Effects of clinorotation-induced weightlessness on the metabolism of type Ⅰ collagen in cultured cardiac fibroblasts

摘要

Re searches on the effects of weightlessness/simulated weightlessness on the heart have focused on the contractile function, and little is known about changes in myocardial interstitial matrix. We investigated whether gene expression and protein deposition of type Ⅰ collagen in cardiac fibroblasts were affected by clinorotation-induced weightlessness. Neonatal rat cardiac fibroblasts were obtained by stepwise trypsin dissociation and cultured in Dulbecco’s minimal essential medium (DMEM) supplemented with 10% fetal bovine serum in a humidified atmosphere of 5% CO₂ at 37℃. The flasks with 2nd or 3rd passage cells were filled up with medium, then the clinorotation flasks were attached to the clinostat base and rotated at 30 r/min for 72 hours in a 37℃ incubator, the control flasks were put into the same incubator for stationary culture. For immunocytochemistry, the cell culture coverslips fixed in flasks were removed, washed twice with PBS and fixed with 4% paraformaldehyde. After blocking nonspecific binding sites with 1% BSA, cells were incubated with rabbit anti-collagen type Ⅰ polyclonal antibody, and HRP-conjugated goat anti-rabbit antibody was then applied. The collagen type Ⅰ was stained brown when observed under a light microscope. In addition, total RNA was extracted from the control and clinorotated cells with TRIzol Reagent, the OD260/280 ratios of total RNA were in the range of 1.8 - 2.0. Primers of type Ⅰ collagen α1 chain (ColⅠA1) and tissue inhibitors of metalloproteinase (TIMP) were designed using the software of Primer 5.0. The total RNA and primers were incubated in one tube for RT-PCR using the “Access RT-PCR system” (Promega) according to the manufacturer’s instructions. PCR products and standard DNA ladder were run on a 2% agarose gel stained with ethidium bromide. The gels were analyzed by the imaging system of ImageMaster VDS (Pharmacia) and TotalLab Software. Com pared with the control group, the cardiac fibroblasts in clinorotation exhibited increased staining intensity of type Ⅰ collagen, no significant differences in gene expression of ColⅠA1, and increased gene expression of TIMP-1, TIMP-2 and TIMP-3. These results suggest that the accumulation of type Ⅰ collagen under clinorotation conditions could not be the direct result of gene expression of ColⅠA1. Considering that TIMPs are inhibitors of collagen degradation and the gene expression of TIMPs was upregulated in clinorotation, we propose that TIMP might contribute to the accumulation of type Ⅰ collagen by depressing the extracellular degradation of collagen[ Acta Zoologica Sinica 50 (2): 252 - 257 , 2004].