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  本文選題：錳　切入點：牛磺酸　出處：《廣西醫(yī)科大學》2012年碩士論文　論文類型：學位論文

【摘要】：[目的]應用新生大鼠丘腦神經(jīng)細胞原代培養(yǎng)的方法,探討谷氨酸(Glu)、谷氨酰胺(Gln)、γ-氨基丁酸(GABA)三種氨基酸類神經(jīng)遞質(zhì),在�；撬�(Tau)拮抗錳(Mn)誘導丘腦神經(jīng)細胞損傷中的作用。 [方法]取新生24小時以內(nèi)的SPF級Wistar大鼠丘腦組織進行細胞原代培養(yǎng),培養(yǎng)至最佳狀態(tài)后,用于實驗。先通過MTT實驗篩選Mn和Tau處理濃度,將用于培養(yǎng)的丘腦神經(jīng)細胞分組為：①空白對照組；②�；撬峤M；③低濃度染錳組；④中濃度染錳組；⑤高濃度染錳組；⑥低錳預防組；⑦中錳預防組；⑧高錳預防組。在各處理組加入處理因素24小時后終止培養(yǎng),于倒置熒光顯微鏡下觀察并拍照后進行以下檢測：BCA法進行蛋白定量用于后續(xù)實驗參考、高效液相色譜法(HPLC)測定細胞內(nèi)液及細胞外液中三種氨基酸類神經(jīng)遞質(zhì)的濃度。 [結(jié)果]1、通過MTT實驗將牛磺酸干預濃度確定為4mmol/L,將染錳的低、中、高濃度分別確定為0.1mmol/L、0.2mmol/L、0.4mmol/L。2、與空白對照組比較,單純�；撬岣深A條件下細胞鋪展更充分、細胞密度更高；染錳可以導致丘腦神經(jīng)細胞形態(tài)改變、活細胞數(shù)量減少,且細胞形態(tài)、活細胞數(shù)量的改變程度隨著染錳濃度的增加而增大；相對于對應的染錳組,�；撬犷A防組細胞形態(tài)較完整、活細胞數(shù)量較多,但細胞形態(tài)改變、活細胞數(shù)量減少的程度隨著染錳濃度的增加逐漸增大；3、HPLC檢測顯示：①細胞外：與空白對照組比較,牛磺酸組Glu濃度降低,差異有統(tǒng)計學意義(P0.01)；與空白對照組比較,染錳各組Glu濃度均明顯增高,差異均有統(tǒng)計學意義(P0.01)；Glu濃度隨著染錳濃度的增加逐漸提高,各染錳組之間差異均有統(tǒng)計學意義(P0.01)；牛磺酸預防各組Glu濃度均低于對應的染錳組,差異均有統(tǒng)計學意義(P0.01)；各預防組之間,Glu濃度隨著染錳濃度的增加逐漸提高,差異均有統(tǒng)計學意義(P0.01)。與空白對照組比較,�；撬峤MGln濃度降低,差異有統(tǒng)計學意義(P0.01)；與空白對照組比較,染錳各組Gln濃度均明顯降低,差異均有統(tǒng)計學意義(P0.01)；Gln濃度隨著染錳濃度的增加逐漸降低,各染錳組間差異均有統(tǒng)計學意義(P0.01)；與對應的染錳組比較,�；撬犷A防各組Gln濃度均較低,差異均有統(tǒng)計學意義(P0.01)；各預防組之間,Gln濃度隨著染錳濃度的增加逐漸降低,差異均有統(tǒng)計學意義(P0.01)。與空白對照組比較,牛磺酸組GABA濃度顯著提高,差異有統(tǒng)計學意義(P0.01)與空白對照組比較,染錳各組GABA濃度均有所提高,但只有高濃度染錳組差異有統(tǒng)計學意義(P0.05)；GABA濃度隨著染錳濃度的增加逐漸提高,但各染錳組間差異均無統(tǒng)計學意義(P0.05)；與對應的染錳組比較,牛磺酸預防各組GABA濃度均明顯提高,差異均有統(tǒng)計學意義(P0.01)；各預防組之間,GABA濃度隨著染錳濃度的增加逐漸提高,差異均有統(tǒng)計學意義(P0.01)。②細胞內(nèi)：與空白對照組比較,牛磺酸組Glu濃度明顯降低,差異有統(tǒng)計學意義(P0.01)；與空白對照組比較,染錳各組Glu濃度均明顯增高,差異均有統(tǒng)計學意義(P0.01)；Glu濃度隨著染錳濃度的增加而逐漸提高,各染錳組間差異均有統(tǒng)計學意義(P0.01)；與對應的染錳組比較,牛磺酸預防各組Glu濃度均較低,差異均有統(tǒng)計學意義(P0.01)；各預防組之間,Glu濃度隨著染錳濃度的增加逐漸提高,差異均有統(tǒng)計學意義(P0.01)。與空白對照組比較,�；撬峤MGln濃度降低,差異有統(tǒng)計學意義(P0.01)；與空白對照組比較,染錳各組Gln濃度均明顯降低,差異均有統(tǒng)計學意義(P0.01)；隨著染錳濃度的增加,Gln濃度逐漸降低,各染錳組間差異均有統(tǒng)計學意義(P0.01)；�；撬犷A防各組Gln濃度均高于對應的染錳組,差異均有統(tǒng)計學意義(P0.01)各預防組之間,Gln濃度隨著染錳濃度的增加逐漸降低,差異均有統(tǒng)計學意義(P0.01)。與空白對照組比較,�；撬峤MGABA濃度顯著提高,差異有統(tǒng)計學意義(P0.01)；與空白對照組比較,染錳各組GABA濃度有所下降,差異均有統(tǒng)計學意義(P0.01)；隨著染錳濃度的增加,GABA濃度逐漸下降,各染錳組間差異均有統(tǒng)計學意義(P0.01)；與對應的染錳組比較,�；撬犷A防各組GABA濃度均明顯提高,差異均有統(tǒng)計學意義(P0.01)；各預防組之間,GABA濃度隨著染錳濃度的增加逐漸提高,差異均有統(tǒng)計學意義(P0.01)。③細胞外液與細胞內(nèi)液3種氨基酸類神經(jīng)遞質(zhì)的平均濃度：與空白對照組比較,牛磺酸組Glu平均濃度降低,差異有統(tǒng)計學意義(P0.01)；與空白對照組比較,染錳各組Glu平均濃度均明顯增高,差異均有統(tǒng)計學意義(P0.01)；隨著染錳濃度的增加,Glu平均濃度逐漸提高,各染錳組間差異均有統(tǒng)計學意義(P0.01)；�；撬犷A防各組Glu平均濃度均低于對應的染錳組,差異均有統(tǒng)計學意義(P0.01)；各預防組之間,Glu平均濃度隨著染錳濃度的增加逐漸提高,差異均有統(tǒng)計學意義(P0.01)。與空白對照組比較,牛磺酸組Gln平均濃度降低,差異有統(tǒng)計學意義(P0.01)；與空白對照組比較,染錳各組Gln平均濃度均明顯降低,差異均有統(tǒng)計學意義(P0.01)；隨著染錳濃度的增加,Gln平均濃度逐漸降低,各染錳組間差異均有統(tǒng)計學意義(P0.01)；與對應的染錳組比較,牛磺酸預防各組Gln平均濃度均較低,差異均有統(tǒng)計學意義(P0.01)：各預防組之間,Gln平均濃度隨著染錳濃度的增加逐漸降低,差異均有統(tǒng)計學意義(P0.01)。與空白對照組比較,牛磺酸組GABA平均濃度顯著提高,差異有統(tǒng)計學意義(P0.01)；與空白對照組比較,染錳各組GABA平均濃度有所提高,但差異均無統(tǒng)計學意義(P0.05)；隨著染錳濃度的增加,GABA平均濃度逐漸提高,但各染錳組間差異均無統(tǒng)計學意義(P0.05)；與對應的染錳組比較,牛磺酸預防各組GABA平均濃度均明顯提高,差異均有統(tǒng)計學意義(P0.01)；各預防組之間,GABA平均濃度隨著染錳濃度的增加逐漸提高,差異均有統(tǒng)計學意義(P0.01)。④Glu平均濃度/GABA平均濃度：與空白對照組比較,�；撬峤M(Glu平均濃度/GABA平均濃度)的值降低,差異有統(tǒng)計學意義(P0.01)；與空白對照組比較,染錳各組(Glu平均濃度/GABA平均濃度)的值均明顯增高,差異均有統(tǒng)計學意義(P值均0.01)；與低濃度染錳組比較,中、高濃度染錳組(Glu平均濃度/GABA平均濃度)的值顯著提高,差異均有統(tǒng)計學意義(P0.01)；中、高濃度染錳組之間,(Glu平均濃度/GABA平均濃度)的值差異無統(tǒng)計學意義(P0.05)；牛磺酸預防各組(Glu平均濃度/GABA平均濃度)值均低于對應的染錳組,差異均有統(tǒng)計學意義(P0.01)；各預防組之間,(Glu平均濃度/GABA平均濃度)的值隨著染錳濃度的增加逐漸提高,但差異均無統(tǒng)計學意義(P0.05)。 [結(jié)論]錳可致體外原代培養(yǎng)的丘腦神經(jīng)細胞損傷,�；撬峥稍谝欢ǔ潭壬限卓勾硕咀饔茫籊lu可能在錳致體外原代培養(yǎng)的丘腦神經(jīng)細胞損傷機制中起到重要作用,GABA可能在�；撬徂卓瑰i致體外原代培養(yǎng)的丘腦神經(jīng)細胞損傷中機制起到重要作用。
[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.

【學位授予單位】：廣西醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R151

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