CN114869873A - 谷氨酰胺在制备治疗干眼症的药物中的应用 - Google Patents
谷氨酰胺在制备治疗干眼症的药物中的应用 Download PDFInfo
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Abstract
本发明公开了谷氨酰胺在制备治疗干眼症的药物中的应用。本发明首次将L‑谷氨酰胺和D‑谷氨酰胺用于改善人角膜上皮细胞干眼模型，能够有效地改善干眼导致的角膜上皮细胞损伤以及炎症状态，且不同构型的谷氨酰胺对于干眼导致的角膜上皮损伤有同等的治疗效果。本发明探索新的治疗方法改善干眼疾病中角膜上皮的损伤，为干眼的治疗奠定基础。
Description
技术领域
本发明涉及生物医药领域，具体地说，涉及谷氨酰胺在制备治疗干眼症的药物中的应用。
背景技术
干眼是目前世界范围内最常见的眼病之一，其主要特征是泪膜稳态失衡伴发的多种眼表症状。过去研究发现干眼是一种慢性炎症性疾病，在某些情况下，急性干燥触发了免疫炎症介质的产生，刺激基质金属蛋白酶产生以及炎症细胞的募集。目前针对干眼的治疗，主要有两种环孢素和lifitegrast两种FDA批准的药物，它们通过一致T细胞激活和细胞因子产生从而达到治疗效果，但这两种治方法并不能有效改善所有患者的角膜上皮损伤。临床前研究已经确定代谢异常是干眼的主要病理生理变化，因此补充或干预小分子代谢物对于角膜上皮细胞损伤的改善具有重大临床意义。
发明内容
本发明的目的是提供谷氨酰胺在制备治疗干眼症的药物中的应用。
本发明的另一目的是提供一种干眼症细胞模型的构建方法。
为了实现本发明目的，第一方面，本发明提供谷氨酰胺在制备治疗干眼症的药物中的应用。
所述谷氨酰胺为L-谷氨酰胺和/或D-谷氨酰胺。
第二方面，本发明提供一种干眼症细胞模型的构建方法，体外培养人角膜上皮细胞，并对细胞进行高渗处理，将处理后的细胞作为干眼症细胞模型。
进一步地，所述方法包括以下步骤：
(1)人角膜上皮细胞的培养：将人角膜上皮细胞置于含有10％胎牛血清和1％青霉素-链霉素双抗的DMEM/F12培养基中，于37℃5％CO2条件下培养，每隔2-3天进行换液；
(2)干眼症细胞模型的建立：待细胞融合度至80％-90％后，换无血清DMEM/F12培养基培养24h后，将培养基更换为含90mmol/L NaCl的无血清DMEM/F12培养基，使培养基渗透到达到500±2mOsm，培养12h。
第三方面，本发明提供按照所述方法构建得到的干眼(高渗)细胞模型。
第四方面，本发明提供按照所述方法构建得到的细胞模型在干眼症治疗药物筛选或干眼症研究中的应用。
借由上述技术方案，本发明至少具有下列优点及有益效果：
(一)本发明首次将L-谷氨酰胺和D-谷氨酰胺用于改善人角膜上皮细胞干眼模型，能够有效地改善干眼导致的角膜上皮细胞损伤以及炎症状态。
(二)不同构型的谷氨酰胺对于干眼导致的角膜上皮损伤有同等的治疗效果。
(三)本发明探索新的治疗方法改善干眼疾病中角膜上皮的损伤，为干眼的治疗奠定基础。
附图说明
图1为本发明较佳实施例中人角膜上皮细胞干眼造模后凋亡情况观察，进行高渗处理后12小时细胞凋亡显著增加。**表示不同处理组之间的差异具有统计学意义，**表示P<0.01。
图2为本发明较佳实施例中人角膜上皮细胞干眼造模后炎症指标观察，进行高渗处理后12小时炎症因子mRNA水平显著增加。*、**和****表示不同处理组之间的差异具有统计学意义，*表示P<0.05，**表示P<0.01，****P<0.0001。
图3为本发明较佳实施例中L-谷氨酰胺及D-谷氨酰胺治疗干眼模型，人角膜上皮细胞凋亡情况观察。
图4为本发明较佳实施例中L-谷氨酰胺及D-谷氨酰胺治疗干眼模型，人角膜上皮细胞凋亡情况观察。*和**表示不同处理组之间的差异具有统计学意义，*表示P<0.05，**表示P<0.01。
具体实施方式
本发明提供谷氨酰胺治疗干眼的新治疗策略。本发明利用人角膜上皮细胞株建立体外干眼(高渗)模型，利用D-谷氨酰胺及L-谷氨酰胺进行治疗并探索有效治疗浓度。
本发明采用如下技术方案：
(1)上皮细胞系的培养：在75cm2的培养瓶中加入12ml含有10％胎牛血清(FBS)和1％青霉素-链霉素双抗的DMEM/F12培养基，37℃5％CO2环境下培养，每隔2-3天进行换液。
(2)上皮细胞系干眼模型的建立：细胞融合度至80％-90％后，换无血清培养基培养24h后(饥饿处理)，培养基换为含90mmol/L NaCl的无血清DMEM/F12培养基，使培养基渗透到达到500±2mOsm(渗透压仪检测)，培养12h后人角膜上皮细胞凋亡和炎症因子分泌最多(图1)。
(3)氨酰胺及D-谷氨酰胺治疗干预：
对照组：饥饿处理后，培养基继续是无血清DMEM/F12培养基，培养12h后收集细胞；
干眼组：饥饿处理后，培养基换为无血清DMEM/F12培养基(含90mmol/L NaCl)，培养12h后收集细胞；
L-谷氨酰胺处理组：饥饿处理后，培养基换为无血清DMEM/F12培养基(含90mmol/LNaCl，15-20mmol/L L-谷氨酰胺)，培养12h后收集细胞；
D-谷氨酰胺处理组：饥饿处理后，培养基换为无血清DMEM/F12培养基(含90mmol/LNaCl，15-20mmol/L D-谷氨酰胺)，培养12h后收集细胞；
(4)损伤评价：经Tunnel染色以及炎症因子PCR评价人角膜上皮细胞的炎症状态及损伤程度。
以下实施例用于说明本发明，但不用来限制本发明的范围。若未特别指明，实施例中所用的技术手段为本领域技术人员所熟知的常规手段，所用原料均为市售商品。
以下实施例中使用人角膜上皮细胞株为HCE-T，购自GENETIC TESTINGBIOTECHNOLOGY。
实施例1 L-谷氨酰胺和D-谷氨酰胺在干眼疾病中的应用
1、人角膜上皮细胞系的培养：在75cm2的培养瓶中加入12ml含有10％胎牛血清(FBS)和1％青霉素-链霉素双抗的DMEM/F12培养基，37℃5％CO2环境下培养，每隔2-3天进行换液。
2、人角膜上皮细胞系干眼模型的建立：细胞融合度至80％-90％后，换无血清培养基培养24h后(饥饿处理)，培养基换为含90mmol/L NaCl的无血清DMEM/F12培养基，使培养基渗透到达到500±2mOsm(渗透压仪检测)，12h后人角膜上皮细胞凋亡(图1)和炎症因子分泌最多(图2)。
3、L-谷氨酰胺及D-谷氨酰胺治疗干预
对照组：饥饿处理后，培养基继续是无血清DMEM/F12培养基，培养12h后收集细胞；
干眼组：饥饿处理后，培养基换为无血清DMEM/F12培养基(含90mmol/L NaCl)，培养12h后收集细胞；
L-谷氨酰胺处理组：饥饿处理后，培养基换为无血清DMEM/F12培养基(含90mmol/LNaCl，15-20mmol/L L-谷氨酰胺)，培养12h后收集细胞；
D-谷氨酰胺处理组：饥饿处理后，培养基换为无血清DMEM/F12培养基(含90mmol/LNaCl，15-20mmol/L D-谷氨酰胺)，培养12h后收集细胞；
TUNEL染色提示，D-谷氨酰胺及L-谷氨酰胺均能有效的改善高渗干眼模型所诱导的角膜上皮细胞的凋亡(图3)，同时能够明显降低TNF-α、IL-1β以及IFN-γ的表达(图4)，改善了角膜上皮细胞的炎症状态。
从图3可以看出，相比于模型组，L-谷氨酰胺和D-谷氨酰胺均能明显改善干眼模型下人角膜上皮细胞的凋亡情况。从图4可以看出，相比于模型组，L-谷氨酰胺和D-谷氨酰胺均能明显改善干眼模型下人角膜上皮细胞的炎症因子表达情况，干眼模型组相比于正常组，TNFα、IL-1β及IFN-γmRNA表达量明显增加，而L-谷氨酰胺及D-谷氨酰胺治疗组相比于干眼模型组上述mRNA表达量明显降低。
以上实验结果表明，L-谷氨酰胺和D-谷氨酰胺在15-20mmol/L均能有效改善角膜上皮细胞的损伤。
虽然，上文中已经用一般性说明及具体实施方案对本发明作了详尽的描述，但在本发明基础上，可以对之做一些修改或改进，这对本领域技术人员而言是显而易见的。因此，在不偏离本发明精神的基础上所做的这些修改或改进，均属于本发明要求保护的范围。
Claims (5)
1.谷氨酰胺在制备治疗干眼症的药物中的应用。
2.根据权利要求1所述的应用，其特征在于，所述谷氨酰胺为L-谷氨酰胺和/或D-谷氨酰胺。
3.干眼症细胞模型的构建方法，其特征在于，体外培养人角膜上皮细胞，并对细胞进行高渗处理，将处理后的细胞作为干眼症细胞模型。
4.根据权利要求3所述的方法，其特征在于，包括以下步骤：
(1)人角膜上皮细胞的培养：将人角膜上皮细胞置于含有10％胎牛血清和1％青霉素-链霉素双抗的DMEM/F12培养基中，于37℃5％CO2条件下培养，每隔2-3天进行换液；
(2)干眼症细胞模型的建立：待细胞融合度至80％-90％后，换无血清DMEM/F12培养基培养24h后，将培养基更换为含90mmol/L NaCl的无血清DMEM/F12培养基，使培养基渗透到达到500±2mOsm，培养12h。
5.按照权利要求3或4所述方法构建得到的细胞模型在干眼症治疗药物筛选或干眼症研究中的应用。
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| WO2010107069A1 (ja) * | 2009-03-17 | 2010-09-23 | 千寿製薬株式会社 | アミノ酸含有眼科用組成物 |
| CN108785251A (zh) * | 2018-01-23 | 2018-11-13 | 厦门大学 | 一种眼用药物组合物及其制备方法和应用 |
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| WO2010107069A1 (ja) * | 2009-03-17 | 2010-09-23 | 千寿製薬株式会社 | アミノ酸含有眼科用組成物 |
| CN108785251A (zh) * | 2018-01-23 | 2018-11-13 | 厦门大学 | 一种眼用药物组合物及其制备方法和应用 |
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| 李慧: "虾青素对高渗诱导体内外干眼模型的保护作用及其抗炎机制研究", 《中国优秀硕士学位论文全文数据库 医药卫生科技辑》, no. 1, pages 057 - 625 * |
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