本文選題：催產(chǎn)素 + 齒狀回��； 參考：《第三軍醫(yī)大學(xué)》2017年碩士論文

【摘要】：研究背景:隨著社會(huì)進(jìn)步,經(jīng)濟(jì)發(fā)展和人們生活水平改善,飲酒變得越發(fā)普遍。酒精是一種中樞神經(jīng)抑制劑,長(zhǎng)期或一次過(guò)量酒精攝入均可以引起酒精中毒,損害多系統(tǒng)器官,例如消化和心血管系統(tǒng),以及神經(jīng)系統(tǒng)等,甚至可引起癌癥發(fā)生[1-4]。在發(fā)育過(guò)程中,中樞神經(jīng)系統(tǒng)(Central nervous system,CNS)對(duì)酒精刺激非常敏感[5,6]。孕期高劑量酒精暴露后,胎兒可以表現(xiàn)為出生后生長(zhǎng)發(fā)育遲緩、面容異常、CNS功能障礙等[7-9],即胎兒酒精綜合征(Fetal alcohol syndrome,FAS)。有研究表明,新生期小鼠在單次酒精注射后可引起新生期小鼠大腦海馬區(qū)域神經(jīng)干細(xì)胞損傷,小腦蒲肯野細(xì)胞和伯格曼膠質(zhì)細(xì)胞損傷,以及神經(jīng)細(xì)胞丟失[10,11];酒精對(duì)青春期邊緣系統(tǒng)尤其對(duì)青春期海馬更容易產(chǎn)生損傷作用,導(dǎo)致記憶受損[12-14]。磁共振成像研究顯示酗酒和酒精依賴的青少年海馬體積減小,同時(shí)相關(guān)功能研究顯示海馬的學(xué)習(xí)和記憶受到損害[15-19]。海馬持續(xù)進(jìn)行神經(jīng)發(fā)生,并參與海馬學(xué)習(xí)記憶等認(rèn)知功能。有研究也顯示,青春期大鼠酒精暴露容易造成海馬結(jié)構(gòu)損傷和行為學(xué)功能異常[20],甚至可影響神經(jīng)發(fā)生[21]。放射狀膠質(zhì)細(xì)胞(Radial glial cells,RGCs)是組織形態(tài)、生化特性和功能方面獨(dú)特的一類細(xì)胞,也是胚胎期間最早出現(xiàn)的具有干細(xì)胞功能的膠質(zhì)細(xì)胞。其胞體位于腦室區(qū)(Ventricular zone,VZ),即早期神經(jīng)管內(nèi)壁的神經(jīng)上皮層,細(xì)胞一端伸出一短的頂突附著于室管膜表面,另一端伸出細(xì)長(zhǎng)且有短側(cè)突的基底突起,并分出許多分支并終止于軟腦膜側(cè)。RGCs在CNS的發(fā)育過(guò)程中扮演著非常重要的角色,被認(rèn)為是神經(jīng)干細(xì)胞[22],它的發(fā)育與分化受到許多因素誘導(dǎo)調(diào)控[23]。另外,RGCs作為干細(xì)胞也存在于成年CNS內(nèi)兩個(gè)增殖區(qū)域:側(cè)腦室壁的腦室下區(qū)(Subventricular zone,SVZ)和海馬齒狀回(Dentate gyrus,DG)的顆粒下區(qū)(Subgranular zone,SGZ)[24]。RGCs作為一種神經(jīng)祖細(xì)胞,在板層結(jié)構(gòu)(皮層、海馬和小腦)發(fā)育過(guò)程中,可作為神經(jīng)前體細(xì)胞產(chǎn)生膠質(zhì)細(xì)胞和神經(jīng)元,其膠質(zhì)長(zhǎng)纖維還可以充當(dāng)“腳手架”作用引導(dǎo)神經(jīng)元進(jìn)行放射狀遷移[23,25,26]。RGCs在海馬齒狀回(DG)可分為初級(jí)RGCs和次級(jí)RGCs。其中,初級(jí)RGCs與大腦皮質(zhì)內(nèi)的RGCs具有相似的結(jié)構(gòu)形態(tài),大部分位于海馬DG區(qū)域(出生前),分布范圍:從海馬傘部至DG的軟腦膜表面。RGCs放射狀膠質(zhì)纖維可引導(dǎo)新生神經(jīng)元或前體細(xì)胞從神經(jīng)上皮向齒狀回原基進(jìn)行遷移。而次級(jí)RGCs來(lái)自于海馬DG區(qū)域(出生后早期)的非放射狀前體細(xì)胞,維持生后海馬DG的形態(tài)發(fā)生。次級(jí)RGCs充分發(fā)揮它的放射狀膠質(zhì)纖維“支架”作用,引導(dǎo)顆粒神經(jīng)元遷移,并部分分化為星形膠質(zhì)細(xì)胞。只有少數(shù)次級(jí)RGCs保留至成年而且作為神經(jīng)干細(xì)胞參與到成年DG的神經(jīng)發(fā)生中[23][15]。伯格曼膠質(zhì)細(xì)胞(Bergman glial cell,BG)作為小腦內(nèi)的RGCs,其結(jié)構(gòu)形態(tài)與海馬及皮層內(nèi)的RGCs相似,放射狀長(zhǎng)纖維向軟腦膜表面延伸[11,26]。在小腦皮質(zhì)發(fā)生過(guò)程中,BG為不同類型的細(xì)胞提供“腳手架”遷移,促進(jìn)小腦葉狀結(jié)構(gòu)和分層模式的正常發(fā)育,并且可以調(diào)節(jié)軸突和樹突的生長(zhǎng)方向[26]。近年來(lái)研究顯示BG影響蒲肯野細(xì)胞的樹突發(fā)育,BG的纖維引導(dǎo)蒲肯野細(xì)胞的樹突生長(zhǎng),影響樹突棘的形成;而隨著發(fā)育的進(jìn)程,伯BG的纖維能夠促進(jìn)蒲肯野細(xì)胞的突觸形成,確保信號(hào)之間的正常傳導(dǎo)[27]。也有報(bào)道指出,保持BG的長(zhǎng)纖維“支架”可以維持蒲肯野細(xì)胞的存活[28]。為探究如何改善酒精暴露導(dǎo)致的腦發(fā)育損傷,我們用催產(chǎn)素(Oxytocin,OT)和白藜蘆醇(Resveratrol,RSV)進(jìn)行干預(yù),分別觀察OT和RSV對(duì)酒精暴露后青春期小鼠大腦和新生期小鼠小腦發(fā)育的影響,以期這些干預(yù)能改善酒精暴露后的腦發(fā)育損傷,并為臨床上提供一種新的治療手段。OT是一種神經(jīng)肽(由9個(gè)氨基酸構(gòu)成),大部分由下丘腦視上核和室旁核合成。它的分泌受下丘腦—垂體—性腺軸的調(diào)控,在青春期,下丘腦—垂體—性器官的發(fā)育趨于成熟,催產(chǎn)素分泌達(dá)到新的水平。最初臨床應(yīng)用催產(chǎn)素來(lái)加強(qiáng)子宮收縮來(lái)治療滯產(chǎn),又稱“縮宮素”;隨著研究的深入,人們發(fā)現(xiàn)催產(chǎn)素的功能和作用十分廣泛,近年來(lái)有研究報(bào)道催產(chǎn)素可以促進(jìn)細(xì)胞增殖以及海馬神經(jīng)發(fā)生[29],而且在哺乳動(dòng)物的母性行為、配偶結(jié)合、社會(huì)認(rèn)同、壓力和焦慮等社交行為中也發(fā)揮重要的作用[30,31]。目前有關(guān)催產(chǎn)素對(duì)酒精暴露后青春期小鼠大腦發(fā)育的作用研究不多。因此,在青春期小鼠酒精暴露后,我們用OT進(jìn)行干預(yù),以期可以改善酒精暴露導(dǎo)致的大腦發(fā)育損傷。關(guān)于RSV,主要存在于紅葡萄皮和某些藥用植物中的一種天然的對(duì)二苯代乙烯類物質(zhì)[32,33]。我們實(shí)驗(yàn)室前期研究顯示,酒精暴露后RSV可以保護(hù)新生期小鼠大腦海馬齒狀回的RGCs,促進(jìn)其神經(jīng)發(fā)生,減輕酒精暴露導(dǎo)致的新生期小鼠大腦發(fā)育損傷,而有關(guān)RSV是否能夠改善酒精暴露后新生期小鼠小腦發(fā)育損傷的研究不多,相關(guān)作用途徑及機(jī)制尚不明確。因此,基于我們的前期研究,在本課題中,新生期小鼠酒精暴露后,我們采用RSV進(jìn)行處理,以期可以改善酒精暴露后引起的新生期小鼠小腦發(fā)育損傷。研究方法與研究結(jié)果:第一部分OT對(duì)酒精暴露后青春期小鼠大腦發(fā)育的作用1.應(yīng)用免疫組織化學(xué)方法,采用溴脫氧核苷尿嘧啶(Bromodeoxyuridine,BrdU)對(duì)大腦海馬齒狀回SGZ區(qū)域的增殖細(xì)胞進(jìn)行標(biāo)記,顯示OT使酒精暴露后青春期小鼠大腦海馬齒狀回SGZ區(qū)域增殖細(xì)胞數(shù)量增加。2.應(yīng)用免疫組織化學(xué)方法,采用小清蛋白(Parvalbumin,PV)標(biāo)記大腦海馬齒狀回SGZ區(qū)域的PV陽(yáng)性中間抑制性神經(jīng)元,顯示OT使酒精暴露后青春期小鼠大腦海馬齒狀回SGZ區(qū)域PV陽(yáng)性中間神經(jīng)元數(shù)量增加。3.應(yīng)用免疫組織化學(xué)方法,采用性別決定相關(guān)HMG盒2(Sry-related HMG box2,Sox2)和膠質(zhì)纖維酸性蛋白(Glial fibrillary acidic protein,GFAP)熒光共標(biāo)對(duì)大腦海馬齒狀回SGZ區(qū)域的RGCs進(jìn)行標(biāo)記,顯示OT可能會(huì)使酒精暴露后青春期小鼠大腦海馬齒狀回RGCs數(shù)量增加。第二部分RSV對(duì)酒精暴露后新生期小鼠小腦發(fā)育的作用1.應(yīng)用免疫組織化學(xué)方法,采用鈣結(jié)合蛋白D-28K(Calbindin D-28k,CBD-28K或CB)標(biāo)記小腦的蒲肯野細(xì)胞,顯示RSV使酒精暴露后新生期小鼠小腦CBD-28K標(biāo)記蒲肯野細(xì)胞數(shù)量增加。2.應(yīng)用免疫組織化學(xué)方法,分別采用腦脂質(zhì)結(jié)合蛋白(Brain lipid-binding protein,BLBP)和GFAP標(biāo)記小腦的伯格曼膠質(zhì)細(xì)胞,顯示RSV使酒精暴露后新生期小鼠小腦伯格曼膠質(zhì)細(xì)胞數(shù)量增加。3.應(yīng)用免疫組織化學(xué)方法,采用小膠質(zhì)細(xì)胞標(biāo)志物Iba1(Ionized calcium-binding adapter molecule 1,Iba1)標(biāo)記小腦的小膠質(zhì)細(xì)胞和蛋白免疫印跡法(Western blot,WB)檢測(cè)小腦內(nèi)核因子-κ結(jié)合基因(Nuclear factor-κ-gene binding,NF-κB)表達(dá)水平,顯示RSV使酒精暴露后新生期小鼠小腦內(nèi)小膠質(zhì)細(xì)胞數(shù)量減少和NF-κB表達(dá)水平下降。研究結(jié)論研究結(jié)果提示:OT和RSV可以通過(guò)對(duì)腦RGCs的保護(hù),降低酒精暴露后腦發(fā)育中細(xì)胞損傷和丟失,改善酒精暴露后的腦發(fā)育。
[Abstract]:Background : As social progress , economic development and people ' s living standards improve , alcohol consumption becomes more common . Alcohol is a central nervous inhibitor . Long - term or one - time excessive alcohol intake can cause alcoholism , damage multi - system organs , such as digestive and cardiovascular system , nervous system and so on . In the development process , the central nervous system ( CNS ) is very sensitive to alcohol stimulation . In the development process , the fetus can manifest as growth retardation , facial capacitance abnormality , CNS dysfunction , and so on , which is fetal alcohol syndrome ( FAS ) . The study shows that the damage of hippocampal volume of hippocampus in neonatal mice can be induced in neonatal mice after single alcohol injection , and the damage of hippocampal volume and loss of nerve cells can be caused . The study also shows that the learning and memory of hippocampus is impaired in hippocampus . The study also shows that alcohol exposure in the hippocampus is easy to cause damage to the hippocampus and learning and memory . Radial glial cells ( RGCs ) are a unique class of cells in tissue morphology , biochemical characteristics and function , and are the earliest glia cells with stem cell function during the embryo . The cells are located in the ventricular zone ( VZ ) , that is , the nerve epithelial layer of the inner wall of the early neural tube . One end of the cell extends out a short top protrusion attached to the surface of the dymal membrane , and the other end of the cell protrudes out of the long and short side protruding base protrusion , and the other end of the cell is extended to the soft - head membrane side . RGCs play a very important role in the development of the CNS , and the development and differentiation of RGCs are regulated by many factors . 鍙﹀,RGCs浣滀負(fù)騫茬粏鑳?yōu)涔熷瓨鍦ㄤ簬鎴愹q碈NS鍐呬袱涓孌栧尯鍩，

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