渗透压对角膜上皮细胞的影响及牛磺酸对高渗环境细胞的保护作用
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摘要
目的观察渗透压对体外培养的兔角膜上皮细胞的影响及牛磺酸对高渗环境细胞的保护作用,为人工泪液的处方制定提供理论依据。方法采用分离培养的兔角膜上皮细胞,分为低渗组(143,229和257mOsm·L~(-1))、等渗对照组(286mOsm·L~(-1))和高渗组(315,343和429mOsm·L~(-1)),用不同渗透压的培养液作用3h后,换回正常培养液培养3d,记录作用3h及恢复培养3d时的细胞形态,并用MTT法检测作用3h、恢复培养1d和恢复培养3d后的细胞活性。在正常培养液286mOsm·L~(-1)中加入牛磺酸培养细胞24h后,换为429mOsm·L~(-1)的高渗培养液培养3h后,换回含牛磺酸的等渗培养液恢复培养1d,倒置显微镜下记录作用3h及恢复培养1d时的细胞形态,并用MTT法检测细胞活性。结果高渗环境会导致细胞活性降低,甚至死亡;低渗环境会改变细胞形态及活性。257mOsm·L~(-1)渗透压组与等渗对照组相比,在作用3h及恢复培养1和3d后细胞形态及细胞活性差异无统计学意义。含有不同浓度牛磺酸培养基的各组细胞也有轻度肿胀,高渗培养基作用3h后以及恢复正常培养基培养1d后,细胞活性差异具有显著统计学意义(P<0.05)。结论泪液高渗透压会造成眼表损伤。应用渗透压为257mOsm·L~(-1)的人工泪液,在对正常细胞无影响的条件下,可降低干眼患者泪液的高渗透压,有助于干眼病的治疗。牛磺酸作为细胞保护剂,可降低高渗透压对细胞的损害,在干眼病治疗方面,具有很好的应用前景。
Objective To observe the effect of osmolarity on rabbit corneal epithelial cells cultured in vitro,and the protective effect of taurine on cells in hypertonic environments,therefore to provide a theoretical basis for the choice of the formulation and selection of artificial tears.Methods The rabbit corneal epithelial cells were cultured in hypo-osmolarity group(143,229 and 257mOsm·L~(-1)),iso-osmolarity control group(286mOsm·L~(-1))and hyper-osmolarity group(315,343and 429mOsm·L~(-1))for 3h.Then they were returned to normal culture for 3d,and their morphology were recorded.MTT assay was used to detect the viability of the cells in each group after 3h,1dand 3d in recovery culture.The cells were cultured in the normal cultural medium(286mOsm·L~(-1))with taurine for 24h,then were transferred to the cultural medium with the hyper-osmolarity of 429mOsm·L~(-1)for 3h,finally were recovered the normal cultural medium with taurine for 1d.The cell morphology was recorded by the inverted microscope and the cell activity were detected by MTT assay.Results Hypertonic environment may lead to the reduction of cell activity or even death.Hypotonic environment would change the cell morphology and activity.Compared with the isoosmolarity control group,the osmolarity of 257mOsm·L~(-1 )group had no significant difference in cell morphology and cell viability after 3h and 1d and 3d of restoration.Although the cells cultured in medium with different concentrations of taurine had mild swelling,after immersing in the hypertonic medium for 3h and restoring in normal medium for 1d,the cell activity was significantly different from that of the group without taurine(P<0.05).Conclusion The hyper-osmolarity of lacrimal fluid may lead to ocular surface damage.With no effect on normal cells,the application of the artificial lacrimal fluid with osmotic pressure of 257m Osm·L~(-1)can reduce the hypertonic pressure of tear in dry eyes and help to treat dry eyes.Taurine,as a cytoprotective agent,could reduce the damage of hyperosmotic pressure to cells,and has a good application prospect in the treatment of dry eyes.
Objective To observe the effect of osmolarity on rabbit corneal epithelial cells cultured in vitro,and the protective effect of taurine on cells in hypertonic environments,therefore to provide a theoretical basis for the choice of the formulation and selection of artificial tears.Methods The rabbit corneal epithelial cells were cultured in hypo-osmolarity group(143,229 and 257mOsm·L~(-1)),iso-osmolarity control group(286mOsm·L~(-1))and hyper-osmolarity group(315,343and 429mOsm·L~(-1))for 3h.Then they were returned to normal culture for 3d,and their morphology were recorded.MTT assay was used to detect the viability of the cells in each group after 3h,1dand 3d in recovery culture.The cells were cultured in the normal cultural medium(286mOsm·L~(-1))with taurine for 24h,then were transferred to the cultural medium with the hyper-osmolarity of 429mOsm·L~(-1)for 3h,finally were recovered the normal cultural medium with taurine for 1d.The cell morphology was recorded by the inverted microscope and the cell activity were detected by MTT assay.Results Hypertonic environment may lead to the reduction of cell activity or even death.Hypotonic environment would change the cell morphology and activity.Compared with the isoosmolarity control group,the osmolarity of 257mOsm·L~(-1 )group had no significant difference in cell morphology and cell viability after 3h and 1d and 3d of restoration.Although the cells cultured in medium with different concentrations of taurine had mild swelling,after immersing in the hypertonic medium for 3h and restoring in normal medium for 1d,the cell activity was significantly different from that of the group without taurine(P<0.05).Conclusion The hyper-osmolarity of lacrimal fluid may lead to ocular surface damage.With no effect on normal cells,the application of the artificial lacrimal fluid with osmotic pressure of 257m Osm·L~(-1)can reduce the hypertonic pressure of tear in dry eyes and help to treat dry eyes.Taurine,as a cytoprotective agent,could reduce the damage of hyperosmotic pressure to cells,and has a good application prospect in the treatment of dry eyes.
引文
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[13]Chen M,Hu D N,Pan Z,et al.Curcumin protects against hyperosmoticity-induced IL-1βelevation in human corneal epithelial cell via MAPK pathways[J].Exp Eye Res,2010,90(3):437-443.
[14]Hua X,Deng R,Li J,et al.Protective effects of L-carnitine against oxidative injury by hyperosmolarity in human corneal epithelial cells[J].Invest Ophthalmol Vis Sci,2015,56(9):5503-5511.
[15]Hua X,Su Z,Deng R,et al.Effects of L-carnitine,erythritol and betaine on pro-inflammatory markers in primary human corneal epithelial cells exposed to hyperosmotic stress[J].Curr Eye Res,2015,40(7):657-667.
[16]Chen W,Zhang X,Li J,et al.Efficacy of osmoprotectants on prevention and treatment of murine dry eye[J].Invest Ophthalmol Vis Sci,2013,54(9):6287-6297.
[17]张松,孙丽,李悦.香叶醇在医学领域的应用研究进展[J].西北药学杂志,2017,32(1):124-126.
[18]李金芳,周荫庄,屠淑洁.牛磺酸对细胞的保护功能[J].首都师范大学学报:自然科学版,2006,27(1):63-66,54.
[19]谭乐义,章超桦,薛长湖,等.牛磺酸细胞保护效应的研究概况[J].中国海洋药物,2000,(4):37-39.
[2]刘祖国,张晓博.解读国际泪膜与眼表协会2017年干眼专家共识中的干眼定义与分类[J].中华眼科杂志,2018,54(4):246-248.
[3]徐建江,洪佳旭.解读国际泪膜与眼表协会2017年干眼专家共识中的干眼处理与治疗[J].中华眼科杂志,2018,54(4):249-251.
[4]洪晶.解读国际泪膜与眼表协会2017年干眼专家共识中的干眼病理生理机制[J].中华眼科杂志,2018,54(6):415-418.
[5]潘士印,肖湘华,王养正,等.泪液渗透压与干眼[J].中华眼科杂志,2011,47(5):1-4.
[6]Sullivan B D,Whitmer D,Nichols K K,et al.An objective approach to dry eye disease severity[J].Invest Ophthalmol Vis Sci,2010,51(12):6125-6130.
[7]于春红,王云松.睑板腺功能障碍的治疗及研究进展[J].海南医学,2018,29(4):531-534.
[8]汪波,杨燕宁,黄林英.干眼治疗方法的研究新进展[J].眼科新进展,2017,37(2):179-184.
[9]张常雄,成颖,周四元.环孢菌素A制剂的研究进展[J].西北药学杂志,2018,33(1):136-139.
[10]Papa V,Aragona P,Russo S,et al.Comparison of hypotonic and isotonic solutions containing sodium hyaluronate on the symptomatic treatment of dry eye patients[J].Ophthalmologica,2001,215(2):124-127.
[11]Motolko M,Breslin C W.The effect of pH and osmolarity on the ability of tolerate artificial tears[J].Am J Ophthalmol,1981,91(6):781-784.
[12]Troiano P,Monaco G.Effect of hypotonic 0.4%hyaluronic acid drops in dry eye patients:a cross-over study[J].Cornea,2008,27(10):1126-1130.
[13]Chen M,Hu D N,Pan Z,et al.Curcumin protects against hyperosmoticity-induced IL-1βelevation in human corneal epithelial cell via MAPK pathways[J].Exp Eye Res,2010,90(3):437-443.
[14]Hua X,Deng R,Li J,et al.Protective effects of L-carnitine against oxidative injury by hyperosmolarity in human corneal epithelial cells[J].Invest Ophthalmol Vis Sci,2015,56(9):5503-5511.
[15]Hua X,Su Z,Deng R,et al.Effects of L-carnitine,erythritol and betaine on pro-inflammatory markers in primary human corneal epithelial cells exposed to hyperosmotic stress[J].Curr Eye Res,2015,40(7):657-667.
[16]Chen W,Zhang X,Li J,et al.Efficacy of osmoprotectants on prevention and treatment of murine dry eye[J].Invest Ophthalmol Vis Sci,2013,54(9):6287-6297.
[17]张松,孙丽,李悦.香叶醇在医学领域的应用研究进展[J].西北药学杂志,2017,32(1):124-126.
[18]李金芳,周荫庄,屠淑洁.牛磺酸对细胞的保护功能[J].首都师范大学学报:自然科学版,2006,27(1):63-66,54.
[19]谭乐义,章超桦,薛长湖,等.牛磺酸细胞保护效应的研究概况[J].中国海洋药物,2000,(4):37-39.