Therapeutic Effects of recombinant human Keratinocyte Growth Factor (rHuKGF) in anElastase-Induced Emphysema Model in the Mouse

Pulmonary emphysema is a major manifestation of chronic obstructive pulmonary disease (COPD), which is characterized by persistent inflammation and progressive alveolar destruction. Such destruction in the distal respiratory tract is thought to be irreversible. Despite promising initial experiments using all-trans retinoic acid (ATRA) supplementation to reverse emphysema in rats, contradictory results were obtained by others. A number of studies have shown that the instillation of recombinant human keratinocyte growth factor (rHuKGF) into the lung protects animals from acute lung injury and favourably influences alveolar maintenance and repair. The mechanism of protection, however, is not completely understood. This study aimed at investigating the potential of rHuKGF to induce alveolar regeneration. Two sets of experiments were performed: 1) an in-vivo study to evaluate the therapeutic effects of rHuKGF in the elastase-induced pulmonary emphysema mouse model; 2) an in-vitro study to elucidate the basic molecular mechanism underlying the therapeutic effect of rHuKGF

Systematic comparison of RNA extraction techniques from frozen and fresh lung tissues: checkpoint towards gene expression studies

<p>Abstract</p> <p>Background</p> <p>The reliability of gene expression profiling-based technologies to detect transcriptional differences representative of the original samples is affected by the quality of the extracted RNA. It strictly depends upon the technique that has been employed. Hence, the present study aimed at systematically comparing silica-gel column (SGC) and guanidine isothiocyanate (GTC) techniques of RNA isolation to answer the question which technique is preferable when frozen, long-term stored or fresh lung tissues have to be evaluated for the downstream molecular analysis.</p> <p>Methods</p> <p>Frozen lungs (n = 3) were prepared by long-term storage (2.5 yrs) in -80°C while fresh lungs (n = 3) were harvested and processed immediately. The purity and quantification of RNA was determined with a spectrophotometer whereas the total amounted copy numbers of target sequences were determined with iCycler detection system for assessment of RNA intactness (28S and 18S) and fragment sizes, i.e. short (GAPDH-3' UTR), medium (GAPDH), and long (PBGD) with 200 bp, 700 bp, and 1400 bp distance to the 3'ends of mRNA motif, respectively.</p> <p>Results</p> <p>Total yield of RNA was higher with GTC than SGC technique in frozen as well as fresh tissues while the purity of RNA remained comparable. The quantitative reverse transcriptase-polymerase chain reaction data revealed that higher mean copy numbers of 28S and a longer fragment (1400 bp) were obtained from RNA isolated with SGC than GTC technique using fresh as well as frozen tissues. Additionally, a high mean copy number of 18S and medium fragment (700 bp) were obtained in RNA isolated with SGC technique from fresh tissues, only. For the shorter fragment, no significant differences between both techniques were noticed.</p> <p>Conclusion</p> <p>Our data demonstrated that although the GTC technique has yielded a higher amount of RNA, the SGC technique was much more superior with respect to the reliable generation of an intact RNA and effectively amplified longer products in fresh as well as in frozen tissues.</p

Therapeutic Effects of recombinant human Keratinocyte Growth Factor (rHuKGF) in anElastase-Induced Emphysema Model in the Mouse

Pulmonary emphysema is a major manifestation of chronic obstructive pulmonary disease (COPD), which is characterized by persistent inflammation and progressive alveolar destruction. Such destruction in the distal respiratory tract is thought to be irreversible. Despite promising initial experiments using all-trans retinoic acid (ATRA) supplementation to reverse emphysema in rats, contradictory results were obtained by others. A number of studies have shown that the instillation of recombinant human keratinocyte growth factor (rHuKGF) into the lung protects animals from acute lung injury and favourably influences alveolar maintenance and repair. The mechanism of protection, however, is not completely understood. This study aimed at investigating the potential of rHuKGF to induce alveolar regeneration. Two sets of experiments were performed: 1) an in-vivo study to evaluate the therapeutic effects of rHuKGF in the elastase-induced pulmonary emphysema mouse model; 2) an in-vitro study to elucidate the basic molecular mechanism underlying the therapeutic effect of rHuKGF