【作者】 徐尤年；

【导师】 张诗海；

【作者基本信息】 华中科技大学 ， 麻醉学， 2010， 博士

【摘要】 目的克隆人血管生成素1基因(hAng1),采用AdMax包装系统构建含人血管生成素1的重组腺病毒质粒,为研究血管生成素1对急性肺损伤中炎症消退的作用奠定基础。方法将hAng1 cDNA克隆于真核表达载体pDC315-EGFP,得到重组质粒pDC315-EGFP-Ang1,采用位点特异性重组系统(Cre/Loxp)将pDC315-EGFP-Angl与pBHG lox△E1,3Cre辅助包装质粒共转染至HEK293细胞,生成带有Angl基因的重组腺病毒载体Ad-EGFP-Ang1,重组腺病毒质粒在HEK293细胞中扩增,氯化铯梯度离心纯化。检测转染的HEK293细胞中以及细胞培养上清中的Ang1蛋白表达。结果经酶切鉴定和测序证实载体构建的正确性,病毒滴度为1.5×1012 PFU·mL-1。Ad-EGFP-Ang1转染的HEK293细胞和细胞培养上清中均检测出Ang1蛋白。结论带有hAng1 cDNA的重组腺病毒载体构建成功,可用于对急性肺损伤炎症消退的影响的研究。目的气管滴入脂多糖复制小鼠急性肺损伤模型,为进一步探讨炎症反应过程奠定基础。方法将20只SPF级雄性BALB/c小鼠(20-25g)随机分为对照组和脂多糖组,每组10只小鼠,麻醉后分别经气管插管滴入生理盐水(对照组)和脂多糖3mg·kg-1(脂多糖组)。观察24小时死亡率、肺组织湿干重比、病理改变,肺泡灌洗液中的白细胞计数、中性粒细胞计数和总蛋白浓度。结果脂多糖组小鼠死亡率33.3%,对照组没有小鼠死亡。脂多糖组肺组织病理评分、肺组织湿干重比、肺泡灌洗液中的白细胞计数、中性粒细胞计数和总蛋白浓度均高于对照组,(P=0.001)。结论气管滴入脂多糖3 mg·kg-1成功模拟了ALI的过程,是进一步探讨炎症反应过程的可靠的ALI动物模型。目的通过基因转染增加血清中血管生成素1(Angl)的浓度,探讨血管生成素1基因治疗对急性肺损伤炎症消退的影响。方法SPF级BALB/c小鼠气管内滴入脂多糖构建急性肺损伤动物模型前,经尾静脉注入空载体病毒(Ad-GFP, GFP组)或者携带血管生成素1的腺病毒(Ad-GFP-Ag1, Ang1组)。经气管滴入脂多糖后4h、12h、24h、48h、72h、96h处死小鼠,取肺泡灌洗液收集细胞,行细胞计数分类,并用流式检测中性粒细胞凋亡和被巨噬细胞吞噬情况。收集肺组织和肝脏组织以及血清,检测血管生成素1的基因和蛋白表达。检测肺泡灌洗液中的粒细胞巨噬细胞集落刺激因子浓度变化。另外取4只小鼠,静脉注入空载体病毒,24小时后气管滴入生理盐水作对照组。结果LPS气管滴入后大量的白细胞浸润到肺组织和肺泡腔,早期以中性粒细胞为主,在48h白细胞和中性粒细胞达到最大值。Angl预处理增加了angl在肝脏的表达,分泌到血清中,减少了炎性细胞浸润并促进了中性粒细胞的凋亡和吞噬,从而加速了中性粒细胞从组织的清除,同时,增加了肺泡灌洗液中的粒细胞单核细胞集落刺激因子浓度。结论血管生成素1预处理促进了急性肺损伤炎症消退,这与其促进中性粒细胞的凋亡和巨噬细胞吞噬有关。1经颈透照明视下完成并证实小鼠气管插管成功,经气管插管滴入3mg·kg-1脂多糖可以成功复制ALI模型。2脂多糖3mg·kg-1气管滴入后48小时,肺组织炎症达高峰,随后进入炎症消退期。血管生成素1预处理有效的稳定血管内皮细胞,减少蛋白渗出,抑制了炎症细胞组织浸润。3血管生成素1促进了急性肺损伤炎症消退。其机制是血管生成素1通过稳定血管内皮细胞促进炎症组织中性粒细胞凋亡,促进巨噬细胞吞噬凋亡的中性粒细胞。4血管内皮细胞参与了炎症消退过程。5血管内皮细胞可能是急性肺损伤防治的一个重要的潜在的靶点。更多还原

【Abstract】 Objective To construct the recombinant adenovirus carrying human Angl (hAngl) in order to explore the effect of Angl on the inflammation resolution of acute lung injury induced by lipopolysaccharide. Methods The cDNA of hAngl was cloned into adenovirus shuttle vector pDC315-EGFP by standard procedure to obtain the recombinant adenoviral plasmid pDC315-EGFP-Ang1. Genomic plasmid pBHG lox AE1,3 Cre and pDC315-EGFP-Angl were cotransfected into 293 cells by using site-specific recombinant system Cre/Lox. The Ad-EGFP-hAngl recombinant adenovirus vector bearing hAngl was then amplified in 293 cells and purified by cesium chloride. The hAngl protein in the transfected HEK 293 cells and the medium of transfented HEK293 cells were detected by western blot and Elisa respectively. Results The cloned hAngl cDNA was confirmed correction by sequencing and enzyme cutting. The titer of Ad-GFP-hAngl was 1.50×1012 PFU·mL-1. The hAngl protein was detected in the Ad-GFP-hAngl transfected HEK 293 cells and time-dependent expressed in the medium of Ad-GFP-hAngl transfented HEK293 cells. Conclusion The recombinant adenovirus carrying human hAngl was successfully constructed by this method and ex vivo assays suggested the expression of human Angl with high efficiency and specificity.Objective To establish a model of acute lung injury (AL1) in mice by LPS intratracheal instillation for studying the inflammation process. Methods Twenty male SPF BALB/c mice weighing 20-25g were randomly divided into two groups:Normal saline group (Control group) and lipopolysaccharide group(LPS group) with 10 mice in each group. Control group and LPS group received intratracheal injection of 2 mL·kg-1 normal saline or 3 mg·kg-1 popolysaccharide. All the mice were killed at 24 hours after injection. The wet-to-dry ratio and pathological change of lung were examined. In addition, the Leukocyte, PMN count and the total protein in BALwere analyzed with the incidence of mortality of each group observed. Results 3 mice died in the LPS group except one that was mis-intubated into the esophagus while no mouse died in the control group (P=0.001). The pathologic score of lung, the leukocyte, PMN and total protein in the BAL of the LPS group were significantly higher with the pathologic score of lung lower than that of the control group. Conclusion It is successful to imitate the development of ALI by injection of lipopolysaccharide. This is a suitable model for the following study of ALI in mice.Objective:To investigate the effect of angiopoietinl on the inflammation resolution in acute lung injury. Methods:Animals were randomly assigned to GFP/saline group (control group), Ad-GFP/LPS group (GFP group) and Ad-GFP-Ang1/LPS group (Angl group).3-4 mice in GFP or Angl group were killed at 4 h,12 h,24 h,48 h,72 h and 96 h respectively. The cells in the BAL were counted and differentiated. The inflammation resolution indices between the GFP group and the Angl group were analyzed. Apoptotic polymorphonuclear leucocyte and its phagocytosis by macrophage cells were determined by fluorescent activated cell sorter. The angiopoietin-1 in serum and the Granulocyte macrophage colony-stimulating factor levels in the BAL were assayed following the manufacturer’s instructions. Results:ALI for 4 h,12 h,24 h,48 h,72 h and 96 h induced evident leukocytes infiltration into the lung tissues, with a maximal infiltration (13.5×106) at 48 h. Pretreatment with adenovirus-GFP-angiopoietinl markedly increased serum Angl, reduced the leukocytes and polymorphonuclear cells infiltration and shortened the time interval from the maximal PMN point to the 50% reduction point (Ri) in inflammation resolution compared with the GFP group. At the same time, pretreatment with Ad-GFP significantly promoted the apoptosis of PMN cells and its clearance by macrophage cells. GMCSF levels in the BAL were augmentated without altering the time course in Angl group. Conclusions:This study provides evidence, for the first time, that angiopoietin-1 pretreatment promotes inflammation resolution of endotoxin-induced ALI in mice. 1. It is successful to imitate the development of ALI by intratracheal injection of lipopolysaccharide at the dose of 3 mg·kg-1. This is a suitable model for the following study of ALI in mice.2. The PMN infiltration into the lung tissue in the ALI mice reached maximum at 48 hours after intratracheal injection of lipopolysaccharide at dose of 3 mg·kg-1. Then the inflammation resoluted and the PMN number in the BAL gradually reduced. Pretreatment with Ad-GFP-Angl significantly reduced the PMN infiltration and protein exudation by stabilizing the endothelium cells.3. Pretreatment with Ad-GFP-Angl promoted the inflammation resolution of ALI. The mechanism is that the Angl accelerated the apoptosis of PMN in the tissues and the phagocytosis of apoptotic PMN by macrophage cells.4. The endothelium cell takes part in the inflammation resolution.5. The endothelium cell is perhaps an important potential target in pretreatment and treatment of ALI.更多还原