慢病毒载体介导的整合素β1基因沉默对体外难治性癫痫细胞神经网络形成的作用及分子机制研究

发布时间：2018-06-28 08:36

本文选题：海马神经元 + Integrinβ1　；参考：《广西医科大学》2014年博士论文

【摘要】：第一部分Real-time qPCR检测大鼠Sombati难治性癫痫细胞模型中Integrin β1基因表达变化目的：整合素-跨膜系统主要介导细胞与细胞外基质的相互作用，本实验初步探讨在海马神经元的体外癫痫细胞模型中的细胞表面受体整合素β1（Integrin β1）在癫痫致痫性形成中的可能作用。方法：利用Neurobasal Medium无血清神经培养基和B-27因子体外培养SD新生乳鼠的海马神经元，对培养至第14天的细胞，参照Sombati的方法制备体外难治性癫痫模型，，随机分为正常对照组（Control）、Sombati癫痫细胞模型组（低Mg2+），分别在用含有1mM Mg2+的pBRS液或低Mg2+的pBRS液培养细胞3h后，各组神经元在恢复维持培养基继续培养，在之后的第4天，采用Real-time qPCR方法检测Integrin β1的表达。结果：Sombati癫痫细胞造模后4天与正常海马神经细胞相比，Integrinβ1基因表达水平均明显高于正常海马神经细胞。结论：Integrin β1基因在Sombati癫痫细胞模型中表达增高，可能参与了难治性癫痫的形成过程。第二部分Integrin β1shRNA重组慢病毒载体的构建及其对Sombati难治性癫痫细胞Integrin β1基因表达的影响目的：构建Integrin β1基因RNA干扰慢病毒载体，为研究Sombati癫痫细胞模型Integrin β1的作用机制提供载体。方法:（1）针对Integrin β1目的基因序列，利用的RNA干扰序列设计软件，设计4个针对Integrin β1的RNA干扰靶点序列，构建4个shRNA慢病毒重组质粒，测序鉴定阳性克隆，常规培养HEK293T细胞，将慢病毒shRNA载体及其辅助包装原件载体质粒共转染进HEK293T细胞进行慢病毒包装、浓缩、滴度测定，并收集病毒。（2）常规培养PC12细胞，将构建的Integrin β1shRNA1-4转染PC12细胞，分为Integrin β1shRNANC组、Integrin β1shRNA1组、 Integrin β1shRNA2组、Integrin β1shRNA3组和Integrin shRNA4组，在倒置荧光显微镜下观察，估算慢病毒感染PC12细胞的效率，并采用Western blotting检测Integrin β1shRNA系统在PC12靶细胞中的沉默效果，以筛选出沉默效果最好的其中一条Integrin β1shRNA慢病毒载体。（3）常规培养至第11天的海马神经元，分别转染Integrin β1shRNA NC和筛选出来的沉默效果最好的Integrin β1shRNA2慢病毒，继续培养至第14天，参照Sombati方法制备体外难治性癫痫模型的方法，将已经转染Integrin β1shRNANC的细胞，用低Mg2+的pBRS液或者含有1mMMg2+的pBRS液分别培养细胞3h，同时将已经转染Integrin β1shRNA2慢病毒的细胞，用低Mg2+的pBRS液或者含有1mM Mg2+的pBRS液分别培养细胞3h，具体分组如下：正常海马神经元+Integrin β1shRNA NC、正常海马神经元+Integrin β1shRNA2、Sombati癫痫细胞模型组+Integrin β1shRNANC，Sombati癫痫细胞模型组+Integrin β1shRNA2，各组神经元均恢复维持培养基后继续培养，通过Western blotting检测其对海马神经元和Sombati癫痫细胞Integrin β1的基因沉默效果。结果：经测序证实RNAi慢病毒载体构建成功，病毒达到有效滴度；Western blotting证实，重组慢病毒Integrin β1shRNA2感染海马神经元和Sombati癫痫细胞后Integrin β1蛋白表达显著下降。结论：成功构建Integrin β1RNA干扰慢病毒载体，并在新生大鼠海马神经元和Sombati癫痫细胞中实现有效的基因沉默效应。第三部分Integrin β1沉默后Sombati难治性癫痫细胞模型的FAK依赖相关信号通路蛋白表达变化目的：利用Integrin β1慢病毒载体转染Sombati癫痫细胞模型，探讨Integrin β1相关信号通路在致癫痫中的作用及其机制，为进一步阐明癫痫的发病机理提供科学依据。方法：利用Neurobasal Medium无血清神经培养基和B-27因子体外培养SD新生乳鼠的海马神经元，随机分为以下四组：正常海马神经元、Sombati癫痫细胞模型、Sombati癫痫细胞模型+Integrin β1shRNA NC、Sombati癫痫细胞模型+Integrin β1shRNA2，培养至第11天时，用Integrin β1shRNA NC感染Sombati癫痫细胞模型+Integrin β1shRNA NC组的细胞，用Integrin β1shRNA2感染Sombati癫痫细胞模型+Integrin β1shRNA2组的细胞，至培养第14天时，参照Sombati的方法制备体外难治性癫痫模型，对Sombati癫痫细胞模型组、Sombati癫痫细胞模型+Integrin β1shRNA NC组、Sombati癫痫细胞模型+Integrin β1shRNA2组用低Mg2+的pBRS液培养细胞3h，恢复维持培养基，再继续培养4天，正常海马神经元组则用含有1mM Mg2+的pBRS液培养细胞3h，恢复维持培养基，再继续培养4天，各组细胞均采用Western blotting检测信号通路蛋白Phospho-FAK(Tyr397)、Phospho-Rac1/cdc42(Ser71)、Phospho-PAK1（Ser199/204）/PAK3蛋白表达水平。结果：与Sombati癫痫细胞+Integrinβ1shRNA2组相比，正常海马神经元、Sombati癫痫细胞和Sombati癫痫细胞+Integrin β1RNA NC的Phospho-FAK水平表达升高（P0.01）；与正常海马神经元相比，Sombati癫痫细胞和Sombati癫痫细胞+Integrin β1RNA NC的Phospho-FAK水平表达升高（P0.01），Sombati癫痫细胞和Sombati癫痫细胞+Integrin β1RNA NC组的Phospho-FAK水平无明显差异；在正常原代海马神经元、Sombati癫痫细胞模型以及转染了Integrin β1shRNA NC、Integrin β1shRNA2的Sombati癫痫细胞中，各组细胞的Phospho-Rac1/cdc42(Ser71)、Phospho-PAK1(Ser199/204)/PAK3蛋白表达水平无明显变化。结论：Integrin β1在Sombati癫痫细胞中发挥作用依赖于FAK相关通路的激活，主要是启动了Tyr397磷酸化位点，但与Phospho-Rac1/cdc42(Ser71)和Phospho-PAK1（Ser199/204）/PAK3蛋白的激活无关。
[Abstract]:Part Real-time qPCR detection of intractable epilepsy in rats with Sombati
Change of Integrin beta 1 gene expression in cell model
Objective: the integrin transmembrane system mainly mediates the interaction between the cells and the extracellular matrix, and the possible role of the cell surface receptor integrin beta 1 (Integrin beta 1) in epileptic formation in the epileptic cell model of hippocampal neurons in vitro is preliminarily investigated.
Methods: the Neurobasal Medium serum-free nerve medium and B-27 factor were used to culture the hippocampal neurons of SD neonatal rats in vitro. In vitro, the intractable epilepsy models were prepared for fourteenth days by Sombati, and were randomly divided into normal control group (Control), Sombati epileptic cell model group (low Mg2+), and 1mM Mg respectively. After 3h of 2+ pBRS solution or low Mg2+ pBRS solution, the neurons in each group continued to be cultured on the maintenance culture medium. After fourth days, the Real-time qPCR method was used to detect the expression of Integrin beta 1.
Results: compared with normal hippocampal neurons, the expression level of Integrin beta 1 in Sombati epileptic cells was significantly higher than that in normal hippocampal neurons on the 4 day after modeling.
Conclusion: the increased expression of Integrin beta 1 gene in Sombati epilepsy cell model may be involved in the formation of intractable epilepsy.
The second part is the construction of Integrin beta 1shRNA recombinant lentiviral vector and its effect on the expression of Integrin beta 1 gene in Sombati refractory epilepsy cells.
Objective: to construct the RNA interference lentiviral vector of Integrin beta 1 gene and provide a vector for studying the mechanism of Integrin beta 1 in Sombati epilepsy cell model.
Methods: (1) according to the Integrin beta 1 gene sequence and the RNA interference sequence design software, 4 RNA interference target sequences for Integrin beta 1 were designed and 4 shRNA lentivirus recombinant plasmids were constructed. The positive clones were sequenced and the HEK293T cells were routinely cultured, and the lentivirus shRNA vector and its auxiliary package plasmid plasmid were transfected into HEK. 293T cells were packed, concentrated, titer, and collected viruses. (2) normal PC12 cells were cultured and the constructed Integrin beta 1shRNA1-4 was transfected into PC12 cells, which were divided into Integrin beta 1shRNANC group, Integrin beta 1shRNA1 group, Integrin beta 1shRNA2 group, Integrin beta group and group, observed under inverted fluorescence microscope. To calculate the efficiency of PC12 cells infected by lentivirus, and to detect the silencing effect of Integrin beta 1shRNA system in PC12 target cells by using Western blotting, in order to screen out a Integrin beta 1shRNA lentivirus carrier with the best silence effect. (3) the normal culture to eleventh days of hippocampal neurons, respectively, transfected Integrin beta 1shRNA NC and screened out The Integrin beta 1shRNA2 lentivirus, which has the best effect of silence, continues to be cultured for fourteenth days, using the Sombati method to prepare the intractable epilepsy model in vitro. The cells that have been transfected with Integrin beta 1shRNANC are cultured with low Mg2+ pBRS solution or pBRS solution containing 1mMMg2+, respectively, and Integrin beta 1shRNA2 slow disease will be transfected at the same time. Cell 3H was cultured with low Mg2+ pBRS solution or pBRS solution containing 1mM Mg2+ respectively. The specific groups were as follows: normal hippocampal neurons +Integrin beta 1shRNA NC, normal hippocampal neurons +Integrin beta 1shRNA2, Sombati epileptic cell model group The cells were restored to maintain medium and then cultured. The gene silencing effect of Integrin beta 1 on hippocampal neurons and Sombati epileptic cells was detected by Western blotting.
Results: by sequencing, the RNAi lentivirus vector was successfully constructed and the virus reached an effective titer. Western blotting confirmed that the expression of Integrin beta 1 protein in the hippocampal neurons and Sombati epileptic cells of the recombinant lentivirus Integrin beta 1shRNA2 decreased significantly.
Conclusion: we successfully constructed Integrin beta 1RNA interference lentiviral vector, and achieved effective gene silencing effect in neonatal rat hippocampal neurons and Sombati epileptic cells.
The third part is the expression of FAK dependent signal pathway protein in Sombati refractory epilepsy cell model after Integrin beta 1 silencing.
Objective: To explore the role and mechanism of Integrin beta 1 related signal pathway in epilepsy and to provide a scientific basis for further elucidate the pathogenesis of epilepsy by transfection of Integrin beta 1 lentivirus vector to Sombati epileptic cell model.
Methods: the hippocampal neurons of SD neonatal rats were cultured with Neurobasal Medium non serum-free medium and B-27 factor in vitro. The neurons were randomly divided into four groups: normal hippocampal neurons, Sombati epileptic cell model, Sombati epileptic cell model +Integrin beta 1shRNA NC, Sombati epileptic epileptic cell model +Integrin beta 1shRNA2, culture to eleventh days. When Integrin beta 1shRNA NC was used to infect the cells of the +Integrin beta 1shRNA NC group of the Sombati epileptic cell model, the cells of the +Integrin beta 1shRNA2 group were infected with Integrin beta 1shRNA2 in Sombati epileptic cell model +Integrin beta 1shRNA2 group, and the intractable epilepsy model in vitro was prepared according to the method, and the epileptic cell model group was prepared for epilepsy. In the cell model +Integrin beta 1shRNA NC group, the Sombati epileptic cell model +Integrin beta 1shRNA2 group used the low Mg2+ pBRS solution to cultivate the cells 3h, resumed the maintenance of the culture medium, and then continued to culture for 4 days. The normal hippocampal neurons were cultured with 1mM Mg2+ pBRS liquid, and resumed the maintenance of the cultured cells, and then continued to culture for 4 days. N blotting detected the expression level of signal pathway protein Phospho-FAK (Tyr397), Phospho-Rac1/cdc42 (Ser71) and Phospho-PAK1 (Ser199/204) /PAK3 protein.
Results: the expression of +Integrin beta 1RNA NC in normal hippocampal neurons, Sombati epileptic cells and Sombati epileptic cells increased (P0.01) compared with the +Integrin beta 1shRNA2 group of Sombati epileptic cells (P0.01); Sombati epilepsy cells and epileptic epileptic cells were compared with normal hippocampal neurons. There was no significant difference in the level of Phospho-FAK between the Sombati epileptic cells and the +Integrin beta 1RNA NC group of the Sombati epileptic cells, and the normal primary hippocampal neurons, the Sombati epileptic cell model and the Integrin beta 1shRNA NC. There was no significant change in the expression level of pho-PAK1 (Ser199/204) /PAK3 protein.
Conclusion: the role of Integrin beta 1 in Sombati epileptic cells depends on the activation of the FAK related pathway, mainly starting the Tyr397 phosphorylation site, but is not related to the activation of Phospho-Rac1/cdc42 (Ser71) and Phospho-PAK1 (Ser199/204) /PAK3 protein.
【学位授予单位】：广西医科大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R742.1

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