CD47对白血病干细胞增殖和凋亡的影响
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摘要
目的通过人白血病干细胞实验探究CD47对白血病发生发展的影响。方法构建慢病毒载体,将慢病毒CD47基因转染至白血病干细胞;MTT法检测白血病干细胞和CD47高表达白血病干细胞增殖状态;蛋白免疫印迹检测正常白血病干细胞和CD47高表达白血病干细胞PCNA蛋白、凋亡相关蛋白Bax以及Bcl-2的变化;Annexin/PI流式检测白血病干细胞和CD47转染后白血病干细胞凋亡率的变化。结果 CD47转染组细胞增殖率明显高于对照组(P <0.05),细胞增殖相关蛋白PCNA在CD47转染组高于正常白血病干细胞组(P <0.05);促凋亡相关蛋白Bax在CD47转染组低于正常白血病干细胞组(P <0. 05)以及抗凋亡相关蛋白Bcl-2在CD47转染组高于正常白血病干细胞组(P <0. 05);细胞凋亡率在CD47转染组低于正常白血病干细胞组(P <0.05)。结论 CD47能够促进白血病干细胞增殖并抑制细胞凋亡,白血病干细胞的增殖过度和凋亡失衡在子宫内膜癌的发生发展中具有重要意义。
Objective To explore the effects of CD47 genetic effect on the occurrence and development of leukemia through human leukemia stem cells study. Methods With Lentivirus vector, CD47 gene was transfected into leukemia stem cells. The cell proliferations in CD47-transfected leukemia stem cells group(CD47 group) and normal leukemia stem cells group(control group) were detected by MTT; Levels of PCNA protein and the marker proteins of apoptosis(Bax and Bcl-2)in CD47-transfected leukemia stem cells group(CD47 group) and normal leukemia stem cells group(control group) were evaluated by western blotting; The rates of apoptosis in CD47-transfected leukemia stem cells group(CD47 group) and normal leukemia stem cells group(control group) were observed by Flow cytometry( FCM) after Annexin/PI staining. Results After leukemia stem cells were transfected by CD47 lentiviral plasmid,the cell proliferation rate was significantly higher than the control group(P < 0. 05), and the expression of cell proliferationassociated protein PCNA was significantly higher than the control group(P <0. 05); while the pro-apoptotic protein Bax was lower in CD47 transfecting leukemia stem cells than the control group(P <0. 05) and the anti-apoptotic protein Bcl2 in CD47 transfecting leukemia stem cells was higher than the control group( P <0. 05); also the apoptotic rate of leukemia stem cells in CD47 transfecting leukemia stem cells was lower than the control group(P <0.05). Conclusion Using lentivirus vectors can significantly increase the expression of CD47 in leukemia stem cells, and further increase the proliferation of leukemia stem cells, as well as inhibit the apoptosis of leukemia stem cells, which has significant influence in the development of leukemia.
Objective To explore the effects of CD47 genetic effect on the occurrence and development of leukemia through human leukemia stem cells study. Methods With Lentivirus vector, CD47 gene was transfected into leukemia stem cells. The cell proliferations in CD47-transfected leukemia stem cells group(CD47 group) and normal leukemia stem cells group(control group) were detected by MTT; Levels of PCNA protein and the marker proteins of apoptosis(Bax and Bcl-2)in CD47-transfected leukemia stem cells group(CD47 group) and normal leukemia stem cells group(control group) were evaluated by western blotting; The rates of apoptosis in CD47-transfected leukemia stem cells group(CD47 group) and normal leukemia stem cells group(control group) were observed by Flow cytometry( FCM) after Annexin/PI staining. Results After leukemia stem cells were transfected by CD47 lentiviral plasmid,the cell proliferation rate was significantly higher than the control group(P < 0. 05), and the expression of cell proliferationassociated protein PCNA was significantly higher than the control group(P <0. 05); while the pro-apoptotic protein Bax was lower in CD47 transfecting leukemia stem cells than the control group(P <0. 05) and the anti-apoptotic protein Bcl2 in CD47 transfecting leukemia stem cells was higher than the control group( P <0. 05); also the apoptotic rate of leukemia stem cells in CD47 transfecting leukemia stem cells was lower than the control group(P <0.05). Conclusion Using lentivirus vectors can significantly increase the expression of CD47 in leukemia stem cells, and further increase the proliferation of leukemia stem cells, as well as inhibit the apoptosis of leukemia stem cells, which has significant influence in the development of leukemia.
引文
[1]杨小娜,郭建美,姚海英,等.中医药治疗白血病的研究进展[J].现代中西医结合杂志,2016,25(10):1134-1137.
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[3]刘玉琴,赵凤菊,陈万青,等.中国2009年白血病发病和死亡资料分析[J].中国肿瘤,2013,22(7);528-534.
[4] LINDBERG F P, LUBLIN D M, TELEN M J, et al. Rh-related antigen CD47 is the signal-transducer integrin-associated protein[J]. J Biol Chem, 1994,269(3):1567-1570.
[5] JAISWAL S, JAMIESON C H, PANG W W, et al. CD47 is upregulated on circulating hematopoietic stem cells and leukemia cells to avoid phagocytosis[J]. Cell,2009,138(2):271-285.
[6] CHENG Q S,WANG X B. CD47 and leukemia stem cells[J].Zhongguo shi yan xue ye xue za zhi,2010,18(4):1088-1091.
[7]陈国强,赵倩,吴英理,等.白血病细胞分化与凋亡的化学生物学研究[J].上海交通大学学报(医学版),2012,(9):1145-1154.
[8] ANNETT S, ROBSON T. Targeting cancer stem cells in the clinic:Current status and perspectives[J]. Pharmacol Ther,2018:13-30.
[9] TESTA U. Leukemia stem cells[J]. Ann Hematol, 2011,90(3):245-271.
[10] BONNET D,DICK J E. Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell[J]. Nat Med,1997,3(7):730-737.
[11] AILLES L E, GERHARD B, KAWAGOE H, et al. Growth characteristics of acute myelogenous leukemia progenitors that initiate malignant hematopoiesis in nonobese diabetic/severe combined immunodeficient mice[J]. Blood, 1999, 94(5):1761-1772.
[12] AZCUTIA V,ROUTLEDGE M,WILLIAMS M R,et al. CD47 plays a critical role in T-cell recruitment by regulation of LFA-1 and VLA-4 integrin adhesive functions[J]. Mol Biol Cell, 2013,24(21):3358-3368.
[13] KONDRATICK C M,LITMAN J M, SHAFFER K V,et al. Crystal structures of PCNA mutant proteins defective in gene silencing suggest a novel interaction site on the front face of the PCNA ring[J].PLoS One,2018,13(3):e0193333.
[14] TELLIER-LEBEGUE C,DIZET E, MA E, et al. Correction:The translesion DNA polymerases Pol zeta and Revl are activated independently of PCNA ubiquitination upon UV radiation in mutants of DNA polymerase delta[J]. PLoS Genet,2018,14(2):e1007236.
[15]李超,伏圣博,刘华玲,等.细胞凋亡研究进展[J].世界科技研究与发展,2007,29(3):45-53.
[16]王莲子,李涛,徐恩君,等.肿瘤细胞凋亡研究进展[A].第十一届全国免疫学学术大会论文集[C]中国安徽合肥,中国免疫学会,2016:315-316.
[17]赵彦超,顾耘.细胞凋亡通路研究进展[J].现代医学,2013,41(4):285-288.
[18]阎海.细胞凋亡研究进展.中山大学研究生学刊(自然科学.医学版),2012,(3):8-13.
[2]耿珊.当归多糖调控CML患者骨髓间充质干细胞衰老抑制白血病干细胞增殖的机理研究[D].重庆医科大学,2016.
[3]刘玉琴,赵凤菊,陈万青,等.中国2009年白血病发病和死亡资料分析[J].中国肿瘤,2013,22(7);528-534.
[4] LINDBERG F P, LUBLIN D M, TELEN M J, et al. Rh-related antigen CD47 is the signal-transducer integrin-associated protein[J]. J Biol Chem, 1994,269(3):1567-1570.
[5] JAISWAL S, JAMIESON C H, PANG W W, et al. CD47 is upregulated on circulating hematopoietic stem cells and leukemia cells to avoid phagocytosis[J]. Cell,2009,138(2):271-285.
[6] CHENG Q S,WANG X B. CD47 and leukemia stem cells[J].Zhongguo shi yan xue ye xue za zhi,2010,18(4):1088-1091.
[7]陈国强,赵倩,吴英理,等.白血病细胞分化与凋亡的化学生物学研究[J].上海交通大学学报(医学版),2012,(9):1145-1154.
[8] ANNETT S, ROBSON T. Targeting cancer stem cells in the clinic:Current status and perspectives[J]. Pharmacol Ther,2018:13-30.
[9] TESTA U. Leukemia stem cells[J]. Ann Hematol, 2011,90(3):245-271.
[10] BONNET D,DICK J E. Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell[J]. Nat Med,1997,3(7):730-737.
[11] AILLES L E, GERHARD B, KAWAGOE H, et al. Growth characteristics of acute myelogenous leukemia progenitors that initiate malignant hematopoiesis in nonobese diabetic/severe combined immunodeficient mice[J]. Blood, 1999, 94(5):1761-1772.
[12] AZCUTIA V,ROUTLEDGE M,WILLIAMS M R,et al. CD47 plays a critical role in T-cell recruitment by regulation of LFA-1 and VLA-4 integrin adhesive functions[J]. Mol Biol Cell, 2013,24(21):3358-3368.
[13] KONDRATICK C M,LITMAN J M, SHAFFER K V,et al. Crystal structures of PCNA mutant proteins defective in gene silencing suggest a novel interaction site on the front face of the PCNA ring[J].PLoS One,2018,13(3):e0193333.
[14] TELLIER-LEBEGUE C,DIZET E, MA E, et al. Correction:The translesion DNA polymerases Pol zeta and Revl are activated independently of PCNA ubiquitination upon UV radiation in mutants of DNA polymerase delta[J]. PLoS Genet,2018,14(2):e1007236.
[15]李超,伏圣博,刘华玲,等.细胞凋亡研究进展[J].世界科技研究与发展,2007,29(3):45-53.
[16]王莲子,李涛,徐恩君,等.肿瘤细胞凋亡研究进展[A].第十一届全国免疫学学术大会论文集[C]中国安徽合肥,中国免疫学会,2016:315-316.
[17]赵彦超,顾耘.细胞凋亡通路研究进展[J].现代医学,2013,41(4):285-288.
[18]阎海.细胞凋亡研究进展.中山大学研究生学刊(自然科学.医学版),2012,(3):8-13.