【摘要】 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。更多还原

【Abstract】 Objective To investigate the effects of mCD99L2 gene silencing on the transformation of mice B lymphoma cell line A20 to H/RS-like cells. Methods Recombinant plasmid LV-mCD99L2 was transfected into A20 cells which endogenetically express mCD99L2. The stably expressed cell line was selected by Blasticidin. The expression of CD30 in transformed cells was detected by immunofluorescense and FCM method. Observation of A20-LV-mCD99L2 cells was made by transmission electron microscopy. The proliferation of the tumor cells (the diameter of H/RS-like ≥25 μm) of A20-LV-mCD99L2 and A20 in vitro and in nude mice were examined by continuously cell counting, with human Hodgkin lymphoma cell line L428 as control. The cell cycle was detected by FCM method. Results Stable expression cell clone A20-LV-mCD99L2 was obtained after recombinant plasmid transfection. The expression of CD30 in transformed cells was positive, and the rate of expression of CD30 in A20-LV-mCD99L2 was 54.4%. The observation of transformed cells showed larger nucleus and H/RS-like cells with obvious nucleoli which were uninucleated, binucleated and multinucleated cells. The rate of H/RS-like cells of A20-LV-mCD99L2 was obviously higher than that of A20(P<0.01)in vitro and in vivo. Cell cycle analysis showed that no obvious differences was found between A20-LV-mCD99L2 and A20 in S stage and no obvious apoptosis was detected in A20-LV-mCD99L2 and A20. Conclusion mCD99L2 gene silencing may transform mice B lymphoma cell line A20 into H/RS-like cells both in vitro and in vivo.更多还原