本文選題：納米氧化鋁 + 氧化應激�。� 參考：《山西醫(yī)科大學》2014年碩士論文

【摘要】：目的通過采用不同粒徑的納米氧化鋁在不同時點對ICR小鼠進行急性、亞慢性染毒，來探討納米氧化鋁對小鼠學習記憶能力損傷的粒徑效應、時間效應以及其可能機制。 方法 Ⅰ.選取72只健康成年ICR小鼠，分為4組（對照組(生理鹽水)、10μm Al2O3組、50nmAl2O3組、13nm Al2O3組），每組18只，染毒后分為3個觀察期（3天、7天、14天），每個觀察期、每組小鼠6只。用灌胃法進行急性染毒，染毒劑量均為5g/kg，每個觀察期結束后處死小鼠，取腦，測腦體比，分離皮質(zhì)，檢測炎癥因子（TNF-α、IL-1β）和氧化應激水平（MDA、SOD），其中觀察期為14天小鼠，觀察結束后先用Morris水迷宮對小鼠進行學習記憶能力檢測，而后同上。 Ⅱ.選取120只健康成年ICR小鼠，分為4組(對照組(生理鹽水)、10μmAl2O3組（50mg/ml）、50nm Al2O3組（50mg/ml）、13nmAl2O3組（50mg/ml）)，每組30只，經(jīng)滴鼻染毒法染毒，連續(xù)染毒時間分為3個時間段（30天、60天、90天），其中，每個時間段每個處理組平均10只小鼠。各時間段組染毒結束后用Morris水迷宮對小鼠進行學習記憶能力檢測。行為學檢測結束后每組取兩只用4%多聚甲醛灌注，取腦做病理切片，進一步做HE染色、硫堇染色，剩余小鼠處死后取腦組織，測定炎癥因子反應（TNF-α、IL-1β）和氧化應激水平（MDA、SOD、GSH、GSH-PX），Western-blot法檢測溶酶體相關蛋白（Cathepsin-B、Cathepsin-L），凋亡相關蛋白（C-Caspase3），自噬相關蛋白（LC3- Ⅱ、Beclin-1），壞死相關蛋白（RIP）的蛋白表達水平。 結果 Ⅰ.急性染毒結果顯示：水迷宮結果顯示：13nmAl2O3組與其余各組相比，學習記憶能力顯著下降，差異有統(tǒng)計學意義（P0.05）。炎癥因子結果顯示：與對照組相比，炎癥因子首先在第3天（TNF-α）、第7天（IL-1β）顯著升高，各組差異有統(tǒng)計學意義（P0.05），但在14天各染毒組與對照組相比，差異無統(tǒng)計學意義（P0.05）。氧化應激結果顯示：與對照組相比，各染毒組的MDA含量在第3天差異無統(tǒng)計學意義（P0.05），從第7天開始顯著增加，差異有統(tǒng)計學意義（P0.05），到第14天達到峰值；與對照組相比，各染毒組SOD活性從第3天開始隨時間延長逐漸降低，到第14天降到最低水平，差異有統(tǒng)計學意義（P0.05），且有隨粒徑減小其活性顯著降低（P0.05）。 Ⅱ.亞慢性染毒結果顯示：水迷宮結果顯示：與對照組相比，各染毒組逃避潛伏期差別有統(tǒng)計學意義（P0.05），50nm Al2O3組、13nm Al2O3組逃避潛伏期顯著高于10μm Al2O3組和對照組，差異有統(tǒng)計學意義（P0.05）；各組不同時間段逃避潛伏期差別有統(tǒng)計學意義（P0.05），隨時間的延長，逃避潛伏期時間逐漸延長。 病理結果顯示：HE染色光學顯微鏡下觀察顯示：對照組海馬CA3區(qū)神經(jīng)細胞形態(tài)正常，排列整齊，連接緊密；10μm Al2O3組海馬CA3區(qū)神經(jīng)細胞數(shù)量減少；50nm Al2O3組小鼠海馬CA3區(qū)神經(jīng)細胞數(shù)量進一步減少，胞漿出現(xiàn)腫大，染色質(zhì)比較疏松；13nm Al2O3組小鼠海馬CA3區(qū)神經(jīng)細胞數(shù)量明顯減少，胞核固縮；隨著時間的延長，各染毒組小鼠海馬CA3區(qū)神經(jīng)細胞數(shù)量減少，錐體層細胞稀疏、松散且排列無序，層次不清晰，連接不緊密。硫堇染色光學顯微鏡下觀察顯示：對照組海馬CA3區(qū)細胞數(shù)量多排列規(guī)則，普遍染色較深，細胞形態(tài)清晰，可見尼氏體顆粒；10μm Al2O3組海馬CA3區(qū)細胞數(shù)量減少，部分細胞腫脹呈空泡樣，胞漿染色深淺不一，50nmAl2O3組海馬CA3區(qū)細胞排列雜亂，細胞空泡樣改變，染色更淺，13nm Al2O3組海馬CA3區(qū)空泡現(xiàn)象更嚴重，神經(jīng)元呈圓形；隨著時間延長，各染毒組小鼠海馬CA3區(qū)神經(jīng)細胞數(shù)量減少，染色變淺，尼氏體逐漸溶解并減少。 炎癥因子結果顯示：與對照組相比，各染毒組TNF-a含量，差異有統(tǒng)計學意義（P0.05），隨粒徑的減小TNF-a含量逐漸升高；隨時間的延長各染毒組TNF-a含量差別有統(tǒng)計學意義（P0.05），30天與60天、90天相比差異有統(tǒng)計學意義（P0.05）；60天與90天相比差異無統(tǒng)計學意義（P0.05）；各組IL-1β表達水平在粒徑水平、時間水平均無顯著差異（P0.05）。氧化應激結果顯示：與對照組相比，各染毒組MDA含量差異均有統(tǒng)計學意義（P0.05），隨粒徑減小含量增加，隨時間延長含量增加；與對照組相比，各染毒組SOD活性差異有統(tǒng)計學意義（P0.05），隨粒徑減小活性降低，隨時間延長活性降低；與對照組相比，各染毒組GSH含量在粒徑水平、時間水平均無顯著差異（P0.05）；與對照組相比，各染毒組GSH-PX活性差異有統(tǒng)計學意義（P0.05），隨粒徑減小活性降低，隨時間的延長活性降低。 Western blot法檢測氧化鋁染毒小鼠大腦皮質(zhì)內(nèi)相關蛋白：溶酶體的相關蛋白（Cathepsin-B、Cathepsin-L）結果顯示：與對照組相比，各染毒組蛋白表達水平，差異有統(tǒng)計學意義（P0.05），隨粒徑減小蛋白表達水平增高，隨時間延長表達水平增高。凋亡相關蛋白（C-Caspase3）結果顯示：與對照組相比，各染毒組蛋白表達水平差異有統(tǒng)計學意義（P0.05），隨粒徑減小蛋白表達水平增高，隨時間延長表達水平增高。自噬相關蛋白（LC3-Ⅱ、Beclin-1）結果顯示：與對照組相比，各染毒組蛋白表達水平差異有統(tǒng)計學意義（P0.05），隨粒徑減小蛋白表達水平增高，隨時間延長表達水平增高。壞死相關蛋白（RIP）結果顯示：與對照組相比，各染毒組蛋白變化率差異有統(tǒng)計學意義（P0.05），隨粒徑減小蛋白表達水平增高，隨時間延長表達水平增高。 結論 Ⅰ.納米氧化鋁經(jīng)灌胃法一次急性染毒可以造成腦組織氧化應激損傷和炎癥因子反應，降低小鼠的學習記憶能力，隨粒徑的減小，時間的延長損傷增大，，存在粒徑效應、時間效應。炎癥因子是這一過程中的早期事件，而氧化應激水平的升高可能是學習記憶能力下降的主要原因之一。 Ⅱ.納米氧化鋁經(jīng)滴鼻法亞慢性染毒，可以導致海馬神經(jīng)元的結構和功能損傷，造成氧化應激損傷和炎癥因子反應，誘發(fā)了溶酶體相關蛋白表達的升高，進而導致細胞死亡，凋亡、自噬、壞死均參與了這一過程。神經(jīng)細胞的死亡是造成學習記憶能力死亡的主要原因之一。 Ⅲ.納米氧化鋁染毒提示存在慢性毒性，其中50nm Al2O3組在30天到60天毒性較大，13nmAl2O3組在不同時間點毒性一直呈上升狀態(tài)。
[Abstract]:Objective To investigate the effect of nano - alumina on the effects of nano - alumina on learning and memory ability of mice and its possible mechanism by using nano - alumina with different particle sizes to study the effects of nano - alumina on the damage of learning and memory ability of mice .

method

I . 72 healthy adult ICR mice were divided into 4 groups ( control group ( normal saline ) , 10 渭m Al2O3 group , 50 nm Al2O3 group and 13 nm Al2O3 group . Each group was divided into 3 observation periods ( 3 days , 7 days , 14 days ) . Each group of mice was divided into 3 observation periods ( 3 days , 7 days , 14 days ) .

Methods 120 healthy adult ICR mice were divided into 4 groups ( control group ( normal saline ) , 10 渭m Al2O3 group ( 50 mg / ml ) , 50 nm Al2O3 group ( 50 mg / ml ) and 13 nm Al2O3 group ( 50 mg / ml ) .

II . Beclin - 1 ) , protein expression level of necrosis - related protein ( RIP ) .

Results

Compared with the control group , there was no significant difference in the levels of MDA in the groups compared with the control group ( P0.05 ) . The results showed that the MDA content of each group increased significantly in the third day ( P0.05 ) , and the difference was statistically significant ( P0.05 ) .
Compared with the control group , SOD activity decreased gradually from the third day to the lowest level on day 14 , and the difference was statistically significant ( P0.05 ) .

The results of sub - chronic toxicity showed that the water maze showed that the difference of escape latency was statistically significant ( P0.05 ) , 50 nm Al2O3 group and 13 nm Al2O3 group were significantly higher than that in control group ( P0.05 ) .
The escape latency of each group was statistically significant ( P0.05 ) , and the escape latency gradually increased with the extension of time .

The pathological results showed that the morphology of nerve cells was normal in the CA 3 region of the control group , the arrangement was regular and the connection was close ;
The number of nerve cells decreased in the hippocampus of 10 渭m Al _ 2O _ 3 group .
The number of nerve cells in hippocampus CA 3 of 50 nm Al _ 2O _ 3 group decreased further , and the cytoplasm appeared enlarged and chromatin was loose ;
The number of nerve cells in the hippocampal CA 3 region of the 13nm Al2O3 group was significantly decreased , and the nuclei were pyknosis .
With the extension of time , the number of nerve cells decreased and the pyramidal layer cells were sparse , loose and disordered , the level was not clear , and the linkage was not compact . The results showed that the number of cells in the hippocampus of the control group was more regular , the general dyeing was deeper , the morphology of the cells was clear , and the microstructure of the cells was visible .
The number of cells in hippocampal CA 3 area of 10 渭m Al _ 2O _ 3 group was decreased , some cells swelled in vacuoles , and the cells in the hippocampus of 50 nm Al2O3 group were disordered . The cells in the CA 3 area of 50 nm Al _ 2O _ 3 group were disrupted and the cells were stained more light . The vacuoles in the CA 3 area of the 13nm Al2O3 group were more serious , and the neurons were circular ;
As the time was prolonged , the number of nerve cells in the hippocampal CA 3 area of each group decreased , the staining became shallow , and the bainite gradually dissolved and decreased .

Compared with the control group , the TNF - a content of each group was significantly higher than that of the control group ( P0.05 ) , and the content of TNF - a gradually increased with the particle size .
The content of TNF - a in each group was significantly different with time ( P0.05 ) , and the difference was statistically significant ( P0.05 ) in 30 days and 60 days .
There was no significant difference between 60 and 90 days ( P0.05 ) .
The levels of IL - 1尾 in each group were not significantly different from those in control group ( P0.05 ) .
Compared with the control group , the activity of SOD in each group had significant difference ( P0.05 ) , the activity of SOD decreased with the decrease of particle size , and the activity of SOD decreased with time .
Compared with the control group , the GSH content of each group had no significant difference ( P0.05 ) .
Compared with the control group , the activity of GSH - PX in each group was statistically significant ( P0.05 ) . The activity of GSH - PX decreased with the decrease of particle size , and the activity of GSH - PX decreased with time .

Compared with the control group , there was significant difference in the expression level of the protein in the infected mice ( P0.05 ) . The results showed that compared with the control group , the difference of the expression level of the protein of each group increased . The results showed that compared with the control group , the difference of the expression level of the protein of each group increased . The results of the necrosis - related protein ( RIP ) showed that the expression level of the protein of the infected group increased with the increase of the expression level of the protein .

Conclusion

I . The acute toxicity of nano - alumina by gavage can cause oxidative stress injury and inflammatory factor reaction in brain tissue , decrease the learning and memory ability of mice , increase with the decrease of particle size , increase the time , and have particle size effect and time effect . The inflammatory factor is an early event in this process , and the increase of oxidative stress level may be one of the main reasons for the decline of learning and memory ability .

The results showed that the cell death , apoptosis , autophagy and necrosis were one of the main causes of death in learning and memory .

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本文編號：1899856