【作者】 陈力；

【导师】 黎檀实； 于益芝；

【作者基本信息】 中国人民解放军军医进修学院 ， 急诊医学， 2007， 硕士

【摘要】 背景：角质化细胞生长因子（Keratinocyte Growth Factor, KGF）是一种备受关注的细胞生长因子，其对肺泡上皮细胞（Alveolar Epithelial Cells，AECs）的多途径、多水平的保护作用已经广为认可，既往研究表明KGF对急性肺损伤／急性呼吸窘迫综合征（Acute Lung Injury／Acute Respiratory Distress Syndrome，ALI／ARDS），以及肺纤维化（Pulmonary Fibrosis，PF）均有预防作用，能够明显缓解病情改善预后。但是，就其应用而言，主要存在如下问题：（1）KGF蛋白昂贵；（2）KGF目前仅限于预防。因此，构建KGF、角质化细胞生长因子受体（Keratinocyte Growth Factor，KGFR）的重组腺病毒载体对于探索肺部疾病的治疗；探讨KGF，及其他细胞生长因子在肺损伤免疫进程中的作用都将是一个有力的工具。目的：构建表达小鼠KGF、KGFR基因的重组腺病毒。方法：（1）根据小鼠KGF、KGFR基因序列，设计正向、反向引物；同时，提取小鼠肺组织RNA，进行逆转录聚合酶链反应（reverse transcription-polymerase chain raction，RT-PCR）反应，构建cDNA文库。构建聚合酶链式反应（polymerase chain reaction，PCR）反应体系，扩增目的基因片段。（2）选择pShuttle-CMV（卡那霉素抗性）作为载体质粒，根据引物设计所选择的核酸内切酶，对载体质粒和目的基因片段进行双酶切。将线性化的两条DNA连接后，进行扩增，提取穿梭质粒pShuttle-CMV-KGF（pKGF）、pShuttle-CMV-KGF（pKGFR）。（3）核酸内切酶PmeⅠ线性化pKGF、pKGFR，通过转化含病毒骨架质粒pAdeasy-1（氨苄西林抗性）的大肠杆菌BJ5183以连接病毒骨架DNA，提取病毒质粒pAdeasy-1-pShuttle-CMV-KGF（pAd-KGF），pAdeasy-1-pShuttle-CMV-KGFR（pAd-KGFR），PacⅠ酶切鉴定后，大量扩增质粒，转染HEK293细胞。提取重组腺病毒Adeasy-1-pShuttle-CMV-KGF（Ad-KGF），Adeasy-1-pShuttle-CMV-KGFR（Ad-KGFR），大量扩增，氯化铯梯度离心纯化，测量病毒滴度。结果：（1）DNA测序结果表明pKGF所载基因仅于第48bp处G突变为A，考虑氨基酸翻译简并性，不影响KGF表达；pKGFR则完全符合，提示成功构建穿梭质粒。（2）线性化穿梭质粒转化BJ5183细菌后，细菌生长于含Kan的LB平板，同时PacⅠ酶切显示目的条带，提示成功构建含KGF、KGFR基因的病毒质粒。（3）显微镜下观察HEK293细胞出现病理形态变化，表明病毒合成并进行复制。（4）测得Ad-KGF、Ad-KGFR病毒滴度，分别为3.0×10¹¹PFU／ml、2.25×10¹¹PFU／ml。结论：本实验成功构建小鼠KGF、KGFR基因重组腺病毒载体Ad-KGF、Ad-KGFR，测得病毒滴度，表明可以作为进一步的体外细胞、体内动物实验的实验工具。本实验是在DNA水平上初步证实病毒的成功构建，进一步的体内、体外实验中，将在蛋白水平上明确病毒构建成功，明确目的基因的稳定表达。更多还原

【Abstract】 Background: As a classic growth factor, KGF has multiple protective effects on AECs to obviate future harm, which has been proven in the past experiments, from the acute course of Acute Lung Injury/ Acute Respiratory Distress Syndrome ALI/ARDS to the chronic course of PF.However, there are still many problems for its cost in protein therapy, for its only contribution of prevention but not protection which may have something with the decrease of KGFR on epithelial cells after the injuries to the lung, and its effects of a kind of cytokines associated with immune network which has been ignored. Therefore, successful construction of recombinant adenovirus vector of KGF and KGFR may be a better way to cure the pulmonary diseases such as PF, and to discover the roles of KGF and even other growth factor in the immune course of lung injuries.Objective: To construct the recombinant adenovirus vectors expressing mouse KGF or KGFR.Methods: (1)According to the gene order of mouse KGF and KGFR provided by the gene Bank in National Center for Biotechnology Information, we had designed the sense and the antisense primer, with which we amplified the KGF/KGFR gene by PCR, with cDNA acquired from mouse lung RNA using RT-PCR. (2)pShuttle-CMV with the drug resistance of kanamycin to be the carrying plasmid, we inserted the KGF or KGFR gene into it, to construct the shuttle vector pKGF and pKGFR. (3) We then linearized pKGF and pKGFR, transformed them, respectively, into the E.Coli BJ5183 which contain virus backbones, followed by the step transfection of HEK293cells after identification of endonuclease Pac I and amplification. Finally, we harvested the virus after amplification, caesium chloride density gradient centrifugation purification and measurement of virus titers.Results: (1)The results of DNA sequencing suggested the success of construction of shuttle vector pKGF and pKGFR. (2)The bacteria could grow in the culture with kanamycin, as well as the agarose gel electrophoresis showed the proper strap, which had shown the success of construction of virus plasmid expressing KGF or KGFR. (3)HFK293 cells showed the pathologic morphologic changes, under microscope, which suggested the synthesis and duplication of recombinant adenovirus. (4)Finally, we had measured the virus titers, which had achieved the requirement of the future experiments in vivo and in vitro.Conclusion: We have successfully constructed the recombinant adenovirus vector of KGF or KGFR, which had supplied a tool to understand more about the role of KGF in pulmonary disease, especially as a cytokine in immune reaction, and even might be a new, a lower cost way to cure PF.更多还原