| ObjectiveTrauma is one of the dangerous diseases occurs widely in advanced society. So the problem of the wound healing is still the emphasis in medical research. The wound healing is a kind of complex course involving many factors and cells. Among them, the effect of growth factors has become a focus of the research. Fibroblast growth factor (FGF), especially the basic fibroblast growth factor (bFGF) have extensively biological activity, which could influence every aspects in repair. Thus, it's important to study its mechanisms deeply for the applying of FGF in clinics. While so far, the report about this is quite a few. In the current study, human umbilical vein endothelial cells ( HUVECs) were stimulated with bFGF, and proliferation was measured. To explore the possible mechanisms of endothelial cell (EC) proliferation stimulated by FGF, we determined the expression of nuclear factor - kappaB ( NF - KB ) and ki - 67, as well as the effect of bFGF on both TNP -470( an antiangiogenic agent) and dexa-methasone( Dex).MethodsAt first, the HUVECs were cultured in our experiments. Thenthe complete media were removed and replaced with serum - free media as the cells were sub - confluent to allow for growth arrest. The cultured HUVECs were divided into 6 groups according to different experimental agents: control ( serum - free DMEM ) ; bFGF ( 500ng/ ml); Dex(0.1 M); TNP -470(Kr7g/ml) ; bFGF + Dex; bFGF + TNP -470. 48 hours later, the following experiments were carried out respectively. The proliferation was quantified by a colorimetric assay using MTT reagent; the EC cycle distribution was analyzed with flow cytometer; the expression of NF - KB and ki -67 was detected with SABC immunohistochemical method. The data was analyzed by SPSS.ResultsCompared with control, HUVECs were markedly stimulated with bFGF(P <0.05) ; and they were inhibited with Dex or TNP -470 ; bFGF could relieve their inhibition(P <0.05). By flow cytometric a-nalysis, the presence of bFGF decreased the proportion of cells in the G0/G1 phase and increased the proportion of cells in S and G2/M phases, raised PI(P <0. 05) ; on the contrary, TNP -470 increased the proportion of cells in the G0/G1 phase and decreased the proportion of cells in S and G2/M phases, reduced PI ( P < 0. 05 ) ; while bFGF could reverse this inhibition ( P < 0. 05 ). The immunohistochemical results suggest that ECs in control, the ratio of positive on ki - 67 was low, and NF - KB was localized in cytoplasm with brown - yellow particles ; in bFGF group, ki - 67 was positive with amount of brown -yellow particles in. nuclei, and stains of NF - KB shifted from cytoplasm to the nucleus, the number of positive cells increased ( P < 0. 01); in TNP -470 or Dex group, the ratio of positive on ki -67 andNF - KB reduced in nuclei (P < 0. 01) , and this inhibition could reversed by bFGF( P < 0.05 ).DiscussionIt's well known that topical application of FGF could promotes the wound healing, but the mechanisms associated are still unclear. In this study, we demonstrated the regulation of bFGF on HUVEC proliferation. HUVEC were stimulated with bFGF and the proliferation was quantified by a colorimetric assay, ki - 67, the cell proliferation - associated antigen, could express on every phases of the cell cycle. We utilized the antibody ki -67 to examine the effect of bFGF on EC. The result shows that bFGF upregulated the expression of ki - 67 in nucle-i, therefor stimulated EC proliferation. Flow cytometric analysis found that bFGF decreased the proportion of cells in the GQ/G! phase and increased the proportion of cells in S and G2/M phases. All these results indicate that bFGF has notable mitogenic effects on EC.The present study also explored the mechanisms of bFGF induced proliferation. bFGF, bound to two receptors(HSPG and bFGFR) on the membrane, could convey the signals into the nucleus through a series signal cascade pathway. NF - KB has an important role in the signals that control HUVEC function. When bound by its inhibitory protein , I - KB, classic...
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