CN116617367A - 中脑星形胶质细胞来源神经营养因子在制备治疗单纯疱疹病毒性角膜炎药物中的应用 - Google Patents
中脑星形胶质细胞来源神经营养因子在制备治疗单纯疱疹病毒性角膜炎药物中的应用 Download PDFInfo
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Abstract
本发明涉及生物医药技术领域，特别是涉及中脑星形胶质细胞来源神经营养因子(Manf)在制备治疗单纯疱疹病毒性角膜炎药物中的应用。本发明所述的Manf具有抗Ⅰ型单纯疱症病毒(HSV‑1)的作用，可以抑制HSV‑1感染后造成的角膜血管增生、角膜炎症反应以及角膜神经消退，可用于制备治疗单纯疱疹病毒性角膜炎药物。
Description
技术领域
本发明涉及生物医药技术领域，特别是涉及中脑星形胶质细胞来源神经营养因子在制备治疗单纯疱疹病毒性角膜炎药物中的应用。
背景技术
Ⅰ型单纯疱疹病毒(HSV-1)是双链线性DNA病毒，基因组长度在125～240kb之间。HSV-1感染力强，人群普遍易感，主要侵犯面、脑及腰以上部位。感染人体后可潜伏于三叉神经胞体，并可终生携带，是世界上流行率最高的病毒之一。HSV-1能够引起口腔粘膜炎症、唇疱疹、脑炎等疾病。感染HSV-1的患者也常伴有全身症状，以簇集性小水疱为表现形式。原发型单纯疱疹皮肤黏膜损害常需2～3周愈合，而复发型单纯疱疹的皮损大多于1周内即可消失。
HSV-1常侵犯皮肤、黏膜、脑和感觉神经等，引起多种眼病如睑缘单纯疱疹炎、急性滤泡性结膜炎、树枝状角膜炎和基质角膜炎等。其中单纯疱疹病毒性角膜炎会多次复发恶化，严重者可导致失明。单纯疱疹病毒性角膜炎原发感染仅有1％的患者表现出皮肤水疱症状，但是复发性感染可造成基质角膜炎和角膜溃疡。严重者可发生角膜穿孔，预后极差。目前临床上治疗单纯疱疹病毒性角膜炎的方法主要以抗病毒药物为主，如阿昔洛韦和更昔洛韦。但此类抗病毒药物需要长时间给药，由此导致耐药性的产生。又因为药物穿透率较差，对深层的病毒性角膜炎疗效欠佳。此外，临床上还会使用糖皮质激素等皮质类固醇类激素来治疗单纯疱疹病毒性角膜炎，具有抗炎和抑制免疫反应的作用，但不适用于上皮型和角膜浅层炎症以及出现溃疡的炎症。皮质类激素药物的作用较局限，滥用激素还可加重角膜穿孔。因此单纯疱疹病毒性角膜炎的治疗仍是一个至关重要的问题，迫切需要研发治疗单纯疱疹病毒性角膜炎的药物。
发明内容
为了解决上述问题，本发明提供了中脑星形胶质细胞来源神经营养因子(Manf)在制备治疗单纯疱疹病毒性角膜炎药物中的应用。本发明所述的Manf具有抗HSV-1的作用，可以抑制HSV-1感染后造成的角膜血管增生、角膜炎症反应以及角膜神经消退所带来的泪液分泌降低和角膜敏感度降低，可用于制备治疗单纯疱疹病毒性角膜炎药物。
为了实现上述目的，本发明提供如下技术方案：
本发明提供了中脑星形胶质细胞来源神经营养因子在制备单纯疱疹病毒抑制剂中的应用。
优选的，所述单纯疱疹病毒抑制剂中中脑星形胶质细胞来源神经营养因子的浓度为1ng/ml～100μg/ml。
优选的，所述单纯疱疹病毒包括Ⅰ型单纯疱疹病毒。
本发明提供了中脑星形胶质细胞来源神经营养因子在制备治疗单纯疱疹病毒性角膜炎药物中的应用。
优选的，所述治疗单纯疱疹病毒性角膜炎的症状包括抑制角膜血管增生、抑制角膜炎症反应、抑制角膜神经消退、恢复角膜敏感度和促进泪液分泌中的一种或多种。
优选的，所述治疗单纯疱疹病毒性角膜炎药物中中脑星形胶质细胞来源神经营养因子的浓度为1ng/ml～100μg/ml。
有益效果：
本发明提供了中脑星形胶质细胞来源神经营养因子在制备单纯疱疹病毒抑制剂中的应用。本发明通过实验证明了Manf具有抗HSV-1的作用，包括：Manf可以显著降低HSV-1感染小鼠角膜炎症因子的浸润，维持角膜上皮的完整性；Manf可直接抑制HSV-1感染人角膜细胞，抑制HSV-1病毒的复制，降低人角膜细胞的感染率；Manf可以显著恢复HSV-1感染所导致的小鼠角膜神经退行和新生血管长入，可用于制备单纯疱疹病毒抑制剂和/或制备治疗单纯疱疹病毒性角膜炎药物。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例中所需要使用的附图作简单地介绍。
图1为正常小鼠和感染HSV-1小鼠的Manf表达结果；
图2为不同处理组小鼠在不同时间的裂隙灯拍照结果(A)、感染角膜临床得分(B)、显微镜切片结果(C)、小鼠泪液分泌量(D)和小鼠角膜敏感度(E)；
图3为不同处理组人角膜上皮细胞的荧光显微镜成像显色结果(A)和相对荧光强度(B)；***为TFT组或MANF组相对于CON组具有显著性差异；
图4为实施例4中共聚焦显微镜拍照结果；
其中*为p＜0.05，**为p＜0.01，***为p＜0.001。
具体实施方式
本发明提供了中脑星形胶质细胞来源神经营养因子在制备单纯疱疹病毒抑制剂中的应用。
在本发明中，所述单纯疱疹病毒优选包括Ⅰ型单纯疱疹病毒。
在本发明中，所述单纯疱疹病毒抑制剂中中脑星形胶质细胞来源神经营养因子的浓度优选为1ng/ml～100μg/ml，进一步优选为10～20μg/ml，更优选为20μg/ml。
本发明所述Manf不仅具有保护细胞和促进神经修复的作用，还可调节炎症相关通路来抑制炎症反应；本发明通过实验证明了Manf具有抗HSV-1的作用，包括：Manf可以显著降低感染小鼠角膜炎症因子的浸润，维持角膜上皮的完整性；Manf可直接抑制HSV-1感染人角膜细胞，降低人角膜细胞的感染率；Manf可以显著恢复HSV-1感染所导致的小鼠角膜神经退行和血管新生，可用于制备单纯疱疹病毒抑制剂。
本发明还提供了中脑星形胶质细胞来源神经营养因子在制备治疗单纯疱疹病毒性角膜炎药物中的应用。
在本发明中，所述治疗单纯疱疹病毒性角膜炎优选包括抑制角膜血管增生、抑制角膜炎症反应、抑制角膜神经消退、恢复角膜敏感度和促进泪液分泌中的一种或多种。
在本发明中，所述治疗单纯疱疹病毒性角膜炎药物中中脑星形胶质细胞来源神经营养因子的浓度优选为1ng/ml～100μg/ml，进一步优选为10～20μg/ml，更优选为20μg/ml。
为了进一步说明本发明，下面结合附图和实施例对本发明提供的中脑星形胶质细胞来源神经营养因子在制备治疗单纯疱疹病毒性角膜炎药物中的应用进行详细地描述，但不能将它们理解为对本发明保护范围的限定。
实施例1
感染HSV-1后小鼠角膜中Manf的改变
1.将12只小鼠的角膜基质中分别注射1μl HSV-1(病毒滴度为2×108PFU/mL)后，分别在第一天、第四天、第六天和第九天颈椎脱臼法处死小鼠，取下小鼠角膜；同时设置注射1μl生理盐水的小鼠做对照(CON)。
使用TransZol Up Plus RNA试剂盒(全式金，中国北京)提取小鼠角膜中的RNA。使用HiScript II 1st Strand cDNA合成试剂盒(雅酶，中国上海)将提取的RNA合成cDNA。使用ChamQ Universal SYBR qPCR MasterMix(中国南京雅酶生物技术有限公司)和RotorGene Q Real time PCR cycler(德国)进行实时PCR。实时PCR的条件为：95℃30秒；95℃10秒，60℃30秒，共40个循环。Manf基因引物由Invitrogen Biotechnology Co.，Ltd.(中国上海)合成，具体引物序列为：m-Manf-F：5’-CTGCGGCCAGGAGACTGT-3’，SEQ ID NO.1和m-Manf-R：5’CAACCGATTCTCTTTGCCTCTT-3’，SEQ ID NO.2。通过对小鼠β-肌动蛋白表达进行归一化，计算每个样品中的mRNA水平。2-ΔΔct法分析相对mRNA表达水平。结果见图1中的A。
结果显示感染HSV-1的小鼠角膜中，Manf的mRNA表达是显著上调的。
2.将9只小鼠的角膜基质中分别注射1μl HSV-1(病毒滴度为2×108PFU/mL)后，在第七天颈椎脱臼法处死小鼠，取下小鼠角膜。用RIPA裂解液提取小鼠角膜组织中的总蛋白(记为HSK)；同时设置未注射HSV-1的小鼠做对照(CON)。在10％SDS-PAGE凝胶上运行30μg蛋白质，并将其转移至聚偏氟乙烯膜(Millipore，Billerica，MA)。在室温下用5％BSA封闭PVDF膜1小时。用TBST洗涤三次后，加入一抗Manf(稀释比例为1:2000，Abcam，ab126321)；第二天，加入二抗(稀释比例为1:3000；AbsinBioscience，Inc.，Shanghai，China)在室温下孵育2小时。使用Bio-Rad分子成像仪ChemiDoc XRS系统(Bio-Rad Laboratories，Inc.，Hercules，CA，USA)观察条带。Β-actin用作对照。结果见图1中的B。
蛋白印迹定量分析：将蛋白印迹实验重复三次后，比较分析实验组(记为MANF)和对照组(记为CON)的统计学差异，结果见图1中的C。
结果显示感染HSV-1的小鼠角膜中，Manf蛋白的表达显著升高。
实施例2
Manf对小鼠单纯疱疹病毒性角膜炎的影响
1.实验组(记为HSK+MANF)：将13只小鼠的角膜基质中分别注射1μl HSV-1(病毒滴度为2×108PFU/mL)，在小鼠角膜感染HSV-1当时、感染HSV-1后的第一天(记为Day1)、感染HSV-1后的第三天(记为Day3)、感染HSV-1后的第五天(记为Day5)和感染HSV-1后的第七天(记为Day7)，分别在小鼠结膜下注射5μl 20μg/ml Manf蛋白(购自R&D systems，货号3748-MN)；
对照组(记为HSK)：与实验组相似，区别在于，感染HSV-1但未注射Manf蛋白。
在Day1、Day3、Day5和Day7，分别对小鼠角膜进行裂隙灯拍照，并评估感染角膜的临床得分。具体的评分规则如下：
临床得分为综合考虑角膜混浊度和新生血管的评分
具体评分如下(1)：0、正常角膜；1、轻度角膜混浊；2、中等不透明度，虹膜可见；3、严重混浊，虹膜不可见；4、严重混浊伴角膜溃疡；5、角膜穿孔。在角膜混浊的基础下，裂隙灯下血管明显充盈加一分，角膜缘到瞳孔缘内出现新生血管加两分，角膜中心出现新生血管加三分。
结果见图2中的A、B和表1。
表1不同处理组小鼠在不同时间的感染角膜临床得分
注：HSV为对照组，总计22只角膜纳入评分；HSV+MANF为注射Manf蛋白的实验组，总计26只角膜纳入评分；平均分为各组评分的平均值。
结果显示Manf蛋白可显著降低小鼠单纯疱疹病毒性角膜炎的症状。
2.采用步骤1相同的方法设置实验组和对照组，同时设置未感染HSV-1的小鼠做空白对照(记为CON)。在Day7注射Manf蛋白后，将三组小鼠眼球取下，脱水包埋入蜡块。用石蜡切片机将眼球切成4μm厚度后，烤片30min(至蜡融化)。玻片放入二甲苯中脱蜡，酒精中脱水。将玻片放入盛有柠檬酸钠缓冲液的玻片盒中，高压加热10min进行抗原修复。10％驴血清封闭1小时。加入一抗Manf(1:100，Abcam，ab126321)，4℃过夜。次日，加入HRP标记的二抗(1：200，Absin Bioscience)。后用DAB混合液显色。苏木素染3min，然后流水冲洗10分钟返蓝，树脂封片。显微镜下观察染色结果，结果见图2中的C。
结果显示，Manf可以显著降低感染小鼠角膜炎症因子的浸润，维持角膜上皮的完整性。
3.采用实施例2中步骤1相同的方法设置实验组和对照组，同时设置未感染HSV-1的小鼠做空白对照(记为CON)。在Day7注射Manf蛋白后，将三组小鼠取出，测量小鼠泪液分泌量和角膜敏感度。
其中小鼠泪液分泌量采用酚红棉线(天津晶明新科技公司)测量，测量方法如下：暴露小鼠下眼睑，将酚红棉线迅速插入小鼠眼睑20秒；随后将酚红棉线取出，依照卡尺读出酚红棉线变色长度，结果见图2中的D。
小鼠角膜敏感度获取采用Cochet氏角膜知觉测量计测量，测量方法如下：首先将小鼠眼球完全暴露；将角膜知觉测量计调至合适长度，随后对小鼠角膜进行点触；观察小鼠是否眨眼或者眯眼，连续点触5次，若其中三次及以上出现眨眼或眯眼反应，则认为此长度对小鼠角膜敏感；直至测量出不敏感反应时，记录此时的角膜知觉测量计长度，结果见图2中的E。
结果显示，Manf可以显著增加感染小鼠角膜的泪液分泌和角膜敏感度，来降低感染小鼠干眼的严重程度。
实施例3
Manf对感染HSV-1的人角膜上皮细胞的影响
设置3个处理组：CON、TFT和MANF，进行如下处理：
CON：对人角膜上皮细胞系进行培养，培养液选用含10％胎牛血清(Gibco)和青霉素-链霉素双抗100U/ml(Gibco)的DMEM/F12(Gibco)；在细胞密度为90％时，加入MOI为0.1和病毒滴度为2×106TU/mL的HSV-1病毒，感染人角膜上皮细胞；
TFT(三氟胸苷)：对人角膜上皮细胞系进行培养，培养液选用含10％胎牛血清(Gibco)和青霉素-链霉素双抗100U/ml(Gibco)的DMEM/F12(Gibco)；在细胞密度为90％时，加入MOI为0.1和病毒滴度为2×106TU/mL的HSV-1病毒和终浓度为50μmol/L的TFT药物(购自Selleck，产品货号为S1778)，与人角膜上皮细胞共培养；
MANF：对人角膜上皮细胞系进行培养，培养液选用含10％胎牛血清(Gibco)和青霉素-链霉素双抗100U/ml(Gibco)的DMEM/F12(Gibco)；在细胞密度为90％时，加入MOI为0.1和病毒滴度为2×106TU/mL的HSV-1病毒和终浓度为10μg/ml的Manf蛋白，与人角膜上皮细胞共培养。
在培养三天后，用4％多聚甲醛固定细胞10min，随后透化和封闭细胞。用GFP-HSV-1直标抗体(Abcam,ab20437)对感染细胞进行染色，随后荧光显微镜成像显色并测定相对荧光强度。结果见图3。
结果显示，Manf可直接抑制HSV-1感染人角膜上皮细胞。
实施例4
采用实施例2中步骤1相同的方法设置实验组和对照组，同时设置未感染HSV-1的小鼠做空白对照(记为CON)。在Day7注射Manf蛋白后，将三组小鼠处死并取下小鼠角膜。剪下的角膜置96孔板中，4℃通透24小时；通透液为含有0.3％(V/V)TX-100和3％(V/V)BSA的PBS缓冲液。96孔板中加入直标抗体Tubulin(Abcam，ab190575)，4℃过夜，静置即可。抗体都配在封闭液中。48孔板中用PBST洗一抗，摇床洗6次，每次10分钟；所述PBST为含有0.3％(V/V)TX-100和0.05％TW-20(V/V)的PBS缓冲液。用含70％(V/V)甘油的PBS封片，共聚焦显微镜拍照。结果见图4。
结果显示，Manf可以显著恢复HSV-1感染所导致的小鼠角膜神经退行。
综上所述，本发明所述的Manf具有抗HSV-1的作用，可以抑制HSV-1感染后的造成的角膜血管增生、角膜炎症反应以及角膜神经消退所带来的泪液分泌降低和角膜敏感度降低，可用于制备治疗单纯疱疹病毒性角膜炎药物。
尽管上述实施例对本发明做出了详尽的描述，但它仅仅是本发明一部分实施例，而不是全部实施例，人们还可以根据本实施例在不经创造性前提下获得其他实施例，这些实施例都属于本发明保护范围。
Claims (6)
1.中脑星形胶质细胞来源神经营养因子在制备单纯疱疹病毒抑制剂中的应用。
2.根据权利要求1所述的应用，其特征在于，所述单纯疱疹病毒抑制剂中中脑星形胶质细胞来源神经营养因子的浓度为1ng/ml～100μg/ml。
3.根据权利要求1或2所述的应用，其特征在于，所述单纯疱疹病毒为Ⅰ型单纯疱疹病毒。
4.中脑星形胶质细胞来源神经营养因子在制备治疗单纯疱疹病毒性角膜炎药物中的应用。
5.根据权利要求4所述的应用，其特征在于，所述治疗单纯疱疹病毒性角膜炎的症状包括抑制角膜血管增生、抑制角膜炎症反应、抑制角膜神经消退、恢复角膜敏感度和促进泪液分泌中的一种或多种。
6.根据权利要求4或5所述的应用，其特征在于，所述治疗单纯疱疹病毒性角膜炎药物中中脑星形胶质细胞来源神经营养因子的浓度为1ng/ml～100μg/ml。
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| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| CB03 | Change of inventor or designer information | ||
| CB03 | Change of inventor or designer information |
Inventor after: Yang Lingling Inventor after: Zou Zongzheng Inventor after: Wang Xiaochuan Inventor after: Chen Rong Inventor after: Feng Jing Inventor after: Wang Huifeng Inventor before: Yang Lingling Inventor before: Zou Zongzheng Inventor before: Chen Rong Inventor before: Feng Jing Inventor before: Wang Huifeng |