IκB-α转基因治疗急性肺损伤的研究

【作者】 朱自路；

【导师】 陈丙莺；

【作者基本信息】 南京医科大学 ， 生物化学， 2003， 硕士

【摘要】 急性肺损伤(Acute lung injury,ALI)是由感染等多种因素引起的以中性粒细胞浸润为主的肺组织的炎症反应。以前炎症因子大量释放和抗炎因子减少为特征的过度全身炎症反应，是危重病患者发生急性肺损伤的重要机制。核因子-κB(nuclear factor-kappa B,NF-κB)是大多数细胞因子表达的一个关键性转录调控因子，NF-κB的活化与急性肺损伤发病关系密切，阻断NF-κB活化有可能是治疗急性肺损伤的重要手段。NP-κB抑制蛋白(inhibitory NF-κB,IκB)是NF-κB的抑制剂，本研究克隆了中国人IκB-α基因，通过腺病毒介导IκB-α基因从体外、体内两个水平观察其对NF-κB抑制作用，为将来，临床应用IκB-α转基因治疗急性肺损伤提供实验依据。 研究方法： 1．应用脂多糖(LPS)诱导制备小鼠急性肺损伤模型，观察NF-κB在脂多糖诱发的急性肺损伤中的表达。 2．应用RT-PCR从胎盘组织克隆中国人IκB-α基因，并对其进行优化，去除N端32、36位两个编码丝氨酸的密码子(即突变型的IκB-α，ΔN IκB-α)，构建pShuttle／ΔN IκB-α真核表达载体。 3．在以上工作基础上，进一步构建Ad-ΔNIκB-α重组腺病毒载体；同时构建Ad-LacZ重组腺病毒载体。 4．从体外、体内两个水平通过Westernblotting、ELISA、免疫组化观察ΔNIκB-α对NF-κB抑制作用。南京医科大学硕士学位论文研究结果:1 .NF一KB在脂多糖诱发的急性肺损伤中呈动态表达。脂多糖腹腔注射 后NF一KB表达在3，J、时达到高峰，随后开始下降。2.△N工KB一a在细胞水平能明显抑制NF一KB活化。3.腺病毒载体可以感染肺内的主要细胞(肺泡上皮细胞)，并高效表 达目的基因。4.通过Ad一△N 1 KB一a转基因可以抑制小鼠急性肺损伤炎症模型NF- KB活化及支气管肺泡灌洗液中性粒细胞比例和TNF一a、工L一6含量的 增高。结论: △N IKB一a通过抑制NF一KB的活化，下调TNF一a、IL一6炎症因子的表达而发挥杭炎和防治急性肺损伤的作用。更多还原

【Abstract】 Acute lung injury occurs as a result of lung inflammation initiated by either infectious or noninfectious stimuli, accompanied with lung neutrophil accumulation . The excessive SIRS (systemic inflammatory response syndyome), induced by an elevated level of proinflammatory mediators combined with a decreased expression of anti-inflammatory molecules is a critical component of acute lung injury . Expression of proinflammatory genes is regulated by transcript ional mechanisms. Nuclear factor-κB (NF-κ B) is one critical transcription factor required for maximal expression of many cytokines involved in the pathogenesis of acute lung injury. The activation of NF- K B is associated with the generation of acute lung injury. Blocking the activation of NF-κ B and down-regulating production of inflammatory mediators could prove to be an important methods to the therapy of acute lung injury .1κB is the inhibitor of NF-κ B. In our research, we clone the Chinese IKB-α gene, construct the eukaryotic express vectors Ad-AN I KB-α and investigate its inhibitory effect to NF-κB in vitro and in vivo, aim to provide clinical information for gene therapy of acute lung injury .Contents:1. Prepare the model of acute lung injury by IPS ip, investigate the expression of NF-κB.2. Clone the Chinese I κB-α gene by RT-PCR from placenta, modify its gene by delete the NH2-terminal 32 and 36 amino acids(AN IKB-OC) .Constructe its eukaryotic express vectors pShuttle/N I κB-α.3. Constructe adenovirus vectors: Ad-N I κ B-α and Ad-LacZ.4. Investigate the inhibitory effect of N I κB-α to NF-κB in vitro and in vivo by western blotting , ELISA and immunohistochemistry.Results:1. The espression of NF-κB in lung tissue of mouse with ALIchanges according to the time after IPS ip.It becomes strongest 3 hours after IPS stimulation, and then decreases.2. The activation of NF-κB induced by LPS is limitedsignificantly after transferring N IκB-α into A549 cells.3. Adenovirus vectors can infect alveolar epithelial cells effectively and express the target gene highly.4. Both NF-κB activity and neutrophil differential count , TNF-α, IL-6 content in BALF of mouse with ALI induced by LPS after AN IκB-α transgene for lung are decreased significantly.Conclusion:N IκB-α transgene for lung can inhibit NF-κ B activity and downregulate the expression of TNF-α IL-6 in BALF of mouse with ALI, suggesting that AN I κB-α might play an anti-inflammatory role in ALI induced by LPS .更多还原