不同冻存保护剂对绵羊前体脂肪细胞冻存效果的影响

doi:10.3864/j.issn.0578-1752.2012.07.024

中国农业科学 ›› 2012, Vol. 45 ›› Issue (7): 1439-1446.doi: 10.3864/j.issn.0578-1752.2012.07.024

不同冻存保护剂对绵羊前体脂肪细胞冻存效果的影响

蔡勇, 阿依木古丽, 臧荣鑫, 刘翊中, 杨具田, 乔自林, 曹忻, 徐红伟, 吴建平

1.西北民族大学实验中心，兰州 730030
2.甘肃农业大学动物科学技术学院/国家绒毛用羊产业体系环境控制实验室，兰州 730070
3.西北民族大学生命科学与工程学院，兰州 730030
４.甘肃省动物细胞工程技术研究中心，兰州 730030

收稿日期:2011-03-09 出版日期:2012-04-01 发布日期:2011-10-11
通讯作者: 通信作者吴建平，E-mail：wujp@gsau.edu.cn
作者简介:蔡勇，E-mail：caiyong1979@163.com
基金资助:
国家自然科学基金项目（30871811、31160440）、甘肃省科技行业专项（0801NKDP037）、西北民族大学校企合作项目（H2009-12、H2010-7）

Effects of Different Cryoprotectants on Ovine Preadipocytes Cryopreservation

CAI Yong, A Yi-Mu-Gu-Li, ZANG Rong-Xin, LIU Yi-Zhong, YANG Ju-Tian, QIAO Zi-Lin, CAO Xin, XU Hong-Wei, WU Jian-Ping

1.西北民族大学实验中心，兰州 730030
2.甘肃农业大学动物科学技术学院/国家绒毛用羊产业体系环境控制实验室，兰州 730070
3.西北民族大学生命科学与工程学院，兰州 730030
４.甘肃省动物细胞工程技术研究中心，兰州 730030

Received:2011-03-09 Online:2012-04-01 Published:2011-10-11

摘要/Abstract

摘要： 【目的】探讨绵羊前体脂肪细胞冷冻保存的最佳冻存保护剂种类和浓度。【方法】在含20%胎牛血清的DMEM/F12培养液中分别添加不同浓度的DMSO、EG、PG、G和PVP作为冻存液，对原代培养的绵羊前体脂肪细胞进行冷冻保存后，检测复苏细胞存活率、细胞增殖能力、细胞中脂肪沉积量，测量GPDH活性，并用实时荧光定量PCR检测PPARγ和LPL mRNA的表达水平，最后对复苏细胞进行染色体核型分析。【结果】各种冻存保护液中，10% DMSO和5% DMSO+5% PVP组细胞存活率最高，与对照组相比差异不显著（P＞0.05），其它各试验组复苏细胞存活率均显著低于对照组（P＜0.05）；各试验组中10% DMSO组细胞增殖能力最强，且与对照组相比差异不显著（P＞0.05）；细胞复苏后培养6 d，各试验组脂肪沉积量与对照组之间没有显著差异（P＞0.05），而第11天时，10 % DMSO组脂肪沉积量显著高于其它试验组（P＜0.05），且与对照组相比差异不显著（P＞0.05）；各试验组GPDH活性和LPL、PPARγ mRNA 的表达量与对照组之间没有显著差异（P＞0.05）；染色体核型分析发现，复苏细胞二倍体细胞占主体，与原代细胞没有显著差异（P＞0.05）。【结论】含20% FBS的DMEM/F12培养液中添加10% DMSO或10%PVP、5% DMSO+5% PVP均能有效保存绵羊前体脂肪细胞，但以10% DMSO最佳。

关键词: 冷冻保存, 前体脂肪细胞, 绵羊, 冷冻保护剂

Abstract: 【Objective】 The aim of this experiment is to investigate a suitable cryoprotectant and its optimal concentration for ovine preadipocytes from omental. 【Method】Trypan blue exclusion test, MTT, oil red O staining and real-time PCR were used to test the effects of following cryoprotective agents (CPAs) with different concentrations on post-thaw survival, proliferation, differentiation capacity, PPARγ and LPL mRNA expression of ovine preadipocytes from omental, dimethyl sulphoxide (DMSO), ethylene glycol (EG), propylene glycol (PG), glycerol (G) and polyvinylpyrrolidone (PVP). In addition to the CPAs, the basic medium is DMEM/F12 medium plus 20% FBS. Then karyotype was analyzed. 【Result】Trypan blue exclusion test showed that the highest survival rate, no significant difference with the primary cells, was obtained when cryopreserved with 10% DMSO or 5% DMSO plus 5% PVP among all the CPA treatments in this study. The highest survival rate (94.96%) and cell viability were obtained when cryopreserved with 10% PVP, and showed no significant difference compared with primary cells. Oil red O staining showed no significant difference in lipogenesis among all the CPAs groups and primary cells on 6 th day (P＞0.05), while on 11th day, cells cryopreserved with 10% DMSO turned up significantly greater lipogenesis than other CPAs (P＜0.05), but had no significant difference with primary cells (P＞0.05). Activity of the GPDH, mRNA expression of LPL and PPARγ showed no significant difference among all the groups (P＞0.05). Karyotype analysis showed that diploid cells were dominant post thaw and showed no significant difference compared with primary cells (P＞0.05). 【Conclusion】Results of the present study indicate that ovine preadipocytes could successfully be cryopreserved with DMEM/F12 medium containing 10% DMSO or 10 % PVP, 5% DMSO plus 5% PVP, while 10% DMSO is a suitable CPA for ovine preadipocytes.

Key words: cryopreservation, preadipocyte, ovine, cryoprotectants

蔡勇, 阿依木古丽, 臧荣鑫, 刘翊中, 杨具田, 乔自林, 曹忻, 徐红伟, 吴建平. 不同冻存保护剂对绵羊前体脂肪细胞冻存效果的影响[J]. 中国农业科学, 2012, 45(7): 1439-1446.

CAI Yong, A Yi-Mu-Gu-Li, ZANG Rong-Xin, LIU Yi-Zhong, YANG Ju-Tian, QIAO Zi-Lin, CAO Xin, XU Hong-Wei, WU Jian-Ping. Effects of Different Cryoprotectants on Ovine Preadipocytes Cryopreservation[J]. Scientia Agricultura Sinica, 2012, 45(7): 1439-1446.

0
/ / 推荐

导出引用管理器 EndNote|Reference Manager|ProCite|BibTeX|RefWorks

链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2012.07.024

https://www.chinaagrisci.com/CN/Y2012/V45/I7/1439

参考文献

[1]Verbeke W, Pérez-Cueto F J, Barcellos M D, Krystallis A, Grunert K G. European citizen and consumer attitudes and preferences regarding beef and pork. Meat Science, 2010, 84(2): 284-292.

[2]Jiang T, Busboom J R, Nelson M L, O'Fallon J, Ringkob T P, Rogers-Klette K R, Joos D, Piper K. The influence of forage diets and aging on beef palatability. Meat Science, 2010, 86(3): 642-650.

[3]?ahin H, Çiçek B, Y?lmaz M, Ongan D, ?nanç N, Aykut M, Elmali F. Obesity prevalence, waist-to-height ratio and associated factors in adult Turkish males. Obesity Research and Clinical Practice, 2011, 5(1): 29-35.

[4]Catalano P M, Mouzon S H D. Is it time to revisit the Pedersen hypothesis in the face of the obesity epidemic? American Journal of Obstetrics and Gynecology, 2011, 204(6): 479-487.

[5]Ashwood-Smith M J, Farrant J. Low Temperature Preservation in Medecine and Biology. Turnbridge Wells: University Park Press, 1980.

[6]Bwanga C O, Ekwall H, Rodriguez-Martinez H. Cryopreservation of boar semen. III: Ultrastructure of boar spermatozoa frozen ultra-rapidly at various stages of conventional freezing and thawing. Acta Veterinaria Scandinavica, 1991, 32(4): 463-471.

[7]李影, 杨公社, 卢荣华. 不同冷冻保护剂对猪前体脂肪细胞冷冻后细胞活力的影响. 分子细胞生物学报, 2006, 39(3): 285-288.

Li Y, Yang G S, Lu R H. Effects of different cryoprotectants on postthaw viability of porcine preadipocyte. Journal of Molecular Cell Biology, 2006, 39(3): 285-288. (in Chinese)

[8]Li Y, Lu R H, Luo G F, Pang W J, Yang G S. Effects of different cryoprotectants on the viability and biological characteristics of porcine preadipocyte. Cryobiology, 2006, 53(2): 240-247.

[9]Chen G R, Ge R S, Lin H, Dong L, Sottas C M, Hardy M P. Development of a cryopreservation protocol for Leydig cells. Human Reproduction, 2007, 22(8): 2160-2168.

[10]蔡永国, 张翼军. F68及其联合DMSO对脐血造血细胞的低温保护作用. 第三军医大学学报, 2002, 24(11): 1293-1295.

Cai Y G, Zhang Y J. Effect of F68 and its combination with dimethyl sulfoxide on cryopreserved hematopoietic stem cell from umbilical cord blood. Acta Academiae Medicinae Militaris Tertiae, 2002, 24(11): 1293-1295. (in Chinese)

[11]蔡勇, 阿依木古丽, 杨具田, 马忠仁, 卢建雄, 臧荣鑫, 吴建平. 绵羊前体脂肪细胞的原代培养及分化. 动物营养学报, 2010, 22(6): 1768-1774.

Cai Y, Ayimuguli, Yang J T, Ma Z R, Lu J X, Zang R X, Wu J P. Primary culture and differentiation of ovine preadipocyte. Chinese Journal of Animal Nutrition, 2010, 22(6): 1768-1774. (in Chinese)

[12]蔡勇, 阿依木古丽, 臧荣鑫, 马忠仁, 乔自林, 卢建雄, 杨具田, 吴建平. 罗格列酮和血清脂对绵羊前体脂肪细胞分化的影响. 中国实验动物学报, 2011, 19(1): 7-12.

Cai Y, Ayimuguli, Zang R X, Ma Z R, Qiao Z L, Lu J X, Yang J T, Wu J P. Effects of rosiglitazone and serum lipid on differentiation of ovine preadipocytes. Acta Laboratorium Animalis Scientia Sinica, 2011, 19(1): 7-12. (in Chinese)

[13]Keros V, Rosenlund B, Hultenby K, Aghajanova L, Levkov L, Hovatta O. Optimizing cryopreservation of human testicular tissue: comparison of protocols with glycerol, propanediol and dimethylsulphoxide as cryoprotectants. Human Reproduction, 2005, 20(6): 1676-1687.

[14]Nishigaki T, Teramura Y, Suemori H, Iwata H. Cryopreservation of primate embryonic stem cells with chemically-defined solution without Me2SO. Cryobiology, 2010, 60(2): 159-164.

[15]Brandebourg T D, Hu C Y. Regulation of differentiating pig preadipocytes by retinoic acid. Journal of Animal Science, 2005, 83(1): 98-107.

[16]Nakamura T, Shiojima S, Hirai Y, Iwama T, Tsuruzoe N, Hirasawa A, Katsuma S, Tsujimoto G. Temporal gene expression changes during adipogenesis in human mesenchymal stem cells. Biochemical and Biophysical Research Communications, 2003, 303(1): 306-312.

[17]Hung S C, Chang CF, Ma H L, Chen T H, Low-Tone H L. Gene expression profiles of early adipogenesis in human mesenchymal stem cells. Gene, 2004, 340(1): 141-150.

[18]Ailhaud G. Early adipocyte differentiation. Biochemical Society Transactions, 1996, 24(2): 400-402.

[19]Kajimura S, Seale P, Tomaru T, Erdjument-Bromage H, Cooper M P, Ruas J L, Chin S, Tempst P, Lazar M A, Spiegelman B M. Regulation of the brown and white fat gene programs through a PRDM16/CtBP transcriptional complex. Genes Development, 2008, 22(10): 1397-1409.

[20]Hauner H, Entenmann G, Wabitsch M, Gaillard D, Ailhaud G, Negrel R, Pfeiffer E F. Promoting effect of glucocorticoids on the differentiation of human adipocyte precursor cells cultured in a chemically defined medium. Journal of Clinical Investigation, 1989, 84(5): 1663-1670.

[21]门正明, 刘霞, 马海明, 韩建林. 兰州大尾羊染色体组型分析. 甘肃农业大学学报, 2002, 37(2): 158-160.

Men Z M, Liu X, Ma H M, Han J L. Karyotype analysis of Lanzhou fat-tailed sheep. Journal of Gansu Agricultural University, 2002, 37(2): 158-160. (in Chinese)

不同冻存保护剂对绵羊前体脂肪细胞冻存效果的影响

Effects of Different Cryoprotectants on Ovine Preadipocytes Cryopreservation

PDF

赞

可视化

被引次数

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 15

Metrics

本文评价

推荐阅读 0

[1]	董复成,马淑丽,时娟娟,张俊梅,崔岩,任有蛇,张春香. LCN5蛋白在雄性绵羊生殖器官及精子中的表达定位[J]. 中国农业科学, 2022, 55(7): 1445-1457.
[2]	冉宏标,赵丽玲,王会,柴志欣,王吉坤,王嘉博,武志娟,钟金城. LncFAM200B对牦牛肌内前体脂肪细胞脂质沉积的影响[J]. 中国农业科学, 2022, 55(13): 2654-2666.
[3]	梁鹏,张天闻,孟科,邵顺成,邹诗凡,荣轩,强浩,冯登侦. 绵羊ADIPOQ多态性与生长性状的关联分析[J]. 中国农业科学, 2022, 55(11): 2239-2256.
[4]	柯娜,郝志云,王建清,甄慧敏,罗玉柱,胡江,刘秀,李少斌,赵志东,黄兆春,梁维炜,王继卿. miR-221靶向IRS1抑制绵羊乳腺上皮细胞活力和增殖[J]. 中国农业科学, 2022, 55(10): 2047-2056.
[5]	李松美,仇雨歌,陈胜男,王晓萌,王春生. CRISPR/Cas9介导的绵羊示踪脐带间充质干细胞系的建立[J]. 中国农业科学, 2021, 54(2): 400-411.
[6]	王继卿,郝志云,沈继源,柯娜,黄兆春,梁维炜,罗玉柱,胡江,刘秀,李少斌. 小尾寒羊泌乳性状重要lncRNAs的筛选、鉴定及功能分析[J]. 中国农业科学, 2021, 54(14): 3113-3123.
[7]	石田培,王欣悦,侯浩宾,赵志达,尚明玉,张莉. 基于全转录组测序的绵羊胚胎不同发育阶段骨骼肌circRNA的分析与鉴定[J]. 中国农业科学, 2020, 53(3): 642-657.
[8]	李讨讨,王霞,马友记,尹德恩,张勇,赵兴绪. 藏绵羊BOLL的分子特征及其在睾丸中的表达调控与功能分析[J]. 中国农业科学, 2020, 53(20): 4297-4312.
[9]	郑玮才,郝小燕,张宏祥,项斌伟,张文佳,张春香,张建新. 饲粮添加酿酒酵母和地衣芽孢杆菌对绵羊生长性能与瘤胃发酵的影响[J]. 中国农业科学, 2020, 53(16): 3385-3393.
[10]	王欣悦,石田培,赵志达,胡文萍,尚明玉,张莉. 基于绵羊胚胎骨骼肌蛋白质组学的PI3K-AKT信号通路分析[J]. 中国农业科学, 2020, 53(14): 2956-5963.
[11]	田志龙,汤继顺,孙庆,王玉琴,张效生,张金龙,储明星. 绵羊SMAD1基因组织表达及其多态性与产羔数关联分析[J]. 中国农业科学, 2019, 52(4): 755-766.
[12]	富丽霞,马涛,刁其玉,成述儒,宋雅喆,孙卓琳. 肉羊精料可代谢蛋白质预测模型的建立[J]. 中国农业科学, 2019, 52(3): 539-549.
[13]	朱江江,林亚秋,王永,林森. 山羊Kruppel样转录因子家族在前体脂肪细胞分化中的表达模式及相关性分析[J]. 中国农业科学, 2019, 52(13): 2341-2351.
[14]	张通,栗瑞兰,范晓梅,刘春洁,海日汗,霍敏,张家新. p66Shc在绵羊卵母细胞中的表达及其与胞质氧化还原稳态的关系[J]. 中国农业科学, 2019, 52(12): 2183-2192.
[15]	周艳, 董利锋, 邓凯东, 许贵善, 刁其玉. 肉用绵羊生长期甲烷排放特点与预测模型的建立[J]. 中国农业科学, 2019, 52(10): 1797-1806.