Three-dimensional culture of human lung fibroblasts in pulmonary fibrosis remodeling process / 中国组织工程研究

ABSTRACT

BACKGROUND:Contraction of three-dimensional collagen gels has been used as a model for the contraction which characterizes both normal wound healing and the development of fibrosis in the tissue. Several factors and cytokines,such as tumor necrosis factor alpha (TNF-α), interleukin-1, prostaglandin E2 and insulin have been proved to play important roles in collagen remodeling in vitro as well as serum extravasation during the fibrotic progress.OBJECTIVE: To observe extracellular collagen matrix contraction and apoptosis of fetal lung fibroblasts in TNF-α,interleukin-1, insulin, prostaglandin E2, albumin and globum under three-dimensional culture, and investigate the effects of cytokines, insuin, serum and serum protein on the remodeling and fibrotic formation of lung tissue.DESIGN: A randomized controlled experiment.SETTING: Department of Respiratory Medicine, Lanzhou General Hospital of Lanzhou Military Area Command of Chinese PLA.MATERIALS The experiment was carried out The experiments were carried out in the respiratory laboratory of Lanzhou General Hospital of Lanzhou Military Area Command of Chinese PLA from August 2005 to January 2006. Human fetal lung fibroblasts (American Type Culture Collection), Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum, insulin, transforming growth factor (TGF) (R&D), prostaglandin E2, type Ⅰ collagen was extracted from rat-tail tendons.METHODS: In order to investigate the effect of initial collagen concentration in the gels on the contractility of fibroblasts,the appropriate amount of collagen was mixed with distilled water, four fold-concentrated DMEM, and cells were suspended so that the mass concentration was 0.75-2.0 g/L, with a physiological ionic strength and the desired cell concentration. In order to investigate the effect of cell number in the gels on the contraction, the cellular concentration fibroblasts in the gels were prepared to 0.2×107-4×107 L-1. The areas of floating gels were measured daily and the contraction was calculated by contrasting the initial size (％ of initial area). Different serum concentrations (0.01％-0.5％)in the medium were prepared, the serum albumin (0.1％) or globulin (0.1％) were added to the serum-free culture medium to observe the gel contraction. TGF (10 mg/L), interleukin-1 (10 mg/L), insulin (1 mg/L) and prostaglandin E2 (0.1 μmol/L) were added to observe the effects of cytokines and insulin on fibroblasts-mediated collagen gel contraction.The DNA content and cellular survivability in gels in collagen were determined.MAIN OUTCOME MEASURES: Effect of lung fibroblasts on collagen contraction with or without the presence of cytokines in three-dimensional culture; Effect of collagen with different concentration on the proliferation and apoptosis.RESULTS: ① Collagen gel contraction showed a dependence on the number of fibroblasts. ② Collagen gel contraction was augmented by increasing serum concentration in culture medium, and albumin increased the contraction dramatically. ③TGF and insulin significantly increased the contraction, whereas prostaglandin E2 and interleukin-1 significantly inhibited the gel contraction. ④ The lower the initial collagen concentration was, the more gel contracted and smaller final size were observed, and cell apoptosis increased.CONCLUSION: During the fibrotic process, fibroblast population migrated into the injured lung tissue may play an important role in the development of pulmonary fibrosis and serum infiltrating into injury lung tissue may play an role in stimulating the fibrotic progress. Infiltrating fluids and edema result in the dilution of the collagen concentration in the pulmonary interstitial which may lead to stronger contraction and serious fibrosis. In the dense fibrosis area, cells were hard to survive. In consequence, the final structure of fibrotic lung could not been changed and lung fibrosis progressed.