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目的探讨树突状细胞(DC)在肾小管间质炎症损伤中的作用,以及抗P-选择素功能域单抗(PsL-EGFmAb)对DC浸润及体外成熟与功能的干预调节。方法(1)建立大鼠单侧输尿管梗阻(UUO)模型。分别采用免疫组化和免疫双染与图像分析,观察P-选择素及CD1a~+ CD80~+DC在肾组织表达和分布变化。(2)从脐血CD34~+造血干细胞中诱导扩增DC,并于成熟过程中采用流式细胞仪分析细胞表面分子表达；RT-PCR检测细胞NF-kB P50、P65 mRNA表达；混合淋巴细胞反应(MLR)检测DC对T细胞刺激能力；以及ELISA测定MLR上清液IL-12 p70分泌含量。结果(1)与假手术组比较,UUO大鼠从第1天起,随着P-选择素以肾小管上皮细胞为主的小管间质表达,CD1a~+CD80~+DC以肾间质为主浸润；至第7天P-选择素上调且CD1a~+ CD80~+DC显著聚集,两者明显相关且与肾小管间质病变程度显著相关。经PsL-EGFmAb处理后,大鼠肾组织P-选择素表达下调,CD1a~+CD80~+DC浸润减少,且肾小管间质损害程度减轻。(2)经TNF-α刺激炎性状态下,培养人DC成熟过程中基本不表达或低表达P-选择素,但持续高表达与P-选择素同属C型凝集素的DC-SIGN。经PsL-EGFmAb处理后,可明显抑制DC-SIGN及细胞内NF-kB基因表达,并相应抑制DC黏附共刺激分子表达、IL-12分泌及刺激T细胞增殖能力。结论DC也是肾小管间质炎症病变启动因素,针对P-选择素功能域的单抗对其浸润具抑制作用。此外,该单抗对人DC成熟与功能有调节效应,提示与抑制作为DC模式识别及黏附受体的DC-SIGN有关,并可能通过影响NF-kB途径起作用。
Objective To investigate the role of dendritic cells (DCs) in the tubulointerstitial inflammatory injury and the interventional regulation of anti-PsL-EGFmAb on DCs infiltration and in vitro maturation and function. Methods (1) To establish unilateral ureteral obstruction (UUO) model in rats. The expression of P-selectin and CD1a ~ + CD80 ~ + DC in renal tissues were observed by immunohistochemistry and double immunofluorescence and image analysis respectively. (2) DCs were induced from cord blood CD34 + hematopoietic stem cells, and cell surface molecules were analyzed by flow cytometry during maturation. The expression of NF-kB P50 and P65 mRNA was detected by RT-PCR. Mixed lymphocytes Response (MLR) was used to detect the ability of DCs to stimulate T cells. ELISA was used to determine the secretion of IL-12 p70 in supernatant of MLR. Results (1) Compared with the sham operation group, UUO rats began to express on the first day, with the expression of P-selectin in tubulointerstitial of tubular epithelial cells. The expression of CD1a ~ + CD80 ~ + DC was The main infiltration of P-selectin up to the seventh day and CD1a ~ + CD80 ~ + DC significantly aggregated, both significantly correlated with the degree of tubulointerstitial lesions significantly related. After PsL-EGFmAb treatment, the expression of P-selectin in renal tissue was decreased, the infiltration of CD1a ~ + CD80 ~ + DC was reduced and the degree of tubulointerstitial lesion was reduced. (2) P-selectin was not expressed or overexpressed in mature human DCs during the course of TNF-α stimulation, but continued to be highly expressed in DC-SIGN which belongs to C-type lectin. PsL-EGFmAb treatment can significantly inhibit DC-SIGN and intracellular NF-kB gene expression, and the corresponding inhibition of DC adhesion co-stimulatory molecules, IL-12 secretion and stimulate T cell proliferation. Conclusion DCs are also the initiators of tubulointerstitial inflammation, and the monoclonal antibody against P-selectin domain can inhibit its infiltration. In addition, the monoclonal antibody has a regulatory effect on the maturation and function of human DCs, suggesting that it is related to the inhibition of DC-SIGN as a DC pattern recognition and adhesion receptor and may play a role in influencing the NF-kB pathway.