低表达mCD99L2鼠B淋巴瘤细胞亚系的建立及鉴定

Construction and identification of subseries stably expressing low level of mCD99L2 gene from mouse B lymphoma

目的:建立和鉴定稳定的低表达mCD99L2鼠B淋巴瘤细胞亚系,以探究mCD99L2基因在Hodgkin′s淋巴瘤H/RS细胞形成中的作用。方法:对前期构建的慢病毒质粒稳定干扰的A20-LV-mCD99L2克隆株进行体外培养,分别选择第10、20、30、40代细胞,DNA-PCR检测shRNA干扰载体的整合,RT-PCR及荧光定量PCR检测目的基因mCD99L2的表达水平,倒置显微镜观察A20-LV-mCD99L2细胞的形态变化并进行计数,免疫荧光观察H/RS样大细胞mCD30分子的表达。结果:第10、20、30、40代A20-LV-mCD99L2细胞(1)抽提DNA进行PCR均能检测出shRNA干扰载体稳定整合至A20细胞基因组;(2)RT-PCR及荧光定量PCR检测目的基因mCD99L2均较A20细胞呈低水平表达;(3)各代A20-LV-mCD99L2细胞中均发现有类似人H/RS细胞的大细胞(A20-H/RS);(4)免疫荧光检测H/RS样细胞mCD30呈阳性表达。结论:鉴定和建立了低表达mCD99L2基因的A20细胞亚系,该细胞亚系中存在类似人H/RS细胞的大细胞。

AIM： To construct and identify the subseries which express low level of mouse CD99 antigen -like 2 gene （mCD99L2） from mouse B lymphoma A20, in order to investigate the importance of mCD99L2 in the formation of Hodgkin/Reed -Sternberg （H/RS） cell of Hodgkin＇s lymphoma＋ METHODS： During continuous passage culture, Lenti- Virus - mCD99L2 vector was stably transfected into the A20 cells, named A20 - LV - mCD99L2 cells. The integrated status of vector was examined using DNA - PCR. RNA interference efficiency was analyzed by RT - PCR and real time RT - PCR＋ The morphological characteristics of the cells were observed under light microscope. The transformation rate was evaluated by net counting method. The A20 - RS like cells were detected by immunofluorescence labeling of mouse CD30 molecules. RESULTS： During 10, 20, 30 and 40 passages, 282 bp fragments of shRNA vector were detected in A20 - LV - mCD99L2 cells by electrophoresis. Expressions of mCD99L2 gene mRNA in A20 - LV - mCD99L2 cells were lower than those in A20 control cells revealed by RT - PCR and real - time PCR. Giant cell like Hodgkin/Reed - Sternberg cells （A20- H/RS） in A20- LV- mCD99L2 group were significantly more than those in A20 control group, especially in the 72 h after passage. Cells in A20 - LV - mCD99L2 group were positive labeled by mouse CD30 antibody. CONCLUSION： The subseries of A20 cells have been constructed and identified during continuous passage culture, which show low expression of mCD99L2 gene stably, with more giant cells of H/RS like morph and immunophenotype.