本文選題：miRNA + miR-204��； 參考：《中南大學(xué)》2014年博士論文

【摘要】：驚厥是嬰幼兒期最常見的急危重癥,驚厥持續(xù)發(fā)作可產(chǎn)生嚴(yán)重神經(jīng)系統(tǒng)后遺癥,影響患兒智力發(fā)育和身心健康,甚至危及患兒生命。小兒驚厥的發(fā)病率很高,約5-6%的小兒曾有過一次或多次驚厥發(fā)作史,早產(chǎn)兒甚至高達(dá)22.7%～25%。若驚厥發(fā)作頻繁或持續(xù)時間較長,約有15%～30%患兒將最終轉(zhuǎn)變?yōu)榘d癇。發(fā)育期驚厥能夠抑制大腦的生長發(fā)育,改變神經(jīng)環(huán)路,提高神經(jīng)元興奮性,加重大腦損傷,導(dǎo)致視覺能、空間能、學(xué)習(xí)記憶和情感障礙,若不及時干預(yù),將嚴(yán)重危害到患兒的生命及遠(yuǎn)期生活質(zhì)量,給家庭與社會帶來沉重負(fù)擔(dān)。就我國人口眾多這一國情來說,所面臨床的困難較其他國家更為嚴(yán)峻。因此,探索發(fā)育期驚厥的發(fā)生發(fā)展機(jī)制及開發(fā)新抗驚厥藥物成了我們目前迫切需要解決的醫(yī)學(xué)難題。 本研究以發(fā)育期反復(fù)驚厥大鼠為模型,開展驚厥發(fā)病機(jī)制的相關(guān)研究。三氟乙醚吸入致驚是經(jīng)典的發(fā)育期驚厥模型。在先前的實驗中,本研究應(yīng)用TaqMan MicroRNA Arrays對發(fā)育期反復(fù)驚厥后大鼠海馬內(nèi)niRNAs表達(dá)譜進(jìn)行檢測,在766種已知miRNA中篩選出驚厥后大鼠海馬內(nèi)具有表達(dá)差異的miRNA,結(jié)果發(fā)現(xiàn),與正常對照組比較,發(fā)育期反復(fù)驚厥后大鼠海馬內(nèi)有20個已知miRNAs表達(dá)下調(diào)0.5倍以上,9個niRNAs表達(dá)明顯上調(diào)2倍以上。其中miR-204在海馬表達(dá)豐富,在反復(fù)驚厥后24h表達(dá)顯著上調(diào)至正常組的7倍左右。因此,本實驗將進(jìn)一步觀察miR-34b-5p、-204、-582-3p、-672在大鼠海馬及大腦皮質(zhì)間的差異性表達(dá),同時選取miR-204做為研究對象,更深入探索其在驚厥性腦損傷中的作用及相關(guān)機(jī)制。 第一部分反復(fù)驚厥性腦損傷后腦組織內(nèi)特異性miRNAs表達(dá)的改變 實驗?zāi)康模禾剿靼l(fā)育期反復(fù)驚厥后大鼠海馬及大腦皮質(zhì)內(nèi)特異性miRNAs的表達(dá)變化及可能機(jī)制。 實驗方法：1)21日齡SD大鼠70只,將實驗仔鼠隨機(jī)分為2大組,即對照組及驚厥組,每組又隨機(jī)分為5個時間點,每時間點各6只大鼠。其中驚厥組通過三氟乙醚反復(fù)吸入(每天1次,連續(xù)6天)制作發(fā)育期反復(fù)驚厥動物模型,而對照組則經(jīng)歷相同步驟,但不吸入三氟乙醚,最終入組大鼠60只。各組大鼠分別于末次驚厥后2h、6h、24h、72h及7d斷頭取腦,分離海馬及大腦皮質(zhì)組織。2)分離組織后,提取各時間點海馬及大腦皮質(zhì)組織總RNA,隨后進(jìn)行miR-34b-5p、 miR-204、miR-582-3p、miR-672的逆轉(zhuǎn)錄,實時定量PCR檢測兩組織中四種miRNAs的表達(dá)。3)體外應(yīng)用海人酸(100μmol/l)干預(yù)體外培養(yǎng)的PC12神經(jīng)元樣細(xì)胞,實時定量PCR檢測干預(yù)后Oh及24h時細(xì)胞內(nèi)miR-204的表達(dá)。4)應(yīng)用公共靶基因預(yù)測軟件starBase (http://starbase.sysu. edu.cn/starbasevl/clipSeq Intersection.php)對在腦組織中具有豐富表達(dá)的miR-204靶基因進(jìn)行預(yù)測,進(jìn)一步篩選出參與驚厥性腦損傷過程的靶基因。 實驗結(jié)果：海馬及大腦皮質(zhì)內(nèi)miR-34b-5p及miR-204在末次驚厥后2h均開始表達(dá)上調(diào)(p0.05),6h時海馬及皮質(zhì)內(nèi)miR-34b-5p的表達(dá)較2h時下調(diào)(p0.05),但仍明顯高于正常對照組(p0.05),而miR-204僅在海馬內(nèi)表達(dá)較2h時下降,6h后皮質(zhì)內(nèi)的miR-204表達(dá)較2h升高。海馬及大腦皮質(zhì)內(nèi)miR-34b-5p及miR-204的表達(dá)均在末次驚厥后24h均達(dá)頂峰,隨后逐漸下降,72h恢復(fù)正常,7d時海馬內(nèi)miR-204的表達(dá),大腦皮質(zhì)內(nèi)miR-34b-5p及miR-204的表達(dá)均較對照組顯著下調(diào)(p0.05),而海馬內(nèi)的miR-34b-5p的表達(dá)則呈上調(diào)趨勢。不同的是,末次驚厥后不同時間點miR-582-3p及miR-672在海馬內(nèi)的表達(dá)均呈下降趨勢,而在大腦皮質(zhì)內(nèi)的表達(dá)均上調(diào),在24h時達(dá)到高峰(p0.05),隨后逐漸下降,至7d時達(dá)正常水平,與對照組相比無明顯差異(p0.05)。不僅如此,我們還發(fā)現(xiàn),反復(fù)驚厥后海馬及大腦皮質(zhì)內(nèi)miR-34b-5p及miR-204的表達(dá)在不同時間點具有高度相關(guān)性,而miR-582-3p及miR-672在不同部位的相關(guān)性并不明顯。因此可以推測,miRNAs在驚厥性腦損傷中的表達(dá)具有組織差異性。為了進(jìn)一步探索特異性miRNA調(diào)控發(fā)育期驚厥性腦損傷的具體機(jī)制,本實驗選取了在大鼠海馬內(nèi)具有豐富表達(dá)的miR-204作為研究目標(biāo)。結(jié)果發(fā)現(xiàn),miR-204在海人酸干預(yù)后的PC12神經(jīng)元樣細(xì)胞的表達(dá)較對照組顯著上調(diào)(p0.05)。進(jìn)而我們選用starBase對三種靶基因預(yù)測軟件(Targetscan、PITA和PicTar)結(jié)果進(jìn)行綜合分析。為了降低假陽性率,選擇同時出現(xiàn)在三種預(yù)測軟件中的靶基因,并進(jìn)一步篩選出參與了發(fā)育期驚厥性腦損傷過程的靶mRNAs。結(jié)果發(fā)現(xiàn),在信息沉默因子1(SIRT1)的3'-UTR可能存在與miR-204的結(jié)合位點,而前者是一種NAD依賴性的去乙酰化酶,廣泛參與了腦損傷后神經(jīng)細(xì)胞凋亡及突觸可塑性的調(diào)控過程。因此,我們推測SIRT1可能是miR-204調(diào)控發(fā)育期驚厥性腦損傷的靶位點之一。 結(jié)論：1)大鼠海馬及大腦皮質(zhì)內(nèi)niR-34b-5p、miR-204、miR-582-3p及miR-672的表達(dá)在發(fā)育期驚厥后均發(fā)生了改變,且具有組織相關(guān)性,從體外水平驗證miRNAs參與了驚厥性腦損傷過程。2)SIRT1可能是miR-204調(diào)控驚厥性腦損傷過程的靶基因之一。 第二部分驚厥性腦損傷后大鼠神經(jīng)元凋亡及突觸可塑性的變化 實驗?zāi)康模簭捏w內(nèi)及體外水平探索驚厥后神經(jīng)元凋亡及神經(jīng)元突觸可塑性的改變,以及SIRT1參與驚厥后性腦損傷的調(diào)控機(jī)制。 實驗步驟：1)如前述,制作發(fā)育期反復(fù)驚厥大鼠模型,在末次驚厥后2h、6h、24h、72h及7d斷頭取腦,分離海馬組織；2)提取各時間點對照組及驚厥組的總蛋白及RNA,應(yīng)用Western blot檢測各實驗組大鼠海馬GAP-43及p53的蛋白表達(dá),qRT-PCR及Western blot檢測各實驗組大鼠SIRT1mRNA及蛋白含量；3)應(yīng)用TUNEL法檢測驚厥后2h、6h、24h、72h及7d大鼠海馬神經(jīng)元的凋亡；4)體外培養(yǎng)PC12神經(jīng)元樣細(xì)胞,應(yīng)用海人酸(100μmo1/l)干預(yù)模擬體外驚厥模型,24小時后收集細(xì)胞,應(yīng)用Western blot分別檢測干預(yù)后Oh及24h細(xì)胞內(nèi)GAP-43、SIRT1及p53的蛋白含量；5)應(yīng)用TUNEL法檢測不同濃度(0、50、100、250μmol/l)海人酸干預(yù)后PC12神經(jīng)元樣細(xì)胞的凋亡。 實驗結(jié)果：1)與對照組相比,海馬內(nèi)SIRT1的蛋白水平在驚厥后2h明顯下降(p0.05),至6h時進(jìn)一步下調(diào)(p0.05),72h時達(dá)最低值(p0.05),7d時逐漸恢復(fù)正常(p0.05)；與之相反,驚厥后2h海馬內(nèi)p53的表達(dá)較對照組明顯上調(diào)(p0.05),至6h時進(jìn)一步上升(p0.05),24h達(dá)頂峰(p0.05),72h時維持高水平表達(dá),與24h比較并無統(tǒng)計學(xué)意義(p0.05),至7d時逐漸恢復(fù)正常,但仍顯著高于正常組(p0.05)；而GAP-43在末次驚厥后6h表達(dá)開始下調(diào)(p0.05),至72h時達(dá)最低值(p0.05),7d時逐漸恢復(fù)正常,而在驚厥后2h與對照組相比并無顯著差異(p0.05)。2)驚厥后2h,海馬內(nèi)神經(jīng)元凋亡較對照組顯著增多,6h進(jìn)一步增加,至72h時達(dá)頂峰,7d時逐漸下降,各實驗組與對照組比較差異均具有統(tǒng)計學(xué)意義(p0.05)。3)海人酸干預(yù)24h后PC12神經(jīng)元樣細(xì)胞內(nèi)SIRT1、GAP-43的蛋白水平均較Oh時顯著下調(diào),而p53的蛋白水平則顯著升高(p0.05)。4)不同濃度海人酸干預(yù)后PC12神經(jīng)元樣細(xì)胞均發(fā)生了凋亡,且呈劑量依賴性,在250μmol/l時凋亡最顯著,各劑量組之間差異均具有統(tǒng)計學(xué)意義(p0.05)。 結(jié)論：本實驗從體內(nèi)及體外實驗證實,驚厥性腦損傷在誘發(fā)神經(jīng)元凋亡的同時,還抑制了神經(jīng)元突觸的生長與延伸。不僅如此,驚厥性腦損傷還顯著抑制了SIRT1的表達(dá),其機(jī)制可能與其下游因子p53發(fā)生去乙�；嘘P(guān),通過上調(diào)凋亡相關(guān)因子p53的表達(dá),加速細(xì)胞的凋亡。 第三部分miR-204通過SIRT1調(diào)控發(fā)育期驚厥性腦損傷的機(jī)制 實驗?zāi)康模簭纳飳W(xué)水平驗證SIRT1是miR-204參與驚厥性腦損傷調(diào)控的靶點以及其相關(guān)機(jī)制。 實驗步驟：1)體外化學(xué)合成miR-204mimics、inhibitors及相應(yīng)的陰性對照；2)如前所述,原代培養(yǎng)PC12神經(jīng)元樣細(xì)胞,將細(xì)胞隨機(jī)分為5組：即正常對照組,miR-204mimics干預(yù)組、miR-204mimics陰性對照組、miR-204inhibitors干預(yù)組及miR-204inhibitors陰性對照組；3)應(yīng)用瞬時轉(zhuǎn)染體外制作miR-204過表達(dá)及低表達(dá)模型,實時定量PCR檢測轉(zhuǎn)染成功率；4)分別應(yīng)用Western blot及實時定量PCR技術(shù)檢測各實驗組內(nèi)SIRT1蛋白及mRNA表達(dá)的變化；5)制作miR-204過表達(dá)及低表達(dá)模型,應(yīng)用海人酸干預(yù)各組PC12神經(jīng)元樣細(xì)胞,隨后TUNEL法檢測各實驗組細(xì)胞的凋亡率,Western blot檢測各組細(xì)胞GAP-43的蛋白表達(dá)；6)應(yīng)用Western blot檢測5組細(xì)胞內(nèi)p53的蛋白表達(dá)；7)體外瞬時轉(zhuǎn)染SIRT1siRNA,制作PC12神經(jīng)元樣細(xì)胞的SIRT1沉默模型,應(yīng)用、Western blot檢測轉(zhuǎn)染成功率；8)應(yīng)用Western blot檢測SIRT1抑制后]miR-204inhibitors對PC12神經(jīng)元樣細(xì)胞內(nèi)p53蛋白表達(dá)的變化。 實驗結(jié)果：1)轉(zhuǎn)染了mimics后的PC12神經(jīng)元樣細(xì)胞內(nèi)miR-204的表達(dá)較空白對照組顯著上升,而轉(zhuǎn)染了inhibitors后的PC12神經(jīng)元細(xì)胞內(nèi)miR-204的表達(dá)明顯低于空白對照組,差異具有統(tǒng)計學(xué)意義(p0.05),提示轉(zhuǎn)染成功。2)miR-204過表達(dá)組細(xì)胞內(nèi)SIRT1蛋白表達(dá)水平較對照組明顯下調(diào),而在低表達(dá)組內(nèi)顯著上(p0.05),但在mRNA水平變化并不顯著,與對照組相比無統(tǒng)計學(xué)差異(p0.05)；3)miR-204低表達(dá)組細(xì)胞內(nèi)p53表達(dá)水平較對照組明顯下調(diào),差異具有統(tǒng)計學(xué)意義(p0.05)；4)miR-204過表達(dá)組細(xì)胞在海人酸誘導(dǎo)驚厥后凋亡較正常組顯著上調(diào),而在低表達(dá)組細(xì)胞凋亡明顯減少,差異具有統(tǒng)計學(xué)意義(p0.05)；5)miR-204達(dá)組細(xì)胞在海人酸誘導(dǎo)驚厥后神經(jīng)元GAP-43的水平顯著下調(diào),而過表在低表達(dá)組則GAP-43的表達(dá)較對照組明顯上調(diào),差異具有統(tǒng)計學(xué)意義(p0.05)；6)轉(zhuǎn)染了SIRTl siRNA后的PC12神經(jīng)元樣細(xì)胞內(nèi)SIRT1的表達(dá)較正常對照組及陽性對照組均顯著下調(diào),差異具有統(tǒng)計學(xué)意義(p0.05)；7)在同時轉(zhuǎn)染了miR-204inhibitors+SIRT1siRNA的PC12神經(jīng)元樣細(xì)胞內(nèi)我們發(fā)現(xiàn),p53蛋白表達(dá)水平與正常對照及相應(yīng)的陽性對照組比較顯著下調(diào)(p0.05)。 結(jié)論：miR-204通過負(fù)調(diào)控SIRT1的蛋白表達(dá)實現(xiàn)對驚厥性腦損傷的調(diào)控。SIRT1介導(dǎo)miR-204調(diào)控驚厥性腦損傷過程可能與影響其下游因子p53的活化有關(guān)。 綜上所述,可以初步得出結(jié)論：miR-34b-5p、-204、-582-3p、-672均參與了發(fā)育期反復(fù)驚厥性腦損傷的調(diào)控,miR-204在反復(fù)驚厥后的海馬及大腦皮質(zhì)中表達(dá)均較對照組顯著上調(diào),通過負(fù)調(diào)控SIRT1的表達(dá)調(diào)控驚厥后神經(jīng)元的凋亡有突觸可塑性的改變,其機(jī)制可能與負(fù)性調(diào)控凋亡相關(guān)因子p53的表達(dá)有關(guān)。因此,下調(diào)miR-204的表達(dá)能在轉(zhuǎn)錄后水平減弱了對SIRT1的抑制,使其表達(dá)顯著增高,抑制驚厥性腦損傷誘發(fā)的神經(jīng)元凋亡,促進(jìn)軸突的生長與延伸,進(jìn)而延緩腦損傷進(jìn)程,為今后治療驚厥性腦損傷提供了新的靶點。
[Abstract]:Convulsion is the most common severe severe disease in infantile period. The persistent seizures can produce severe neurological sequelae, affecting the mental development and physical and mental health of the children, even endangering the life of children. The incidence of convulsion in children is very high, about 5-6% of children have once or many times of convulsion history, preterm infants even up to 22.7% to 25%. if convulsions hair With frequent or longer duration, about 15% to 30% children will eventually change into epilepsy. Developmental seizures can inhibit the growth and development of the brain, change the nerve loop, improve the neuronal excitability, aggravate the brain damage, and lead to visual, spatial, learning, memory and emotional obstacles. If not intervened in time, it will seriously jeopardize the life of the child and The long-term quality of life has brought a heavy burden to the family and the society. As far as our country's population is concerned, the difficulty of the bed is more severe than that of other countries. Therefore, exploring the mechanism of development and the development of new anticonvulsant drugs have become the medical problems that we are urgently needed to solve.
The present study conducted a related study of the mechanism of convulsion in rats with recurrent seizures. Three fluoro ethyl ether inhalation induced convulsion was a classic model of developmental seizures. In previous experiments, this study used TaqMan MicroRNA Arrays to detect the niRNAs expression profiles in the hippocampus of rats after recurrent seizures and in the 766 known miRNA. The expression of miRNA in hippocampus of rats after convulsion was screened. It was found that, compared with the normal control group, 20 known miRNAs expressions were down regulated by 0.5 times in hippocampus after recurrent seizures, and the expression of 9 niRNAs was up to above 2 times. The expression of miR-204 was abundant in the hippocampus, and the expression of 24h was up to a significant increase after repeated convulsion. 7 times of the normal group. Therefore, this experiment will further observe the differential expression of miR-34b-5p, -204, -582-3p, -672 in the hippocampus and cerebral cortex of rats, and select miR-204 as the research object, and explore its role and mechanism in the brain damage of convulsion.
Part one changes in the expression of specific miRNAs in brain tissue after recurrent convulsive brain injury
Objective: To explore the expression and possible mechanism of specific miRNAs in hippocampus and cerebral cortex of rats after recurrent seizures.
Methods: 1) 70 rats of 21 day old SD rats were randomly divided into 2 groups, namely the control group and the convulsive group. Each group was randomly divided into 5 time points and 6 rats at each time point. The convulsions group was repeatedly inhaled by three fluorine ether (1 times a day for 6 days), and the control group underwent phase synchronization. Suddenly, three fluorine ether was not inhaled, and 60 rats were finally enrolled in the group. After the last convulsion, the rats were divided into 2h, 6h, 24h, 72h and 7d head to take the brain, and the hippocampus and cerebral cortex.2 were separated. The total RNA of hippocampus and cerebral cortex was extracted at each time point. Then miR-34b-5p, miR-204, miR-582-3p, miR-672 were reverse transcriptase, real-time quantitative PCR was used to detect the expression of four kinds of miRNAs in two tissues.) in vitro application of sea human acid (100 mu mol/l) to PC12 neuron like cells cultured in vitro, and real-time quantitative PCR detection of miR-204 expression in Oh and 24h when Oh and 24h were detected by PCR. We predicted the target genes with abundant expression in the brain tissue, and further screened out the target genes involved in the process of convulsive brain injury. MiR-204
The results showed that the expression of miR-34b-5p and miR-204 in the hippocampus and cerebral cortex began to increase (P0.05) after the last convulsion, and the expression of miR-34b-5p in hippocampus and cortex was lower than that of 2H (P0.05) at 6h, but it was still significantly higher than that in the normal control group (P0.05), but the expression of miR-204 in the hippocampus was lower than that in the normal control group (P0.05). The expression of miR-204 in the hippocampus was lower than that of 2h in the hippocampus, and the expression in the cortex after 6h was higher than that in the 6h. The expression of miR-34b-5p and miR-204 in hippocampus and cerebral cortex all reached the peak of 24h after the last convulsion, and then gradually decreased and 72h returned to normal. The expression of miR-204 in the hippocampus and the expression of miR-34b-5p and miR-204 in the cerebral cortex were significantly lower than those of the control group (P0.05), while the expression of miR-34b-5p in the hippocampus was up trend. The expression of miR-582-3p and miR-672 in the hippocampus decreased in different time points after the last convulsion, while the expression in the cerebral cortex was up up, reached the peak at 24h (P0.05), and then gradually decreased to 7d to the normal level, no significant difference compared with the control group (P0.05). Not only that, but also, we found repeated convulsions in Houhai. The expression of miR-34b-5p and miR-204 in the horse and cerebral cortex is highly correlated at different time points, while the correlation of miR-582-3p and miR-672 in different parts is not obvious. Therefore, it is possible to speculate that the expression of miRNAs in the brain damage of convulsive brain is different. In order to further explore the convulsive brain damage of the specific miRNA in the regulation of the developmental period of the brain The specific mechanism of injury was selected in the experiment of miR-204 with rich expression in the hippocampus of rats. The results showed that the expression of PC12 neuron like cells in the miR-204 after the sea human acid drying was significantly higher than that of the control group (P0.05). Then we selected the results of starBase for three target gene prediction software (Targetscan, PITA and PicTar). In order to reduce the false positive rate, select the target genes that appear in the three prediction software and further screen out the target mRNAs. that participates in the process of brain damage in the development period. It is found that in the 3'-UTR of information silencing factor 1 (SIRT1), there may be a binding site with miR-204, while the former is a NAD dependence. Acetyltransferase is widely involved in the regulation of neuronal apoptosis and synaptic plasticity after brain injury. Therefore, we speculate that SIRT1 may be one of the target sites for miR-204 to regulate the development of convulsive brain damage.
Conclusion: 1) the expression of niR-34b-5p, miR-204, miR-582-3p and miR-672 in the hippocampus and cerebral cortex of rats changes after the development of convulsion, and has tissue correlation. It is demonstrated that miRNAs participates in.2 in the process of convulsive brain injury in vitro. SIRT1 may be one of the target genes in the process of regulating convulsive brain damage in miR-204.
The second part is the change of neuronal apoptosis and synaptic plasticity after convulsive brain injury in rats.
Objective: To explore the changes in neuronal apoptosis and neuronal synaptic plasticity after convulsion in vivo and in vitro, as well as the regulatory mechanism of SIRT1 involved in sexual brain injury after convulsion.
The experimental steps: 1) as mentioned above, the rat model of recurrent seizures was made in the development period. After the last convulsion, 2h, 6h, 24h, 72h and 7d broke the head, separated the hippocampus; 2) the total protein and RNA of the control group and the convulsive group were extracted, and the protein expression of the GAP-43 and p53 in the hippocampus of the rats in the experimental groups was detected by Western blot, qRT-PCR and Western detection. SIRT1mRNA and protein content of rats in the experimental group were measured; 3) the apoptosis of hippocampal neurons in 2H, 6h, 24h, 72h and 7d rats after convulsion was detected by TUNEL; 4) cultured PC12 neuron like cells in vitro, and used sea human acid (100 micron) to simulate the model of convulsion in vitro, collect the cells after 24 hours, and detect the Oh and finely after the intervention respectively with Western blot. Intracellular GAP-43, SIRT1 and p53 protein content; 5) TUNEL method was used to detect the apoptosis of PC12 neuron like cells after different concentrations (0,50100250 mol/l mol/l) of kainic acid.
The results were as follows: 1) compared with the control group, the protein level of SIRT1 in the hippocampus decreased significantly (P0.05) after convulsion (P0.05), further down to 6h (P0.05), 72h at the lowest value (P0.05) and 7d when 7d gradually returned to normal (P0.05). On the contrary, the p53 expression in the hippocampus was up significantly up after convulsion (P0.05), up to 24. H reached the peak (P0.05) and 72h at a high level. There was no statistical significance compared with 24h (P0.05), and gradually recovered to normal at 7d, but it was still significantly higher than that in normal group (P0.05), and GAP-43 began to decrease (P0.05) after the last convulsion (P0.05), to 72h (P0.05), and gradually returned to normal at the time of 72h, and compared with the control group after convulsion. No significant difference (P0.05).2) after convulsion, 2h, neuronal apoptosis in the hippocampus increased significantly compared with the control group, 6h increased further, reached the peak at 72h, and decreased gradually when 7d, and the differences in the experimental group and the control group were statistically significant (P0.05).3). Sea human acid was involved in SIRT1 in PC12 neuron like cells after 24h, and the protein level of GAP-43 was significantly higher than that of the control group. Down regulation, while the protein level of p53 increased significantly (P0.05).4) the apoptosis of PC12 neuron like cells after different concentrations of sea human acid was in a dose-dependent manner. The apoptosis was most significant at 250 u mol/l, and the difference between each dose group was statistically significant (P0.05).
Conclusion: in this experiment, the experiment in vivo and in vitro proved that the seizure brain injury also inhibited the growth and extension of neuron synapse at the same time, and inhibited the growth and extension of neuron synapse. Not only so, the seizure brain injury also significantly inhibited the expression of SIRT1, and its mechanism may be related to the deacetylation of the downstream factor p53, by up regulation of apoptosis related factors. The expression of subp53 accelerates the apoptosis of cells.
The third part is the mechanism of miR-204 regulating the development of convulsive brain injury through SIRT1.
Objective: to verify that SIRT1 is the target and mechanism of miR-204 in the regulation of convulsive brain injury from biological level.
Experimental steps: 1) in vitro chemical synthesis of miR-204mimics, inhibitors and corresponding negative control; 2) as mentioned earlier, the primary cultured PC12 neuron like cells were randomly divided into 5 groups: the normal control group, the miR-204mimics intervention group, the miR-204mimics negative control group, the miR-204inhibitors intervention group and the miR-204inhibitors negative control group. 3) using transient transfection in vitro to make miR-204 overexpression and low expression model, real-time quantitative PCR detection of transfection success rate; 4) use Western blot and real-time quantitative PCR technique to detect the changes of SIRT1 protein and mRNA expression in each experiment group; 5) make miR-204 overexpression and low expression model, use sea human acid to intervene PC12 neuron sample in each group Cell apoptosis rate was detected by TUNEL method, Western blot was used to detect the protein expression of GAP-43 in each group; 6) Western blot was used to detect the protein expression of p53 in the 5 groups; 7) transient transfection of SIRT1siRNA in vitro, SIRT1 silent model of PC12 neuron like cells, application, Western blot test success rate of transfection; 8) Western blot was used to detect the changes of p53 protein expression in PC12 neuron like cells after inhibition of SIRT1 by SIRT1.
The results were as follows: 1) the expression of miR-204 in the PC12 neuron like cells after mimics transfection was significantly higher than that in the blank control group, and the expression of miR-204 in PC12 neurons transfected after inhibitors was significantly lower than that in the blank control group. The difference was statistically significant (P0.05), and the expression of SIRT1 protein in the miR-204 overexpression group was shown in the transfected.2). The expression level in the low expression group was significantly lower than that in the control group (P0.05), but there was no significant difference between the mRNA level and the control group (P0.05); 3) the expression level of p53 in the miR-204 low expression group was significantly lower than that in the control group (P0.05), and 4) the miR-204 overexpressed group cells were in the sea people. After acid induced convulsion, apoptosis was significantly higher than that in the normal group, but the apoptosis decreased significantly in the low expression group, and the difference was statistically significant (P0.05); 5) the level of GAP-43 in the neurons of the miR-204 group was significantly down down after the sea human acid induced convulsion, while the expression of GAP-43 in the low expression group was up significantly higher than that in the control group. The difference was statistically significant. Learning significance (P0.05); 6) the expression of SIRT1 in PC12 neuron like cells after transfected with SIRTl siRNA was significantly lower than that in the normal control group and the positive control group. The difference was statistically significant (P0.05); 7) we found that the expression level of p53 protein and the normal pair in the PC12 neuron like cells transfected to miR-204inhibitors+SIRT1siRNA at the same time. The corresponding positive control group was significantly lower than that of the control group (P0.05).
Conclusion: miR-204 can regulate the expression of SIRT1 protein by regulating the expression of SIRT1 in rats.

【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2014
【分類號】：R720.597

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本文編號：1813433