本文關(guān)鍵詞： 轉(zhuǎn)化生長因子激活激酶1 RAW264.7 凋亡 轉(zhuǎn)化生長因子 β1 脂多糖 Toll樣受體4　出處：《天津醫(yī)科大學(xué)》2012年碩士論文　論文類型：學(xué)位論文

【摘要】：研究內(nèi)容與目的： TGF-β-activated kinase1(TAK1)是MAPKKK家族成員之一,因首先被報道作為TGF-β1激活的MAPK信號通路的條件蛋白而獲名。TAK1在所有組織中廣泛表達(dá),尤其在胸腺、大腦和肝臟組織中表達(dá)豐富,它受很多細(xì)胞因子和應(yīng)激因素的調(diào)節(jié),是促炎反應(yīng)和細(xì)胞應(yīng)激信號轉(zhuǎn)導(dǎo)中的關(guān)鍵調(diào)節(jié)蛋白,其下游信號通路調(diào)控著細(xì)胞的激活和凋亡。有多篇文獻(xiàn)報道TAK1的活性與多種類型細(xì)胞的抗凋亡作用密切相關(guān),這其中包括淋巴細(xì)胞和中性粒細(xì)胞,同時TAK1在固有免疫和特異性免疫中發(fā)揮著不可或缺的作用。然而關(guān)于其與髓系巨噬細(xì)胞的存活及Toll樣受體(TLR)介導(dǎo)的固有免疫的關(guān)系至今還沒有報道。本課題研究發(fā)現(xiàn)LPS. TGF-β1可以刺激巨噬細(xì)胞TAK1的激活,過表達(dá)TAK1對巨噬細(xì)胞具有抗凋亡作用。 研究方法與結(jié)果： 本課題實(shí)驗(yàn)分三部分進(jìn)行： 一、檢測TAK1在巨噬細(xì)胞系的表達(dá)和活性,包括兩個實(shí)驗(yàn)： 1. Real-time PCR檢測TAK1在不同免疫細(xì)胞系中的表達(dá)情況,檢測結(jié)果顯示,TAK1在巨噬細(xì)胞RAW264.7中表達(dá)豐富； 2. Real-time PCR和Western blot分別從基因和蛋白水平上檢測LPS和TGF-β1對RAW264.7中TAK1的激活作用,結(jié)果顯示LPS和TGF-β1可以激活巨噬細(xì)胞RAW264.7中TAK1的活性并且具有時間依賴性； 二、優(yōu)化RAW264.7的電轉(zhuǎn)染條件,包括三個實(shí)驗(yàn)： 1.制備感受態(tài)大腸桿菌細(xì)胞并轉(zhuǎn)化相應(yīng)的質(zhì)粒,結(jié)果顯示感受態(tài)大腸桿菌細(xì)胞狀態(tài)良好,并提取出了高純度、高濃度的質(zhì)粒； 2.巨噬細(xì)胞RAW264.7的培養(yǎng)及電轉(zhuǎn)染條件的優(yōu)化,結(jié)果顯示胰酶和細(xì)胞聯(lián)合使用可使細(xì)胞傳代時損傷最小,電轉(zhuǎn)染時質(zhì)粒量為20ug,電壓值為250V,電容值為960uF,電阻值為∞時可使轉(zhuǎn)染效率達(dá)到最大； 3.根據(jù)優(yōu)化條件轉(zhuǎn)染含目的基因的質(zhì)粒,并用Real-time PCR和Western blot分別從基因和蛋白水平上檢測轉(zhuǎn)染效果,結(jié)果顯示質(zhì)�；虺晒Φ貙�(dǎo)入RAW264.7中。 第三部分預(yù)實(shí)驗(yàn)檢測過表達(dá)TAK1對巨噬細(xì)胞凋亡的影響。 按照分組轉(zhuǎn)染相應(yīng)目的質(zhì)粒60小時后,加入LPS或TGF-β1刺激細(xì)胞,流式細(xì)胞術(shù)檢測細(xì)胞的凋亡情況,結(jié)果顯示過表達(dá)TAK1可能抑制LPS和TGF-β1誘導(dǎo)的巨噬細(xì)胞凋亡效應(yīng)。 結(jié)論： 綜上所述,我們的研究結(jié)果表明,轉(zhuǎn)化生長因子激活激酶1(TAK1)參與了巨噬細(xì)胞TLR4和TGF-β1激活的信號轉(zhuǎn)導(dǎo)通路并可能發(fā)揮抗巨噬細(xì)胞凋亡的作用。我們的研究對于進(jìn)一步了解TAK1與巨噬細(xì)胞凋亡關(guān)系以及相關(guān)機(jī)制奠定了實(shí)驗(yàn)基礎(chǔ),也對進(jìn)一步了解TLR4信號轉(zhuǎn)導(dǎo)的調(diào)控機(jī)制、了解病原體的致病機(jī)理和控制病原感染有一定的理論意義。
[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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