锌指蛋白A20对急性肺损伤炎症反应的调控及机制研究

发布时间：2018-05-15 10:16

本文选题：脂多糖所致急性肺损伤 + A20蛋白　；参考：《复旦大学》2012年博士论文

【摘要】：第一部分A20蛋白在静脉注射LPS所致大鼠急性肺损伤中的变化及意义 [摘要]目的本实验通过建立大鼠LPS所致急性肺损伤模型,旨在研究A20蛋白在LPS急性肺损伤的表达变化及意义。方法实验选用SPF级雄性SD大鼠54只,随机分为2个实验组：正常对照组(尾静脉注射PBS,1ml/只,定义为0h)；LPS组(尾静脉注射LPS,10mg/kg,浓度为2mg/ml,根据不同时间点1h,2h,4h,6h,8h,12h,24h,48h分为8个亚组)。所有动物在注射LPS相应时间点放血处死,收集标本,观察光镜(HE染色),检测肺湿干比(W/D),行支气管肺泡灌洗,检测支气管肺泡灌洗液(BALF)中蛋白浓度,BALF中有核细胞总数,BALF中TNF-α、IL-1β水平,肺组织NF-κB DNA结合活性及核蛋白p65水平,肺组织中A20蛋白及mRNA含量,A20定位。结果与正常对照相比,注射LPS24小时后肺W/D、BALF中有核细胞总数(×104/ml)及BALF中蛋白浓度(ug/ml)均明显升高(p0.05),光镜下可见肺泡间隔增厚,有较多炎症细胞浸润,肺泡腔内可见红细胞和中性粒细胞渗出；注射LPS后,BALF液中TNF-α、IL-1β水平及肺组织中NF-κB DNA结合活性及核蛋白p65含量随时间升高,于注射后2h达高峰,之后逐渐下降,但仍高于注射前,且在24h又发生一过性升高(p0.05)；肺组织中A20蛋白及mRNA水平也逐渐升高,于注射后2h达高峰,之后下降,但仍高于注射前(p0.05)；免疫组化示A20在LPS注射后2小时主要表达于肺泡巨噬细胞胞浆。结论1.健康大鼠静脉注射LPS10ml/kg24h后可出现急性肺损伤。2.急性肺损伤时,A20蛋白表达升高,且高峰期与ALI早期细胞因子及NF-κB活性一致。3.A20在急性肺损伤早期明显升高,且表达于肺泡巨噬细胞,由此推测A20可能通过调控肺泡巨噬细胞炎症活性影响肺损伤。第二部分A20对LPS刺激后肺泡巨噬细胞炎症反应的作用及机制 [摘要]目的本实验通过建立A20基因沉默及过表达肺泡巨噬细胞株研究A20对LPS刺激后肺泡巨噬细胞炎症活性影响,旨在探讨A20对LPS所致肺损伤的作用及机制。方法实验选用大鼠肺泡巨噬细胞株(NR8383),慢病毒法构建A20基因沉默及过表达细胞株,Western Bblot及RT-PCR鉴定A20表达,并行体外培养慢病毒包装后的A20干扰(A20-SH)、干扰空载(SCR)、过表达(A20)及过表达空载(VEC)细胞株。向培养基中加入LPS进行干预(lug LPS/ml培养基),于刺激后0.5、1、2、4小时收集细胞培养上清液及细胞,ELISA法检测上清液中TNF-α、IL-1β水平及细胞中NF-κB DNA结合活性；Western Blot法检测A20蛋白及核内p65含量；RT-PCR检测A20、TNF-a、IL-1βmRNA含量。结果构建的A20-SH肺泡巨噬细胞株中,A20蛋白及mRNA含量较SCR明显降低(p0.05),干扰效率达80%；过表达肺泡巨噬细胞中A20蛋白及mRNA含量较VEC明显升高(p0.05),表达增加10倍以上。LPS刺激后,SCR组及VEC组A20蛋白及mRNA含量升高,且于1h达高峰,之后逐渐下降,而A20-SH组较SCR组明显降低(p0.05),且始终维持在较低水平,A20组较VEC明显升高(p0.05)。LPS刺激后各组肺泡巨噬细胞中细胞因子(TNF-α、IL-1β)mRNA及培养上清液中含量、肺组织中NF-κB DNA结合活性及核内p65含量都随时间升高,于1h达高峰,之后逐渐下降；但与SCR相比,A20-SH组明显升高(p0.05)；与VEC组相比,A20组水平明显降低(p0.05)。结论1.慢病毒RNAi表达载体及A20过表达载体可分别有效地抑制大鼠肺泡巨噬细胞株中的A20基因表达或增加该基因表达。2.A20基因沉默导致肺泡巨噬细胞NF-κB活性升高,促进TNF-α、IL-1β表达及分泌；反之A20基因过表达能够抑制肺泡巨噬细胞NF-κB活性及TNF-α、IL-1β表达和分泌。3.A20蛋白能够抑制肺泡巨噬细胞炎症反应活性,进而减轻肺损伤。
[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.

【学位授予单位】：复旦大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R563.8

【引证文献】