本文選題：miR-134 + OL細(xì)胞　； 參考：《重慶醫(yī)科大學(xué)》2014年碩士論文

【摘要】：背景與目的： 抑郁癥是一類常見病，臨床上主要表現(xiàn)為患者對(duì)日�；顒�(dòng)和娛樂的興趣減退、對(duì)前途悲觀失望、容易焦慮、睡眠質(zhì)量較差等，，迄今，人們對(duì)抑郁癥的發(fā)病原因以及發(fā)病機(jī)制還不是很清楚，但是可以肯定的是，其發(fā)病有生物、心理以及社會(huì)環(huán)境等內(nèi)外因素的參與。近年來，有研究提示miRNAs與精神疾病有關(guān)聯(lián)，其可能在精神疾病的發(fā)生、發(fā)展中起著十分重要的角色。 有研究發(fā)現(xiàn)miR-134的表達(dá)水平在BD躁狂相病人的血液中降低，然而miR-134的表達(dá)水平在癲癇持續(xù)狀態(tài)（status epilepticus，SE）造模成功的大鼠模型中上升，并且在內(nèi)側(cè)顳葉癲癇（Mesial Temporal LobeEpilepsy，MTLE）病人中也同樣檢測(cè)到miR-134表達(dá)水平的升高。目前的研究主要是證實(shí)了miR-134異常表達(dá)與精神疾病有關(guān)，但是對(duì)精神疾病中miR-134的精細(xì)調(diào)控機(jī)制的研究還十分有限。如果想要更加深入地了解miR-134發(fā)揮的作用，我們需要建立細(xì)胞或動(dòng)物模型，在細(xì)胞及動(dòng)物水平檢測(cè)其引起的病理生理改變。 本課題的目的在于構(gòu)建miR-134真核質(zhì)粒表達(dá)載體和慢病毒載體，并且將構(gòu)建好的質(zhì)粒和慢病毒轉(zhuǎn)染至OL（oligodendroglia，人類少突膠質(zhì)瘤）細(xì)胞、SD大鼠海馬神經(jīng)元中，為研究抑郁癥的分子機(jī)制提供有效的實(shí)驗(yàn)工具。 方法： 1.用PCR的方法從OL細(xì)胞基因組中擴(kuò)增含有miR-134基因的目的片段，并將miR-134基因的PCR產(chǎn)物和載體pEGFP-N1連接以構(gòu)建pEGFP-miR-134載體，經(jīng)酶切、測(cè)序證實(shí)之后采用Invitrogen公司的Lipofectamine LTX和Plus Reagents轉(zhuǎn)染試劑盒將質(zhì)粒瞬時(shí)轉(zhuǎn)染至OL細(xì)胞中，提取總RNA,在基因水平檢測(cè)miR-134在OL細(xì)胞中的表達(dá)變化。 2.取出生24h內(nèi)的清潔級(jí)SD乳鼠，斷頭后分離提取海馬神經(jīng)元，采用無血清培養(yǎng)基培養(yǎng)神經(jīng)元，為后期在海馬神經(jīng)元中進(jìn)行的細(xì)胞活力實(shí)驗(yàn)、細(xì)胞凋亡實(shí)驗(yàn)及突觸可塑性實(shí)驗(yàn)奠定良好基礎(chǔ)。 3.構(gòu)建Lenti-miR134-EX慢病毒表達(dá)載體，并包裝、純化慢病毒，經(jīng)鑒定慢病毒包裝成功后將其感染SD大鼠海馬神經(jīng)元，提取總RNA,在基因水平檢測(cè)miR-134在海馬神經(jīng)元中的表達(dá)變化。 結(jié)果： 1.從OL細(xì)胞基因組中成功擴(kuò)增出含有miR-134基因的目的片段，并將其連接到載體pEGFP-N1，命名為pEGFP-miR-134，且轉(zhuǎn)染OL細(xì)胞效果較好。 2.用簡單的方法即可分離培養(yǎng)出高純度（可達(dá)90%以上）的海馬神經(jīng)元。 3.成功包裝了能夠表達(dá)綠色熒光蛋白的慢病毒載體，命名為Lenti-miR134-EX。 結(jié)論： 本課題成功構(gòu)建了miR-134真核質(zhì)粒表達(dá)載體和慢病毒載體，并獲得瞬時(shí)轉(zhuǎn)染的OL細(xì)胞以及SD大鼠原代海馬神經(jīng)元，為深入研究miR-134在抑郁癥的精細(xì)調(diào)控機(jī)制提供了有效的工具和平臺(tái)。
[Abstract]:Background and purpose:Depression is a kind of common disease, the main clinical manifestation is the decrease of patients' interest in daily activities and entertainment, pessimistic disappointment about the future, easy anxiety, poor sleep quality, etc. So far,The cause and mechanism of depression are not well understood, but it is certain that there are internal and external factors, such as biological, psychological and social environment.In recent years, some studies have suggested that miRNAs may play an important role in the occurrence and development of mental illness.Some studies found that the expression of miR-134 decreased in the blood of patients with BD mania, but the expression of miR-134 increased in the successful model of epileptic status epilepticus.An increase in miR-134 expression was also detected in patients with medial temporal lobe epilepsy (Mesial Temporal Lobe Epilepsym MTLEs).The present studies mainly confirm that the abnormal expression of miR-134 is related to mental illness, but the study on the fine regulation mechanism of miR-134 in mental disease is still very limited.To better understand the role of miR-134, we need to establish cell or animal models to detect pathophysiological changes at the cellular and animal levels.The purpose of this study was to construct miR-134 eukaryotic expression vector and lentivirus vector, and transfect the constructed plasmids and lentiviruses into the hippocampal neurons of SD rats.To provide an effective experimental tool for studying the molecular mechanism of depression.Methods:1.The target fragment containing miR-134 gene was amplified by PCR from the OL cell genome. The PCR product of miR-134 gene and the vector pEGFP-N1 were ligated to construct pEGFP-miR-134 vector.After sequencing, the plasmid was transiently transfected into OL cells with Lipofectamine LTX and Plus Reagents transfection kit of Invitrogen Company. The total RNAs were extracted and the expression of miR-134 in OL cells was detected at the gene level.2.The newborn rats of clean grade within 24 hours were taken out and the hippocampal neurons were isolated and extracted after the head cut. The neurons were cultured in serum-free medium for the later experiment of cell viability in the hippocampal neurons.Apoptosis test and synaptic plasticity test lay a good foundation.3.The expression vector of Lenti-miR134-EX lentivirus was constructed, and the lentivirus was packaged and purified. The lentivirus was infected into the hippocampal neurons of SD rats after packaging successfully. The total RNAs were extracted and the changes of miR-134 expression in hippocampal neurons were detected at the gene level.Results:1.The target fragment containing miR-134 gene was successfully amplified from the genome of OL cells and ligated to the vector pEGFP-N1, named pEGFP-miR-134.2.Hippocampal neurons with high purity (up to 90%) can be isolated by simple method.3.The lentivirus vector which can express green fluorescent protein was successfully packaged and named Lenti-miR134-EX.Conclusion:In this study, miR-134 eukaryotic expression vector and lentivirus vector were successfully constructed, and transient transfected OL cells and primary hippocampal neurons of SD rats were obtained, which provided an effective tool and platform for further study on the fine regulation mechanism of miR-134 in depression.
【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】：2014
【分類號(hào)】：R749.4

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 龐曉輝;劉明;畢鋒;;胃癌相關(guān)MicroRNA研究進(jìn)展[J];生物醫(yī)學(xué)工程學(xué)雜志;2012年04期

本文編號(hào)：1764618