【摘要】： 目的:中性粒細胞抑制因子(NIF)是整合素β2的專一性拮抗劑,能抑制中性粒細胞與內(nèi)皮細胞的粘附和聚集,阻止白細胞的滲透和浸潤。角質(zhì)細胞生長因子(KGF)是成纖維細胞生長因子家族中的一員,由間充質(zhì)細胞分泌并特異性地作用于上皮細胞促進其生長繁殖。許多實驗均證明KGF和NIF在肺臟的發(fā)育、炎癥和修復中發(fā)揮重要的作用。針對NIF能夠抑制病理性自身免疫反應和KGF能夠修復損傷的肺泡上皮及抑制纖維化的功效,我們構建了這兩種細胞因子的基因嵌合重組體,并進行表達,目的是研制能夠治療急性肺損傷和肺纖維化的重組蛋白,為進一步的研究和開發(fā)奠定基礎。方法:以8個甘氨酸作為鉸鏈區(qū),運用over-lap PCR的方法將NIF與KGF連接,獲得雙重抗肺纖維化基因嵌合重組體NKM的基因序列。利用pET原核表達系統(tǒng),在BL21 Star (DE3)plysS大腸桿菌中高效表達NKM。經(jīng)過測序、誘導表達、純化和Western-Blot等確證后,再對NKM的體內(nèi)外活性進行測定。結果:本研究成功構建了雙重抗肺纖維化基因重組嵌合體NKM;并在BL21 Star (DE3)plysS大腸桿菌中表達量達到25%;經(jīng)Western-Blot檢測有免疫活性;通過DEAE層析柱純化得到純度為80%的NKM;體外細胞活性測定表明,NKM對白細胞粘附有劑量依賴性的抑制作用,對成纖維細胞生長也有劑量依賴性的抑制作用;動物體內(nèi)活性測定顯示,NKM抑制急性肺損傷后循環(huán)內(nèi)白細胞的增加;病理解剖顯示NKM能明顯抑制博萊霉素誘導的肺炎和肺纖維化。結論:本實驗獲得了具有雙重功能的抗肺纖維化嵌合體蛋白,可用于治療急性肺損傷和肺纖維化,并有望用于治療其它與白細胞活化浸潤及組織纖維化有關的多種臨床疾病。
[Abstract]:Aim: (NIF) is a specific antagonist of integrin 尾 2, which can inhibit the adhesion and aggregation of neutrophils to endothelial cells and prevent the infiltration and infiltration of leukocytes. Keratinocyte growth factor (KGF) is a member of fibroblast growth factor family, secreted by mesenchymal cells and acting specifically on epithelial cells to promote their growth and reproduction. Many experiments have demonstrated that KGF and NIF play an important role in lung development, inflammation and repair. In view of the inhibition of pathological autoimmune response by NIF and the ability of KGF to repair damaged alveolar epithelium and inhibit fibrosis, we constructed and expressed the chimeric recombinant of these two cytokines. The aim is to develop recombinant protein which can treat acute lung injury and pulmonary fibrosis and lay the foundation for further research and development. Methods: using 8 glycine as hinge regions, NIF and KGF were connected by over-lap PCR to obtain the gene sequence of double anti-pulmonary fibrosis gene chimeric recombinant NKM. Expression of NKM. in BL21 Star (DE3) plysS Escherichia coli using pET prokaryotic expression system After sequencing, induced expression, purification and Western-Blot, the activity of NKM in vivo and in vitro was determined. Results: in this study, double anti-pulmonary fibrosis gene recombinant chimeric NKM; was successfully constructed and expressed in BL21 Star (DE3) plysS Escherichia coli. The cell activity of NKM; with purity of 80% was determined by DEAE column purification in vitro. The results showed that NKM inhibited leukocyte adhesion in a dose-dependent manner and the growth of fibroblasts in a dose-dependent manner. In vivo, NKM inhibited the increase of leukocyte in circulation after acute lung injury, and the pathological anatomy showed that NKM could inhibit bleomycin induced pneumonia and pulmonary fibrosis. Conclusion: this study has obtained double function anti-pulmonary fibrosis chimeric proteins which can be used in the treatment of acute lung injury and pulmonary fibrosis and may be used in the treatment of other clinical diseases related to leukocyte activation infiltration and tissue fibrosis.
【學位授予單位】：蘇州大學
【學位級別】：碩士
【學位授予年份】：2006
【分類號】：R392

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