本文選題：海馬神經(jīng)元 + Integrinβ1　； 參考：《廣西醫(yī)科大學》2014年博士論文

【摘要】：第一部分Real-time qPCR檢測大鼠Sombati難治性癲癇 細胞模型中Integrin β1基因表達變化 目的：整合素-跨膜系統(tǒng)主要介導細胞與細胞外基質(zhì)的相互作用，本實驗初步探討在海馬神經(jīng)元的體外癲癇細胞模型中的細胞表面受體整合素β1（Integrin β1）在癲癇致癇性形成中的可能作用。 方法：利用Neurobasal Medium無血清神經(jīng)培養(yǎng)基和B-27因子體外培養(yǎng)SD新生乳鼠的海馬神經(jīng)元，對培養(yǎng)至第14天的細胞，參照Sombati的方法制備體外難治性癲癇模型，，隨機分為正常對照組（Control）、Sombati癲癇細胞模型組（低Mg2+），分別在用含有1mM Mg2+的pBRS液或低Mg2+的pBRS液培養(yǎng)細胞3h后，各組神經(jīng)元在恢復維持培養(yǎng)基繼續(xù)培養(yǎng)，在之后的第4天，采用Real-time qPCR方法檢測Integrin β1的表達。 結果：Sombati癲癇細胞造模后4天與正常海馬神經(jīng)細胞相比，Integrinβ1基因表達水平均明顯高于正常海馬神經(jīng)細胞。 結論：Integrin β1基因在Sombati癲癇細胞模型中表達增高，可能參與了難治性癲癇的形成過程。 第二部分Integrin β1shRNA重組慢病毒載體的構建及其對Sombati難治性癲癇細胞Integrin β1基因表達的影響 目的：構建Integrin β1基因RNA干擾慢病毒載體，為研究Sombati癲癇細胞模型Integrin β1的作用機制提供載體。 方法:（1）針對Integrin β1目的基因序列，利用的RNA干擾序列設計軟件，設計4個針對Integrin β1的RNA干擾靶點序列，構建4個shRNA慢病毒重組質(zhì)粒，測序鑒定陽性克隆，常規(guī)培養(yǎng)HEK293T細胞，將慢病毒shRNA載體及其輔助包裝原件載體質(zhì)粒共轉染進HEK293T細胞進行慢病毒包裝、濃縮、滴度測定，并收集病毒。（2）常規(guī)培養(yǎng)PC12細胞，將構建的Integrin β1shRNA1-4轉染PC12細胞，分為Integrin β1shRNANC組、Integrin β1shRNA1組、 Integrin β1shRNA2組、Integrin β1shRNA3組和Integrin shRNA4組，在倒置熒光顯微鏡下觀察，估算慢病毒感染PC12細胞的效率，并采用Western blotting檢測Integrin β1shRNA系統(tǒng)在PC12靶細胞中的沉默效果，以篩選出沉默效果最好的其中一條Integrin β1shRNA慢病毒載體。（3）常規(guī)培養(yǎng)至第11天的海馬神經(jīng)元，分別轉染Integrin β1shRNA NC和篩選出來的沉默效果最好的Integrin β1shRNA2慢病毒，繼續(xù)培養(yǎng)至第14天，參照Sombati方法制備體外難治性癲癇模型的方法，將已經(jīng)轉染Integrin β1shRNANC的細胞，用低Mg2+的pBRS液或者含有1mMMg2+的pBRS液分別培養(yǎng)細胞3h，同時將已經(jīng)轉染Integrin β1shRNA2慢病毒的細胞，用低Mg2+的pBRS液或者含有1mM Mg2+的pBRS液分別培養(yǎng)細胞3h，具體分組如下：正常海馬神經(jīng)元+Integrin β1shRNA NC、正常海馬神經(jīng)元+Integrin β1shRNA2、Sombati癲癇細胞模型組+Integrin β1shRNANC，Sombati癲癇細胞模型組+Integrin β1shRNA2，各組神經(jīng)元均恢復維持培養(yǎng)基后繼續(xù)培養(yǎng)，通過Western blotting檢測其對海馬神經(jīng)元和Sombati癲癇細胞Integrin β1的基因沉默效果。 結果：經(jīng)測序證實RNAi慢病毒載體構建成功，病毒達到有效滴度；Western blotting證實，重組慢病毒Integrin β1shRNA2感染海馬神經(jīng)元和Sombati癲癇細胞后Integrin β1蛋白表達顯著下降。 結論：成功構建Integrin β1RNA干擾慢病毒載體，并在新生大鼠海馬神經(jīng)元和Sombati癲癇細胞中實現(xiàn)有效的基因沉默效應。 第三部分Integrin β1沉默后Sombati難治性癲癇細胞模型的FAK依賴相關信號通路蛋白表達變化 目的：利用Integrin β1慢病毒載體轉染Sombati癲癇細胞模型，探討Integrin β1相關信號通路在致癲癇中的作用及其機制，為進一步闡明癲癇的發(fā)病機理提供科學依據(jù)。 方法：利用Neurobasal Medium無血清神經(jīng)培養(yǎng)基和B-27因子體外培養(yǎng)SD新生乳鼠的海馬神經(jīng)元，隨機分為以下四組：正常海馬神經(jīng)元、Sombati癲癇細胞模型、Sombati癲癇細胞模型+Integrin β1shRNA NC、Sombati癲癇細胞模型+Integrin β1shRNA2，培養(yǎng)至第11天時，用Integrin β1shRNA NC感染Sombati癲癇細胞模型+Integrin β1shRNA NC組的細胞，用Integrin β1shRNA2感染Sombati癲癇細胞模型+Integrin β1shRNA2組的細胞，至培養(yǎng)第14天時，參照Sombati的方法制備體外難治性癲癇模型，對Sombati癲癇細胞模型組、Sombati癲癇細胞模型+Integrin β1shRNA NC組、Sombati癲癇細胞模型+Integrin β1shRNA2組用低Mg2+的pBRS液培養(yǎng)細胞3h，恢復維持培養(yǎng)基，再繼續(xù)培養(yǎng)4天，正常海馬神經(jīng)元組則用含有1mM Mg2+的pBRS液培養(yǎng)細胞3h，恢復維持培養(yǎng)基，再繼續(xù)培養(yǎng)4天，各組細胞均采用Western blotting檢測信號通路蛋白Phospho-FAK(Tyr397)、Phospho-Rac1/cdc42(Ser71)、Phospho-PAK1（Ser199/204）/PAK3蛋白表達水平。 結果：與Sombati癲癇細胞+Integrinβ1shRNA2組相比，正常海馬神經(jīng)元、Sombati癲癇細胞和Sombati癲癇細胞+Integrin β1RNA NC的Phospho-FAK水平表達升高（P0.01）；與正常海馬神經(jīng)元相比，Sombati癲癇細胞和Sombati癲癇細胞+Integrin β1RNA NC的Phospho-FAK水平表達升高（P0.01），Sombati癲癇細胞和Sombati癲癇細胞+Integrin β1RNA NC組的Phospho-FAK水平無明顯差異；在正常原代海馬神經(jīng)元、Sombati癲癇細胞模型以及轉染了Integrin β1shRNA NC、Integrin β1shRNA2的Sombati癲癇細胞中，各組細胞的Phospho-Rac1/cdc42(Ser71)、Phospho-PAK1(Ser199/204)/PAK3蛋白表達水平無明顯變化。 結論：Integrin β1在Sombati癲癇細胞中發(fā)揮作用依賴于FAK相關通路的激活，主要是啟動了Tyr397磷酸化位點，但與Phospho-Rac1/cdc42(Ser71)和Phospho-PAK1（Ser199/204）/PAK3蛋白的激活無關。
[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.
【學位授予單位】：廣西醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2014
【分類號】：R742.1

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