本文選題：缺糖缺氧損傷 + 全反式視黃酸��； 參考：《重慶醫(yī)科大學(xué)》2012年碩士論文

【摘要】：背景維生素A缺乏（VitaminAdeficiency, VAD）、鐵缺乏、碘缺乏已被WHO和聯(lián)合國(guó)兒童基金會(huì)確定為世界三大營(yíng)養(yǎng)素缺乏性疾病。VAD在世界范圍內(nèi)普遍存在，其中孕婦和兒童是其高危人群。新生兒缺氧缺血性腦損傷(hypoxic-ischemic brain damage，HIBD)是新生兒期常見(jiàn)的中樞神經(jīng)系統(tǒng)高發(fā)病、危重病，是導(dǎo)致新生兒死亡和殘疾的主要原因之一。有研究表明，HIBD的主要發(fā)病機(jī)制包括腦血流動(dòng)力學(xué)變化、腦細(xì)胞能量代謝衰竭、再灌注損傷與氧自由基的產(chǎn)生、鈣內(nèi)流、興奮性氨基酸導(dǎo)致神經(jīng)毒性作用、遲發(fā)性神經(jīng)元死亡和凋亡，其中細(xì)胞凋亡是HIBD神經(jīng)細(xì)胞死亡的主要形式。本課題組的前期研究發(fā)現(xiàn)，HIBD新生大鼠體內(nèi)VA水平與其神經(jīng)功能的修復(fù)存在密切的相關(guān)性，但其具體分子機(jī)制目前還不清楚。 目的體外探討全反式視黃酸（ATRA）對(duì)缺氧缺糖損傷（OGD）后的PC12細(xì)胞的修復(fù)作用。 方法建立PC12細(xì)胞OGD損傷模型，給予不同ATRA濃度（0.5、2、4和20μmol/L）誘導(dǎo)，利用Annexin V-PI流式細(xì)胞術(shù)檢測(cè)PC12細(xì)胞的凋亡發(fā)生率，熒光探針JC-1測(cè)定細(xì)胞線粒體膜電位的變化，，同時(shí)運(yùn)用Real-time PCR技術(shù)和Western blot技術(shù)分析ATRA對(duì)OGD損傷后的PC12細(xì)胞線粒體凋亡通路關(guān)鍵性凋亡因子表達(dá)水平的影響。 結(jié)果光鏡下觀察顯示PC12細(xì)胞OGD損傷后，細(xì)胞聚團(tuán)生長(zhǎng)，胞體皺縮，折光性下降；Annexin V-PI、JC-1檢測(cè)顯示細(xì)胞凋亡率明顯增加，線粒體膜電位下降明顯；加入2、4μmol/LATRA后均可降低細(xì)胞凋亡率，并保持線粒體膜電位的相對(duì)穩(wěn)定，尤以4μmol/LATRA作用效果明顯；與對(duì)照組相比較，0.5μmol/L ATRA組沒(méi)有顯著差異；但20μmol/L ATRA組的細(xì)胞凋亡率及線粒體膜電位下降率增高。Real-timePCR和Western blot檢測(cè)顯示，4μmol/L ATRA組較OGD損傷組中的促凋亡因子Bax的表達(dá)水平顯著下調(diào)，抗凋亡因子Bcl-2明顯升高，蛋白表達(dá)水平與mRNA水平變化一致。 結(jié)論4μmol/L ATRA對(duì)OGD損傷后的PC12細(xì)胞具有抑制凋亡的作用，其機(jī)制可能是ATRA通過(guò)上調(diào)Bcl-2和下調(diào)Bax的表達(dá)，影響線粒體凋亡通路，從而實(shí)現(xiàn)對(duì)缺糖缺氧損傷細(xì)胞的保護(hù)作用。 背景HIBD后神經(jīng)元的死亡形式主要為細(xì)胞凋亡，位于線粒體的Bcl-2家族及其編碼的蛋白質(zhì)表達(dá)失衡是線粒體凋亡通路中的關(guān)鍵事件，最終導(dǎo)致細(xì)胞結(jié)構(gòu)和功能的廣泛損害。本課題組前期的研究表明，體內(nèi)正常VA水平更有效地促進(jìn)HIBD大鼠的神經(jīng)細(xì)胞的功能恢復(fù)，其機(jī)制可能是由于VAD抑制了RAR的表達(dá)，降低了神經(jīng)細(xì)胞Ca~(2+)的興奮性，從而影響學(xué)習(xí)記憶能力。我們前一部分研究結(jié)果顯示，4μmol/LATRA能夠通過(guò)調(diào)節(jié)線粒體凋亡通路，抑制神經(jīng)細(xì)胞的凋亡，其機(jī)制是否是通過(guò)RAR的調(diào)節(jié)而發(fā)揮作用，將作進(jìn)一步研究。 目的探討RA信號(hào)通路中RAR在缺糖缺氧損傷的PC12細(xì)胞抗凋亡的調(diào)節(jié)作用。 方法采用本課題組自行構(gòu)建的視黃酸核受體siRAR重組腺病毒（Ad-siRAR）感染PC12細(xì)胞，應(yīng)用RT-PCR方法檢測(cè)Ad-siRAR對(duì)RAR受體的抑制效率。利用Ad-siRAR技術(shù)，進(jìn)一步觀察4μmol/LATRA對(duì)損傷的PC12細(xì)胞抗凋亡的影響，同時(shí)以空腺病毒載體為對(duì)照。Annexin V-PI和熒光探針JC-1染色，流式細(xì)胞技術(shù)分別檢測(cè)siRAR感染PC12細(xì)胞后細(xì)胞凋亡率及線粒體膜電位下降率的變化，進(jìn)一步運(yùn)用Real-time PCR和Western blot技術(shù)分析RAR受體與線粒體凋亡通路中關(guān)鍵因子基因和蛋白表達(dá)水平的變化。 結(jié)果PC12細(xì)胞缺糖缺氧損傷后，RAR mRNA表達(dá)水平降低。Ad-siRAR感染36h后，可有效抑制RAR基因表達(dá)。經(jīng)Annexin V-PI和熒光探針JC-1染色，流式細(xì)胞技術(shù)檢測(cè)顯示，4μmol/LATRA誘導(dǎo)的OGD損傷PC12細(xì)胞在Ad-siRAR感染條件下，其細(xì)胞凋亡率及線粒體膜電位下降率均明顯增高。同時(shí)，Real-time PCR和Western blot結(jié)果表明，感染Ad-siRAR重組腺病毒的PC12細(xì)胞OGD損傷后，Bax表達(dá)水平顯著增高，Bcl-2的表達(dá)降低。 結(jié)論重組腺病毒Ad-siRAR顯著抑制了ATRA對(duì)OGD損傷的PC12細(xì)胞的抗凋亡保護(hù)作用，提示ATRA通過(guò)調(diào)節(jié)RA信號(hào)通路中的RAR，影響線粒體凋亡信號(hào)通路中關(guān)鍵因子Bax和Bcl-2的表達(dá)水平，從而發(fā)揮對(duì)OGD損傷的PC12細(xì)胞的保護(hù)作用。
[Abstract]:Background Vitamin A deficiency ( VAD ) , iron deficiency , iodine deficiency have been identified by WHO and UNICEF as the world ' s three major nutritional deficiencies . VAD is prevalent worldwide , among which pregnant women and children are high - risk populations . The study shows that the main pathogenesis of HIBD includes brain hemodynamic changes , brain cell energy metabolism failure , reperfusion injury and oxygen free radical generation , calcium influx , excitatory amino acids leading to neurotoxicity , delayed neuronal death and apoptosis .

Objective To investigate the effect of all - trans retinoic acid ( ATRA ) on PC12 cells after hypoxia and glucose deprivation ( OGD ) .

Methods PC12 cell OGD injury model was established and induced by different concentrations of ATRA ( 0.5 , 2 , 4 and 20 渭mol / L ) . The apoptosis rate of PC12 cells was determined by flow cytometry . The effects of ATRA on the expression level of apoptosis in PC12 cells after OGD injury were analyzed by Real - time PCR and Western blot .

Results After the PC12 cell OGD injury was observed under the light microscope , the cell clusters were grown , the cell bodies shrink and the refractive index decreased ;
The apoptosis rate of the cells was significantly increased and the mitochondrial membrane potential decreased .
After addition of 2,4 - 渭mol / LATRA , the apoptosis rate was decreased , and the relative stability of mitochondrial membrane potential was maintained , especially in 4 渭mol / LATRA .
Compared with the control group , there was no significant difference in 0.5 渭mol / L ATRA group .
However , the apoptosis rate and mitochondrial membrane potential decreased significantly in the 20 渭mol / L ATRA group . Real - time PCR and Western blot showed that the expression level of pro - apoptotic factor Bax was down - regulated in 4 渭mol / L ATRA group compared with that in OGD group , and the expression level of anti - apoptotic factor Bcl - 2 increased significantly , and the level of protein expression was consistent with that of mRNA level .

Conclusion 4 渭mol / L ATRA can inhibit the apoptosis of PC12 cells after OGD injury . The mechanism may be that ATRA can regulate the expression of Bcl - 2 and down - regulate Bax and influence the apoptosis pathway of mitochondria .

Background : The death form of neurons in HIBD is mainly apoptosis . The expression imbalance of Bcl - 2 family and its encoded protein in mitochondria is a key event in the apoptosis pathway of mitochondria , which results in the extensive damage of cell structure and function .

Objective To investigate the effect of RARs on anti - apoptosis of PC12 cells induced by glucose deprivation and hypoxia .

Methods The effect of 4 渭mol / LATRA on the anti - apoptosis of PC12 cells was investigated by RT - PCR . The effects of 4 渭mol / LATRA on the anti - apoptosis of PC12 cells were investigated by RT - PCR .

Results After 36 hours of Ad - sirar infection , the apoptosis rate and mitochondrial membrane potential of PC12 cells infected with Ad - sirar were significantly increased , and the expression of Bax was significantly increased and the expression of Bcl - 2 decreased .

Conclusion The effect of ATRA on the anti - apoptotic effect of ATRA on PC12 cells induced by OGD was significantly inhibited by recombinant adenovirus Ad - sirar , suggesting that ATRA could play a role in protecting PC12 cells injured by OGD .
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類(lèi)號(hào)】：R363

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本文編號(hào)：2000147