本文關鍵詞：雄激素對SAMP8小鼠學習記憶能力和海馬神經(jīng)元的影響　出處：《河北醫(yī)科大學》2008年碩士論文　論文類型：學位論文

  更多相關文章： 去勢 雄激素 學習記憶 海馬CA1區(qū) 凋亡 Aβ SAMP8小鼠

【摘要】： 目的:應用SAMP8小鼠為研究對象,觀察去勢及雄激素補充治療對其學習記憶能力和海馬神經(jīng)元的影響,初步探討雄激素改善學習記憶能力的機制,為雄激素替代治療提供實驗依據(jù)。 方法:選用健康雄性7月齡快速老化SAMP8小鼠48只。隨機分為假手術對照組(簡稱P8組)、去勢組、去勢+雄激素補充治療組(簡稱雄激素組)。選取16只雄性SAMR1小鼠作為同源正常對照組(簡稱R1組)。去勢組和雄激素組小鼠切除睪丸。雄激素組于去勢術1w后肌肉注射十一酸睪酮(TU)37.4mg?kg-1?15d-1,其余各組注射等量無菌芝麻油,注射3次,共45d。 1. Morris水迷宮試驗:給藥結束后,小鼠在迷宮實驗室環(huán)境下飼養(yǎng)2d。訓練在每天上午8:00～12:00間進行。定位航行試驗:將Morris水迷宮均分為4個象限,平臺放入其中1個象限中。小鼠自第一象限中點面向池壁入水,視頻采集系統(tǒng)跟蹤并記錄找到平臺所需的時間。5d后撤掉平臺進行探索試驗,記錄小鼠120s內(nèi)跨越平臺的次數(shù)。 2.組織切片制備及觀察:每組取10只小鼠,將輸液針刺入左心室,同時剪開右心耳,用生理鹽水沖洗,4%多聚甲醛灌注。自上丘至視交叉節(jié)段取腦組織,制備五套石蠟切片,分別用于HE染色,Nissl染色,TUNEL染色,抗Aβ免疫組化染色和陰性對照。 3.流式細胞儀檢測海馬細胞凋亡:每組取5只小鼠,斷頭取腦,迅速于冰盤上剝離海馬組織,4℃70%乙醇中固定。采用網(wǎng)搓法制備單細胞懸液,碘化丙啶進行染色,流式細胞儀進行檢測。 4.海馬CA1區(qū)神經(jīng)元超微結構觀察:迅速游離海馬,置于4%預冷的戊二醛溶液中固定,將海馬切成厚約1mm的橫斷薄片,1h后再次切成1mm3的海馬CA1區(qū)組織塊,鋨酸固定,脫水,樹脂包埋,LKB-5超薄切片機切片,厚度50nm。醋酸鈾-檸檬酸鉛染色,透射電鏡觀察。 結果: 1. Morris水迷宮實驗結果:去勢組定位航行試驗潛伏期明顯長于其他組(P0.05),探索試驗跨越平臺次數(shù)減少(P0.05)。TU補充治療能改善學習記憶能力,與假手術組比較差異無統(tǒng)計學意義(P0.05)。 2. HE染色結果:R1組海馬CA1區(qū)結構正常,神經(jīng)元排列緊密整齊,層次清楚。P8組神經(jīng)元排列較疏松紊亂,細胞層數(shù)減少。去勢組海馬CA1區(qū)神經(jīng)元彌漫性空泡變性,細胞排列疏松、紊亂,細胞腫脹,核深染、固縮現(xiàn)象明顯。雄激素組神經(jīng)元病理改變較去勢組有明顯改善。 3. Nissl染色結果:R1組海馬神經(jīng)元胞漿中尼氏體豐富致密,細胞數(shù)較多,與其它各組相比差異有統(tǒng)計學意義(P0.01)。P8組海馬神經(jīng)元尼氏體較稀疏。去勢組海馬神經(jīng)元數(shù)目較P8組明顯減少,細胞核固縮、濃密深染,胞漿內(nèi)尼氏體減少,部分缺失。雄激素補充治療后,細胞數(shù)較去勢組增加,差異有統(tǒng)計學意義(P0.05),但與P8組相比差異無統(tǒng)計學意義(P0.05)。 4. TUNEL染色結果:R1組存在少量散在的TUNEL陽性細胞。P8組陽性細胞數(shù)較R1組明顯增加(P0.05)。去勢組存在大量強陽性細胞,且核邊界清晰,大小不一,多有核周空暈,TUNEL陽性細胞較P8組明顯增加,差異有統(tǒng)計學意義(P0.05)。雄激素組陽性細胞表達較去勢組減弱(P0.05),與P8組相比無統(tǒng)計學差異(P0.05)。 5.抗Aβ免疫組化染色結果:去勢組Aβ陽性神經(jīng)元染色深,其數(shù)目及吸光度明顯高于其他組(P0.05)。雄激素組Aβ陽性細胞數(shù)目及吸光度較去勢組明顯減少(P0.05)。 6.流式細胞儀檢測結果:去勢組出現(xiàn)了一個較高的亞二倍體峰,細胞凋亡率達到32.66±4.60%,與P8組凋亡率21.19±3.96%以及雄激素組凋亡率20.78±3.08%相比明顯升高,有統(tǒng)計學意義(P0.05),而P8組和雄激素組間凋亡率比較,差異無統(tǒng)計學意義(P0.05)。 7.電鏡超微結構觀察結果:電鏡觀察R1組神經(jīng)元核膜完整光滑,核內(nèi)染色質均勻,電子密度低,線粒體豐富。P8組神經(jīng)元核膜雙層結構較完整,線粒體正�；蜉p微腫脹,有少量脂褐素沉積,核內(nèi)染色質電子密度略高。去勢組神經(jīng)元核膜雙層結構消失融散,線粒體腫脹變性,嵴斷裂,脂褐素沉積,核內(nèi)染色質聚集成團、邊集,形成不規(guī)則的高電子密度團塊,有些出現(xiàn)凋亡小體。雄激素組超微結構損傷較去勢組減輕,神經(jīng)元核膜較清晰,核內(nèi)染色質邊集不明顯,線粒體形態(tài)未見明顯異常,脂褐素沉積減少。 結論: 1. SAMP8小鼠去勢后,雄激素缺乏導致學習記憶能力下降;雄激素補充治療能減輕內(nèi)源性雄激素下降對學習記憶功能的損害,改善學習記憶能力。 2. SAMP8小鼠去勢后,雄激素缺乏導致海馬神經(jīng)元疏松紊亂、腫脹變性,超微結構受損,凋亡數(shù)目增加,神經(jīng)元大量丟失;雄激素補充治療能改善病理損傷,減輕神經(jīng)元的凋亡和丟失。 3. SAMP8小鼠去勢后,雄激素缺乏導致海馬CA1區(qū)神經(jīng)元Aβ沉著增加;雄激素補充治療能減少Aβ的生成。
[Abstract]:Objective: To observe the effects of castration and androgen supplementation on learning and memory ability and hippocampal neurons in SAMP8 mice, and to preliminarily explore the mechanism of androgen improving learning and memory ability, so as to provide experimental evidence for androgen replacement therapy.
Methods: healthy male July age fast aging SAMP8 mice 48. Were randomly divided into sham operation group (P8 group), ovariectomized group, ovariectomized and androgen replacement therapy group (the androgen group). A total of 16 male SAMR1 mice were used as homologous normal control group (R1 group), ovariectomized group and androgen group mouse testis androgen group. Resection of castration 1W after intramuscular injection of eleven acid testosterone (TU) 37.4mg? Kg-1? 15d-1, other groups were injected with sterile sesame oil, 3 injection, 45d.
1. Morris water maze test: after injection, mice in the maze laboratory environment feeding 2D. training in the morning at 8:00 ~ 12:00. The place navigation test: the Morris water maze was divided into 4 quadrants, the platform into which of the 1 quadrants. Mice from the first quadrant midpoint to the wall into the water, video acquisition system to track and record the time required to find the platform after.5d removed platform experiment, record mouse 120s across platform number.
2. tissue sections were prepared and observed: each group of 10 mice, the infusion needle is inserted into the left ventricle, and cut the right atrial appendage, flushed with saline and 4% paraformaldehyde perfusion. From the superior colliculus to optic chiasma segment brain tissues were collected, prepared five sets of paraffin sections were used for HE staining, Nissl staining color, TUNEL staining, immunohistochemical staining of anti A beta and negative control.
Detection of apoptosis of the hippocampus cells. 3. flow cytometry: each group 5 mice were decapitated rapidly, in Bingpan stripping the hippocampus, fixed 4 C 70% ethanol. The rubbing preparation of single cell suspension, propidium iodide staining and flow cytometry.
To observe the ultrastructure of neurons in hippocampal CA1 area: 4. fast free hippocampus, fixed in 4% glutaraldehyde solution precooling, will cut into transverse slice thickness of 1mm hippocampus, 1H again after cut 1mm3 in CA1 area of hippocampus tissue, osmic acid fixation, dehydration, resin embedding, LKB-5 ultrathin sections, the thickness of 50nm. acetate uranium - lead citrate staining and transmission electron microscopy.
Result:
The results of 1. Morris water maze test showed that the latency of positioning test in ovariectomized group was significantly longer than that in other groups (P0.05), and the number of crossing platforms was reduced (P0.05)..TU supplementation treatment could improve learning and memory ability, and there was no significant difference between sham operation group and sham operation group (P0.05).
2. HE staining: group R1 normal neuronal structure in hippocampal CA1 area, arranged, clear.P8 group of neurons arranged in loose disorder, cell layers decreased. Castration groups of neurons in hippocampal CA1 region of diffuse vacuolar degeneration, cells arranged in loose and disorder, cell swelling, hyperchromatic nuclei pyknosis obviously. Androgen group of neurons the pathological change is significantly better than that of OVX group.
3. Nissl staining: R1 group of hippocampal neurons Nissl body rich and dense, cell number compared with the other groups there were statistically significant differences (P0.01).P8 group Nissl body in hippocampus were sparse. The number of neurons in the hippocampus of ovariectomized group decreased significantly compared with group P8, karyopyknosis, dense hyperchromatic cytoplasm tigroid the body is reduced, partial deletion. Androgen replacement therapy, cell number compared with OVX group increased, the difference was statistically significant (P0.05), but compared with the P8 group, the difference was not statistically significant (P0.05).
4. TUNEL staining: in group R1, TUNEL positive cells in.P8 group the number of positive cells in a few scattered was higher than R1 group (P0.05). A large number of strong positive cells in the ovariectomized group, and the nuclear size, clear boundary, a perinuclear halo, TUNEL positive cells were significantly increased compared with group P8, there were significant differences (P0.05). The expression of positive cells compared with androgen group (P0.05), ovariectomized group decreased compared with the P8 group showed no significant difference (P0.05).
5., anti A beta immunohistochemical staining results: the A beta positive neurons in the ovariectomized group were deep stained, the number and absorbance of them were significantly higher than those in other groups (P0.05). The number and absorbance of A beta positive cells in androgen group were significantly decreased compared with those in ovariectomized group (P0.05).
The testing results of 6. flow cytometry: the OVX group had a higher hypodiploid peak, the apoptosis rate of 32.66 + 4.60%, compared with the P8 group the apoptosis rate of 21.19 + 3.96% and 20.78 + 3.08% in the testosterone group apoptosis rate increased significantly, with statistical significance (P0.05), and P8 group and male hormone group apoptosis rate comparison, the difference was not statistically significant (P0.05).
7. ultrastructural observations: observation group R1 neurons nuclear membrane smooth and complete, the nuclear chromatin is uniform, low electron density, abundant mitochondria in neurons of the.P8 group double membrane structure is more complete, normal or slightly swollen mitochondria, a small amount of lipofuscin deposition, nuclear chromatin slightly higher electron density. The OVX group neurons double membrane structure disappearance of melting powder, mitochondria swelling, cristae, lipofuscin deposition, nuclear chromatin aggregation, margination, formation of high electron density were irregular, some apoptotic bodies were observed. The ultrastructure injury of androgen group than the OVX group decreased, nuclear membrane was clear, the nuclear chromatin is not obvious, mitochondria there was no obvious morphological abnormalities, lipofuscin deposition decreased.
Conclusion:
1., after SAMP8 castration, androgen deficiency leads to a decrease in learning and memory ability. Androgen supplementation therapy can reduce the impairment of endogenous androgen and damage learning and memory function, and improve learning and memory ability.
2. after SAMP8 castration, androgen deficiency resulted in loose, disorder, swelling, degeneration and ultrastructure of hippocampal neurons. The number of apoptotic neurons increased and the number of neurons was lost. Androgen supplementation therapy can improve pathological damage and alleviate neuronal apoptosis and loss.
After the castration of 3. SAMP8 mice, androgen deficiency leads to the increase of A beta in the hippocampal CA1 region neurons, and androgen supplement therapy can reduce the formation of A beta.

【學位授予單位】：河北醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2008
【分類號】：R341

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本文編號：1420397