【作者】 吕学军；

【导师】 钱桂生；

【作者基本信息】 第三军医大学 ， 内科学， 2010， 博士

【摘要】 急性肺损伤(acute lung injury, ALI)/急性呼吸窘迫综合征(acute respiratory distress syndrome, ARDS)为临床常见的危重症,主要表现为急性呼吸窘迫、顽固性低氧血症和非心源性肺水肿。ALI/ARDS可发生于任何年龄段的患者,通常由严重感染、创伤、休克等因素诱发。但迄今为止其发病机制尚未完全阐明。近年来,病理研究结果显示,ALI/ARDS发生时受损伤最明显的结构是肺泡上皮,尤其是肺泡Ⅰ型上皮细胞(AT-1)对损伤更敏感,而且这种损伤常常发生在ALI/ARDS的早期阶段。小窝蛋白(Caveolin, Cln)是一个相对分子质量为21-24kD的膜蛋白,高度富集于蛋白小窝膜(Caveolea, Cle),是Cle的标志性蛋白,具有多种潜在功能,在维持Cle形态、结构、功能中起重要作用。目前发现,哺乳动物中至少存在3种Cln基因家族产物,即Cln-1,Cln-2及Cln-3。其中Cln-1广泛存在于肺泡Ⅰ型上皮细胞膜,并有证据表明Cln-1/Cle在肺泡上皮屏障功能变化中扮演重要角色。本研究应用pRNAi-u2.6-Cln-1慢病毒载体转染原代培养肺泡Ⅰ型上皮细胞(AT-1)和c57BL6小鼠,并观察Caveolin-1基因对LPS致炎AT-1细胞和小鼠的影响及作用机制,为临床防治ALI/ARDS提供新的思路。研究内容和方法:1.小鼠Cln-1基因的克隆及载体构建:PCR法扩增Cln-1 cDNA全长基因序列,将其克隆至慢病毒表达载体pRNAi-u2.6上并进行慢病毒包装。2.肺泡Ⅰ型上皮细胞分离、培养:PE标记的AT-1细胞特异性抗体RTI40标记细胞,采用抗PE的免疫磁珠阳性筛选AT-1细胞,流式细胞仪测定纯度。3.慢病毒载体pRNAi-u2.6-Cln-1转染AT-1及鉴定:慢病毒载体pRNAi-u2.6-Cln-1病毒上清转染AT-1细胞,转染后在倒置荧光显微镜下观察绿色荧光,流式细胞仪测定转染效率,用Western-blotting法鉴定转染AT-1细胞中目的蛋白表达情况。4. Cln-1基因对LPS致炎AT-1细胞的影响及作用机制:(1) LPS致炎AT-1细胞模型:AT-1细胞分为转染目的基因的实验组及转染空载体的对照组,均给予LPS刺激,浓度10μg/ml,分2hr和4hr两个时相点。(2) ELISA法测定LPS致炎细胞上清液的炎性因子TNF-α、IL-6表达水平。(3)荧光定量PCR法检测LPS致炎AT-1细胞cPLA2和p38 MAPK mRNA表达变化。(4) Western blotting法检测LPS致炎AT-1细胞cPLA2、p38 MAPK蛋白表达变化。(5) EMSA法检测LPS致炎AT-1细胞核蛋白NF-κB表达变化。(6)特异性阻断cPLA2、p38 MAPK、NF-κB后,细胞上清液TNF-α、IL-6表达水平变化及相互作用。5.观察Cln-1基因对LPS致炎小鼠的影响及作用机制:(1)慢病毒载体pRNAi-u2.6-Cln-1转染c57BL6小鼠及LPS致炎小鼠模型:c57BL6小鼠分为转染目的基因的实验组及转染空载体的对照组,经尾静脉注入caveolin-1慢病毒载体,1月后给予腹腔注射LPS,2mg/kg,分2hr和4hr两个时相点。(2) ELISA法测定LPS致炎小鼠血清的TNF-α、IL-6表达水平。(3) Western blotting法检测LPS致炎小鼠肺组织cPLA2、p38 MAPK蛋白表达变化。(4)病理形态学观察LPS致炎小鼠肺组织HE染色切片。结果:1. PCR法成功扩增出Cln-1 cDNA全长序列,并将其克隆至慢病毒表达载体pRNAi-u2.6,病毒包装后得pRNAi-u2.6-Cln-1慢病毒。2.获得原代培养的肺泡Ⅰ型上皮细胞,纯度可达85%~98%,细胞总量为3-5×10~6。3.慢病毒载体转染肺泡Ⅰ型上皮细胞后,在倒置荧光显微镜下可观察到绿色荧光,细胞转染效率>95%。Western blotting法可检测到相应目的蛋白的表达,24hr后Cln-1表达开始升高,36hr的表达量比转染前表达量显著升高,72hr达到峰值。4. LPS干预AT-1细胞后,在2hr及4hr时相点,高表达Cln-1实验组细胞上清液TNF-α、IL-6的表达水平均显著高于对照组,P<0.05。实验组AT-1细胞cPLA2和p38 MAPK mRNA的表达均显著高于对照组,P<0.05;Western blotting结果显示,cPLA2和p38 MAPK总蛋白及磷酸化蛋白水平也同步升高,P<0.05。EMSA法测定核蛋白的表达结果显示:实验组NF-κB的表达均显著高于对照组,P<0.05。阻断cPLA2/p38 MAPK及下游转录因子NF-κB均可引起炎症因子(TNF-α、IL-6)显著下降,p<0.05。cPLA2抑制剂可以下调磷酸化的p38 MAPK及NF-κB的表达,p<0.05;p38 MAPK抑制剂能够降低磷酸化的cPLA2及NF-κB的表达,p<0.05,而NF-κB的抑制剂对磷酸化的cPLA2和p38 MAPK表达无明显影响,p>0.05。5. LPS干预c57BL6小鼠后,在2hr及4hr时相点,高表达Cln- 1实验组小鼠血清TNF-α、IL-6的表达均显著高于对照组,P<0.05。实验组小鼠肺组织cPLA2和p38 MAPK蛋白表达水平均显著高于对照组,P<0.05。小鼠肺组织病理切片显示实验组小鼠肺部渗出、水肿更明显,损伤更严重。结论:1.成功构建Cln-1基因高表达的肺泡Ⅰ型上皮细胞模型。2. Caveolin-1加重LPS引起的肺泡I型上皮细胞和小鼠肺部损伤。3. Caveolin-1上调cPLA2及其信号通路下游分子p38 MAPK、NF-κB,是LPS引起的肺泡I型上皮细胞和小鼠肺部损伤的可能机制。4.特异性阻断cPLA2、p38 MAPK及NF-κB可以减轻LPS引起的细胞炎性反应。更多还原

【Abstract】 Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are well defined and readily recognized as clinical critical disorders which commonly induced by severe infection, trauma and shock directly and indirectly. The major clinical manifestations include acute respiratory distress, refractory hypoxemia and non-cardiac source pulmonary edema. Mortality rates of acute lung injury is about 30-40%. At present, the pathogenesis of ALI/ARDS has not been fully clarified. Pathological studies have shown that when the diffuse alveolar damage occurs in the ALI/ARDS, the most obvious structure change is the alveolar epithelium, especially typeⅠalveolar epithelial cells, which is more sensitive to injury than typeⅡepithelial cells and the damage often occurs in the early stage.Caveolins are the chief structural proteins of caveolae, which are vesicular invaginations of the plasma membrane. Molecular cloning has identified three distinct caveolin genes, caveolin-1(Cln-1), caveolin-2(Cln-2), and caveolin-3(Cln-3). Cln-1, 22kD, appears to have principal functions in lipid transport, membrane traffic, and cell signaling. There is evidence that Cln-1 is widely present in typeⅠalveolar epithelial cell membrane and that Cln-1/Cle may play an important role in the function of the alveolar epithelial barrier.In the present study, we increased specifically and transiently elevated caveolin-1 expression in AT-1 cells using gene clone technique and studied the effects of over-expression of caveolin-1 on cPLA2/p38 MAPK and the consequent role in the AT-1 cells and the mouse lung injury and in regulating pro-inflammatory cytokines synthesis, nuclear transcription factor activation following LPS challenge.Methods1. The full length cDNA of mouse Cln-1 was cloned by PCR and constructed into lentiviral expression vector pRNAi-u2.6. Recombinant lentivirus vector were packaged using a four-plasmid transient transfection procedure.2. Immunoselection was undertaken to isolate and culture AT-1 cells by incubating cells with the PE-labeled AT-1 cell specific monoclonal antibody (MAb) RTI40. Cell purity was determined by Flow Cytometry.3. The recombinant pRNAi-u2.6-Cln-1 was transfected into AT-1 cells by lentiviral vector. The green fluorescence protein expression was confirmed by inverted fluorescence microscope and the proportion of cells expressing GFP was determined by Flow Cytometry. The expression of Cln-1 were identified by Western blotting.4. AT-1 cells, either transfected with lentiviral vector as experiment group or empty vector (only encoding GFP) as control group, were treated with LPS at concentration 10μg/ml at 2hrs and 4hrs. The level of cytokines (TNF-α, IL-6) released by LPS-stimulated AT-1 cells into the culture supernatant were measured by ELISA kits. The mRNA expression of cPLA2 and p38 MAPK were measured by real-time PCR. Western blotting was used to verify the result of real-time PCR. The expression of NF-κB was detected by EMSA. MAFP (the inhibitor of cPLA2), SB203580 (the inhibitor of p38 MAPK) and BAY11-7082 (the inhibitor of NF-κB) were added to examine the effect on the production of cytokines (TNF-α, IL-6) and the inter-action among cPLA2, p38 MAPK, and NF-κB.5. c57BL6 mice, injected of caveolin-1 lentiviral vector via tail vein, were treated with LPS at concentration 2mg/kg at 2hrs and 4hrs one month later. The level of cytokines (TNF-α, IL-6) in serum were measured by ELISA kits. The expression of cPLA2 and p38 MAPK were detected by Western blotting and lung histological sections were analyzed.Results1. The Cln-1 gene full length cDNA was amplified by PCR and cloned into pRNAi-u2.6, and sequencing result showed that the sequence of Cln-1 cDNA was in coincidence with the sequence in Genbank. Lentiviral transfer vector encoding the GFP marker gene was packaged by a standard four-plasmid cotransfection procedure. The titers of concentrated vector supernatants generated by this procedure are typically on the order of 2.5×10~8 TU/ml.2. Type I alveolar epithelial cells were obtained. The purity of type I cells ranged from 85% to 98%. Yields were 3-5×10~6 cells. 3. Lentiviral vector was transfected into AT-1 cells. The green fluorescence protein was confirmed by inverted fluorescence microscope. Representative FACS analysis at 3 days post-transfection shows a shifted population of cells exhibiting higher fluorescence intensity specifically in the GFP wavelength (FL1 channel), with >95% GFP-positive cells, demonstrating that AT-1 cells are efficiently transduced by this vector. At 36hr post-transfection, caveolin-1 protein levels were significantly increased with the peak value at 72hrs compared with that of the control, as evaluated by Western blotting analysis.4. The over-expression of caveolin-1 caused a significant increase in pro-inflammatory cytokines TNF-αand IL-6 at 2hr and 4hr. The mRNA expression of cPLA2 and p38 MAPK were significantly increased in caveolin-1 over-expressing cells at 2hrs and 4hrs induced by LPS. The activation of the total and phosphorylated cPLA2 and p38 MAPK were significantly increased correspondingly. In the experiment group, the expression of NF-κB significantly increased as comparison to that in control group. MAFP (the inhibitor of cPLA2), SB203580 (the inhibitor of p38 MAPK) and BAY11-7082 (the inhibitor of NF-κB) decreased TNF-αand IL-6 production compared with DMSO/LPS treatment. Pre-treatment with MAFP reduced the activation of phosphorylated p38 MAPK and NF-κB, compared with vector control (DMSO). When the SB203580 was administered to AT-1 cells, the phosphorylated cPLA2 and NF-κB were inhibited, whereas the phosphorylated cPLA2 and p38 MAPK were not affected when BAY11-7082 was added.5. The expression of pro-inflammatory cytokines (TNF-αand IL-6) in the serum of over-expression of caveolin-1 mice increased significantly than that in the control induced by LPS. So did as to the expression of cPLA2 and p38 MAPK according to the change in the AT-1 cells. The analysis of lung tissue pathology showed that the pulmonary effusion, edema is more evident, and the damage is more serious in the experiment group than that in the control.Conclusion1. The model of over-expression caveolin-1 in the AT-1 and c57BL6 cells was successfully constructed.2. The over-expression of caveolin-1 aggravates injury in the AT-1 cells by up-regulation of pro-inflammatory cytokine (TNF-αand IL-6) production. We speculate that the increased activation of cPLA2 and p38 MAPK and downstream molecule lead to inflammation aggravation.3. The specific blocking of cPLA2, p38 MAPK and NF-κB can reduce inflammatory response induced by LPS.更多还原