[Effects of alveolar macrophage conditioned media from interstitial lung disease patients on the procollagen mRNA expression in human lung fibroblasts].
We investigated the effects of alveolar macrophage (Am) conditioned media from interstitial lung disease (ILD) patients on lung fibroblast proliferation and procollagen mRNA expression in cells. 13 patients with ILD and 8 patients with bronchial carcinoma (BC) received bronchoalveolar lavage (BAL). The BAL fluid was collected and centrifuged to obtain cells of two groups. The cells were washed and incubated at 37 degrees C and 5% CO2 for 2 hours in DMEM with 10% FCS. Then the adherent Am was added with DMEM with 0.1% FCS. (2ml/10(6) cells), and cultured at 37 degrees C and 5% CO2 for 24 hours. The supernatant was obtained by centrifugation and stored in -20 degrees C for experiment. Lung fibroblasts were planted in 96 well microtitle plate and cultured to confluence. Each well was added 100 microliters Am conditioned medium and incubated for 16 hours, then added 5 uci 3H-TdR for measuring the fibroblast proliferation and incubated till 24 hours. The cells were collected and counted. The DMEM with 0.1% FCS was used as control. When stimulated with Am conditioned media from ILD patients, fibroblast proliferation increased 71%, but for media from BC patients, it just increased 14% (P < 0.05). After stimulated with conditioned media for 24 hours, the cells were trypzied, collected, ashed and isolated total RNA with guanine method. Several samples' total RNA was subjected to electrophoresis in 1% agaroseformaldehyde gel to test specificity of procollagen al (I) and al (III) cDNA probes. 10 micrograms total RNA of each sample was bloted to nitrit membrane and baked at 80 degrees C for 2 hours.
Gao
Z
Z
Department of Medicine, PUMC Hospital, Beijing.
Zhu
Y
Y
Liu
B
B
D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D017077:Culture Media, Conditioned / Q000494:pharmacology; D005347:Fibroblasts / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D008168:Lung / Q000378:metabolism / Q000473:pathology*; D017563:Lung Diseases, Interstitial / Q000235:genetics / Q000473:pathology*; D016676:Macrophages, Alveolar / Q000473:pathology*; D011347:Procollagen / Q000235:genetics*; D012333:RNA, Messenger / Q000378:metabolism*
D017077:Culture Media, Conditioned; D011347:Procollagen; D012333:RNA, Messenger