| Acute lung injury (ALI) is a complicated process with severe respiratory dysfunction. How to therapy the damage caused by ALI and to reduce the mortality are always a hot spot in medical science. Nowadays, beyond traditional treatment of ALI, a progress has been made in ALI gene therapy. The objective gene for the using of ALI gene therapy comprise of the coding gene of anti-oxidant, anti-inflammation, protecting and maintaining alveolar epithelial and endothelial function, inhibition of fibrosis proliferation in ARDS, and so on. Among these genes, the coding genes of protecting and maintaining alveolar epithelial function play a key role in repairing alveolar epithelial damage, which are one of focal points to rescue ALI. When ALI occurred, alveolar epithelial cells appear not only structural and functional injury, but also severe apoptosis. So the recovery of alveolar epithelium in ALI involves in repairing of cellular structure and function, also involves in regenerating of alveolar epithelial cells. Many cytokines yielded by mesenchymal cells can act as signal molecule to regulate epithelial proliferation and differentiation. Among these cytokines, keratinocyte growth factor (KGF) is the one of the most important factors. KGF is not only the mitogen of epithelial cells,but also the cytokine which can enhance regeneration and sodium-water transport of alveolar epithelial cells. KGF can only conjugate KGF receptor (KGFR), which expressed by epithelial cells, and bring into full play biological functions. These biological functions include acceleration of Na+-K+-ATP enzyme expression and sodium passage quantities on membrane; enhancement of surfactant which synthesis and excretion by alveolar type â…¡cell; facilitating alveolar epithelial cells' regeneration, differentiation and injury repair. Due to the significance of KGF-KGFR pathway in repairing and regenerating alveolar epithelial cells injured by ALI, we studied KGFR as following steps. Firstly, we observed changes of KGFR expression in mouse lung of ALI model and A549 cell of LPS injury. Secondly, we constructed adenovirus expression vector of KGFR. Finally, we transferred exogenous gene encoding KGFR into A549 cell by using of adenovirus expression vector. It can help A549 cell increasing the expression of KGFR, can also make it possible that A549 cell still express KGFR under condition of ALI. The A549 cell infected KGFR adenovirus expression vector was provided with preventible ability, therefore we can achieve the aim of ALI gene therapy.Our study had achieved the results as following: (1) ALI models of mouse lung and A549 had been constructed by inducing LPS. The changes of KGFR mRNA expression in mouse lung and A549 cell had been observed by PCR and FQ-PCR respectively. Regulation of KGFR expression under condition of ALI were also understood elementally. (2) Total RNA of mouse lung was extracted by using TriPure reagent. Fragment of exon3~exon11 in mouse FGFR2 coding gene was produced by RT-PCR. The fragment was cloned into TA-cloning and screened by restricted enzyme and PCR. This step could be confirmed that exon8 DNA was included in the fragment. Positive fragment replaced another FGFR2 exon3~exon11 DNA which comprise exon9 DNA in pSK-FGFR2-â…¢c. After that, the plasmid of pSK-FGFR2-â…¢b was established (FGFR2-â…¢b is also named as KGFR). (3) To amplify pSK-FGFR2-â…¢b and clone FGFR2-â…¢b into adenovirus vector, a shuttle plasmid--- pAdTrack-FGFR2-â…¢b was constructed. This plasmid plus adenovirus vector (AdEasy-1) were homologously recombined as AdEasy- FGFR2-â…¢b DNA. (4) AdEasy- FGFR2-â…¢b DNA was transfected into HEK293 cell line and synthesized adenovirus expression vector of AdEasy- FGFR2-â…¢b. After amplifying and purifying, the expression vector's titer was evaluated. This expression vector infected A549 cell line, we could finally observe the effect of transgene. (5) Expression of KGFR transgene into A549 was evaluated by using of SDS-PAGE and Western-Blot. Results indicated that KGFR expression had descent tendency under conditions of...
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