下调PAK1对MPLW515L突变的MPN细胞分化、凋亡及6133/MPL移植小鼠存活的影响  

Effects of Down-Regulation of PAKl on Differentiation and Apop-tosis of MPN Cells with MPLW515L Gene Mutation and Survival of 6133/MPI Mice

作　　者：张启岗 王淑瑾 于翔茹 张丽伟 徐开林 付春玲 ZHANG Qi-Gang;WANG Shu-Jin;YU Xiang-Ru;ZHANG Li-Wei;XU Kai-Lin;FU Chum-Ling(Blood Diseases Institute,Xuzhou Medical University,Department of Hematology,The Affiliated Hospital of Xuzhou Medical University,Xuzhou 221000,Jiangsu Province,China)

机构地区：[1]徐州医科大学血液病研究所,徐州医科大学附属医院血液科,江苏徐州221000

出　　处：《中国实验血液学杂志》2024年第5期1472-1478,共7页Journal of Experimental Hematology

基　　金：国家自然科学基金(82070131);江苏省自然科学基金(BK2020022348)。

摘　　要：目的:探讨下调p21蛋白激活激酶1(PAK1)对血小板生成素受体(MPL)密码子515突变(MPLW515L)的MPN细胞(6133/MPL)增殖、分化、凋亡及移植小鼠存活的影响。方法:使用慢病毒介导的shRNA转染技术干扰6133/MPL细胞中PAK1的蛋白表达水平;CCK-8法检测下调PAK1对6133/MPL细胞增殖能力的影响,细胞计数法检测其集落形成能力;流式细胞术检测敲低PAK1对6133/MPL细胞中多倍体DNA形成能力和细胞凋亡的影响;Western blot法检测细胞周期蛋白cyclin D1、cyclin D3和细胞凋亡相关蛋白Bax的表达;HE染色法观察移植小鼠脾脏和骨髓的肿瘤细胞浸润情况。结果:下调PAK1能显著抑制6133/MPL细胞的增殖并降低细胞集落形成能力;敲低PAK1后,6133/MPL细胞中多倍体DNA含量从31.8%增加到57.5%和48.0%,凋亡比例约增至10.8%;下调PAK1能够减少6133/MPL移植小鼠脾脏和骨髓肿瘤细胞的浸润,从而延长其生存期。结论:下调PAK1能显著抑制6133/MPL细胞生长促进多倍体DNA的形成,诱导6133/MPL细胞凋亡,延长6133/MPL移植小鼠的生存时间。Objective:To investigate the effects of down-regulation of p21 activated kinase 1(PAK1)on the:proliferation,differentiation,and apoptosis of myeloproliferative neoplasm(MPN)cells(6133/MPL)with thrombopoietin receptor MPL mutation at codon 515(MPLW5I5L)and survival of 6133/MPL mice.Methods:Interference with the protein level of PAKI in 6133/MPL cells was assessed using lentivirus-mediated shRNA transfection technology.CCK-8 assay was used to detect the effect of down-regulation of PAKI on the proliferation viability of 6133/MPL cells,and colony-forming ability was measured by cell counting.Flow cytometry was used to detect the PAKI kinase activity on the ability of polyploid DNA formation and cell apoptosis in 6133/MPL cells.The expression of cyclin D1,cyclin D3 and apoptosis-related protein Bax was detected by Western blot.The infiltration of tumor cells in spleen and bone marrow of 6133/MPL mice were detected by HE staining.Results:Down-regulation of PAKI inhibited the proliferation and reduced the ability of cell colony formation of 6133/MPL cells.After knocking down PAKI,the content of polyploid DNA in 6133/MPL cells increased from 31.8 to 57.5%and 48.0%,and the proportion of apoptosis increased approximately to 10.8%.Down-regulation of PAKI led to a reduction of infiltration of tumor cells in liver and bone marrow of 6133/MPL mice,thereby prolonging survival time.Conclusion:Down-regulation of PAKI can significantly inhibit the growth of 6133/MPL cells,promote the formation of polyploid DNA,induce 6133/MPL cell apoptosis,and prolong the survival time of 6133/MPL mice.

关 键 词：骨髓增殖性肿瘤 巨核细胞 p21蛋白激活激酶1 细胞凋亡 多倍化 

分 类 号：R733.3[医药卫生—肿瘤]