本文選題：急性呼吸窘迫綜合征 + 基因芯片　； 參考：《濱州醫(yī)學(xué)院》2012年碩士論文

【摘要】：[目的] 采用基因芯片技術(shù)檢測油酸型急性呼吸窘迫綜合征(acute respiratory distress syndrome ARDS)大鼠肺組織中炎癥免疫相關(guān)基因的表達(dá)；應(yīng)用AG490(氰-3,4-二羥基-N-芐基肉桂酰胺)和GCs(糖皮質(zhì)激素)干預(yù)ARDS大鼠,觀察其RhoA基因表達(dá)變化；通過建立體外脂多糖(LPS)誘導(dǎo)RAW264.7巨噬細(xì)胞系,采用RhoA特異干擾RNA、AG490、GCs干預(yù),觀察巨噬細(xì)胞中RhoA基因和炎癥因子的表達(dá)變化,從基因表達(dá)差異以及RhoA基因調(diào)控的角度探討ARDS發(fā)生機(jī)制。 [方法] 1、采用舌下靜脈注射油酸復(fù)制ARDS大鼠模型。 2、基因芯片技術(shù)檢測ARDS大鼠肺組織炎癥免疫相關(guān)基因,LuxScan3.0圖像分析軟件對芯片圖像進(jìn)行分析,標(biāo)準(zhǔn)化處理,用Sam3.0進(jìn)行統(tǒng)計(jì)分析。并選擇兩個差異表達(dá)基因(Cyp2b3、RhoA),用Real time-PCR驗(yàn)證。 3、Real-time PCR法檢測ARDS大鼠肺組織RhoA mRNA, ELISA法檢測白血病抑制因子(LIF)的表達(dá)。 4、建立LPS誘導(dǎo)RAW264.7巨噬細(xì)胞系,Real time-PCR檢測RhoA mRNA在2h、6h、12和24h四個時間點(diǎn)的表達(dá),用RhoA特異干擾RNA、AG490和GCs干預(yù),在6h、12h、24h三個時間點(diǎn)Real-time PCR法檢測RhoA mRNA, ELISA法檢測IL-6的表達(dá)變化。 [結(jié)果] 1.建立油酸型ARDS大鼠模型,并經(jīng)血?dú)夥治龊头谓M織病理學(xué)驗(yàn)證； 2.基因芯片技術(shù)檢測ARDS大鼠肺組織炎癥免疫相關(guān)基因,發(fā)現(xiàn)表達(dá)上調(diào)1.5倍以上的炎癥免疫相關(guān)基因有4個：RhoA、Lif、Serpinel和Tacl,下調(diào)的有10個：RT1-T24-1、RT1-S3、RT1-14-、Cfh、Casp12、Hspa8、Scin、Cyp2e1、 Cyp2b3和Itpka。Real time-PCR結(jié)果證實(shí)芯片檢測結(jié)果可靠； 3. AG490和GCs干預(yù)ARDS大鼠結(jié)果表明,肺組織損傷程度ARDS組最嚴(yán)重,AG490和GCs組較輕,正常對照組無變化(表現(xiàn)正常)；大鼠肺組織RhoA mRNA表達(dá)水平ARDS組最高,AG490和GCs組下降明顯但仍高于對照組；LIF表達(dá)水平與RhoA趨勢相似； 4.LPS誘導(dǎo)RAW264.7巨噬細(xì)胞后,RhoA mRNA表達(dá)水平在2h LPS處理組和正常對照組之間差別不明顯,6h、12h LPS處理組高于正常對照組,到達(dá)24h后兩組間差異又不顯著； 5.LPS處理組RhoA mRNA表達(dá)水平在6h最高,12h時顯著降低但仍高于正常組,到達(dá)24h后降至正常組水平；體外SiRNA、AG490和GCs干預(yù)結(jié)果表明,三干預(yù)組RhoAmRNA表達(dá)水平在6h、12h兩個時間點(diǎn)顯著低于LPS處理組,同時SiRNA-1組在12和24h顯著低于正常組；IL-6表達(dá)水平在各時間點(diǎn)LPS組最高,SiRNA-1組、AG490和GCs組下降明顯但仍高于對照組。
[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.
【學(xué)位授予單位】：濱州醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R563.8

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