本文選題：脂多糖所致急性肺損傷 + A20蛋白�。� 參考：《復(fù)旦大學(xué)》2012年博士論文

【摘要】：第一部分A20蛋白在靜脈注射LPS所致大鼠急性肺損傷中的變化及意義 [摘要]目的本實(shí)驗(yàn)通過(guò)建立大鼠LPS所致急性肺損傷模型,旨在研究A20蛋白在LPS急性肺損傷的表達(dá)變化及意義。方法實(shí)驗(yàn)選用SPF級(jí)雄性SD大鼠54只,隨機(jī)分為2個(gè)實(shí)驗(yàn)組：正常對(duì)照組(尾靜脈注射PBS,1ml/只,定義為0h)；LPS組(尾靜脈注射LPS,10mg/kg,濃度為2mg/ml,根據(jù)不同時(shí)間點(diǎn)1h,2h,4h,6h,8h,12h,24h,48h分為8個(gè)亞組)。所有動(dòng)物在注射LPS相應(yīng)時(shí)間點(diǎn)放血處死,收集標(biāo)本,觀察光鏡(HE染色),檢測(cè)肺濕干比(W/D),行支氣管肺泡灌洗,檢測(cè)支氣管肺泡灌洗液(BALF)中蛋白濃度,BALF中有核細(xì)胞總數(shù),BALF中TNF-α、IL-1β水平,肺組織NF-κB DNA結(jié)合活性及核蛋白p65水平,肺組織中A20蛋白及mRNA含量,A20定位。結(jié)果與正常對(duì)照相比,注射LPS24小時(shí)后肺W/D、BALF中有核細(xì)胞總數(shù)(×104/ml)及BALF中蛋白濃度(ug/ml)均明顯升高(p0.05),光鏡下可見肺泡間隔增厚,有較多炎癥細(xì)胞浸潤(rùn),肺泡腔內(nèi)可見紅細(xì)胞和中性粒細(xì)胞滲出；注射LPS后,BALF液中TNF-α、IL-1β水平及肺組織中NF-κB DNA結(jié)合活性及核蛋白p65含量隨時(shí)間升高,于注射后2h達(dá)高峰,之后逐漸下降,但仍高于注射前,且在24h又發(fā)生一過(guò)性升高(p0.05)；肺組織中A20蛋白及mRNA水平也逐漸升高,于注射后2h達(dá)高峰,之后下降,但仍高于注射前(p0.05)；免疫組化示A20在LPS注射后2小時(shí)主要表達(dá)于肺泡巨噬細(xì)胞胞漿。結(jié)論1.健康大鼠靜脈注射LPS10ml/kg24h后可出現(xiàn)急性肺損傷。2.急性肺損傷時(shí),A20蛋白表達(dá)升高,且高峰期與ALI早期細(xì)胞因子及NF-κB活性一致。3.A20在急性肺損傷早期明顯升高,且表達(dá)于肺泡巨噬細(xì)胞,由此推測(cè)A20可能通過(guò)調(diào)控肺泡巨噬細(xì)胞炎癥活性影響肺損傷。 第二部分A20對(duì)LPS刺激后肺泡巨噬細(xì)胞炎癥反應(yīng)的作用及機(jī)制 [摘要]目的本實(shí)驗(yàn)通過(guò)建立A20基因沉默及過(guò)表達(dá)肺泡巨噬細(xì)胞株研究A20對(duì)LPS刺激后肺泡巨噬細(xì)胞炎癥活性影響,旨在探討A20對(duì)LPS所致肺損傷的作用及機(jī)制。方法實(shí)驗(yàn)選用大鼠肺泡巨噬細(xì)胞株(NR8383),慢病毒法構(gòu)建A20基因沉默及過(guò)表達(dá)細(xì)胞株,Western Bblot及RT-PCR鑒定A20表達(dá),并行體外培養(yǎng)慢病毒包裝后的A20干擾(A20-SH)、干擾空載(SCR)、過(guò)表達(dá)(A20)及過(guò)表達(dá)空載(VEC)細(xì)胞株。向培養(yǎng)基中加入LPS進(jìn)行干預(yù)(lug LPS/ml培養(yǎng)基),于刺激后0.5、1、2、4小時(shí)收集細(xì)胞培養(yǎng)上清液及細(xì)胞,ELISA法檢測(cè)上清液中TNF-α、IL-1β水平及細(xì)胞中NF-κB DNA結(jié)合活性；Western Blot法檢測(cè)A20蛋白及核內(nèi)p65含量；RT-PCR檢測(cè)A20、TNF-a、IL-1βmRNA含量。結(jié)果構(gòu)建的A20-SH肺泡巨噬細(xì)胞株中,A20蛋白及mRNA含量較SCR明顯降低(p0.05),干擾效率達(dá)80%；過(guò)表達(dá)肺泡巨噬細(xì)胞中A20蛋白及mRNA含量較VEC明顯升高(p0.05),表達(dá)增加10倍以上。LPS刺激后,SCR組及VEC組A20蛋白及mRNA含量升高,且于1h達(dá)高峰,之后逐漸下降,而A20-SH組較SCR組明顯降低(p0.05),且始終維持在較低水平,A20組較VEC明顯升高(p0.05)。LPS刺激后各組肺泡巨噬細(xì)胞中細(xì)胞因子(TNF-α、IL-1β)mRNA及培養(yǎng)上清液中含量、肺組織中NF-κB DNA結(jié)合活性及核內(nèi)p65含量都隨時(shí)間升高,于1h達(dá)高峰,之后逐漸下降；但與SCR相比,A20-SH組明顯升高(p0.05)；與VEC組相比,A20組水平明顯降低(p0.05)。結(jié)論1.慢病毒RNAi表達(dá)載體及A20過(guò)表達(dá)載體可分別有效地抑制大鼠肺泡巨噬細(xì)胞株中的A20基因表達(dá)或增加該基因表達(dá)。2.A20基因沉默導(dǎo)致肺泡巨噬細(xì)胞NF-κB活性升高,促進(jìn)TNF-α、IL-1β表達(dá)及分泌；反之A20基因過(guò)表達(dá)能夠抑制肺泡巨噬細(xì)胞NF-κB活性及TNF-α、IL-1β表達(dá)和分泌。3.A20蛋白能夠抑制肺泡巨噬細(xì)胞炎癥反應(yīng)活性,進(jìn)而減輕肺損傷。
[Abstract]:Part I changes and significance of A20 protein in acute lung injury induced by intravenous injection of LPS in rats
[Abstract] Objective The aim of this experiment was to establish a rat model of acute lung injury induced by LPS in order to study the expression and significance of A20 protein in LPS acute lung injury. Methods 54 SPF male SD rats were selected and divided into 2 experimental groups randomly: the normal control group (tail vein injection PBS, 1ml/ only, 0h); LPS group (LPS, 10mg intravenously by tail vein) /kg, the concentration was 2mg/ml, according to the different time points 1H, 2h, 4h, 6h, 8h, 12h, 24h, 48h were divided into 8 subgroups. All animals were killed at the corresponding time point of injection, collecting specimens, observing the light microscopy (HE staining), detecting the lung wet dry ratio, the bronchoalveolar lavage, detection of the protein concentration in bronchoalveolar lavage fluid and the total number of nuclear cells. TNF- alpha, IL-1 beta level, NF- kappa B DNA binding activity and nuclear protein p65 level in lung tissue, A20 protein and mRNA content in lung tissue, and A20 localization in lung tissue. Results compared with normal control, lung W/D, the number of nuclear cells and protein concentration increased significantly in LPS24 hours after injection, and the alveolar septum increased under light microscope. After injection of LPS, the TNF- alpha, IL-1 beta level and the NF- kappa B DNA binding activity and the nucleoprotein p65 content increased with time after injection of LPS. After injection, the 2H reached the peak and decreased gradually after injection, but it was still higher than before the injection, and another sex rise occurred in 24h. High (P0.05), the level of A20 protein and mRNA in lung tissue also increased gradually. After the injection, the 2H reached its peak and then decreased, but was still higher than that before injection (P0.05). Immunohistochemistry showed that A20 was mainly expressed in alveolar macrophage cytoplasm 2 hours after LPS injection. Conclusion acute lung injury of acute lung injury in acute lung injury after intravenous injection of LPS10ml/kg24h in 1. healthy rats could be found. At the time of injury, the expression of A20 protein increased, and the peak period was in accordance with the early ALI cytokine and NF- kappa B activity..3.A20 was significantly increased in the early stage of acute lung injury, and expressed in alveolar macrophages. Thus, it is suggested that A20 may affect lung injury by regulating the inflammatory activity of alveolar macrophages.
The second part is the effect and mechanism of A20 on inflammatory response of alveolar macrophages after LPS stimulation.
[Abstract] Objective To study the effect of A20 on the inflammatory activity of alveolar macrophages after LPS stimulation by establishing A20 gene silencing and overexpressing alveolar macrophages, and to explore the effect and mechanism of A20 on lung injury induced by LPS. Methods the rat alveolar macrophage strain (NR8383) was selected and the A20 gene silencing and overexpression were constructed by the slow virus method. Cell lines, Western Bblot and RT-PCR identification of A20 expression, A20 interference (A20-SH), interference empty load (SCR), overexpression (A20) and overexpression of empty (VEC) cell lines in vitro, in vitro (A20) and overexpression of empty (VEC) cell lines. The cell culture medium was added to the medium (lug LPS/ml medium), and cell culture supernatant and cells were collected for hours after stimulation. TNF- alpha, IL-1 beta level and NF- kappa B DNA binding activity in the supernatant were detected by the method. Western Blot method was used to detect A20 protein and p65 content in the nucleus; RT-PCR detected A20, TNF-a, and beta content. The interference efficiency was 80%; overexpressed alveolus. The content of A20 protein and mRNA in macrophages was significantly higher than that of VEC (P0.05). After more than 10 times of.LPS stimulation, the content of A20 protein and mRNA increased in SCR and VEC groups, and then reached the peak in 1H, and then decreased gradually, while A20-SH group was lower than that of SCR group. The content of cytokine (TNF- alpha, IL-1 beta) mRNA and culture supernatant in alveolar macrophages in each group, the NF- kappa B DNA binding activity in the lung and the content of p65 in the nucleus all increased with time, at the peak of 1H, then gradually decreased, but compared with SCR (P0.05), the level of the A20-SH group was significantly lower than that of the VEC group. The conclusion was 1. slow. The expression vector of RNAi and A20 overexpression vector can effectively inhibit the expression of A20 gene in the alveolar macrophage of rats, or increase the expression of NF- kappa B activity in alveolar macrophages and promote the expression and secretion of TNF- a, IL-1 beta, and the expression and secretion of TNF- a, and conversely, A20 based overexpression can inhibit NF- kappa B of alveolar macrophages. Activity and TNF- alpha, IL-1 beta expression and secretion of.3.A20 protein can inhibit the inflammatory response of alveolar macrophages, thereby reducing lung injury.

【學(xué)位授予單位】：復(fù)旦大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2012
【分類號(hào)】：R563.8

【引證文獻(xiàn)】

相關(guān)碩士學(xué)位論文 前1條

1 陳娜;升麻素苷抗炎及抗小鼠肺損傷作用的研究[D];吉林大學(xué);2014年

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本文編號(hào)：1892048