本文選題：DHA + 丙泊酚麻醉�。� 參考：《山東大學(xué)》2016年博士論文

【摘要】：第一部分 DHA對(duì)丙泊酚重復(fù)麻醉大鼠學(xué)習(xí)記憶損傷的干預(yù)作用目的研究不同劑量的DHA對(duì)丙泊酚重復(fù)麻醉SD大鼠引起的學(xué)習(xí)記憶功能損傷的干預(yù)作用、量效關(guān)系及作用機(jī)制,以期為臨床使用丙泊酚重復(fù)麻醉產(chǎn)生的副作用提供改善解決的方案。方法將60只SD大鼠隨機(jī)分為6組,每組10只,生理鹽水對(duì)照組(A組),溶劑脂肪乳對(duì)照組(B組),丙泊酚組(C組),低劑量DHA+丙泊酚組(D組),中劑量DHA+丙泊酚組(E組),高劑量DHA+丙泊酚組(F組)。丙泊酚組(C組)連續(xù)腹腔內(nèi)注射丙泊酚5天,每天1次,劑量為75 mg/kg/day;A組、B組與C組方法相同,B組劑量為10 ml/kg/day的脂肪乳；A組劑量為10 ml/kg/day的0.9%生理鹽水；F組在給予丙泊酚前10天開(kāi)始每天灌胃給予3g/kg/day的DHA,然后繼續(xù)給予5天DHA的同時(shí)每天腹腔注射75 mg/kg/day的丙泊酚；E組、D組方法同F(xiàn)組。E組、D組分別為1g/kg/day和0.3g/kg/day的DHA。所有大鼠在第1-5天的訓(xùn)練前腹腔注射丙泊酚或脂肪乳或0.9%生理鹽水,在第6天的測(cè)試前不給予任何藥物(注：丙泊酚麻醉大鼠用新生兒血氧飽和度監(jiān)測(cè),排除因缺氧導(dǎo)致的腦損傷,注射丙泊酚麻醉的大鼠在翻正反射恢復(fù)后20分鐘進(jìn)行訓(xùn)練)。定位航行實(shí)驗(yàn)：SD大鼠在首次游泳時(shí),一般不易發(fā)現(xiàn)隱藏于水下的站臺(tái),在120秒內(nèi)若大鼠仍未發(fā)現(xiàn)水池中的站臺(tái)或者無(wú)法登上站臺(tái)時(shí),將動(dòng)物引導(dǎo)置于站臺(tái)上停留30秒,將大鼠從站臺(tái)上拿下擦干,休息60秒后進(jìn)行下一次訓(xùn)練,每天固定時(shí)間訓(xùn)練4次,連續(xù)訓(xùn)練5天,大鼠當(dāng)日的學(xué)習(xí)成績(jī)以大鼠當(dāng)日4次訓(xùn)練潛伏期的平均值來(lái)計(jì)算�？臻g探索實(shí)驗(yàn)：大鼠進(jìn)行定位航行試驗(yàn)完畢后24小時(shí),將站臺(tái)撤走,將大鼠在某個(gè)隨機(jī)選取的相同的入水點(diǎn)放入水中,記錄大鼠在120s內(nèi)的游泳線路,把大鼠在目標(biāo)象限停留的時(shí)間以及在目標(biāo)象限穿越的次數(shù)記錄下來(lái),探索大鼠的空間學(xué)習(xí)規(guī)律。定位航行試驗(yàn)結(jié)束后將所有大鼠解剖取海馬組織并分成兩份,一部分用高效液相色譜熒光法檢測(cè)氨基酸神經(jīng)遞質(zhì)谷氨酸(GLU)、γ-氨基丁酸(GABA),另一部分用ELISA法進(jìn)行腦源性神經(jīng)營(yíng)養(yǎng)因子(BNDF)、丙二醛(MDA)、超氧化物歧化酶(SOD)與谷胱甘肽過(guò)氧化物酶(GSH-Px)檢測(cè)。結(jié)果 實(shí)驗(yàn)期間各組大鼠皮毛顏色正常,能夠自由進(jìn)食和飲水,未見(jiàn)明顯異常病理狀態(tài)；C組大鼠的活動(dòng)量相比其它各組有明顯的減少,表現(xiàn)為行動(dòng)遲緩、精神萎靡；各DHA組大鼠也有不同程度的活動(dòng)量減少。A組與B組大鼠逃逸潛伏期及穿越平臺(tái)的次數(shù)無(wú)顯著差異(P0.05),與A組大鼠相比,C組大鼠逃逸潛伏期明顯延長(zhǎng)(P0.01),穿越平臺(tái)的次數(shù)明顯減少(P0.01),有顯著差異；與C組大鼠相比,E組(P0.05)與F組(P0.01)大鼠的逃逸潛伏期明顯縮短(P0.01),并且穿越平臺(tái)的次數(shù)明顯增加(P0.01)。顯然,隨著DHA劑量的增加,D組、E組、F組大鼠的逃逸潛伏期呈下降的趨勢(shì),而穿越平臺(tái)的次數(shù)則逐漸增加。A組與B組大鼠無(wú)論是谷氨酸和γ-氨基丁酸的含量還是Glu/GABA比值均無(wú)顯著差異(P0.05)。與A組大鼠相比,C組大鼠谷氨酸含量和Glu/GABA比值顯著增加(P0.01),而γ-氨基丁酸的含量顯著減少(P0.05)。與C組大鼠相比較, D組(P0.05)、E組(P0.05)與F組(P0.01)大鼠的谷氨酸含量顯著降低而γ-氨基丁酸的含量升高(P0.05)。隨著DHA劑量的增加,谷氨酸含量逐漸降低而γ-氨基丁酸含量逐漸升高,表現(xiàn)出了一定的劑量依賴性。A組與B組大鼠S0D和GSH-Px的活力無(wú)顯著差異(P0.05)。與A、B對(duì)照組相比,C組大鼠海馬組織內(nèi)SOD和GSH-Px的活力分別下降了35.1%(P0.01)、39.8%(P0.01)。而E組(P0.05)與F組(P0.01)則使S0D與GSH-Px的活力明顯增強(qiáng)且隨著DHA劑量的增加,DHA的作用明顯增強(qiáng)。A組與B組大鼠的MDA濃度無(wú)顯著差異(P0.05),與A、B對(duì)照組大鼠相比,經(jīng)丙泊酚重復(fù)麻醉處理的各組大鼠MDA濃度明顯升高(P0.01)。D組(P0.05)、E組(P0.05)、F組(P0.01)的MDA濃度與C組大鼠相比顯著降低。A組與B組大鼠的BDNF濃度無(wú)顯著差異(P0.05),與A組大鼠相比,C組大鼠的BDNF濃度下降了34.7%(P0.05),有顯著差異。與C組大鼠相比,D組、E組、F組大鼠給予DHA使得BDNF的濃度分別升高了11.3%(P0.05)、33.9%(P0.05)、47.8%(P0.01),差異有顯著性,說(shuō)明隨著DHA劑量的增加,其干預(yù)作用逐漸增強(qiáng)。結(jié)論DHA通過(guò)調(diào)節(jié)G1u與GABA的濃度平衡,降低氧化應(yīng)激損傷和增加BDNF的水平從而改善丙泊酚重復(fù)麻醉引起的學(xué)習(xí)與記憶障礙,而且DHA劑量增加,干預(yù)作用增強(qiáng)�；谏鲜鲅芯拷Y(jié)果,我們建議DHA可有效的用于丙泊酚重復(fù)麻醉導(dǎo)致的學(xué)習(xí)與記憶障礙的術(shù)后治療,但是需要更多的研究支持這一假設(shè)。第二部分 遠(yuǎn)志皂苷對(duì)丙泊酚重復(fù)麻醉大鼠學(xué)習(xí)記憶損傷的干預(yù)作用目的 研究不同劑量遠(yuǎn)志皂苷對(duì)丙泊酚重復(fù)麻醉大鼠所導(dǎo)致學(xué)習(xí)記憶損傷的干預(yù)作用及作用機(jī)制,為臨床使用丙泊酚重復(fù)麻醉產(chǎn)生的副作用提供更多的解決方案。方法將60只SD大鼠隨機(jī)分為6組,生理鹽水對(duì)照組(A組),溶劑脂肪乳對(duì)照組(B組),丙泊酚組(C組),低劑量組(50mg/kg/day)遠(yuǎn)志皂苷+丙泊酚組(D組),中劑量組(200mg/kg/day)遠(yuǎn)志皂苷+丙泊酚組(E組),高劑量組(500mg/kg/day)遠(yuǎn)志皂苷+丙泊酚組(F組)。C組于實(shí)驗(yàn)開(kāi)始第11～15天連續(xù)腹腔內(nèi)注射5天丙泊酚注射液,每天1次,劑量為75mg/kg/day;B組與C組方法相同,給藥劑量為10ml/kg/day的脂肪乳；A組同上,連續(xù)注射10ml/kg/day的0.9%生理鹽水；F組在實(shí)驗(yàn)第1-10天(即給予丙泊酚麻醉前10天)開(kāi)始每天灌胃遠(yuǎn)志皂苷,然后繼續(xù)給予5天遠(yuǎn)志皂苷的同時(shí)每天腹腔注射75 mg/kg/day的丙洎酚；E組、D組給予方法同F(xiàn)組。各組大鼠在第16～20天的水迷宮測(cè)試前腹腔內(nèi)注射丙泊酚或脂肪乳或生理鹽水,在第21天的測(cè)試前不給予任何藥物(注：丙泊酚麻醉大鼠用新生兒血氧飽和度監(jiān)測(cè),排除因缺氧導(dǎo)致的腦損傷,注射丙泊酚麻醉的大鼠在翻正反射恢復(fù)后20分鐘進(jìn)行訓(xùn)練)。實(shí)驗(yàn)期間觀察動(dòng)物日常狀況,并每周稱量體重；大鼠的學(xué)習(xí)記憶情況采用Morris Water Maze (MWM)即水迷宮測(cè)定；解剖動(dòng)物觀察腦系數(shù)并對(duì)海馬組織進(jìn)行組織病理學(xué)檢查；測(cè)定腦細(xì)胞MDA、SOD、GSH的活性,觀察細(xì)胞損傷的程度；提取各組大鼠海馬細(xì)胞DNA,進(jìn)行片斷化檢測(cè)；檢測(cè)大鼠海馬組織細(xì)胞凋亡相關(guān)因子Bcl-2和Caspase-3表達(dá)量的情況,觀察細(xì)胞凋亡情況。結(jié)果1.各組大鼠皮毛顏色正常,能夠自由進(jìn)食和飲水,無(wú)明顯異常病理狀態(tài)；C組大鼠的活動(dòng)量相比于A組、B組有明顯的減少,表現(xiàn)為行動(dòng)遲緩、精神萎靡；D組、E組、F組大鼠也有不同程度的活動(dòng)量減少。各組大鼠3周內(nèi)的體重逐漸增長(zhǎng),C組、D組、E組、F組與A、B對(duì)照組比較未見(jiàn)明顯統(tǒng)計(jì)學(xué)差異(P0.05),但C組大鼠的體重增長(zhǎng)有緩慢的趨勢(shì)。2.定位航行實(shí)驗(yàn)和空間探索實(shí)驗(yàn)發(fā)現(xiàn),A組與B組大鼠逃逸潛伏期及穿越平臺(tái)的次數(shù)以及目標(biāo)象限停留時(shí)間均無(wú)顯著差異(P0.05)。與A組比較,C組、D組、E組、F組與A組之間存在顯著差異(P0.05),與C組比較,D組、E組、F組差異有顯著性(P0.05),提示隨著遠(yuǎn)志皂苷劑量的增加,其潛伏期逐漸縮短,穿越平臺(tái)的次數(shù)增加以及目標(biāo)象限停留時(shí)間增長(zhǎng)。3.A組與B組大鼠各氧化損傷指標(biāo)無(wú)顯著差異(P0.05)。與A組、B組比較,其余各組大鼠MDA、SOD、GSH有明顯差異(P0.05)。與A組比較,C組MDA含量明顯增加,SOD含量與GSH濃度顯著降低,說(shuō)明C組大鼠腦細(xì)胞氧化損傷嚴(yán)重；與C組比較,D組、E組、F組MDA含量逐漸降低,E組、F組有顯著差異(P0.05)；與C組比較,D組、E組、F組SOD、GSH旨標(biāo)逐漸升高,F組有顯著差異(P0.05)。4.A組與B組大鼠腦系數(shù)無(wú)顯著差異(P0.05)。與A組比較,C組大鼠腦系數(shù)有明顯減小(P0.05),表明C組大鼠腦細(xì)胞有一定程度的萎縮；遠(yuǎn)志皂苷各劑量組大鼠腦系數(shù)有少量減少,E組有顯著差異(P0.05)。與C組比較,遠(yuǎn)志皂苷各劑量組腦系數(shù)有一定程度的增加,有顯著差異(P0.05),F組高于E組與D組。表明低、中、高劑量的遠(yuǎn)志皂苷均能夠一定程度改善丙泊酚重復(fù)麻醉大鼠的腦功能,但是仍然低于對(duì)照組。5.組織病理學(xué)檢查發(fā)現(xiàn),A組和B組大鼠海馬組織結(jié)構(gòu)清晰,神經(jīng)細(xì)胞排列緊密,細(xì)胞染色均勻,其形態(tài)完整無(wú)水腫；細(xì)胞質(zhì)染色清晰；細(xì)胞核呈藍(lán)紫色,核仁清晰可見(jiàn),細(xì)胞核結(jié)構(gòu)完整。C組大鼠海馬組織的神經(jīng)細(xì)胞數(shù)目明顯減少且排列不規(guī)整,細(xì)胞呈萎縮狀態(tài),有濃染現(xiàn)象；部分細(xì)胞核核仁不明顯,細(xì)胞核溶解固縮,大量空泡物質(zhì)出現(xiàn)在細(xì)胞核周圍；細(xì)胞的細(xì)胞質(zhì)染色不均；在神經(jīng)細(xì)胞周圍可見(jiàn)散在的大量炎性細(xì)胞。遠(yuǎn)志皂苷各劑量組與C組比較,海馬組織有所改善,可見(jiàn)細(xì)胞染色均勻,僅少量細(xì)胞形態(tài)固縮,排列不規(guī)整；細(xì)胞核核仁不明顯,且細(xì)胞核體積增大；細(xì)胞質(zhì)染色尚清晰；在細(xì)胞周圍可見(jiàn)散在的少量炎性細(xì)胞,F組優(yōu)于E組與D組。6.A組與B組海馬細(xì)胞DNA電泳都是單條帶,表明DNA結(jié)構(gòu)完整；而C組海馬細(xì)胞DNA電泳出現(xiàn)明顯的梯狀帶表明DNA出現(xiàn)斷裂；遠(yuǎn)志皂苷D、E、F劑量組出現(xiàn)梯狀帶的數(shù)量依次減少,但其數(shù)量都低于丙泊酚組。7. ELISA結(jié)果顯示：A組與B組大鼠凋亡因子表達(dá)無(wú)顯著差異(P0.05)。與A組比較,C組Bcl-2蛋白表達(dá)量顯著降低(P0.05),Caspase-3表達(dá)量顯著升高(P0.05)。與C組比較,D組、E組、F組Bc1-2蛋白表達(dá)量不同程度的逐漸升高,Caspase-3表達(dá)量不同程度的逐漸降低,有顯著差異(P0.05)。結(jié)論遠(yuǎn)志皂苷通過(guò)降低氧化應(yīng)激損傷,調(diào)控凋亡因子Bcl-2和Caspase-3的表達(dá),減少了細(xì)胞凋亡的發(fā)生,減輕了海馬組織的損傷,增強(qiáng)了大鼠的空間學(xué)習(xí)記憶能力從而對(duì)丙泊酚重復(fù)麻醉所導(dǎo)致大鼠學(xué)習(xí)記憶損傷具有保護(hù)作用。
[Abstract]:In this study , 60 SD rats were randomly divided into 6 groups , 10 rats in each group , normal saline control group ( group A ) , solvent fat emulsion control group ( group B ) , propofol group ( group C ) , low - dose DHA + propofol group ( group D ) , middle - dose DHA + propofol group ( group C ) , low - dose DHA + propofol group ( group D ) , middle - dose DHA + propofol group ( group E ) , high - dose DHA + propofol group ( group F ) .
Group A was 0.9 % normal saline at 10 ml / kg / day ;
Group F was given 3 g / kg / day of DHA per day prior to administration of propofol for 10 days , followed by continued administration of 5 days of DHA and 75 mg / kg / day of propofol per day ;
Group E and Group D were treated with propofol or fat emulsion or 0.9 % normal saline at 1 - 5 days . All rats received propofol or fat emulsion or 0.9 % normal saline before training on Day 1 - 5 . No drugs were administered before the 6 - day test ( note : propofol anesthesia rats were trained with neonatal blood oxygen saturation , excluding brain injury due to hypoxia , and 20 minutes after the normal reflex recovery rats were injected ) . The rats were placed on the platform for 30 seconds , the rats were guided on the platform for 30 seconds , the rats were placed on the platform for 30 seconds , the rats were trained 4 times daily , and the rats were trained for 5 days .
Compared with other groups , the activity of the rats in group C decreased significantly compared with other groups .
Compared with group A , the latency of escape latency of group A and group B was significantly prolonged ( P0.01 ) .
Compared with group A , group D ( P0.05 ) , group E ( P0.05 ) and group F ( P0.01 ) showed no significant difference ( P0.05 ) .
In group A , 0.9 % normal saline was injected continuously for 10 ml / kg / day .
Group F was administered daily gavage of hyperoside saponins on day 1 to 10 of the experiment ( i.e . 10 days prior to administration of propofol ) , followed by continued administration of 75 mg / kg / day of propofol per day while continuing to give 5 days of apogenin ;
Group E and Group D were given the same method as group F . Each group received propofol or fat emulsion or physiological saline in the abdominal cavity before the water maze test on Days 16 to 20 . No drug was administered before the first day of test ( note : propofol anesthesia rats used neonatal blood oxygen saturation monitoring to eliminate brain injury due to hypoxia , and rats injected with propofol anesthesia were trained 20 minutes after normal reflex recovery ) . The daily status of animals was observed during the experiment and the body weight was weighed weekly ;
The nucleolus of some nuclei is not obvious , the nucleus dissolves and shrinks , and a lot of vacuoles appear around the nucleus ;
The brain coefficients were observed in the dissected animals and the histopathological examination was performed on the hippocampus .
The activity of MDA , SOD and GSH in brain cells was measured and the extent of cell damage was observed .
extracting the DNA of each group of rat hippocampal cellular DNA and performing fragment detection ;
The expression of apoptosis - related factors Bcl - 2 and Caspase - 3 in hippocampus of rats was detected , and the apoptosis was observed .
Compared with group A and group B , the activity of group C was significantly decreased compared with group A and group B .
Compared with group A , group C , group D , E group , F group and group A had no significant difference ( P0.05 ) . Compared with group A , group C , group D , E group , F group had no significant difference ( P0.05 ) . Compared with group A , group C , group D , E group , F group had no significant difference ( P0.05 ) . Compared with group A , group C , group D , E group , F group had no significant difference ( P0.05 ) . Compared with group A , group C , group D , group E , group F had no significant difference ( P0.05 ) . Compared with group A , group C , group D , group E , group F had no significant difference ( P0.05 ) .
Compared with group C , the content of MDA in group D , E group and F group decreased gradually , and there was significant difference between group E and F ( P0.05 ) .
Compared with group C , there was no significant difference between group D , group E , SOD and GSH of group F ( P0.05 ) . There was no significant difference between group A and group B ( P0.05 ) . Compared with group A , the brain coefficients of group C were significantly decreased ( P0.05 ) .
Compared with group C , there was a significant difference in the brain coefficients ( P0.05 ) and the F group was higher than that in group E and D .
the cytoplasm dyeing is clear ;
The nucleus was blue - purple , the nucleolus was clearly visible , the nucleus structure was intact , the number of nerve cells in the hippocampus of group C was obviously decreased and the arrangement was not regular , the cells were in atrophy state , and there was strong staining phenomenon ;
The learning and memory of rats was determined by Morris Water Maze ( MWM ) , the water maze ;
cell cytoplasm staining was not uniform ;
There was a large amount of inflammatory cells scattered around the nerve cells . Compared with group C , the hippocampal tissue was improved , the cells were stained uniformly , only a small amount of cells were fixed , and the arrangement was irregular ;
Nuclear nucleolus is not obvious , and the nucleus volume increases ;
The cytoplasm dyeing is still clear .
There was a small amount of inflammatory cells scattered around the cells , the F group was superior to that of group E and D . 6 . DNA electrophoresis of hippocampal cells in group B and group B was a single band , indicating that the DNA structure was intact ;
In group C , DNA electrophoresis showed that the DNA appeared to be fractured .
The number of ladder - like bands in D , E and F groups decreased in turn , but the number was lower than that of propofol group . Compared with group A , the expressions of Bcl - 2 and Caspase - 3 in group C and group C were significantly decreased ( P0.05 ) .
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R614

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