支架蛋白JLP对树突状细胞CD40分子跨膜穿梭转运的调控作用

目的:了解支架蛋白JLP分子在树突状细胞(DC)中的表达及其在CD40分子跨膜穿梭转运中的作用。方法:以GM-CSF诱导骨髓干细胞分化为骨髓来源的DC(BMDC)。给予BMDC不同刺激和处理后,通过Western blot检测BMDC中JLP的表达情况。采取慢病毒介导的shRNA干扰技术抑制BMDC中JLP表达,给予BMDC不同刺激或处理后,通过流式细胞技术检测细胞表面及细胞总CD40分子表达水平变化。通过胞内因子染色技术检测不同处理后细胞内IL-12分子的表达。结果:幼稚BMDC表达低水平JLP,Poly I∶C刺激可上调未成熟BMDC表达JLP,但细菌脂多糖(LPS)刺激则不能上调JLP表达。LPS诱导成熟后的BMDC进一步给予抗CD40抗体刺激则可诱导JLP表达。CD40与配体结合导致LPS活化的DC表面CD40分子表达水平明显下调,但细胞总CD40表达水平无变化。在JLP基因敲除的情况下,LPS处理的DC表面CD40分子表达明显减少,但细胞总CD40分子表达水平无变化。CD40抗体或CD40L处理成熟DC后可介导CD40分子内化,在JLP缺失情况下CD40分子内化明显减低。结论:支架蛋白JLP分子参与了BMDC中CD40分子的跨膜穿梭转运。

Scaffold Protein Mediates the CD40 Shuttle Around Cytomembrane and Cytoplasm

Objective:To study the role of scaffold protein JLP in regulating CD40movement around the cytomembrane and cytoplasm.Methods:The murine bone marrow-derived DCs(BMDCs)were generated from bone marrow(BM).JLP expression profile in BMDC with different stimulations was detected by Western blot.Flow cytometry was applied to investigate the alteration of CD40 expression at the cell surface or in the cytoplasm of BMDCs,in which the scaffold JLP was knockdown by lentivirus transduction.The IL-12production in BMDC upon CD40ligation was also studied in the presence or absence of JLP.Results:Immature BMDCs displayed low level of JLP expression,and poly:IC but not LPS stimulation could upregulate the JLP expression.However,LPS pre-matured BMDCs exhibited increased JLP expression after CD40crosslinking.We also found that the surface CD40expression was down-regulated after the CD40ligation,while the total CD40in BMDCs remained stable.LPS-induced surface CD40expression in JLP-silenced BMDCs was obviously lower than in mock-silenced BMDCs,but the total CD40expression in both JLP-silenced and mock-silenced BMDCs were similar.In JLP-deficient BMDCs,the CD40 internalization upon its ligand engagement was decreased to a lower level.Conclusion:Scaffold JLP are involved in the CD40shuttle around the cytomembrane and cytoplasm.