Lung inflammation after bleomycin treatment in mice: Selection of an accurate normalization strategy for gene expression analysis in an ex-vivo and in-vitro model.
Pulmonary fibrosis (PF) is the most common and aggressive interstitial lung disease, characterized by a patchy development of fibrosis leading to progressive destruction of the normal lung architecture which is preceded by an inflammatory process. Gene expression studies are important to understand the development of PF but the accuracy and reproducibility of Real-Time PCR depend on appropriate normalization strategies. This study aimed to analyze the expression variability of eight commonly used reference genes during the initial inflammatory phase of bleomycin-induced PF in a mouse model and to verify whether the selected reference genes could be applied to an in-vitro model of BLM-treated primary murine lung fibroblasts. Wild-type C57BL/6 mice (n=40) were used. Real-Time PCR was carried out on lung tissue of mice either BLM (BLM-tm) or physiological solution-treated (PSS-tm), and in primary lung fibroblasts, isolated from healthy C57BL/6 mice. Histological analysis was performed to confirm the inflammation development. During inflammation, the most stable genes resulted: PPIA, HPRT-1 and SDHA for both models; the normalization strategy was tested analyzing mRNA expression of PTX-3 and TNF-α which resulted up-regulated both in ex-vivo and in-vitro with respect to PSS-tm/fibroblasts. Histological analysis supported the results. This study identified a new set of reference genes expressed both in the in-vitro and ex-vivo models. A higher expression of both markers in BLM-tm with respect to PSS-tm indicated that BLM might lead to increased PTX-3 local production by a co-regulation with TNF-α at lung level.
['Della Latta|Veronica|V|', 'Cabiati|Manuela|M|', 'Burchielli|Silvia|S|', 'Frenzilli|Giada|G|', 'Bernardeschi|Margherita|M|', 'Cecchettini|Antonella|A|', 'Viglione|Federica|F|', 'Morales|Maria-Aurora|MA|', 'Del Ry|Silvia|S|']
D000818:Animals D001761:Bleomycin D005260:Female D005347:Fibroblasts D020869:Gene Expression Profiling D008168:Lung D008297:Male D051379:Mice D011014:Pneumonia D012015:Reference Standards D012984:Software D013223:Statistics as Topic
Bleomycin Pulmonary fibrosis Real-Time PCR Reference genes PTX-3 TNF-α