宫颈癌HeLa细胞株高纯度同步化模型的建立与分析
|
摘要
利用药物抑制法,采用胸苷、含羞草素、秋水仙碱和羟基脲阻断或两种试剂共同阻断HeLa细胞,去除药物后正常培养细胞,根据流式细胞术确认细胞在不同时间点的细胞周期情况,建立宫颈癌HeLa细胞株G_1、S和G_2/M期高纯度的同步化模型。药物阻断实验结果表明:胸苷和羟基脲顺序阻断后释放可获得90.0%以上S期细胞;胸苷和秋水仙碱顺序阻断后释放可获得96.0%的G_1期和99.0%的G_2/M细胞;胸苷单阻断也可获得持续8 h高于85.0%的G_1同步率。此外,细胞种植密度从10~5个/mL提高到10~7个/mL,G_1期百分比也由50.0%提高至75.0%。宫颈癌HeLa细胞株高同步率模型可以通过以下方式建立:高S期细胞可通过胸苷和羟基脲顺序阻断实现;G_2/M细胞可通过胸苷和秋水仙碱顺序阻断后释放获得;高G_1期细胞可通过胸苷和秋水仙碱顺序阻断或胸苷单阻断实现,接触抑制对提高G_1期的比率影响要大于血清饥饿。
One or two of reagents(thymidine, hydroxyurea, L-mimosine and colchicine) were used to interdict HeLa cells by drug inhibition method. Then,the cells were cultured normally after the drug removed. Cell cycle at different time was tested by flow cytometry, and high-purity synchronization model of HeLa cell strain in the phases of G_1, S and G_2/M was established. The results showed that above 90.0% of cells in S phase could be obtained by thymidine and hydroxyurea interdiction sequentially. 96.0% of cells in G_1 phase and can 99.0% of cells in G_2/M phase could be obtained by thymidine and cochicine interdiction sequentially. In addition, over 85.0% of G_1 synchronous rate which could last for 8 h could be gained only by thymidine interdiction. Besides, cell seeding density increased to 10~7/mL from 10~5/mL, and the percentage of G_1 phase also rose to 75.0% from 50.0%. High-purity synchronization model of HeLa cell strain can be established through the following method: the cells in S phase could be achieved through thymidine and hydroxyurea interdiction; the cells in G_2/M phases could be achieved by thymidine and cochicine interdiction sequentially. The cells in G_1 phase could be achieved by thymidine and cochicine interdiction sequentially or single thymidine interdiction. The influence of contact inhibition on improvement of G_1 phase was greater than that of serum starvation.
One or two of reagents(thymidine, hydroxyurea, L-mimosine and colchicine) were used to interdict HeLa cells by drug inhibition method. Then,the cells were cultured normally after the drug removed. Cell cycle at different time was tested by flow cytometry, and high-purity synchronization model of HeLa cell strain in the phases of G_1, S and G_2/M was established. The results showed that above 90.0% of cells in S phase could be obtained by thymidine and hydroxyurea interdiction sequentially. 96.0% of cells in G_1 phase and can 99.0% of cells in G_2/M phase could be obtained by thymidine and cochicine interdiction sequentially. In addition, over 85.0% of G_1 synchronous rate which could last for 8 h could be gained only by thymidine interdiction. Besides, cell seeding density increased to 10~7/mL from 10~5/mL, and the percentage of G_1 phase also rose to 75.0% from 50.0%. High-purity synchronization model of HeLa cell strain can be established through the following method: the cells in S phase could be achieved through thymidine and hydroxyurea interdiction; the cells in G_2/M phases could be achieved by thymidine and cochicine interdiction sequentially. The cells in G_1 phase could be achieved by thymidine and cochicine interdiction sequentially or single thymidine interdiction. The influence of contact inhibition on improvement of G_1 phase was greater than that of serum starvation.
引文
[1] 刘宏,王庆伟,章晓梅,等.体外胆管癌细胞X线照射后细胞周期及放射敏感性变化[J].中国现代普通外科进展,2003,6(3):156-158.
[2] 高志清,海春旭,梁欣,等.过氧化氢诱导同步化HepG2细胞DNA损伤和凋亡的敏感时相研究[J].第四军医大学学报,2004,25(8):744-748.
[3] Pellegrin P,Fernandez A,Lamb,N J,et al.Macromolecular uptake is a spontaneous event during mitosis in cultured fibroblasts:Implications for vector-dependent plasmid transfection[J].Molecular Biology of the Cell,2002,13(2):570-578.
[4] Tacheva-Grigorova S K,Santos A,Boucrot E,et al.Clathrin-mediated endocytosis persists during unperturbed mitosis[J].Cell Reports,2013,4(4):659-668.
[5] 李春艳,张莹,薛晓霞,等.流式细胞仪分选非染色S期CHO细胞有助于提高聚乙烯亚胺的转染效率[J].解剖科学进展,2009,15(2):156-160.
[6] 杨金亮,房殿春,张海荣,等.细胞同步化能显著提高逆转录病毒载体的转染效率[J].第三军医大学学报,2002,24(1):34-34.
[7] Medina-Reyes E I,Bucio-Lopez L,Freyre-Fonseca V,et al.Cell cycle synchronization reveals greater G2/M-phase accumulation of lung epithelial cells exposed to titanium dioxide nanoparticles[J].Environmental Science and Pollution Research,2015,22(5):3976-3982.
[8] 郑神.细胞内荧光量子点的双光子吸收截面及摄取率等的相关研究[D].上海:复旦大学,2011:54-65.
[9] 高世勇,曲笑莹.细胞周期同步化研究进展[J].中国药理学通报,2014,30(1):17-21.
[10] 孔宁.Marc-145细胞周期同步化及PRRSV细胞受体表达与病毒感染的关系[D].泰安:山东农业大学,2013:4-47.
[11] 孔德钦.百草枯引起非同步化与同步化细胞氧化、凋亡损伤效应的比较研究[D].西安:第四军医大学,2016:23-61.
[12] 高志清.同步化肿瘤细胞氧化损伤机理的初步研究[D].西安:第四军医大学,2004:13-49.
[13] Yoshizawa-Sugata N,Masai H.Cell Cycle Synchronization and Flow Cytometry Analysis of Mammalian Cells[M].New York:Humana Press,2014,1170:279-293.
[14] 方娟,王珺,龚坚,等.hFAM92A1在人宫颈癌细胞不同周期时相的表达[J].贵阳医学院学报,2015(3):304-306.
[15] 王昱,卢仲毅,许峰,等.羟基脲对体外HeLa细胞周期同步化作用的研究[J].重庆医学,2010,39(24):3331-3332.
[16] Ma H T,Poon R Y.Synchronization of HeLa Cells[M].New York:Humana Press,2011,761:151-161.
[17] Pedrali-Noy G,Spadari S,Miller-Faurès A,et al.Synchronization of HeLa cell cultures by inhibition of DNA polymerase alpha with aphidicolin[J].Nucleic Acids Research,1980,8(2):377-387.
[18] 张凤秋,杨战国.血清浓度及饥饿时间在HeLa细胞G0/G1期同步化中的影响[J].细胞与分子免疫学杂志,2008,24(9):928-929.
[19] 徐秋芳,余克花,易婷,等.不同浓度Nocodazole对HeLa细胞G2/M期同步化的调节作用[J].上海交通大学学报:医学版,2010,30(3):284-287.
[20] 赵雪芹,任磊,王大巾,等.含羞草素对肝癌细胞QGY-7703周期同步化的作用[J].浙江理工大学学报,2016,35(4):620-624.
[21] 黄蕾,江千令,阙云超,等.家蚕BmN细胞S期同步化体系建立[J].中国农学通报,2014,30(2):39-44.
[22] Wu F,Buckley S,Bui K C,et al.Cell cycle arrest in G0/G1 phase by contact inhibition and TGF-beta 1 in mink Mv1Lu lung epithelial cells[J].American Journal of Physiology,1996,270(5):L879-L888.
[23] Galgano P J,Schildkraut C L.G1/S phase synchronization using mimosine arrest[J].Cold Spring Harbor Protocol,2006(4):869-870.
[24] Mladenov E,Anachkova B.DNA breaks induction by mimosine[J].Zeitschrift Für Naturforschung C,2014,58(9/10):732-735.
[25] Chung L C,Tsui K H,Feng T H,et al.L-Mimosine blocks cell proliferation via upregulation of B-cell translocation gene 2 and N-myc downstream regulated gene 1 in prostate carcinoma cells[J].American Journal of Physiology-Cell Physiology,2012,302(4):C676-C685.
[26] Soon-Young P,Jun-Sub I,Sung-Ryeet P,et al.Mimosine arrests the cell cycle prior to the onset of DNA replication by preventing the binding of human Ctf4/And-1 to chromatin via Hif-1α activation in HeLa cells[J].Cell Cycle,2012,11(4):761-766.
[27] Zu H L,Zhang B L,Yu M R,et al.Effect of phytohaemagglutinin and concanavalin A in different stimulants on mitosis metaphase of cashmere goat lymphocyte[J].China Animal Husbandry & Veterinary Medicine,2014,41(10):118-122.
[28] 姜彦杰,刘兆鹏.微管蛋白聚合抑制剂秋水仙碱衍生物的研究进展[J].中国药物化学杂志,2017,27(2):138-151.
[29] Vankayala S L,Hargis J C,Woodcock H L.Unlocking the binding and reaction mechanism of hydroxyurea substrates as biological nitric oxide donors[J].Journal of Chemical Information and Modeling,2012,52(5):1288-1297.
[30] 宋春娇,吕冰洁,张小玲,等.血清饥饿法用于细胞周期同步化的方法学研究[J].中国地方病学杂志,2003,22(4):362-364.
[31] Aghababazadeh M,Kerachian M A.Cell fasting:Cellular response and application of serum starvation[J].Journal of Fasting & Health,2014,2(4):147-150.
[2] 高志清,海春旭,梁欣,等.过氧化氢诱导同步化HepG2细胞DNA损伤和凋亡的敏感时相研究[J].第四军医大学学报,2004,25(8):744-748.
[3] Pellegrin P,Fernandez A,Lamb,N J,et al.Macromolecular uptake is a spontaneous event during mitosis in cultured fibroblasts:Implications for vector-dependent plasmid transfection[J].Molecular Biology of the Cell,2002,13(2):570-578.
[4] Tacheva-Grigorova S K,Santos A,Boucrot E,et al.Clathrin-mediated endocytosis persists during unperturbed mitosis[J].Cell Reports,2013,4(4):659-668.
[5] 李春艳,张莹,薛晓霞,等.流式细胞仪分选非染色S期CHO细胞有助于提高聚乙烯亚胺的转染效率[J].解剖科学进展,2009,15(2):156-160.
[6] 杨金亮,房殿春,张海荣,等.细胞同步化能显著提高逆转录病毒载体的转染效率[J].第三军医大学学报,2002,24(1):34-34.
[7] Medina-Reyes E I,Bucio-Lopez L,Freyre-Fonseca V,et al.Cell cycle synchronization reveals greater G2/M-phase accumulation of lung epithelial cells exposed to titanium dioxide nanoparticles[J].Environmental Science and Pollution Research,2015,22(5):3976-3982.
[8] 郑神.细胞内荧光量子点的双光子吸收截面及摄取率等的相关研究[D].上海:复旦大学,2011:54-65.
[9] 高世勇,曲笑莹.细胞周期同步化研究进展[J].中国药理学通报,2014,30(1):17-21.
[10] 孔宁.Marc-145细胞周期同步化及PRRSV细胞受体表达与病毒感染的关系[D].泰安:山东农业大学,2013:4-47.
[11] 孔德钦.百草枯引起非同步化与同步化细胞氧化、凋亡损伤效应的比较研究[D].西安:第四军医大学,2016:23-61.
[12] 高志清.同步化肿瘤细胞氧化损伤机理的初步研究[D].西安:第四军医大学,2004:13-49.
[13] Yoshizawa-Sugata N,Masai H.Cell Cycle Synchronization and Flow Cytometry Analysis of Mammalian Cells[M].New York:Humana Press,2014,1170:279-293.
[14] 方娟,王珺,龚坚,等.hFAM92A1在人宫颈癌细胞不同周期时相的表达[J].贵阳医学院学报,2015(3):304-306.
[15] 王昱,卢仲毅,许峰,等.羟基脲对体外HeLa细胞周期同步化作用的研究[J].重庆医学,2010,39(24):3331-3332.
[16] Ma H T,Poon R Y.Synchronization of HeLa Cells[M].New York:Humana Press,2011,761:151-161.
[17] Pedrali-Noy G,Spadari S,Miller-Faurès A,et al.Synchronization of HeLa cell cultures by inhibition of DNA polymerase alpha with aphidicolin[J].Nucleic Acids Research,1980,8(2):377-387.
[18] 张凤秋,杨战国.血清浓度及饥饿时间在HeLa细胞G0/G1期同步化中的影响[J].细胞与分子免疫学杂志,2008,24(9):928-929.
[19] 徐秋芳,余克花,易婷,等.不同浓度Nocodazole对HeLa细胞G2/M期同步化的调节作用[J].上海交通大学学报:医学版,2010,30(3):284-287.
[20] 赵雪芹,任磊,王大巾,等.含羞草素对肝癌细胞QGY-7703周期同步化的作用[J].浙江理工大学学报,2016,35(4):620-624.
[21] 黄蕾,江千令,阙云超,等.家蚕BmN细胞S期同步化体系建立[J].中国农学通报,2014,30(2):39-44.
[22] Wu F,Buckley S,Bui K C,et al.Cell cycle arrest in G0/G1 phase by contact inhibition and TGF-beta 1 in mink Mv1Lu lung epithelial cells[J].American Journal of Physiology,1996,270(5):L879-L888.
[23] Galgano P J,Schildkraut C L.G1/S phase synchronization using mimosine arrest[J].Cold Spring Harbor Protocol,2006(4):869-870.
[24] Mladenov E,Anachkova B.DNA breaks induction by mimosine[J].Zeitschrift Für Naturforschung C,2014,58(9/10):732-735.
[25] Chung L C,Tsui K H,Feng T H,et al.L-Mimosine blocks cell proliferation via upregulation of B-cell translocation gene 2 and N-myc downstream regulated gene 1 in prostate carcinoma cells[J].American Journal of Physiology-Cell Physiology,2012,302(4):C676-C685.
[26] Soon-Young P,Jun-Sub I,Sung-Ryeet P,et al.Mimosine arrests the cell cycle prior to the onset of DNA replication by preventing the binding of human Ctf4/And-1 to chromatin via Hif-1α activation in HeLa cells[J].Cell Cycle,2012,11(4):761-766.
[27] Zu H L,Zhang B L,Yu M R,et al.Effect of phytohaemagglutinin and concanavalin A in different stimulants on mitosis metaphase of cashmere goat lymphocyte[J].China Animal Husbandry & Veterinary Medicine,2014,41(10):118-122.
[28] 姜彦杰,刘兆鹏.微管蛋白聚合抑制剂秋水仙碱衍生物的研究进展[J].中国药物化学杂志,2017,27(2):138-151.
[29] Vankayala S L,Hargis J C,Woodcock H L.Unlocking the binding and reaction mechanism of hydroxyurea substrates as biological nitric oxide donors[J].Journal of Chemical Information and Modeling,2012,52(5):1288-1297.
[30] 宋春娇,吕冰洁,张小玲,等.血清饥饿法用于细胞周期同步化的方法学研究[J].中国地方病学杂志,2003,22(4):362-364.
[31] Aghababazadeh M,Kerachian M A.Cell fasting:Cellular response and application of serum starvation[J].Journal of Fasting & Health,2014,2(4):147-150.