盐酸去氧肾上腺素和丝裂霉素对体外培养兔角膜内皮细胞的毒性影响

发布时间：2018-05-21 14:58

本文选题：盐酸去氧肾上腺素 + 丝裂霉素　；参考：《东南大学》2005年硕士论文

【摘要】： 【目的】观察盐酸去氧肾上腺素和丝裂霉素(MMC)对体外培养家兔角膜内皮的作用,探讨临床应用这两种药物的可能毒性作用。【方法】显微分离兔角膜后弹力层-内皮细胞层,联合贴壁法和(或)消化法培养角膜内皮细胞,利用含小牛血清的高糖DMEM作为培养液,不添加生长因子。培养的角膜内皮细胞,传代接种于96孔板,盐酸去氧肾上腺素设定为4个实验组0.625mg/ml、1.25mg/ml、2.5mg/ml、5mg/ml,4个相应阴性对照组;MMC分为6个实验组1×10~(-6)mg/ml、1×10~(-5)mg/ml、1×10~(-4)mg/ml、1×10~(-3)mg/ml、1×10~(-2)mg/ml、1×10~(-1)mg/ml,均设空白对照2组。不同时相相差倒置显微镜下观察细胞的形态变化和增殖情况,去氧肾上腺素组于加药72h后,MMC组于加药22h后MTT法检测各组细胞增殖情况,对比不同浓度组细胞吸光度并进行统计学处理。去氧肾上腺素组再次设定4个实验组0.625mg/ml、1.25mg/ml、2.5mg/ml、5mg/ml,在加药后5min、10min、30min、1h、1h30min更换实验组的培养液,进一步观察细胞的空泡变性能否逆转。【结果】培养的兔角膜内皮细胞呈单层密集生长,形态为六角形或多边形,具有活体细胞的形态特征。去氧肾上腺素5mg/ml实验组加药4h后细胞出现空泡,2.5mg/ml组加药5h后细胞出现空泡,1.25mg/ml组加药后10h出现明显空泡,加药12h后3个实验组均显示明显空泡,30h后2.5mg/ml、5mg/ml实验组细胞部分死亡,60h后1.25mg/ml、2.5mg/ml实验组原空泡变细胞大部分死亡,72h后3个浓度组几乎全部死亡。0.625mg/ml浓度组和4个阴性对照组均未出现形态学变化。1.25mg/ml、2.5mg/ml、5mg/ml实验组细胞吸光度值与空白及阴性对照的差异经统计学分析有极显著意义(P0.001);0.625mg/ml试验组、4个阴性对照组与空白对照组相比较,差异无统计学意义(P0.05)。去氧肾上腺素接触内皮细胞后,5mg/ml、2.5mg/ml、1.25mg/ml浓度组5min以内换液,细胞均未出现空泡变,加药后6h再次换液,已出现的细胞空泡变性减轻,所有换液复孔细胞均未死亡。MMC组:加药后1h、2h、3h、5h各组细胞形态变化不明显,1×10~(-2)mg/ml、1×10~(-1)mg/ml实验组8h后细胞部分变圆,20h后细胞几乎全部变圆死亡,其余各组变化不明显。1×10~(-6)mg/ml、1×10~(-5)mg/ml组和空白对照组经统计学分析差异无显著性(P0.05),1×10~(-4)组和空白对照组比较差异有显著性(P0.05),1×10~(-3)mg/ml、1×10~(-2)mg/ml、1×10~(-1)mg/ml组和空白对照组比较有极显著差异(P0.001)。【结论】盐酸去氧肾上腺素和丝裂霉素对体外培养兔角膜内皮细胞有损伤作用,并有时间和浓度相关性。尽早和及时的换液可避免或减轻盐酸去氧肾上腺素对角膜内皮细胞的毒性作
[Abstract]:[objective] to observe the effects of noradrenaline hydrochloride and mitomycin MMCon on rabbit corneal endothelium in vitro and to explore the possible toxic effects of these two drugs in clinical application. [methods] the rabbit corneal posterior elastic layer and endothelial cell layer were separated microscopically. Corneal endothelial cells were cultured with adherent method and / or digestion method. High glucose DMEM containing calf serum was used as culture medium without adding growth factor. Cultured corneal endothelial cells were cultured on 96 well plates. Noradrenaline hydrochloride was set at 0.625 mg / ml 1.25 mg / ml ~ (2.5 mg / ml). The four corresponding negative control groups were divided into 6 groups: 1 脳 10 ~ (-1) -6 mg / ml -1 脳 10 ~ (-5) mg / ml -1 脳 10 ~ (-4) mg / ml -1 脳 10 ~ (-3) mg / ml -1 脳 10 ~ (-1) mg / ml, respectively. The morphologic changes and proliferation of the cells were observed under different phase contrast microscope, and the proliferation of the cells in the noradrenaline group was detected by MTT assay after 22 hours of administration after 72 h of administration of noradrenaline. The cell absorbance of different concentration groups was compared and treated statistically. In the noradrenaline group, 0.625 mg / ml 1.25 mg / ml ~ (2.5 mg / ml) 2.5 mg / ml ~ (-1) mg 路ml ~ (-1) 路ml ~ (-1)? [results] the cultured rabbit corneal endothelial cells showed monolayer dense growth, with hexagonal or polygonal morphology, with the morphological characteristics of living cells. Vacuoles appeared in the cells of the 5mg/ml group treated with noradrenaline 2.5 mg / ml for 4 h, and the vacuoles appeared in the cells of the 1.25 mg / ml group 10 h after the addition of the drug for 5 h, and the vacuoles appeared in the cells in the group of 2.5 mg / ml of noradrenaline at 10 h after addition of the drug. After 12 h of administration, all the three experimental groups showed significant vacuolation 30 h, 2.5 mg / ml 5 mg / ml after partial cell death 60 h later, 1.25 mg / ml 2.5 mg / ml after most of the original vacuolar cells died 72 h later, almost all of the 3 groups died. 0.625 mg / ml and 4 negative control groups. There were no morphological changes. 1.25 mg / ml 2.5 mg / ml 5 mg / ml cell absorbance value of the experimental group was significantly different from the blank and negative control groups. There was significant difference between the four negative control groups and the blank control group, compared with the blank control group, the four negative control groups were compared with the blank control group. The difference was not statistically significant (P 0.05). After noradrenaline was exposed to endothelial cells, 5 mg / ml of 2.5 mg / ml ~ (-1) mg 路ml ~ (-1) mg / ml of 5min did not change the vacuolation of the cells, 6 h after adding the drug, the vacuolar denaturation of the cells was alleviated. There was no death in all the cells in the fluid exchange. MMC group: the morphological changes of the cells in each group were not obvious 1 脳 10 ~ (-2) mg / ml ~ (-1) 10~(-1)mg/ml after 8 h, and almost all the cells became round and died after 20 h. There was no significant difference between the other groups (1 脳 10 ~ (-6) mg 路ml ~ (-1) 10~(-5)mg/ml) and the blank control group (P ~ (0.05) 1 脳 10 ~ (-4) and the blank control group (P ~ (0.05) ~ (1 脳 10 ~ (-1) mg 路ml ~ (-1) 路ml ~ (-1) 脳 10 ~ (-1) mg 路ml ~ (-1) 路ml ~ (-1) 10~(-1)mg/ml and the control group (P ~ (0.001). [conclusion] noradrenaline hydrochloride and mitomycin can injure rabbit corneal endothelial cells in vitro and have time and concentration correlation. Early and timely fluid exchange can avoid or reduce the toxicity of noradrenaline hydrochloride to corneal endothelial cells.
【学位授予单位】：东南大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R329

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