NF-κB、MAPKs等信號(hào)通路在TMT致神經(jīng)元損害中的作用與機(jī)制研究
發(fā)布時(shí)間：2018-07-25 06:52
【摘要】：第一部分TMT致細(xì)胞凋亡模型的建立及其對NF-ΚB、MAPKs等信號(hào)通路的影響目的：建立TMT中毒的體外細(xì)胞模型,研究TMT對細(xì)胞的毒性作用及NF-ΚB、MAPKs等信號(hào)通路的影響。方法：MTT法測定細(xì)胞存活率；流式細(xì)胞術(shù)檢測細(xì)胞凋亡；免疫細(xì)胞化學(xué)、免疫熒光技術(shù)、WB檢測NF-ΚB信號(hào)通路的激活；免疫細(xì)胞化學(xué)檢測Ub的變化；WB檢測MAPKs、p-Akt、Bcl-2、XIAP等蛋白的變化。結(jié)果：(1)TMT對SH-SY5Y細(xì)胞存活率與凋亡的影響：與正常對照組相比,5、10、20、40μmol/LTMT分別作用24h、48h,細(xì)胞存活率明顯下降(P0.05),有較好的劑量-時(shí)間-反應(yīng)關(guān)系；2.5、5、10μmol/LTMT能使早期凋亡率明顯增加,10μmol/LTMT能增加晚期凋亡率,TMT可誘導(dǎo)SH-SY5Y細(xì)胞凋亡,并以早期凋亡為主；(2)TMT對PC12細(xì)胞存活率與凋亡率的影響：與正常對照組相比,2.5、5、10、20、40μmol/L TMT分別作用24h、48h,細(xì)胞存活率明顯下降(P0.05),有較好的劑量-時(shí)間-反應(yīng)關(guān)系；1.25、2.5、5、10μmol/LTMT能使早期凋亡率和晚期凋亡率明顯增加,并以晚期凋亡為主；因此將選用SH-SY5Y細(xì)胞作為研究模型,染毒劑量為2.5、5、10μmol/L,作用時(shí)間為24h；(3)NF-ΚB信號(hào)通路激活：免疫細(xì)胞化學(xué)、免疫熒光技術(shù)對NF-ΚB的分布進(jìn)行定位,隨著染毒劑量增加,胞漿中的NF-ΚB p65逐漸向細(xì)胞核轉(zhuǎn)移,WB檢測核蛋白NF-ΚB p65,中、高劑量組核蛋白NF-ΚB p65相對表達(dá)量分別是對照組的2.87、4.69倍(P0.05),中、高劑量組IΚBa水平明顯下降(P0.05)；(4)Ub表達(dá)變化：5、10μmol/LTMT使細(xì)胞胞體變小,核輕微皺縮,胞漿中棕色加深,Ub表達(dá)增加；(5) MAPKs: 10μmol/LTMT可使p-JNK、p-p38增加(P0.05),2.5、5、10μmol/LTMT可使p-ERK1/2增加(P0.05),但各劑量組TMT對總MAPKs表達(dá)變化無明顯影響；(6) p-Akt、Bcl-2、XIAP:隨著TMT劑量增加,p-Akt、Bcl-2、XIAP水平均明顯下降(P0.05)。結(jié)論：在本實(shí)驗(yàn)室條件下,選擇兩種細(xì)胞株為模型,發(fā)現(xiàn)2.5、5、10μmol/L TMT可誘導(dǎo)SH-SY5Y細(xì)胞凋亡,并以早期凋亡為主,而1.25、2.5、5、10μmol/L TMT均可誘導(dǎo)PC12細(xì)胞出現(xiàn)凋亡,以晚期凋亡,即壞死為主。因此,本研究將選用SH-SY5Y細(xì)胞作為研究對象,染毒劑量為2.5、5、10μmol/L,染毒時(shí)間為24h。TMT引起SH-SY5Y細(xì)胞泛素表達(dá)增加,IΚB；α降解,NF-ΚB p65核轉(zhuǎn)位增加,p-ERX1/2、p-JNK、p-p38增加,p-Akt降低,提示NF-ΚB、UPPs、MAPKs、Akt信號(hào)通路可能參與TMT的毒作用機(jī)制。第二部分TMT致SH-SY5Y凋亡模型中NF-ΚB與MAPKs等其他信號(hào)通路的'Cross-talk"目的：以不同信號(hào)通路的特異性抑制劑為工具,從蛋白表達(dá)層面研究TMT致細(xì)胞凋亡模型中NF-ΚB與MAPKs等其他信號(hào)通路的"Cross-talk",并觀察各抑制劑對TMT致細(xì)胞凋亡的影響,試圖探明其毒性途徑。方法：MG132、BAY11-7082、SP600125、U0126、LY294002分別預(yù)處理SH-SY5Y細(xì)胞2h后,TMT染毒24h,提取蛋白,WB檢測蛋白表達(dá)變化；流式細(xì)胞術(shù)檢測各抑制劑對TMT導(dǎo)致的細(xì)胞凋亡的影響。結(jié)果：(1)蛋白酶體抑制劑MG132與NF-ΚB抑制劑BAY11-7082預(yù)處理后再進(jìn)行TMT染毒,早期凋亡率明顯高于單獨(dú)的TMT染毒(P0.05),對TMT誘導(dǎo)的細(xì)胞凋亡有促進(jìn)作用；而SP600125、U0126、LY294002作用后TMT誘導(dǎo)的早期凋亡率明顯降低(P0.05),可拮抗TMT誘導(dǎo)的細(xì)胞凋亡。(2)泛素-蛋白酶體途徑：與單獨(dú)的TMT染毒組相比,蛋白酶體抑制劑MG132預(yù)處理使核蛋白NF-ΚB減少,總蛋白IΚBa增加(P0.05)；磷酸化ERK/總ERK比值降低(P0.05)；磷酸化JNK/總JNK比值升高(P0.05)；磷酸化p38/總p38比值降低(P0.05)；Bcl-2表達(dá)減少(P0.05)。(3) NF-ΚB信號(hào)通路：與單獨(dú)的TMT染毒組相比,NF-ΚB特異性抑制劑BAY11-7082預(yù)處理可使核蛋白NF-ΚB p65減少(P0.05)；總蛋白IΚBα增加(P0.05)；磷酸化ERK/總ERK比值減小(P0.05)；磷酸化JNK/總JNK比值增加(P0.05)；磷酸化p38/總p38比值減小(P0.05)；Bcl-2表達(dá)減少(P0.05)；XIAP表達(dá)減少(P0.05)。(4) JNK/SAPK信號(hào)通路：與單獨(dú)的TMT染毒組相比,20μM JNK特異性抑制劑SP600125預(yù)處理可使核蛋白NF-ΚB p65增加(P0.05)；總蛋白IΚBa減少(P0.05)；磷酸化ERK/總ERK比值增加(P0.05)；磷酸化JNK/總JNK比值降低(P0.05)；磷酸化p38/總p38比值降低(P0.05)；磷酸化Akt水平降低(P0.05)；Bcl-2表達(dá)增加(P0.05)；XIAP表達(dá)增加(P0.05)。(5)ERK1/2信號(hào)通路：與單獨(dú)的TMT染毒組相比,10μM ERK1/2特異性抑制劑U0126預(yù)處理可使核蛋白NF-ΚB p65增加(P0.05)；總蛋白IΚBa減少(P0.05)；磷酸化ERK/總ERK比值降低(P0.05)：磷酸化JNK/總JNK比值增加(P0.05)；磷酸化p38/總p38比值降低(P0.05)；Bcl-2表達(dá)增加(P0.05)；XIAP表達(dá)增加(P0.05)。(6) P13K/Akt信號(hào)通路：與單獨(dú)的TMT染毒組相比,20μM Akt特異性抑制劑LY294002預(yù)處理可使磷酸化ERK/總ERK比值降低(P0.05)；磷酸化JNK/總JNK比值升高(P0.05)：磷酸化p38/總p38比值升高(P0.05)；XIAP表(?)增加(P0.05)。結(jié)論：在TMT致細(xì)胞凋亡模型中,NF-ΚB、泛素蛋白酶體信號(hào)通路的激活使Bcl-2、XIAP表達(dá)增加,具有拮抗細(xì)胞凋亡的作用,泛素蛋白酶體信號(hào)通路(?)NF-ΚB的激活；而JNK、ERK1/2信號(hào)通路的激活使Bcl-2、XIAP表達(dá)減少,進(jìn)細(xì)胞凋亡。途徑研究表明各信號(hào)通路間存在“Cross-talk":NF-ΚB與JNK信號(hào)通路相互抑制,JNK與ERK1/2信號(hào)通路間相互抑制,JNK促進(jìn)p38信號(hào)通路的激活,而ERK1/2與NF-kB則抑制p38信號(hào)通路,ERK1/2對NF-kB信號(hào)通路也有抑制作用。這些信號(hào)通路問相互調(diào)節(jié),最終結(jié)局為TMT使Bcl-2、XIAP蛋白表達(dá)減少,凋亡發(fā)生。第三部分TMT中毒整體動(dòng)物模型的建立目的：建立TMT中毒的體內(nèi)模型,研究TMT對大鼠學(xué)習(xí)記憶能力的影響。方法：40只雄性SD大鼠隨機(jī)分為4組,每組10只；TMT染毒劑量分別為0.75、1.5、3mg/kg以及溶劑對照組(雙蒸水)；每日上午8點(diǎn)經(jīng)口灌胃染毒,下午兩點(diǎn)進(jìn)行水迷宮實(shí)驗(yàn),晚上8點(diǎn)進(jìn)行一般行為學(xué)評分與癲癇評分,共6天；末次染毒24h后斷頭處死動(dòng)物,收集血液,分離血清,測定血液生化指標(biāo)及血清鉀離子濃度；分離臟器,計(jì)算臟器系數(shù)；每只動(dòng)物大腦一分為二,一半用于制備石蠟切片,一半分離海馬、皮層等,凍存?zhèn)溆�。結(jié)果：(1)一般行為改變：TMT可引起大鼠出現(xiàn)震顫、尖叫、易激惹、攻擊性、陣攣等神經(jīng)系統(tǒng)中毒癥狀,高劑量組從第4天開始,出現(xiàn)中毒癥狀,癥狀評分分值增加,第5、6、7天癥狀評分分值持續(xù)升高,明顯高于對照組(P0.05)；并可誘發(fā)癲癇,各劑量組在染毒初期均無明顯癲癇癥狀,均處于癲癇分級0,染毒結(jié)束后(第7天),對照組90%動(dòng)物仍然處于0級,低劑量組90%動(dòng)物也處于0級,中劑量組60%動(dòng)物為0級,40%動(dòng)物為1級,高劑量組所有動(dòng)物均達(dá)到5級；(2)大鼠體重變化：與對照組相比,3mg/kg組第6、7天體重明顯下降(P0.05)；(3)生化指標(biāo)改變：與對照組相比高劑量組AST、ALT明顯升高(P0.05),中、高劑量組BUN、Cr明顯升高(P0.05),高劑量組血清鉀離子濃度明顯降低(P0.05)；(4)學(xué)習(xí)記憶能力改變：訓(xùn)練第四天,各組大鼠逃避潛伏期明顯縮短(P0.05),隨著染毒繼續(xù),低劑量組與對照組持續(xù)縮短,而中、高劑量組逃避潛伏期反而增加(P0.05),第6天訓(xùn)練結(jié)束后撤掉平臺(tái),中、高劑量組在目標(biāo)區(qū)域停留時(shí)間比例較少(P0.05),穿越平臺(tái)次數(shù)較少(P0.05)。結(jié)論：本室成功建立TMT中毒的整體動(dòng)物模型,3mg/kgTMT可引起SD大鼠中毒,觀察到震顫、易激惹、攻擊性、癲癇發(fā)作等癥狀,以及血清鉀離子濃度明顯下降等特異性中毒特征,TMT對學(xué)習(xí)記憶能力也有顯著的影響。第四部分TMT對大鼠海馬神經(jīng)元的損傷及機(jī)制研究目的：觀察TMT對海馬神經(jīng)元的損傷及分子生物學(xué)機(jī)制研究。方法：尼氏染色觀察神經(jīng)元損傷；流式細(xì)胞術(shù)測定大鼠海馬神經(jīng)元凋亡率；免疫組織化學(xué)法檢測泛素水平變化；Western blot檢測NF-ΚB、MAPKs等信號(hào)通路蛋白變化。結(jié)果：(1)神經(jīng)細(xì)胞損傷：尼氏染色表明中、高劑量組海馬各區(qū)神經(jīng)細(xì)胞排列紊亂、尼氏小體減少、細(xì)胞間隙變大、細(xì)胞腫脹、數(shù)量減少；(2)TMT對大鼠海馬神經(jīng)元凋亡的影響：與對照組相比,各劑量組海馬神經(jīng)元早期凋亡率明顯增加(P0.05),并且有劑量-反應(yīng)關(guān)系；(3)泛素表達(dá)變化：中、高劑量組海馬各區(qū)泛素蛋白沉積明顯增加,表現(xiàn)為棕黃色顆粒物增加；(4)蛋白免疫印跡檢測海馬蛋白表達(dá)變化：與對照組相比,高劑量組核蛋白NF-ΚB水平增加,總蛋白IΚBα減少,p-JNK增加,低、中、高劑量組p-p38均增加,中、高劑量組p-ERK1/2增加,差異均有統(tǒng)計(jì)學(xué)意義(P0.05)。結(jié)論：在整體動(dòng)物模型中,TMT可導(dǎo)致神經(jīng)元尼氏體丟失,神經(jīng)細(xì)胞凋亡,其分子生物學(xué)機(jī)制涉及NF-ΚB、MAPKs、泛素-蛋白酶體信號(hào)通路的激活,與體外細(xì)胞模型較為吻合。
[Abstract]:The first part of the TMT induced apoptosis model and its effect on the signal pathways such as NF- B, MAPKs and other signal pathways: to establish the cell model of TMT poisoning in vitro, to study the toxicity of TMT to the cells and the effect of NF- B, MAPKs and other signaling pathways. Methods: MTT method was used to determine cell survival rate; flow cytometry was used to detect cell apoptosis; immunization was used. Study, immunofluorescence technology, WB detection of the activation of NF- B signaling pathway; immunocytochemical detection of Ub changes; WB detection of MAPKs, p-Akt, Bcl-2, XIAP and other proteins. Results: (1) TMT on the survival rate and apoptosis of SH-SY5Y cells: compared with the normal control group Down (P0.05), there is a good dose time response relationship; 2.5,5,10 micron mol/LTMT can increase the early apoptosis rate obviously, 10 mu mol/LTMT can increase the late apoptosis rate, TMT can induce apoptosis of SH-SY5Y cells and early apoptosis; (2) the effect of TMT on the survival rate and apoptosis rate of PC12 cells: 2.5,5,10,20,40 mu mol/L compared with the normal control group The survival rate of 24h and 48h decreased significantly (P0.05), and there was a good dose time response relationship; 1.25,2.5,5,10 micron mol/LTMT could increase the early apoptosis rate and the late apoptosis rate, and was mainly in the late stage of apoptosis. Therefore, SH-SY5Y cells were selected as the research model, the dose was 2.5,5,10 u mol/L, and the action time was 24h. The action time was 24h. (3) the activation of NF- B signaling pathway: immunocytochemistry and immunofluorescence technique to locate the distribution of NF- B, with the increase of the dose, the NF- B p65 in the cytoplasm gradually transferred to the nucleus, WB detection of the nucleoprotein NF- B p65, and the high dose of nucleoprotein NF- The level of I Ba in the dose group decreased (P0.05), and (4) the changes in Ub expression: 5,10 micron mol/LTMT made the cell body smaller, the nucleus slightly crinkled, the cytoplasm brown deepened, and the Ub expression increased; (5) MAPKs: 10 micron could increase p-JNK, p-p38 increase (P0.05). Obviously, (6) p-Akt, Bcl-2, XIAP: decreased significantly with the increase of TMT dose, p-Akt, Bcl-2 and XIAP (P0.05). Conclusion: in the laboratory conditions, two cell lines were selected as models, and 2.5,5,10 micron mol/L TMT could induce apoptosis in SH-SY5Y cells. In this study, SH-SY5Y cells are selected as the research object, and the dose of SH-SY5Y cells is 2.5,5,10 Mu and 24h.TMT causes SH-SY5Y cells to increase the expression of ubiquitin, I B, alpha degradation, NF- B p65 nuclear transposition, p-ERX1/ 2, p-JNK, p-JNK, p-JNK, decreasing, decreasing Signaling pathway may be involved in the toxic mechanism of TMT. Second part TMT induces'Cross-talk in other signaling pathways, such as NF-, B and MAPKs in SH-SY5Y apoptosis model, "objective: To study NF- B and MAPKs and other signaling pathways in TMT induced apoptosis models from the protein expression level by the specific inhibitors of different signaling pathways. K "and observe the effect of each inhibitor on apoptosis induced by TMT, and try to explore the toxic pathway. Methods: MG132, BAY11-7082, SP600125, U0126, LY294002 were pretreated with SH-SY5Y cells 2h, TMT was poisoned 24h, protein extraction, protein expression changes; flow cytometry was used to detect the effect of each inhibitor on apoptosis. (1) the proteasome inhibitor MG132 and the NF- B inhibitor BAY11-7082 were pretreated with TMT, and the early apoptosis rate was significantly higher than that of TMT (P0.05), which could promote the apoptosis induced by TMT, while SP600125, U0126 and LY294002 acted on the apoptosis rate of the early stage induced by TMT, and could antagonize the cells induced by TMT. Apoptosis. (2) the ubiquitin proteasome pathway: compared with the single TMT, proteasome inhibitor MG132 preconditioning reduces the nuclear protein NF- B, the total protein I Ba increases (P0.05); the total ERK ratio of the phosphorylated ERK/ is reduced (P0.05); the JNK/ total JNK ratio of phosphorylation is increased; the expression of phosphorylated ERK/ is reduced; the expression decreases (P0.05) (3) NF- B signaling pathway: compared with a single TMT exposure group, the NF- B specific inhibitor BAY11-7082 preconditioning can reduce the nucleoprotein NF- B p65 (P0.05); the total protein I decreases; Bcl-2 expression decreased (P0.05); XIAP expression decreased (P0.05). (4) JNK/SAPK signaling pathway: compared with a single TMT exposure group, the 20 M JNK specific inhibitor SP600125 preconditioning can increase the nuclear protein NF- B p65; (P0.05); the total p38 ratio of phosphorylated p38/ decreased (P0.05); the level of phosphorylated Akt decreased (P0.05); Bcl-2 expression increased (P0.05); XIAP expression increased (P0.05). (5) ERK1/2 signaling pathway .05); the total ERK ratio of phosphorylated ERK/ decreased (P0.05): the total JNK ratio of phosphorylated JNK/ increased (P0.05), the total p38 ratio of phosphorylated p38/ decreased (P0.05), Bcl-2 expression increased (P0.05); (6) The total ERK ratio of RK/ decreased (P0.05); the total JNK ratio of phosphorylated JNK/ increased (P0.05): the total p38 ratio of phosphorylated p38/ increased (P0.05); XIAP table (?) increased (P0.05). Conclusion: in the apoptosis model of the TMT cell, the activation of the ubiquitin proteasome signaling pathway increases the expression of ubiquitin proteasome, and has the effect of antagonistic apoptosis, ubiquitin protease The activation of the body signal pathway (?) NF- B; while the activation of JNK, ERK1/2 signaling pathway reduces the expression of Bcl-2 and XIAP and enters the cell apoptosis. The pathway study indicates that there is a "Cross-talk" between the signaling pathways, and that the NF- B and JNK signaling pathways are mutually inhibited, and that JNK and ERK1/2 signal pathways are interdependent and promote the activation of the signaling pathway. Inhibition of the p38 signaling pathway and the inhibitory effect of ERK1/2 on the NF-kB signaling pathway. These signaling pathways are mutually regulated, and the final outcome is that TMT causes Bcl-2, XIAP protein expression to decrease, and apoptosis occurs. Third part of the whole animal model of TMT poisoning: establish an intracellular model of TMT poisoning and study the shadow of TMT on learning and memory ability in rats. Methods: 40 male SD rats were randomly divided into 4 groups, with 10 rats in each group; the dose of TMT was 0.75,1.5,3mg/kg and the control group (double water), at 8 p.m. daily, the water maze experiment was carried out at two o'clock in the afternoon, the general behavior score and the epileptic score were 6 days at 8 o'clock in the evening, and the last time after 24h was executed. Animals, collect blood, separate serum, determine blood biochemical index and serum potassium ion concentration, separate organs, calculate viscera coefficient; each animal brain is divided into two, half used to prepare paraffin section, half of hippocampus, cortex, etc., and frozen in reserve. Results: (1) general behavior changes: TMT can cause tremor, screaming, irritability in rats. Intoxication symptoms such as provoking, aggression, clonus and other nervous system poisoning, the high dose group started from fourth days, the symptom of poisoning, the score of symptom increased, the score of symptom score increased continuously on day 5,6,7, obviously higher than that of the control group (P0.05), and could induce epilepsy. After seventh days (seventh days), 90% animals in the control group were still in grade 0, 90% in low dose group were 0, 60% in middle dose group were 0, 40% animals were 1, and all animals in high dose group reached 5. (2) body weight changes in rats: Group 6,7 days weight decreased significantly compared with control group (P0.05); (3) biochemical index changes: and control group Compared to high dose group AST, ALT significantly increased (P0.05), high dose group BUN, Cr significantly increased (P0.05), high dose group potassium ion concentration decreased significantly (P0.05); (4) learning and memory ability changes: training fourth days, rats escape latency significantly shortened (P0.05), with continued exposure, low dose group and the control group continued to shorten, and middle, In high dose group, the escape latency increased (P0.05). After sixth days of training, the platform was withdrawn. In the high dose group, the stay time in the target area was less (P0.05), and the number of crossing platforms was less (P0.05). Conclusion: the whole animal model of TMT poisoning was established successfully in this room. 3mg/kgTMT could cause the poisoning of SD rats, and observed the tremor, irritability and attack. The characteristics of specific poisoning, such as striking, epileptic seizures, and a significant decrease in the concentration of serum potassium ions, and the significant effects of TMT on learning and memory. Fourth the damage and mechanism of TMT on the hippocampus neurons in rats: the study of the damage and molecular biological mechanism of the hippocampal neurons by TMT. Methods: Nissl staining The apoptosis rate of hippocampal neurons in rats was measured by flow cytometry; changes in ubiquitin level were detected by immunohistochemistry; Western blot was used to detect the changes in signal pathway proteins such as NF- B and MAPKs. Results: (1) nerve cell damage: Nissl staining showed that the nerve cells in the hippocampus of the high dose group were in disorder, Nissl was small The effect of TMT on the apoptosis of hippocampal neurons in rats: the early apoptosis rate of hippocampal neurons in each dose group increased significantly (P0.05), and there was a dose-response relationship with the control group. (3) the ubiquitin protein deposition in the hippocampus of the high dose group increased significantly in the high dose group. The expression of brown yellow particles increased; (4) protein immunoblotting detected the changes in the expression of hippocampal protein: compared with the control group, the level of nuclear protein NF- B increased in the high dose group, the total protein I B alpha decreased, the p-JNK increased, and the high dose group p-p38 increased, and the high dose group p-ERK1/2 increased, the difference was statistically significant (P0.05). Conclusion: in the high dose group, the difference is statistically significant (P0.05). Conclusion: in the group (P0.05). In the whole animal model, TMT can lead to the loss of Nissl body and apoptosis of neurons, and its molecular mechanism involves the activation of NF- B, MAPKs, and ubiquitin proteasome signaling pathway, which is more consistent with the cell model in vitro.
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2012
【分類號(hào)】：R363
本文編號(hào)：2142993
[Abstract]:The first part of the TMT induced apoptosis model and its effect on the signal pathways such as NF- B, MAPKs and other signal pathways: to establish the cell model of TMT poisoning in vitro, to study the toxicity of TMT to the cells and the effect of NF- B, MAPKs and other signaling pathways. Methods: MTT method was used to determine cell survival rate; flow cytometry was used to detect cell apoptosis; immunization was used. Study, immunofluorescence technology, WB detection of the activation of NF- B signaling pathway; immunocytochemical detection of Ub changes; WB detection of MAPKs, p-Akt, Bcl-2, XIAP and other proteins. Results: (1) TMT on the survival rate and apoptosis of SH-SY5Y cells: compared with the normal control group Down (P0.05), there is a good dose time response relationship; 2.5,5,10 micron mol/LTMT can increase the early apoptosis rate obviously, 10 mu mol/LTMT can increase the late apoptosis rate, TMT can induce apoptosis of SH-SY5Y cells and early apoptosis; (2) the effect of TMT on the survival rate and apoptosis rate of PC12 cells: 2.5,5,10,20,40 mu mol/L compared with the normal control group The survival rate of 24h and 48h decreased significantly (P0.05), and there was a good dose time response relationship; 1.25,2.5,5,10 micron mol/LTMT could increase the early apoptosis rate and the late apoptosis rate, and was mainly in the late stage of apoptosis. Therefore, SH-SY5Y cells were selected as the research model, the dose was 2.5,5,10 u mol/L, and the action time was 24h. The action time was 24h. (3) the activation of NF- B signaling pathway: immunocytochemistry and immunofluorescence technique to locate the distribution of NF- B, with the increase of the dose, the NF- B p65 in the cytoplasm gradually transferred to the nucleus, WB detection of the nucleoprotein NF- B p65, and the high dose of nucleoprotein NF- The level of I Ba in the dose group decreased (P0.05), and (4) the changes in Ub expression: 5,10 micron mol/LTMT made the cell body smaller, the nucleus slightly crinkled, the cytoplasm brown deepened, and the Ub expression increased; (5) MAPKs: 10 micron could increase p-JNK, p-p38 increase (P0.05). Obviously, (6) p-Akt, Bcl-2, XIAP: decreased significantly with the increase of TMT dose, p-Akt, Bcl-2 and XIAP (P0.05). Conclusion: in the laboratory conditions, two cell lines were selected as models, and 2.5,5,10 micron mol/L TMT could induce apoptosis in SH-SY5Y cells. In this study, SH-SY5Y cells are selected as the research object, and the dose of SH-SY5Y cells is 2.5,5,10 Mu and 24h.TMT causes SH-SY5Y cells to increase the expression of ubiquitin, I B, alpha degradation, NF- B p65 nuclear transposition, p-ERX1/ 2, p-JNK, p-JNK, p-JNK, decreasing, decreasing Signaling pathway may be involved in the toxic mechanism of TMT. Second part TMT induces'Cross-talk in other signaling pathways, such as NF-, B and MAPKs in SH-SY5Y apoptosis model, "objective: To study NF- B and MAPKs and other signaling pathways in TMT induced apoptosis models from the protein expression level by the specific inhibitors of different signaling pathways. K "and observe the effect of each inhibitor on apoptosis induced by TMT, and try to explore the toxic pathway. Methods: MG132, BAY11-7082, SP600125, U0126, LY294002 were pretreated with SH-SY5Y cells 2h, TMT was poisoned 24h, protein extraction, protein expression changes; flow cytometry was used to detect the effect of each inhibitor on apoptosis. (1) the proteasome inhibitor MG132 and the NF- B inhibitor BAY11-7082 were pretreated with TMT, and the early apoptosis rate was significantly higher than that of TMT (P0.05), which could promote the apoptosis induced by TMT, while SP600125, U0126 and LY294002 acted on the apoptosis rate of the early stage induced by TMT, and could antagonize the cells induced by TMT. Apoptosis. (2) the ubiquitin proteasome pathway: compared with the single TMT, proteasome inhibitor MG132 preconditioning reduces the nuclear protein NF- B, the total protein I Ba increases (P0.05); the total ERK ratio of the phosphorylated ERK/ is reduced (P0.05); the JNK/ total JNK ratio of phosphorylation is increased; the expression of phosphorylated ERK/ is reduced; the expression decreases (P0.05) (3) NF- B signaling pathway: compared with a single TMT exposure group, the NF- B specific inhibitor BAY11-7082 preconditioning can reduce the nucleoprotein NF- B p65 (P0.05); the total protein I decreases; Bcl-2 expression decreased (P0.05); XIAP expression decreased (P0.05). (4) JNK/SAPK signaling pathway: compared with a single TMT exposure group, the 20 M JNK specific inhibitor SP600125 preconditioning can increase the nuclear protein NF- B p65; (P0.05); the total p38 ratio of phosphorylated p38/ decreased (P0.05); the level of phosphorylated Akt decreased (P0.05); Bcl-2 expression increased (P0.05); XIAP expression increased (P0.05). (5) ERK1/2 signaling pathway .05); the total ERK ratio of phosphorylated ERK/ decreased (P0.05): the total JNK ratio of phosphorylated JNK/ increased (P0.05), the total p38 ratio of phosphorylated p38/ decreased (P0.05), Bcl-2 expression increased (P0.05); (6) The total ERK ratio of RK/ decreased (P0.05); the total JNK ratio of phosphorylated JNK/ increased (P0.05): the total p38 ratio of phosphorylated p38/ increased (P0.05); XIAP table (?) increased (P0.05). Conclusion: in the apoptosis model of the TMT cell, the activation of the ubiquitin proteasome signaling pathway increases the expression of ubiquitin proteasome, and has the effect of antagonistic apoptosis, ubiquitin protease The activation of the body signal pathway (?) NF- B; while the activation of JNK, ERK1/2 signaling pathway reduces the expression of Bcl-2 and XIAP and enters the cell apoptosis. The pathway study indicates that there is a "Cross-talk" between the signaling pathways, and that the NF- B and JNK signaling pathways are mutually inhibited, and that JNK and ERK1/2 signal pathways are interdependent and promote the activation of the signaling pathway. Inhibition of the p38 signaling pathway and the inhibitory effect of ERK1/2 on the NF-kB signaling pathway. These signaling pathways are mutually regulated, and the final outcome is that TMT causes Bcl-2, XIAP protein expression to decrease, and apoptosis occurs. Third part of the whole animal model of TMT poisoning: establish an intracellular model of TMT poisoning and study the shadow of TMT on learning and memory ability in rats. Methods: 40 male SD rats were randomly divided into 4 groups, with 10 rats in each group; the dose of TMT was 0.75,1.5,3mg/kg and the control group (double water), at 8 p.m. daily, the water maze experiment was carried out at two o'clock in the afternoon, the general behavior score and the epileptic score were 6 days at 8 o'clock in the evening, and the last time after 24h was executed. Animals, collect blood, separate serum, determine blood biochemical index and serum potassium ion concentration, separate organs, calculate viscera coefficient; each animal brain is divided into two, half used to prepare paraffin section, half of hippocampus, cortex, etc., and frozen in reserve. Results: (1) general behavior changes: TMT can cause tremor, screaming, irritability in rats. Intoxication symptoms such as provoking, aggression, clonus and other nervous system poisoning, the high dose group started from fourth days, the symptom of poisoning, the score of symptom increased, the score of symptom score increased continuously on day 5,6,7, obviously higher than that of the control group (P0.05), and could induce epilepsy. After seventh days (seventh days), 90% animals in the control group were still in grade 0, 90% in low dose group were 0, 60% in middle dose group were 0, 40% animals were 1, and all animals in high dose group reached 5. (2) body weight changes in rats: Group 6,7 days weight decreased significantly compared with control group (P0.05); (3) biochemical index changes: and control group Compared to high dose group AST, ALT significantly increased (P0.05), high dose group BUN, Cr significantly increased (P0.05), high dose group potassium ion concentration decreased significantly (P0.05); (4) learning and memory ability changes: training fourth days, rats escape latency significantly shortened (P0.05), with continued exposure, low dose group and the control group continued to shorten, and middle, In high dose group, the escape latency increased (P0.05). After sixth days of training, the platform was withdrawn. In the high dose group, the stay time in the target area was less (P0.05), and the number of crossing platforms was less (P0.05). Conclusion: the whole animal model of TMT poisoning was established successfully in this room. 3mg/kgTMT could cause the poisoning of SD rats, and observed the tremor, irritability and attack. The characteristics of specific poisoning, such as striking, epileptic seizures, and a significant decrease in the concentration of serum potassium ions, and the significant effects of TMT on learning and memory. Fourth the damage and mechanism of TMT on the hippocampus neurons in rats: the study of the damage and molecular biological mechanism of the hippocampal neurons by TMT. Methods: Nissl staining The apoptosis rate of hippocampal neurons in rats was measured by flow cytometry; changes in ubiquitin level were detected by immunohistochemistry; Western blot was used to detect the changes in signal pathway proteins such as NF- B and MAPKs. Results: (1) nerve cell damage: Nissl staining showed that the nerve cells in the hippocampus of the high dose group were in disorder, Nissl was small The effect of TMT on the apoptosis of hippocampal neurons in rats: the early apoptosis rate of hippocampal neurons in each dose group increased significantly (P0.05), and there was a dose-response relationship with the control group. (3) the ubiquitin protein deposition in the hippocampus of the high dose group increased significantly in the high dose group. The expression of brown yellow particles increased; (4) protein immunoblotting detected the changes in the expression of hippocampal protein: compared with the control group, the level of nuclear protein NF- B increased in the high dose group, the total protein I B alpha decreased, the p-JNK increased, and the high dose group p-p38 increased, and the high dose group p-ERK1/2 increased, the difference was statistically significant (P0.05). Conclusion: in the high dose group, the difference is statistically significant (P0.05). Conclusion: in the group (P0.05). In the whole animal model, TMT can lead to the loss of Nissl body and apoptosis of neurons, and its molecular mechanism involves the activation of NF- B, MAPKs, and ubiquitin proteasome signaling pathway, which is more consistent with the cell model in vitro.
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2012
【分類號(hào)】：R363
【引證文獻(xiàn)】
相關(guān)碩士學(xué)位論文 前2條
1 付留中;IL-4、IL-6、TNF-a、NF-κB在椎間盤源性下腰痛發(fā)病中的作用[D];新鄉(xiāng)醫(yī)學(xué)院;2013年
2 杜清清;溴苯腈對SH-SY5Y細(xì)胞毒性及NF-κB、MAPKs通路的影響[D];華中科技大學(xué);2013年
，本文編號(hào)：2142993
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