TAK1介导的小鼠巨噬细胞RAW264.7凋亡的实验研究

发布时间：2018-01-20 10:03

  本文关键词： 转化生长因子激活激酶1 RAW264.7 凋亡 转化生长因子 β1 脂多糖 Toll样受体4　出处：《天津医科大学》2012年硕士论文　论文类型：学位论文

【摘要】：研究内容与目的： TGF-β-activated kinase1(TAK1)是MAPKKK家族成员之一,因首先被报道作为TGF-β1激活的MAPK信号通路的条件蛋白而获名。TAK1在所有组织中广泛表达,尤其在胸腺、大脑和肝脏组织中表达丰富,它受很多细胞因子和应激因素的调节,是促炎反应和细胞应激信号转导中的关键调节蛋白,其下游信号通路调控着细胞的激活和凋亡。有多篇文献报道TAK1的活性与多种类型细胞的抗凋亡作用密切相关,这其中包括淋巴细胞和中性粒细胞,同时TAK1在固有免疫和特异性免疫中发挥着不可或缺的作用。然而关于其与髓系巨噬细胞的存活及Toll样受体(TLR)介导的固有免疫的关系至今还没有报道。本课题研究发现LPS. TGF-β1可以刺激巨噬细胞TAK1的激活,过表达TAK1对巨噬细胞具有抗凋亡作用。 研究方法与结果： 本课题实验分三部分进行： 一、检测TAK1在巨噬细胞系的表达和活性,包括两个实验： 1. Real-time PCR检测TAK1在不同免疫细胞系中的表达情况,检测结果显示,TAK1在巨噬细胞RAW264.7中表达丰富； 2. Real-time PCR和Western blot分别从基因和蛋白水平上检测LPS和TGF-β1对RAW264.7中TAK1的激活作用,结果显示LPS和TGF-β1可以激活巨噬细胞RAW264.7中TAK1的活性并且具有时间依赖性； 二、优化RAW264.7的电转染条件,包括三个实验： 1.制备感受态大肠杆菌细胞并转化相应的质粒,结果显示感受态大肠杆菌细胞状态良好,并提取出了高纯度、高浓度的质粒； 2.巨噬细胞RAW264.7的培养及电转染条件的优化,结果显示胰酶和细胞联合使用可使细胞传代时损伤最小,电转染时质粒量为20ug,电压值为250V,电容值为960uF,电阻值为∞时可使转染效率达到最大； 3.根据优化条件转染含目的基因的质粒,并用Real-time PCR和Western blot分别从基因和蛋白水平上检测转染效果,结果显示质粒基因成功地导入RAW264.7中。 第三部分预实验检测过表达TAK1对巨噬细胞凋亡的影响。 按照分组转染相应目的质粒60小时后,加入LPS或TGF-β1刺激细胞,流式细胞术检测细胞的凋亡情况,结果显示过表达TAK1可能抑制LPS和TGF-β1诱导的巨噬细胞凋亡效应。 结论： 综上所述,我们的研究结果表明,转化生长因子激活激酶1(TAK1)参与了巨噬细胞TLR4和TGF-β1激活的信号转导通路并可能发挥抗巨噬细胞凋亡的作用。我们的研究对于进一步了解TAK1与巨噬细胞凋亡关系以及相关机制奠定了实验基础,也对进一步了解TLR4信号转导的调控机制、了解病原体的致病机理和控制病原感染有一定的理论意义。
[Abstract]:Contents and objectives of the study: TGF- 尾 -activated kinase1 (TAK1) is a member of MAPKKK family. TGF- 尾 1 is widely expressed in all tissues, especially in thymus, brain and liver tissues, because it was first reported as a conditional protein of MAPK signaling pathway activated by TGF- 尾 1. It is regulated by many cytokines and stress factors and is a key regulatory protein in the inflammatory response and cellular stress signal transduction. The downstream signaling pathway regulates the activation and apoptosis of cells. There have been many reports that the activity of TAK1 is closely related to the anti-apoptotic effects of many types of cells, including lymphocytes and neutrophils. At the same time, TAK1 plays an indispensable role in innate and specific immunity. However, it is associated with the survival of myeloid macrophages and Toll like receptor (TLR). The relationship of innate immunity mediated by LPS. TGF- 尾 1 has not been reported yet. In this study, we found that LPS. TGF- 尾 1 can stimulate the activation of TAK1 in macrophages. Overexpression of TAK1 can inhibit the apoptosis of macrophages. Research methods and results: The experiment is divided into three parts: One was to detect the expression and activity of TAK1 in macrophage cell lines, including two experiments: 1. Real-time PCR was used to detect the expression of TAK1 in different immune cell lines. 2. Real-time PCR and Western. Blot was used to detect the activation of TAK1 in RAW264.7 by LPS and TGF- 尾 1 at gene and protein levels, respectively. The results showed that LPS and TGF- 尾 1 could activate the activity of TAK1 in RAW264.7 of macrophages in a time-dependent manner. Second, optimize the electrotransfection conditions of RAW264.7, including three experiments: 1. The competent Escherichia coli cells were prepared and transformed into corresponding plasmids. The results showed that the competent E. coli cells were in good condition, and the high purity and high concentration plasmids were extracted. 2. The culture of macrophage RAW264.7 and the optimization of the electrotransfection conditions. The results showed that the combined use of trypsin and cells could minimize the damage to the passage of the cells, and the amount of plasmids in the electrotransfection was 20ug. When the voltage value is 250V, the capacitance value is 960uF, and the resistance value is 鈭，

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