牛磺酸拮抗锰致大鼠丘脑神经细胞损伤的体外实验研究

发布时间：2018-03-19 12:16

本文选题：锰　切入点：牛磺酸　出处：《广西医科大学》2012年硕士论文　论文类型：学位论文

【摘要】：[目的]应用新生大鼠丘脑神经细胞原代培养的方法,探讨谷氨酸(Glu)、谷氨酰胺(Gln)、γ-氨基丁酸(GABA)三种氨基酸类神经递质,在牛磺酸(Tau)拮抗锰(Mn)诱导丘脑神经细胞损伤中的作用。 [方法]取新生24小时以内的SPF级Wistar大鼠丘脑组织进行细胞原代培养,培养至最佳状态后,用于实验。先通过MTT实验筛选Mn和Tau处理浓度,将用于培养的丘脑神经细胞分组为：①空白对照组；②牛磺酸组；③低浓度染锰组；④中浓度染锰组；⑤高浓度染锰组；⑥低锰预防组；⑦中锰预防组；⑧高锰预防组。在各处理组加入处理因素24小时后终止培养,于倒置荧光显微镜下观察并拍照后进行以下检测：BCA法进行蛋白定量用于后续实验参考、高效液相色谱法(HPLC)测定细胞内液及细胞外液中三种氨基酸类神经递质的浓度。 [结果]1、通过MTT实验将牛磺酸干预浓度确定为4mmol/L,将染锰的低、中、高浓度分别确定为0.1mmol/L、0.2mmol/L、0.4mmol/L。2、与空白对照组比较,单纯牛磺酸干预条件下细胞铺展更充分、细胞密度更高；染锰可以导致丘脑神经细胞形态改变、活细胞数量减少,且细胞形态、活细胞数量的改变程度随着染锰浓度的增加而增大；相对于对应的染锰组,牛磺酸预防组细胞形态较完整、活细胞数量较多,但细胞形态改变、活细胞数量减少的程度随着染锰浓度的增加逐渐增大；3、HPLC检测显示：①细胞外：与空白对照组比较,牛磺酸组Glu浓度降低,差异有统计学意义(P0.01)；与空白对照组比较,染锰各组Glu浓度均明显增高,差异均有统计学意义(P0.01)；Glu浓度随着染锰浓度的增加逐渐提高,各染锰组之间差异均有统计学意义(P0.01)；牛磺酸预防各组Glu浓度均低于对应的染锰组,差异均有统计学意义(P0.01)；各预防组之间,Glu浓度随着染锰浓度的增加逐渐提高,差异均有统计学意义(P0.01)。与空白对照组比较,牛磺酸组Gln浓度降低,差异有统计学意义(P0.01)；与空白对照组比较,染锰各组Gln浓度均明显降低,差异均有统计学意义(P0.01)；Gln浓度随着染锰浓度的增加逐渐降低,各染锰组间差异均有统计学意义(P0.01)；与对应的染锰组比较,牛磺酸预防各组Gln浓度均较低,差异均有统计学意义(P0.01)；各预防组之间,Gln浓度随着染锰浓度的增加逐渐降低,差异均有统计学意义(P0.01)。与空白对照组比较,牛磺酸组GABA浓度显著提高,差异有统计学意义(P0.01)与空白对照组比较,染锰各组GABA浓度均有所提高,但只有高浓度染锰组差异有统计学意义(P0.05)；GABA浓度随着染锰浓度的增加逐渐提高,但各染锰组间差异均无统计学意义(P0.05)；与对应的染锰组比较,牛磺酸预防各组GABA浓度均明显提高,差异均有统计学意义(P0.01)；各预防组之间,GABA浓度随着染锰浓度的增加逐渐提高,差异均有统计学意义(P0.01)。②细胞内：与空白对照组比较,牛磺酸组Glu浓度明显降低,差异有统计学意义(P0.01)；与空白对照组比较,染锰各组Glu浓度均明显增高,差异均有统计学意义(P0.01)；Glu浓度随着染锰浓度的增加而逐渐提高,各染锰组间差异均有统计学意义(P0.01)；与对应的染锰组比较,牛磺酸预防各组Glu浓度均较低,差异均有统计学意义(P0.01)；各预防组之间,Glu浓度随着染锰浓度的增加逐渐提高,差异均有统计学意义(P0.01)。与空白对照组比较,牛磺酸组Gln浓度降低,差异有统计学意义(P0.01)；与空白对照组比较,染锰各组Gln浓度均明显降低,差异均有统计学意义(P0.01)；随着染锰浓度的增加,Gln浓度逐渐降低,各染锰组间差异均有统计学意义(P0.01)；牛磺酸预防各组Gln浓度均高于对应的染锰组,差异均有统计学意义(P0.01)各预防组之间,Gln浓度随着染锰浓度的增加逐渐降低,差异均有统计学意义(P0.01)。与空白对照组比较,牛磺酸组GABA浓度显著提高,差异有统计学意义(P0.01)；与空白对照组比较,染锰各组GABA浓度有所下降,差异均有统计学意义(P0.01)；随着染锰浓度的增加,GABA浓度逐渐下降,各染锰组间差异均有统计学意义(P0.01)；与对应的染锰组比较,牛磺酸预防各组GABA浓度均明显提高,差异均有统计学意义(P0.01)；各预防组之间,GABA浓度随着染锰浓度的增加逐渐提高,差异均有统计学意义(P0.01)。③细胞外液与细胞内液3种氨基酸类神经递质的平均浓度：与空白对照组比较,牛磺酸组Glu平均浓度降低,差异有统计学意义(P0.01)；与空白对照组比较,染锰各组Glu平均浓度均明显增高,差异均有统计学意义(P0.01)；随着染锰浓度的增加,Glu平均浓度逐渐提高,各染锰组间差异均有统计学意义(P0.01)；牛磺酸预防各组Glu平均浓度均低于对应的染锰组,差异均有统计学意义(P0.01)；各预防组之间,Glu平均浓度随着染锰浓度的增加逐渐提高,差异均有统计学意义(P0.01)。与空白对照组比较,牛磺酸组Gln平均浓度降低,差异有统计学意义(P0.01)；与空白对照组比较,染锰各组Gln平均浓度均明显降低,差异均有统计学意义(P0.01)；随着染锰浓度的增加,Gln平均浓度逐渐降低,各染锰组间差异均有统计学意义(P0.01)；与对应的染锰组比较,牛磺酸预防各组Gln平均浓度均较低,差异均有统计学意义(P0.01)：各预防组之间,Gln平均浓度随着染锰浓度的增加逐渐降低,差异均有统计学意义(P0.01)。与空白对照组比较,牛磺酸组GABA平均浓度显著提高,差异有统计学意义(P0.01)；与空白对照组比较,染锰各组GABA平均浓度有所提高,但差异均无统计学意义(P0.05)；随着染锰浓度的增加,GABA平均浓度逐渐提高,但各染锰组间差异均无统计学意义(P0.05)；与对应的染锰组比较,牛磺酸预防各组GABA平均浓度均明显提高,差异均有统计学意义(P0.01)；各预防组之间,GABA平均浓度随着染锰浓度的增加逐渐提高,差异均有统计学意义(P0.01)。④Glu平均浓度/GABA平均浓度：与空白对照组比较,牛磺酸组(Glu平均浓度/GABA平均浓度)的值降低,差异有统计学意义(P0.01)；与空白对照组比较,染锰各组(Glu平均浓度/GABA平均浓度)的值均明显增高,差异均有统计学意义(P值均0.01)；与低浓度染锰组比较,中、高浓度染锰组(Glu平均浓度/GABA平均浓度)的值显著提高,差异均有统计学意义(P0.01)；中、高浓度染锰组之间,(Glu平均浓度/GABA平均浓度)的值差异无统计学意义(P0.05)；牛磺酸预防各组(Glu平均浓度/GABA平均浓度)值均低于对应的染锰组,差异均有统计学意义(P0.01)；各预防组之间,(Glu平均浓度/GABA平均浓度)的值随着染锰浓度的增加逐渐提高,但差异均无统计学意义(P0.05)。 [结论]锰可致体外原代培养的丘脑神经细胞损伤,牛磺酸可在一定程度上拮抗此毒作用；Glu可能在锰致体外原代培养的丘脑神经细胞损伤机制中起到重要作用,GABA可能在牛磺酸拮抗锰致体外原代培养的丘脑神经细胞损伤中机制起到重要作用。
[Abstract]:[Objective] to explore the role of three amino acids neurotransmitters of glutamic acid (Glu), glutamine (Gln) and GABA (GABA) in the primary culture of thalamic neurons in neonatal rats, and to evaluate the effect of taurine (Tau) on antagonizing manganese (Mn) induced neuronal injury in the thalamus.
[Methods] within 24 hours of the new SPF thalamic tissue Wistar rats were primary cultured cells, cultured to the best state, used in the experiment. The first screening of Mn and Tau concentration by MTT experiments, will be used for nerve cells cultured thalamus are as follows: blank control group; the taurine group; the low the concentration of Mn group; the concentration of Mn group; the high concentration of Mn group; the low manganese manganese group and prevention group; prevention; the prevention of high manganese group. In the treatment group treated with factor 24 hours after the termination of the culture, in the inverted fluorescence microscope and photographed using the following methods: BCA method for the reference of follow-up quantitative protein, high performance liquid chromatography (HPLC) determination of the concentration of intracellular fluid and extracellular fluid in three amino acid neurotransmitters.
The]1, through the MTT experiment will be determined as 4mmol/L taurine concentration, the Mn low, high concentration were identified as 0.1mmol/L, 0.2mmol/L, 0.4mmol/L.2, compared with the blank control group, taurine intervention under the condition of simple cell spreading more fully, more high cell density; manganese can cause morphological changes of nerve cells in the thalamus and the number of living cells decreased, and cell morphology, degree of change in the number of viable cells increased with the concentration of Mn Mn group; compared with the corresponding, taurine treatment group cell morphology is more complete, more number of live cells, but cell morphological changes, the number of living cells decrease gradually increases with increasing concentration of Mn the 3; HPLC showed that the cells: compared with the blank control group, taurine group Glu decreased, the difference was statistically significant (P0.01); compared with the control group, the Glu concentration of Mn group Increased significantly, the differences were statistically significant (P0.01); the concentration of Glu increased with the concentration of Mn, Mn between group differences were statistically significant (P0.01); taurine prevention groups Glu concentrations were lower than the corresponding Mn group, the differences were statistically significant (P0.01); the prevention group, Glu the concentration increased with the concentration of Mn, the differences were statistically significant (P0.01). Compared with the blank control group, taurine group Gln decreased, the difference was statistically significant (P0.01); compared with the control group, each group of Mn Gln concentration were significantly decreased, the differences were statistically significant (P0.01); the concentration of Gln decreased gradually with the increase of the concentration of Mn, Mn between group differences were statistically significant (P0.01); compared with the corresponding Mn group, taurine prevention groups Gln concentration was low, the differences were statistically significant (P0.01); The prevention group, the concentration of Gln decreased gradually with the increase of the concentration of Mn, the differences were statistically significant (P0.01). Compared with the blank control group, taurine group GABA concentration significantly increased, the difference was statistically significant (P0.01) compared with the control group, each group with Mn concentration of GABA increased, but only high concentration of dye Mn group differences were statistically significant (P0.05); the concentration of GABA increased with the concentration of Mn, but the differences between the groups with manganese had no statistical significance (P0.05); compared with the corresponding Mn group, taurine concentration GABA prevention groups were significantly improved, the differences were statistically significant (P0.01); the prevention group, the concentration of GABA increased with the concentration of Mn, the differences were statistically significant (P0.01). The cells: compared with the blank control group, taurine group Glu concentration decreased significantly, the difference was statistically significant (P0.01); Compared with the control group, each group with Mn concentration of Glu were significantly increased, the differences were statistically significant (P0.01); the concentration of Glu increase gradually with the concentration of Mn, Mn differences between groups were statistically significant (P0.01); compared with the corresponding Mn group, taurine group were significantly higher than Glu prevention low, the differences were statistically significant (P0.01); the prevention group, the concentration of Glu increased with the concentration of Mn, the differences were statistically significant (P0.01). Compared with the blank control group, taurine group Gln decreased, the difference was statistically significant (P0.01); compared with the control group, Gln groups were exposed to manganese the concentration decreased significantly, the differences were statistically significant (P0.01); with the increase of Mn concentration, Gln concentration decreased, the Mn group differences were statistically significant (P0.01); taurine prevention groups was higher than that of the concentration of Gln The Mn group, there were statistically significant differences (P0.01) between the prevention group, the concentration of Gln decreased gradually with the increase of the concentration of Mn, the differences were statistically significant (P0.01). Compared with the blank control group, taurine group GABA concentration significantly increased, the difference was statistically significant (P0.01); compared with the blank control group, Mn the concentration of GABA decreased, the differences were statistically significant (P0.01); with the increase of Mn concentration, the concentration of GABA decreased gradually, the Mn group differences were statistically significant (P0.01); compared with the corresponding Mn group, taurine concentration GABA prevention groups were significantly improved, the differences were statistically significant (P0.01); the prevention group, the concentration of GABA increased with the concentration of Mn, the differences were statistically significant (P0.01). The extracellular fluid and intracellular fluid of 3 kinds of amino acids by God: the average concentration of neurotransmitters and blank control Group, taurine group average Glu concentration decreased, the difference was statistically significant (P0.01); compared with the control group, the average concentration of Glu Mn groups were significantly increased, the differences were statistically significant (P0.01); with the increase of the concentration of Mn, the average concentration of Glu gradually increased, the Mn group differences were statistically the significance of taurine (P0.01); prevention groups were lower than the average Glu concentration of Mn group respectively, the differences were statistically significant (P0.01); the prevention group, the average concentration of Glu increased with the concentration of Mn, the differences were statistically significant (P0.01). Compared with the blank control group, taurine group average Gln concentration decreased, the difference was statistically significant (P0.01); compared with the control group, each group of Mn average Gln concentration decreased significantly, the differences were statistically significant (P0.01); with the increase of the concentration of Mn, the average Gln concentration gradually Mn decreased, the difference between groups was significant (P0.01); compared with the corresponding Mn group, taurine prevention groups average Gln concentration were lower, the differences were statistically significant (P0.01) between the prevention group, the average concentration of Gln decreased gradually with the increase of the concentration of Mn, the differences were statistically significant (P0.01). Compared with the blank control group, taurine group average GABA concentration significantly increased, the difference was statistically significant (P0.01); compared with the control group, each group of Mn average concentration of GABA increased, but there were no statistically significant differences (P0.05); with the increase of the concentration of Mn, the average concentration of GABA increased gradually, but the differences between the groups with manganese had no statistical significance (P0.05); compared with the corresponding Mn group, taurine prevention groups average GABA concentration were significantly improved, the differences were statistically significant (P0.01); the prevention group, the average concentration of GABA With the increase of Mn concentration gradually increased, the differences were statistically significant (P0.01). The average concentration of Glu /GABA average concentration: compared with the blank control group, taurine group (average Glu concentration the average concentration of /GABA) decreased, the difference was statistically significant (P0.01); compared with the control group, each group (Mn the average concentration of Glu /GABA average concentration) values were significantly increased, the differences were statistically significant (P < 0.01); and the low concentration of Mn group, high concentration of Mn group (average Glu concentration /GABA average concentration) value increased significantly, the differences were statistically significant (P0.01); in between the concentration of Mn group (Glu, the average concentration of /GABA average concentration) was no significant difference (P0.05); taurine prevention groups (Glu average concentration of /GABA average concentration) values were lower than the corresponding Mn group, the differences were statistically significant (P0.01); the prevention group The value of the average concentration of Glu (mean concentration of /GABA) increased gradually with the increase of manganese concentration, but the difference was not statistically significant (P0.05).
[Conclusion] manganese can cause injury of primary cultured thalamic nerve cells, taurine could antagonize the toxic effect to a certain extent; Glu may play an important role in primary cultured nerve cells in the thalamus injury mechanism of Mn, GABA in manganese induced damage mechanism of taurine on cultured nerve cells in the thalamus play an important role.

【学位授予单位】：广西医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R151

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