An Experimental Study On The Activation Of Nuclear Factor-kappaB And The Expression Of Interleukin-8 In Peripheral Blood Mononuclear Cells In Patients With Kawasaki Disease

Objective Kawasaki disease(KD) is now the leading cause of acquired heart disease in children. Its etiology is unknown. In recent years, data have shown that the dysfunction of T cell and monocytes(?)macrophages plays a central role in the development of vasculitis. However the mechanism of vasculitis formation is unknow(?) perfectly. Therefore, the objective of this study is to explore the activation of nuclear factor-kappaB (NF-κB) and the subsequent expression of interleukin-8 (IL-8) in peripheral blood mononuclear cells (PBMCs) in children with Kawasaki disease,and to evaluate their effects on the formation of vasculitis in KD.Methods (1) Culturing PBMCs in vitro: PBMCs were isolated and purified from blood of forty children including 22 KD children and 18 normal ones by density gradient centrifugation, and were then cultured for one hour at 37℃ with 5% CO₂ after cell concentration adjusted to 10⁶/ml with culture .PBMCs in every sample were divided into three groups, the first group was cultured naturally, the second one was stimulated by phorbol 12-myristate 13-acetate (PMA), and the third one was stimulated by PMA and pyrrolidine dithiocarbamate (PDTC). (2) PBMCs cultured for 1h were then prepared into cell smear, fixed in 4% paraformaldehyde for 30 minutes at room temperature, while collecting 200μl supernatant to store at —20℃ for detecting IL-8 protein. (3) Activation of NF-κB in PBMCs was determined by immunocytochemical staining, and enzyme-linked immuosorbent assy (ELISA) was used to measure the concentration of IL-8 in supernatant.