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目的检测树突状细胞(DC)表面钙网蛋白(CRT)的组成性和诱导性表达,并初步分析细胞表面CRT在DC分化及免疫应答中的作用。方法 AriaⅢ流式分选仪分选出C57 BL/6小鼠脾脏来源的DC,以荧光素标记的CRT多抗和CRT单克隆抗体染色,检测静息态DC表面CRT的组成性表达;静息态DC在体外以脂多糖(LPS)刺激培养24 h后,同样以抗体染色法,流式细胞仪检测培养后DC表面CRT表达量的变化;并检测DC诱导活化前后细胞表面CD86表达量的变化,以确定诱导前后DC的分化状态。结果静息态DC表面能检测到CRT的低水平组成性表达;LPS体外刺激能够有效诱导DC成熟,促使其高表达CRT,商品化CRT多抗和CRT单抗流式检测结果趋势一致,LPS能够诱导DC活化,成熟DC表面CD86的表达量较静息态DC显著提高。结论 CRT表达于静息态及活化态DC细胞表面,在LPS诱导下表达量会明显增加,初步提示在某些疾病状态下,DC细胞膜表面CRT表达量明显提高的状态,可能与其免疫学功能及某些病理状态相关,如抗原提呈、诱导T细胞分化等。
Objective To detect the constitutive and inducible expression of calreticulin (CRT) on the surface of dendritic cells (DCs) and to analyze the role of cell surface CRT in DC differentiation and immune response. Methods The splenic DCs of C57 BL / 6 mice were isolated by Aria Ⅲ flow cytometer and stained with fluorescein - labeled CRT polyclonal antibody and CRT monoclonal antibody to detect the constitutive expression of CRT on resting DCs. DCs were stimulated with lipopolysaccharide (LPS) in vitro for 24 h, then the changes of CRT expression on DCs were detected by flow cytometry and antibody staining. The changes of CD86 expression on DCs To determine the differentiation status of DCs before and after induction. Results The low level of constitutive expression of CRT could be detected on resting DC surface. LPS stimulated the maturation of DC effectively and induced the high expression of CRT. The trend of commercialized CRT polyclonal antibody and CRT monoclonal antibody was the same, LPS could Induced DC activation, the expression of CD86 on mature DCs was significantly higher than that of resting DCs. Conclusions CRT is expressed on the surface of resting and activated DCs, and the expression of CRT is significantly increased under the induction of LPS. It is suggested that the expression of CRT on the surface of DCs may be significantly increased in some disease states, which may be related to the immunological function and Some pathological state related, such as antigen presentation, induced T cell differentiation.