目的探索GTP结合蛋白氯苄乙胺2(Septin2)在霍奇金淋巴瘤细胞株L428细胞中的表达及其在H/RS(Hodgkin/Reed—Sternberg)细胞向B细胞再分化中的作用。方法采用实时荧光定量PCR(RT-qPCR)、Westernblot、激光共聚焦显微镜、免疫细胞化学法检测霍奇金淋巴瘤细胞株L428细胞过表达CD99前后Septin2在mRNA和蛋白水平的表达;采用RT-qPCR和Westernblot检测L428细胞转染Septin2干扰片段(Septin2-siRNA)后Septin2的表达;采用激光共聚焦显微镜、CCK8法和流式细胞术检测干扰Septin2基因对L428细胞骨架蛋白、细胞增殖活性和免疫表型的影响。结果与L428细胞空白对照组比较,L428细胞Septin2mRNA表达水平在CD99过表达后(O.329±0.019对1.000,P=0.001)和转染Septin2-0siRNA后(0.276±0.025对1.000,P=0.000)均下降,蛋白表达水平也下降。与转染空载体组比较,转染Septin2-siRNA后的L428细胞较前者胞体缩小,细胞增殖活性下降(F=204.927,P〈0.001),细胞中的微丝骨架发生了重建,CD30和CD15表达下降,CD19、CD10、CD38表达增高。结论Septin2在L428细胞中高表达,干扰Septin2可促进H/RS细胞向B细胞方向再分化。
Objective To explore the role of GTP binding protein 2 (Septin2) in the differentiation of Hodgkin' s Lymphoma H/RS cells (Hodgkin/Reed- Sternberg) to B lymphocytes. Methods The expressions of Septin2 mRNA and protein in Hodgkin' s Lymphoma cell line L428 which CD99 was overexpressed were detected by RT-qPCR and Western blot,confocal laser microscopy and immunocytochemistry (ICC). RT-qPCR and Western blot were used to assay the expression of Septin2 after Septin2-siRNA transfected into L428 cells, and confocal laser microscopy, CCK8 assay and flow cytometry were used to analyze the changes of F-actin cytoskeleton,cell proliferation ability and immunophenotype. Results The low expressions of Septin2 mRNA and protein were detected in L428 cell line after overexpresion of CD99 (0.329±0.019 vs 1.000, P=0.001) and Septin2 siRNA transfection (0.276± 0.025 vs 1.000, P=0.000) compared to controls. Compared to vector group, Septin2 siRNA transfection could lead to decrease of cell size, decline of proliferation activity (F=204.927, P〈0.001 ) and reconstruction of F-actin cytoskeleton, and the expression of specific antigen markers CD30 and CD15 of H/RS cell decreased, B cell antigen marker CD19 as well as germinal center marker CD10 and early plasma cell marker CD38 were up-regulated. Conclusion Septin2 interference promotes H/RS cells' redifferentiation to B lymphocytes