组。
2.F-actin染色检测细胞骨架
如图2,镜下观察正常角膜内皮细胞的细胞边界骨架被染成红色,失活内皮细胞的骨架消失。1h时,实验组和对照组角膜内皮细胞骨架良好,边界清晰,随着时间延长,对照组在2h时角膜内皮细胞丧失正常骨架结构数目增多,出现内皮细胞的剥脱,实验组在2h时角膜内皮细胞骨架形态良好。
3.活体共聚焦检查结果
如图3,正常组角膜内皮细胞呈六边形,大小均等,细胞间连接紧密;实验组在前房灌注1h后,角膜内皮细胞多形性增加,细胞边界清晰,细胞间连接紧密;对照组在前房灌注1h后,角膜内皮细胞丧失正常六边形,细胞间间隙扩大,边界不清。
本发明中的眼内灌注液既保持了原有灌注、平衡眼内压的作用,对眼内组织无毒副作用,加入一定浓度的Ala-Glu,又能对手术过程中缺血缺氧起保护作用,还能提供营养物质,可以减少角膜内皮细胞凋亡数量,能够很好的维持角膜内皮细胞形态,从而得到更好的手术效果,是一种良好的新型眼内手术用前房灌注液。
以上所述,仅为本发明较佳实施例而已,故不能依此限定本发明实施的范围,即依本发明专利范围及说明书内容所作的等效变化与修饰,皆应仍属本发明涵盖的范围内。
Claims (3)
1.一种眼内手术用前房灌注液,其特征在于:为含有0.217~1.090 mg/mL的L-丙氨酰-L-谷氨酰胺的眼科平衡盐溶液;所述眼科平衡盐溶液含有5.95~6.00 mg/mL的氯化钠,0.29~0.30 mg/mL的氯化钾,0.19~0.20 mg/mL的氯化钙,3.05~3.15 mg/mL的乳酸钠。
2.根据权利要求1所述的眼内手术用前房灌注液,其特征在于:为含有1.085~1.087mg/mL的L-丙氨酰-L-谷氨酰胺的眼科平衡盐溶液。
3.一种权利要求1或2所述的眼内手术用前房灌注液在制备术中用前房灌注制剂中的用途。
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