LIF与心肌条件液在小鼠ES细胞分离中的差异比较

摘要/Abstract

摘要： 通过比较LIF和大鼠心肌条件液在小鼠ES细胞分离培养过程中的差异, 从而选择较为合适的培养液用于小鼠ES细胞的分离培养与深入研究。取怀孕3.5 d小鼠囊胚, 培养于小鼠胎儿成纤维细胞饲养层上, 然后根据ES细胞培养液的不同分成2组,一组添加LIF的ES细胞培养液，另一组添加由大鼠心肌条件液组成的ES细胞培养液。结果显示，小鼠ICM的孵出率在心肌条件液中为76.30 %, LIF条件液中为59.35 %,两者差异极显著(p＜0.01)))))) ; 在LIF条件液中比心肌条件液中能较早地分离出ICM，时间差分别为11、11、12、10和12 h，平均为11.2 h; 对传代的ES细胞集落，培养48 h时周边出现分化现象的ES细胞集落所占的比例在心肌条件液中为51.55%, LIF条件液中为31.69 %，两者差异显著(p＜0.05); 第五代小鼠ES细胞核型正常率在心肌细胞条件液中为78.6 %，稍高于LIF条件液中的76 %，差异不显著。

Abstract: By comparing the difference between LIF media conditioned and rat cardiomyocyte media conditioned during isolation and culture of mouse ES cell, suitable media is selected in the future. Mouse embryos of 3.5 d were collected, cocultured with mouse fetal fibroblasts as feeder layer. According to the difference of ES cell media, mouse embryos were divided into two groups, one with LIF media, other with cardiomyocyte media conditioned. Results showed that isolation rate of ICM with cardiomyocyte media conditioned was 76.30 %, LIF media 59.35 %, and difference between two media was significant (p＜0.01). Isolation of mouse ICM with LIF media was earlier than that with cardiomyocyte media conditioned, and difference between two medias were 11, 11, 12, 10 and 12 h respectively, mean difference was 11.2 h. Differentiation rate of ES cell masses of 48th h with cardiomyocyte media conditioned was 51.55 %, LIF media was 31.69 %, and difference between two medias was significant (p＜0.05). In addition, Normal karyotype rate of mouse ES cell with cardiomyocyte media conditioned 78.6 % was slightly higher than that with LIF media 76 %, but difference was not significant.

刘俊平,,曹鸿国,安志兴,张涌. LIF与心肌条件液在小鼠ES细胞分离中的差异比较[J]. 中国农学通报, 2005, 21(3): 30-30.

Liu Junping ,, Cao Hongguo , An Zhixing , Zhang Yong. Comparison to Isolation of Mouse ES Cells With LIF Media and Cardiomyocyte Media Conditioned[J]. Chinese Agricultural Science Bulletin, 2005, 21(3): 30-30.

参考文献

1 Evans M J, Kaufman M H. Establishment in culture of pluripotential cells from mouse embryos. Nature,1981,292(9): 154~ 156
2 Martin G R. Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells. Proc Natl Acad Sci US, 1981, 78(12): 7634~7638
3 Metcalf D. The Unsolved Enigmas of Leukemia Inhibitory Factor. Stem Cells, 2003, 21: 5~14
4 Song H, Lim H, Das S K, et al. Dysregulation of EGF family of growth factors and COX-2 in the uterus during the preattachment and attachment reactions of the blastocyst with the luminal epithelium correlates with implantation failure in LIF-deficient mice. Mol Endocrinol., 2000, 14: 1147~1161
5 Vogiagis D, Salamonsen L A. The role of leukaemia inhibitory factor in the establishment of pregnancy. J Endocrinol.,1999,160: 181~190
6 Sun H, Shi W K. Expression of LIF gene during early development of mouse embryo. J Exp Biol , 1998, 31:105~109
7 Fukada K, Korsching S, Towle M F. Tissue- specific and ontogenetic regulation of LIF protein levels determined by quantitative enzyme im- munoassay. Growth Factors, 1997,14:279~ 295
8 Chesnokova V, Melmed S. Leukemia inhibitory factor mediates the hypothalamic pituitary adrenal axis response to inflammation. Endocrinology, 2000, 141: 4032~4040
9 Ancey C, Corbi P, Froger J, et al. Secretion of IL-6, IL-11 and LIF by human cardiomy -ocytes in primary culture. Cytokine, 2002, 18(4): 199~205
10 Morel D S, Taupin J L, Potier M, et al. Renal synthesis of leukaemia inhibitory factor (LIF), under normal and inflammatory conditions. Cytokine, 2000, 12(3): 265~271
11 Tofaris G K, Patterson P H, Jessen K R, et al. Denervated Schwann cells attract macrophages by secretion of leukemia inhibitory factor (LIF) and monocyte chemoattractant protein-1 in a process regulated by interleukin-6 and LIF. J Neurosci. , 2002, 22: 6696~6703
12 Bonifati C, Mussi A, Dauria L, et al. Spontaneous release of leukemia inhibitory factor and oncostatin-M is increased in supernatants of short-term organ cultures from lesional psoriatic skin. Arch Dermatol Res., 1998, 290(1-2): 9~13
13 Duval D, Reinhardt B, Kedinger C, et al. Role of suppressors of cytokine signaling (Socs) in leukemia inhibitory factor (LIF)–dependent embryonic stem cell survival. FASEB J., 2000, 14:1577~ 1584
14 薛庆善. 体外培养的原理与技术. 北京: 科学出版社, 2003.517~525.
15 韩嵘,柴桂萱,尚克刚.小鼠心肌细胞条件培养基对形成小鼠胚胎干细胞集落的影响.北京大学学报(自然科学版),1997, 33: 185~188
16 Rossant J. Stem cells from the mammalian blastocyst. Stem Cells, 2001,19 (6):477~482
17 Boeuf H, Merienne K, Jacquot S, et al. The Ribosomal S6 Kinases, cAMP- responsive Element-binding, and STAT3 Proteins Are Regulated by Different Leukemia Inhibitory Factor Signaling Pathways in Mouse Embryonic Stem Cells. J. Biol. Chemi., 2001, 276(49): 46204~45211
18 Tofaris G K, Patterson P H, Jessen K R, et al. Leukemia inhibitory factor receptor is structurally related to the IL-6 signal transducer, gp130. EMBO J., 1991, 10: 2839~2848
19 Hibi M, Nakajima K, Hirano T. IL-6 cytokine family and signal transduction: a model of the cytokine system. J. Mol. Med., 1996, 74:1~12
20 Pitard V, Lorgeot V, Taupin J L, et al. The presence in human serum of a circulating soluble leukemia inhibitory factor receptor (sgp190) and its evolution during pregnancy. Eur Cytokine Netw., 1998, 9(4): 599~605
21 Zhang J G, Zhang Y, Owczarek C M, et al. Identification and characterization of two distinct truncated forms of gp130 and a soluble form of leukemia inhibitory factor receptor alphachain in normal human urine and plasma. J Biol Chem., 1998, 273:10798~ 10805
22 Stewart C L, Kaspar P, Brunet L J, et al. Blastocyst implant- tation depends on maternal expression of leukae- mia inhibitory factor. Nature, 1992, 359:76~79
23 Escary J L, Perreau J, Duménil D, et al. Leukaemia inhibitory factor is necessary for maintenance of haematopoietic stem cells and thymocyte stimulation. Nature, 1993, 363:361~364
24 Croy B A, Guilbert L J, Browne M A, et al. Characterization of cytokine production by the metrial gland and granulated metrial gland cells. J Reprod Immunol., 1991, 19(2):149~166
25 Chen J R, Cheng J G, Shatzer T, et al. Leukemia inhibitory factor can substitute for nidatory estrogen and is essential to inducing a receptive uterus for implantation but is not essential for subsequent embryogenesis. Endocrinology, 2000, 141: 4365~4372