大鼠急性呼吸窘迫综合征炎症免疫相关基因表达谱分析及RhoA基因调控作用的实验研究

发布时间：2018-05-26 07:36

  本文选题：急性呼吸窘迫综合征 + 基因芯片　； 参考：《滨州医学院》2012年硕士论文

【摘要】：[目的] 采用基因芯片技术检测油酸型急性呼吸窘迫综合征(acute respiratory distress syndrome ARDS)大鼠肺组织中炎症免疫相关基因的表达；应用AG490(氰-3,4-二羟基-N-苄基肉桂酰胺)和GCs(糖皮质激素)干预ARDS大鼠,观察其RhoA基因表达变化；通过建立体外脂多糖(LPS)诱导RAW264.7巨噬细胞系,采用RhoA特异干扰RNA、AG490、GCs干预,观察巨噬细胞中RhoA基因和炎症因子的表达变化,从基因表达差异以及RhoA基因调控的角度探讨ARDS发生机制。 [方法] 1、采用舌下静脉注射油酸复制ARDS大鼠模型。 2、基因芯片技术检测ARDS大鼠肺组织炎症免疫相关基因,LuxScan3.0图像分析软件对芯片图像进行分析,标准化处理,用Sam3.0进行统计分析。并选择两个差异表达基因(Cyp2b3、RhoA),用Real time-PCR验证。 3、Real-time PCR法检测ARDS大鼠肺组织RhoA mRNA, ELISA法检测白血病抑制因子(LIF)的表达。 4、建立LPS诱导RAW264.7巨噬细胞系,Real time-PCR检测RhoA mRNA在2h、6h、12和24h四个时间点的表达,用RhoA特异干扰RNA、AG490和GCs干预,在6h、12h、24h三个时间点Real-time PCR法检测RhoA mRNA, ELISA法检测IL-6的表达变化。 [结果] 1.建立油酸型ARDS大鼠模型,并经血气分析和肺组织病理学验证； 2.基因芯片技术检测ARDS大鼠肺组织炎症免疫相关基因,发现表达上调1.5倍以上的炎症免疫相关基因有4个：RhoA、Lif、Serpinel和Tacl,下调的有10个：RT1-T24-1、RT1-S3、RT1-14-、Cfh、Casp12、Hspa8、Scin、Cyp2e1、 Cyp2b3和Itpka。Real time-PCR结果证实芯片检测结果可靠； 3. AG490和GCs干预ARDS大鼠结果表明,肺组织损伤程度ARDS组最严重,AG490和GCs组较轻,正常对照组无变化(表现正常)；大鼠肺组织RhoA mRNA表达水平ARDS组最高,AG490和GCs组下降明显但仍高于对照组；LIF表达水平与RhoA趋势相似； 4.LPS诱导RAW264.7巨噬细胞后,RhoA mRNA表达水平在2h LPS处理组和正常对照组之间差别不明显,6h、12h LPS处理组高于正常对照组,到达24h后两组间差异又不显著； 5.LPS处理组RhoA mRNA表达水平在6h最高,12h时显著降低但仍高于正常组,到达24h后降至正常组水平；体外SiRNA、AG490和GCs干预结果表明,三干预组RhoAmRNA表达水平在6h、12h两个时间点显著低于LPS处理组,同时SiRNA-1组在12和24h显著低于正常组；IL-6表达水平在各时间点LPS组最高,SiRNA-1组、AG490和GCs组下降明显但仍高于对照组。
[Abstract]:[purpose] The expression of inflammatory immune-associated genes in lung tissue of acute respiratory distress syndrome ARDS) rats with oleic acid acute respiratory distress syndrome (ARDS) was detected by gene chip technique, and ARDS rats were treated with AG490 (Cyanogen 3-dihydroxy-N-benzyl cinnamamide) and GCs3 (glucocorticoid). The changes of RhoA gene expression and the expression of RhoA gene and inflammatory factors in macrophages induced by lipopolysaccharide (LPS) were observed. To explore the mechanism of ARDS from the perspective of gene expression difference and RhoA gene regulation. [methods] 1. ARDS rat model was induced by sublingual intravenous injection of oleic acid. 2. The image analysis software LuxScan3.0 was used to analyze and standardize the ARDS rat lung tissue inflammation immune-associated gene LuxScan3.0. The microarray image was standardized and analyzed by Sam3.0. Two differentially expressed genes, Cyp2b3, RhoA, were selected and verified by Real time-PCR. Real-time PCR was used to detect the expression of leukemia inhibitory factor (LIF) in lung tissue of ARDS rats by RhoA mRNA, ELISA method. 4. LPS induced RAW264.7 macrophage cell line, Real time-PCR, was established to detect the expression of RhoA mRNA at 12 and 24 hours. RhoA specific interference RNA-AG490 and GCs were used to detect the expression of RhoA mRNA. RhoA mRNA, ELISA was used to detect the expression of IL-6 at three time points (6 h, 12 h and 24 h). [results] 1. The oleic acid ARDS rat model was established and verified by blood gas analysis and lung histopathology. 2. The expression of inflammatory immune-associated genes in lung tissue of ARDS rats was detected by microarray technique. Four inflammatory immune-associated genes were found to be up-regulated by more than 1.5-fold. Four of them were found to be serpinel and Tacl, while 10 were down-regulated by 10: RT1-T24-1 / RT1-S3 / RT1-14-CfhCasp12Hspa8ScinCyp2e1. The results of Cyp2b3 and Itpka.Real time-PCR confirmed the reliability of the microarray. 3. The results of AG490 and GCs intervention on ARDS rats showed that the severity of lung tissue injury in ARDS group was the most severe in AG490 group and in GCs group. The expression level of RhoA mRNA in lung tissue of rats in ARDS group and GCs group was significantly decreased, but still higher than that in control group, which was similar to the trend of RhoA. The expression of RhoA mRNA in RAW264.7 macrophages induced by 4.LPS was not significantly different between the 2 h LPS treatment group and the normal control group at 6 h and 12 h LPS treatment, and there was no significant difference between the two groups after 24 hours. The level of RhoA mRNA expression in the 5.LPS treatment group decreased significantly at the peak of 6 h and remained higher than that in the normal group at 12 h, and decreased to the normal level at 24 h after the treatment. The results of in vitro SiRNA-AG490 and GCs intervention showed that the RhoAmRNA expression level in the three intervention groups was significantly lower than that in the LPS treatment group at 6 h and 12 h, respectively. At the same time, the expression of IL-6 in the SiRNA-1 group was significantly lower than that in the normal group at 12 and 24 hours. At each time point, the expression of IL-6 in the highest siRNA-1 group and GCs group in the LPS group was significantly decreased but still higher than that in the control group.
【学位授予单位】：滨州医学院
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R563.8

【参考文献】

相关期刊论文 前3条

1 石增立,刘凤,石磊,刘海霞,田东,王国祥,董晓青,于小玲;油酸型急性呼吸窘迫综合征大鼠血清及肺组织IL-4、IL-10、IL-12水平的变化[J];中国病理生理杂志;2003年07期

2 王颢,陈玉霞,曹冬梅,张晔,卢建,孟荣贵;RhoA基因在结直肠癌组织中的表达[J];中华医学杂志;2002年05期

3 陈永华,蒋建新,谢国旗,张宇,邱俊,刘大维,周继红,朱佩芳;内毒素血症时小鼠肺局部致炎及抗炎因子与急性肺损伤的关系[J];中国危重病急救医学;2000年06期

，

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