miR-134真核质粒表达载体和慢病毒载体的构建及其初步验证

发布时间：2018-04-17 17:41

本文选题：miR-134 + OL细胞　；参考：《重庆医科大学》2014年硕士论文

【摘要】：背景与目的：抑郁症是一类常见病，临床上主要表现为患者对日常活动和娱乐的兴趣减退、对前途悲观失望、容易焦虑、睡眠质量较差等，，迄今，人们对抑郁症的发病原因以及发病机制还不是很清楚，但是可以肯定的是，其发病有生物、心理以及社会环境等内外因素的参与。近年来，有研究提示miRNAs与精神疾病有关联，其可能在精神疾病的发生、发展中起着十分重要的角色。有研究发现miR-134的表达水平在BD躁狂相病人的血液中降低，然而miR-134的表达水平在癫痫持续状态（status epilepticus，SE）造模成功的大鼠模型中上升，并且在内侧颞叶癫痫（Mesial Temporal LobeEpilepsy，MTLE）病人中也同样检测到miR-134表达水平的升高。目前的研究主要是证实了miR-134异常表达与精神疾病有关，但是对精神疾病中miR-134的精细调控机制的研究还十分有限。如果想要更加深入地了解miR-134发挥的作用，我们需要建立细胞或动物模型，在细胞及动物水平检测其引起的病理生理改变。本课题的目的在于构建miR-134真核质粒表达载体和慢病毒载体，并且将构建好的质粒和慢病毒转染至OL（oligodendroglia，人类少突胶质瘤）细胞、SD大鼠海马神经元中，为研究抑郁症的分子机制提供有效的实验工具。方法： 1.用PCR的方法从OL细胞基因组中扩增含有miR-134基因的目的片段，并将miR-134基因的PCR产物和载体pEGFP-N1连接以构建pEGFP-miR-134载体，经酶切、测序证实之后采用Invitrogen公司的Lipofectamine LTX和Plus Reagents转染试剂盒将质粒瞬时转染至OL细胞中，提取总RNA,在基因水平检测miR-134在OL细胞中的表达变化。 2.取出生24h内的清洁级SD乳鼠，断头后分离提取海马神经元，采用无血清培养基培养神经元，为后期在海马神经元中进行的细胞活力实验、细胞凋亡实验及突触可塑性实验奠定良好基础。 3.构建Lenti-miR134-EX慢病毒表达载体，并包装、纯化慢病毒，经鉴定慢病毒包装成功后将其感染SD大鼠海马神经元，提取总RNA,在基因水平检测miR-134在海马神经元中的表达变化。结果： 1.从OL细胞基因组中成功扩增出含有miR-134基因的目的片段，并将其连接到载体pEGFP-N1，命名为pEGFP-miR-134，且转染OL细胞效果较好。 2.用简单的方法即可分离培养出高纯度（可达90%以上）的海马神经元。 3.成功包装了能够表达绿色荧光蛋白的慢病毒载体，命名为Lenti-miR134-EX。结论：本课题成功构建了miR-134真核质粒表达载体和慢病毒载体，并获得瞬时转染的OL细胞以及SD大鼠原代海马神经元，为深入研究miR-134在抑郁症的精细调控机制提供了有效的工具和平台。
[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.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R749.4

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