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目的探究mCD99L2基因沉默对小鼠B淋巴瘤细胞系A20细胞转化为H/RS样细胞的影响。方法重组SiRNA表达质粒LV-mCD99L2,体外转染内源性mCD99L2表达阳性的A20细胞,筛选出稳定表达LV质粒的细胞株并扩增培养;采用免疫荧光技术和流式细胞仪检测转化前后两组细胞鼠源CD30表达;透射电镜观察转化后细胞超微结构的形态特点;细胞计数方法动态观测培养细胞干扰组A20-LV-mCD99L2和未经干扰组A20细胞的H/RS样细胞(直径≥25μm)转型率,以人霍奇金淋巴瘤细胞系L428作为对照;采用流式细胞仪检测细胞周期变化。结果获得了稳定表达LV质粒的单克隆细胞株A20-LV-mCD99L2;免疫荧光标记显示转化细胞CD30(+);流式细胞仪检测A20-LV-mCD99L2细胞CD30阳性率为54.4%;透射电镜观察转化后细胞核增大,可见单核、双核及多核,核仁明显的H/RS样细胞;干扰组H/RS样细胞的转型率明显高于未经干扰组(P<0.01)。两组处于S期的细胞无明显差异,两组细胞均未见凋亡峰。结论mCD99L2基因沉默可诱导小鼠B淋巴瘤细胞系A20细胞转化为H/RS样细胞。
Objective To investigate the effect of mCD99L2 gene silencing on the transformation of mouse B lymphoma cell line A20 into H / RS-like cells. Methods Recombinant SiRNA expression plasmid LV-mCD99L2 was transfected into A20 cells with endogenous mCD99L2 expression in vitro and cell lines stably expressing LV plasmid were selected and expanded. The immunofluorescence technique and flow cytometry The morphological characteristics of the transformed cells were observed by transmission electron microscopy. The number of H / RS-like cells (diameter≥25μm) of A20-LV-mCD99L2 cells and A20 cells without interference cells ) Transformation rate, the human Hodgkin lymphoma cell line L428 as a control; using flow cytometry to detect cell cycle changes. RESULTS: A20-LV-mCD99L2 cell line stably expressing LV plasmid was obtained. CD30 (+) was detected by immunofluorescence staining. The positive rate of CD30 on A20-LV-mCD99L2 was 54.4% by flow cytometry. Transmission electron microscopy The number of H / RS-like cells with mononuclear, binuclear, multi-nucleus and nucleoli was obviously increased after transformation. The transformation rate of H / RS-like cells in the interference group was significantly higher than that without interference (P <0.01). There was no significant difference between the two groups of cells in S phase, and no peak of apoptosis was found in both groups. Conclusion mCD99L2 gene silencing induces the transformation of mouse B lymphoma cell line A20 into H / RS-like cells.