Establishment Of One Fibroblast Cell Line From Murine Adipose Tissue And Expression Of C/EBP α, PPAR γ And Pref-1 In E.coli., Production Of Polyclonal Antibody

Objective:To investigate the role of adipose-derived fibroblasts in chronic inflammation, the visceral adipose fibroblast cell line was established and its gene expression pattern and endocrine action was systematically studied; exprssion of Pref-1-GST fusion protein, and preparation of C/EBP α , PPAR Y polyclonal antibody was conducted to explore the mechanism by which Pref-1 inhibits preadipocyte differentiation.Methods:1 Establishment of VAFC by tissue culture.2 Morphology evaluation of VAFC by AFM and TEM, characterization of genetic feature by chromosomal analysis, RT-PCR and immunofluorescence staining analysis of gene expression pattern.3 BMCs were cultured in DMEM (control) or VAFC CM for 6 days, and then the phagocytic activity and phagocytic index was examined by phagocytosis assay.4 BMCs were cultured in serum-free DMEM (control) or VAFC CM for 24 h, and then were stained by Annexin-V-PE and 7-AAD. The effect of VAFC CM on BMCs apoptosis was investigated by flow cytometry.5 Lymphocytes were labeled with CFSE and Con A was used to stimulate lymphocyte proliferation. After 72 h, cell proliferation was analyzed by flow cytometry.6 Expression and purification of Pref-1-GST fusion protein and preparation of C/EBP α and PPAR Y polyclonal antibody.Results1 The VAFC was established by tissue culture and stably cultured for more than 150 passages near two years.2 AFM analysis indicated there were a large number of "pits" at the plasma membrane, and several lamellipodias; TEM analysis confirmed the existence of pits and also indicated there was the protrusion structure in the vicinity of pits, where plenty of granules assembled; the VAFC cells were hyperdiploid, with chromosome numbers varying from 48 to 76; the VAFC expressed fibroblast specific genes FSP1, fibronectin and Type I collagen, and synthesized Type I collagen and MCP-1.; the VAFC also expressed cytokines such as TNFa, IL-6. adiponectin, resistin, ASP. MIF, MCP-1, agt , and IGF-1.3 VAFC CM promoted BMCs adhesion and differentiation to macrophage. There were5considerably more adherent cells in VAFC CM than those in control (2x10 cells/well5versus 0.6 x 10 cells/well). In contrast to the mixed spindle cell and polygonal cell type in DMEM, the cells in VAFC CM presented dominantly the large and round appearance. 89 ± 1 % of BMCs treated with VAFC CM had phagocytic activity with a phagocytic index of 12 ± 3, while only 47 ± 3 % in the control group with a phagocytic index of 5 ± 1.4 VAFC CM helped to survive in the serum-free condition with the rate of 74.8 (± 4.10)% versus 46.7 (±4.33)% (P<0.01), and protected cells from apoptosis with the rate of 19.3 (±2.37)% versus 32.4 (± 1.75)% (P<0.05).VAFC CM interfered in lymphocytes proliferation stimulated with Con A. Compared to Con A alone, VAFC CM plus Con A attenuated lymphocyte proliferation with successive division numbers of one versus two and proliferation rate of 44.01 (±1.77) % versus 50.69 (± 1.21) % (P<0.01).6 Pref-1-GST fusion protein and polyclonal rabbit antibodies to mouse C/EBP a and PPAR Y were obtained.Conclusion1 Establishment of one immortal visceral adipose fibroblast cell line which expresses numerous inflammation-related cytokines.2 The VAFC involved in the regulation of macrophage development and lymphocyte proliferation in vitro. It might be one model to study the role of adipose fibroblasts in obesity-associated chronic inflammation.3 Preparation of Pref-GST fusion protein and polyclonal antibodies to mouse C/EBP a and PPAR v lay a basic foundation on the exploration of the pathway for Pref-1 inhibition action.