本文選題：老年癡呆癥 + 快速老化小鼠（SAM）��； 參考：《北京中醫(yī)藥大學(xué)》2012年博士論文

【摘要】：1.背景 老年癡呆癥包括：輕度認(rèn)知障礙、阿爾茨海默病(Alzheimer's disease,AD)、血管性癡呆,以及由其他疾病,如帕金森病等導(dǎo)致的癡呆,其中以AD患者最為多見。該病是以認(rèn)知功能缺損為主要癥狀的疾病,常伴有精神、行為和人格異常,嚴(yán)重影響日常生活或工作能力。全球患病人數(shù)龐大,造成了巨大的社會、經(jīng)濟(jì)、家庭壓力�，F(xiàn)代醫(yī)學(xué)研究表明,AD發(fā)病主要與β-淀粉樣蛋白(amyloid-β, Aβ)在腦組織異常沉積、腦內(nèi)神經(jīng)血管單元(neurovascular unit,NVU)功能異常導(dǎo)致Aβ清除障礙、膽堿能神經(jīng)元受損、氧化應(yīng)激、神經(jīng)炎癥反應(yīng)或基因突變等因素有關(guān),為此研究者針對調(diào)節(jié)Aβ代謝、改善膽堿能神經(jīng)元功能或抗氧化應(yīng)激等多方面研制了多種藥物,不過目前對該病的治療尚無切實(shí)有效的措施。中醫(yī)從整體出發(fā),認(rèn)為老年癡呆癥的病位在腦,發(fā)病與腎、心、脾、肝等臟腑的功能失調(diào)有密切關(guān)系。其病因以內(nèi)因?yàn)橹?或因腎精虧耗、氣血不足、痰瘀交阻,腦髓失養(yǎng)；或因七情內(nèi)傷、久病耗損、年邁體虛,腦髓失充,而致氣、血、痰、瘀等病邪為患。并由此制定了補(bǔ)腎填精,解郁散結(jié)的總體治法,臨床發(fā)現(xiàn)多種中藥復(fù)方對該病顯示了獨(dú)特的療效。 本研究在老年癡呆癥系“腎精不足”或“毒損腦絡(luò)”而發(fā)病的中醫(yī)病機(jī)指導(dǎo)下,結(jié)合兩種擬老年癡呆癥小鼠模型——腎虛精乏腦髓失充證(SAMP8小鼠)模型和毒損腦絡(luò)證(雙海馬注射Aβ1-40的SAMR1小鼠)模型,探討兩種治療老年癡呆癥常用的不同功效中藥復(fù)方——具有補(bǔ)腎填精功效的六味地黃顆粒(LWDH)和解毒通絡(luò)功效的通絡(luò)救腦滴丸(TLJN),對不同擬老年癡呆癥模型的作用特點(diǎn)及可能的藥效靶點(diǎn),并比較這兩種相同品系小鼠,在不同造模因素誘導(dǎo)下的神經(jīng)行為學(xué)差異,尋找不同模型小鼠對造模因素反映的特征,以期為中藥復(fù)方藥效評價(jià)指標(biāo)體系提供證據(jù)。 2.目的 1)確認(rèn)增齡對兩種擬老年癡呆癥小鼠的影響； 2)比較兩種擬老年癡呆癥模型小鼠的神經(jīng)行為學(xué)及發(fā)病相關(guān)病理環(huán)節(jié)的差異； 3)比較兩種不同功效中藥復(fù)方的作用特點(diǎn),并根據(jù)動物模型的相關(guān)病理環(huán)節(jié),尋找中藥復(fù)方可能的藥效靶點(diǎn)。 3.方法 1)使用行為學(xué)Morris水迷宮和跳臺實(shí)驗(yàn),比較2月齡、6月齡及8月齡Aβ注射模型和SAMP8模型與同月齡正常對照組在Morris水迷宮實(shí)驗(yàn)逃避潛伏期、尋臺成功率、游速和跳臺潛伏期、錯(cuò)誤次數(shù)、錯(cuò)誤區(qū)時(shí)間等指標(biāo)的差異。 2)對8月齡Aβ注射模型和SAMP8模型,均設(shè)立對癥給藥和非對癥給藥組,即兩種模型分別均給予LWDH和TLJN治療,使用行為學(xué)Morris水迷宮和避暗實(shí)驗(yàn),比較上述各組在Morris水迷宮實(shí)驗(yàn)逃避潛伏期、尋臺成功率、游速和避暗潛伏期、錯(cuò)誤次數(shù)、錯(cuò)誤區(qū)時(shí)間等指標(biāo)的差異。 3)使用免疫組織化學(xué)方法,標(biāo)記上述各組小鼠腦組織Aβ、APP、BACE1、IDE、LRP1、RAGE、apoE、α2M等Aβ代謝相關(guān)蛋白的表達(dá)情況,并比較不同功效中藥復(fù)方治療組的差異。 4)使用蛋白免疫印跡方法,分別檢測上述各組小鼠海馬和皮層APP、BACE1、IDE、LRP1、RAGE、apoE、α2M等蛋白的表達(dá)情況,并比較不同功效中藥復(fù)方治療組的差異。 4.結(jié)果 1)與對照組比較,8月齡Aβ注射模型和SAMP8模型在水迷宮逃避潛伏期、尋臺成功率,跳臺實(shí)驗(yàn)潛伏期、錯(cuò)誤區(qū)時(shí)間及避暗實(shí)驗(yàn)潛伏期、錯(cuò)誤次數(shù)、錯(cuò)誤區(qū)時(shí)間等行為學(xué)檢測指標(biāo),腦組織Aβ、APP、BACE1、IDE、LRP1、RAGE、apoE和a2M等蛋白表達(dá)上,均表現(xiàn)出顯著性差異。在跳臺實(shí)驗(yàn)和避暗實(shí)驗(yàn)錯(cuò)誤區(qū)時(shí)間這個(gè)指標(biāo)上,8月齡Aβ注射模型與同月齡對照組相比有差異。在水迷宮游速這個(gè)指標(biāo)上,8月齡SAMP8模型與同月齡對照組相比有差異。 2)兩種模型之間,與8月齡Aβ注射模型比較,同月齡SAMP8模型在水迷宮游速,跳臺潛伏期,避暗錯(cuò)誤次數(shù)、錯(cuò)誤區(qū)時(shí)間和腦組織APP(皮層)、BACE1(皮層)、IDE(皮層和海馬)、LRP1(海馬)、RAGE(皮層和海馬)及apoE (皮層)蛋白表達(dá)上,有顯著性差異。 3)對于8月齡Aβ注射模型而言,對癥藥物,即TLJN,在調(diào)節(jié)模型小鼠避暗潛伏期、錯(cuò)誤次數(shù)、錯(cuò)誤區(qū)時(shí)間等行為學(xué)檢測指標(biāo),腦組織APP、BACE1、IDE、LRP1、RAGE和apoE等蛋白表達(dá)上,發(fā)揮了顯著作用。對于8月齡SAMP8模型而言,對癥藥物,即LWDH,在調(diào)節(jié)模型小鼠水迷宮逃避潛伏期、尋臺成功率和跳臺實(shí)驗(yàn)潛伏期等行為學(xué)檢測指標(biāo),腦組織Aβ、APP、BACE1、IDE、LRP1、RAGE和α2M等指標(biāo)上,發(fā)揮了顯著作用。 4)兩種不同功效中藥復(fù)方治療組之間,對于8月齡Aβ注射模型而言,對癥藥物TLJN在改善該模型小鼠避暗潛伏期及腦組織皮層和海馬APP表達(dá)、皮層apoE表達(dá)、海馬BACE1、IDE和RAGE表達(dá)水平上,發(fā)揮了顯著作用,而非對癥藥物L(fēng)WDH對上述指標(biāo)的調(diào)節(jié)作用不如TLJN;對于8月齡SAMP8模型而言,對癥藥物L(fēng)WDH在改善小鼠水迷宮尋臺成功率、避暗潛伏期及腦組織皮層和海馬APP、BACE1、IDE表達(dá)、海馬Aβ表達(dá)水平上,發(fā)揮了顯著作用,而非對癥藥物TLJN對上述指標(biāo)的調(diào)節(jié)作用不如LWDH。 5.結(jié)論 1)增齡對SAMP8小鼠和雙海馬注射Aβ1-40的SAMR1小鼠行為學(xué)改變有顯著影響。 2)不同證型的模型之間,8月齡Aβ注射模型和SAMP8模型,均表現(xiàn)出了空間學(xué)習(xí)記憶、對電刺激的記憶、被動逃避反應(yīng)能力缺失,及Aβ代謝相關(guān)蛋白的表達(dá)異常。但是,與Aβ注射模型比較,SAMP8模型的活動度相關(guān)指標(biāo)(如,水迷宮游速、避暗錯(cuò)誤次數(shù)等)下降；增加腦內(nèi)Aβ水平的相關(guān)蛋白(如APP、BACE1、RAGE等)表達(dá)更高；降低腦內(nèi)Aβ水平的相關(guān)蛋白(如IDE、LRP1、apoE等)表達(dá)更低,反應(yīng)了SAMP8模型Aβ代謝障礙更嚴(yán)重,及腎虛精乏證的行為學(xué)特征。 3)不同功效中藥復(fù)方之間,對于8月齡Aβ注射模型和SAMP8模型,對癥藥物均發(fā)揮了糾正Aβ代謝障礙的作用,表現(xiàn)在降低Aβ生成相關(guān)蛋白APP和BACE1的表達(dá)；增加Aβ降解相關(guān)蛋白IDE的表達(dá)等。非對癥藥物對上述指標(biāo)未發(fā)揮顯著改善作用,甚至還有反作用。提示,腦組織APP、BACE1、IDE等與Aβ生成及降解相關(guān)的蛋白,可作為評價(jià)不同功效中藥復(fù)方的特異性指標(biāo),反應(yīng)藥物的藥效特點(diǎn)。 4)對于腦內(nèi)清除Aβ的重要途徑LRP1系統(tǒng)而言,在調(diào)節(jié)多數(shù)指標(biāo)上,不同功效中藥復(fù)方分別對兩種模型,均發(fā)揮了作用,且作用趨勢一致。僅在8月齡Aβ注射模型的皮層apoE、海馬RAGE表達(dá)上,及SAMP8模型的皮層LRP1表達(dá)上,表現(xiàn)出了對癥藥物的優(yōu)勢。提示,可能因?yàn)長RP1和RAGE及apoE、α2M等蛋白之間有密切的相互作用,發(fā)生病理改變時(shí),相互影響也更復(fù)雜,導(dǎo)致不同功效中藥復(fù)方在不同模型上未顯示出特異性療效。但該系統(tǒng)指標(biāo)可作為評價(jià)藥物整體療效的參考指標(biāo)。 5)對于行為學(xué)相關(guān)指標(biāo)而言,不同功效中藥復(fù)方對兩種證型的模型,在8月齡Aβ注射模型的避暗潛伏期、錯(cuò)誤次數(shù)、錯(cuò)誤區(qū)時(shí)間等指標(biāo),及SAMP8模型的水迷宮逃避潛伏期、尋臺成功率和避暗潛伏期、錯(cuò)誤次數(shù)等指標(biāo)上,表現(xiàn)出了對癥藥物的優(yōu)勢。提示對癥藥物對于改善相應(yīng)證候模型小鼠的空間學(xué)習(xí)記憶和被動逃避反應(yīng)能力上有作用,有助于評價(jià)不同功效中藥復(fù)方的藥效特點(diǎn)。 總之,這兩種擬老年癡呆癥小鼠模型在Aβ代謝相關(guān)蛋白的表達(dá)和行為學(xué)上有各自的病理特點(diǎn),不同功效中藥復(fù)方對不同模型的起效靶點(diǎn)也有特異性,且對癥給藥對于改善模型行為學(xué)和Aβ生成及降解的環(huán)節(jié)上,發(fā)揮特異性作用,因此這些指標(biāo)可作為評價(jià)不同功效中藥復(fù)方藥效的特異性指標(biāo)。而LRP1系統(tǒng)的各指標(biāo),在兩種模型上,未反映出不同功效中藥復(fù)方的藥效特點(diǎn),作為評價(jià)指標(biāo),特異性欠佳。所以,對于中藥復(fù)方藥效評價(jià)指標(biāo)而言,應(yīng)根據(jù)藥效特點(diǎn)和模型證候進(jìn)行選擇,以期更客觀地評價(jià)中藥復(fù)方藥效,更準(zhǔn)確地發(fā)現(xiàn)藥物作用靶點(diǎn)和特點(diǎn)。
[Abstract]:1 . Background
Alzheimer ' s disease ( AD ) , vascular dementia , and dementia caused by other diseases , such as Parkinson ' s disease , have been studied .
or can be caused by seven - case internal injury , long - term disease consumption , aging body deficiency , brain marrow loss , qi , blood , phlegm , blood stasis , etc . , and the general treatment method for tonifying kidney and replenishing essence and resolving stagnation is developed , and various traditional Chinese medicine compounds are found to have unique curative effect on the disease .
The purpose of this study was to explore the effects of two kinds of traditional Chinese medicine compound _ ( LWDH ) and toxin - impaired brain collateral syndrome ( SAMR1 mouse ) model of two kinds of senile dementia mice , and to explore the effects of two kinds of traditional Chinese medicines for the treatment of senile dementia .
2 . Purpose
1 ) Confirm the effect of aging on two kinds of senile dementia mice ;

2 ) comparing the differences of neurobehavioral and pathological links between the two models of Alzheimer ' s disease ;

and 3 ) comparing the functional characteristics of the traditional Chinese medicine compound with two different efficacies , and looking for the possible drug effect target point of the traditional Chinese medicine according to the relevant pathological links of the animal model .
3 . Methodology
1 ) Using the behavioral Morris water maze and the skip experiment , the differences of the latency , the success rate , the latency , the number of errors and the time of the wrong area were compared between the two - month - old , 6 - month - old and 8 - month - old A尾 injection models and the normal control group of the same month in Morris water maze test .
2 ) In the 8 - month - old A - 尾 injection model , the two models were treated with both LWDH and TLJN , the behavioral learning Morris water maze and the avoiding - dark experiment were used to compare the differences between the above groups in Morris water maze test to avoid the latency , the success rate , the swimming speed and the latency of avoiding dark latency , the number of errors , and the time of the wrong area .
3 ) Using immunohistochemical method , the expression of A尾, APP , BACE1 , IDE , LRP1 , RAGE , apoE , 偽2M , and so on in brain tissue of each group were marked .
4 ) Using Western blot method , the expression of APP , BACE1 , IDE , LRP1 , RAGE , apoE , 偽2M in the hippocampus and cortex of the above groups were detected , and the difference of different efficacy Chinese herbal compound treatment groups was compared .
4 . Results
1 ) Compared with the control group , the 8 - month - old A尾 injection model and the 8 - month - old A - 尾 injection model showed significant difference compared with the control group . The 8 - month - old A - 尾 injection model was different from the control group in the same month .
2 ) There was a significant difference between the two models , which was compared with the 8 - month - old A尾injection model , and the same month - old model was significantly different in the water maze tour speed , the latency of the platform , the number of avoiding dark errors , the time of the wrong area and the brain tissue APP ( cortex ) , the BACE1 ( cortex ) , the IDE ( cortex and hippocampus ) , the LRP1 ( hippocampus ) , the RAGE ( cortex and hippocampus ) and apoE ( cortex ) protein expression .
3 ) For the 8 - month - old A尾 injection model , the therapeutic drugs , TLJN , play a prominent role in regulating the expression of protein such as the latent period , the number of errors , the time of the wrong area and so on in the model mice . For the 8 - month - old , the symptomatic drug , i.e . , LWDH , plays a prominent role in the indexes such as the escape latency of the water maze in the model mice , the success rate of the search and the latency of the platform experiment , and the indexes such as A尾, APP , BACE1 , IDE , LRP1 , RAGE and 偽2M in brain tissue .
4 ) For the 8 - month - old A 尾 injection model , TLJN plays a prominent role in improving the expression level of the mouse ' s dark latency and brain cortex and hippocampus , apoE expression , the expression level of BACE1 , IDE and RAGE in the hippocampus , and plays a prominent role in improving the success rate of the mouse water maze , avoiding dark latency and the level of the brain cortex and hippocampus APP , BACE1 , IDE and hippocampus A尾expression , and the non - symptomatic drug TLJN is not as good as the LWDH .
5 . Conclusions
1 ) There was a significant effect on the behavioral changes of SAMR1 mice injected with A尾 1 - 40 by age - increasing mice and hippocampus .
2 ) Between models of different types , 8 - month - old A.beta . injection model and 8 - month - old model showed spatial learning and memory , loss of passive avoidance response ability and abnormal expression of A尾metabolism - related protein . However , compared with A尾injection model , the activity degree - related indexes ( e.g . , water maze tour speed , avoiding dark error number , etc . ) were decreased compared with A尾injection model .
Increased expression of related proteins ( e.g . , APP , BACE1 , RAGE , etc . ) in brain A尾levels was increased ;
The lower expression of related protein ( such as IDE , LRP1 , apoE , etc . ) in brain A 尾 level was lower , and the behavioral characteristics of A尾metabolism disorder and kidney deficiency syndrome were studied .
3 ) For the 8 - month - old A - 尾 injection model and the 8 - month - old A - 尾 injection model , the action of correcting the metabolic disorder of A尾was exerted on the symptomatic drugs , which showed that the expression of APP and BACE1 associated with A尾was decreased .
It is suggested that the protein of APP , BACE1 , IDE and other proteins related to the formation and degradation of A尾can be used as a specific index for evaluating the compound of different efficacies of Chinese medicine and the drug effect of the reaction medicine .
4 ) For the LRP1 system , which is an important pathway for the elimination of A尾in the brain , the effects of different traditional Chinese medicine combinations on the regulation of most indexes are consistent . Only in the cortical apoE , the hippocampus RAGE expression of the 8 - month - old A尾 injection model and the expression of the cortical LRP1 of the model of the 8 - month - old A尾 injection , the interaction between the two traditional Chinese medicine compounds is more complicated , which leads to the lack of specific therapeutic effect on different models .
5 ) For the indexes of behavior - related indexes , the models of the two types of traditional Chinese medicine compound are shown , and the advantages of the drugs are shown in the indexes such as the hidden latency , the number of errors , the time of the wrong area and the like of the 8 - month - old A.beta . injection model , the success rate of the search and the latency of avoiding dark latency and the number of errors , etc . The indications are helpful for improving the spatial learning and memory and the passive avoidance response capability of the corresponding syndrome model mice , and help to evaluate the efficacy characteristics of the Chinese traditional Chinese medicine compound .
In conclusion , the two models of Alzheimer ' s disease have their own pathological characteristics in the expression and behavior of A尾metabolism - related proteins , and the different effects of traditional Chinese medicine are specific for the improvement of model behavior and the generation and degradation of A尾. Therefore , these indexes can be used as the indexes for evaluating the efficacy of Chinese traditional Chinese medicine compound . Therefore , the indexes of the LRP1 system can be selected according to the drug effect characteristics and the model syndrome , so as to objectively evaluate the compound drug effect of the traditional Chinese medicine and more accurately find the target point and the characteristic of the drug action .

【學(xué)位授予單位】：北京中醫(yī)藥大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2012
【分類號】：R277.7;R-332

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