斑节对虾性腺组织、细胞的原代培养条件优化
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摘要
通过观察不同成分培养基及不同培养方法培养的斑节对虾性腺组织及细胞原代培养生长情况,分析细胞活力及生长相关基因表达情况,判断3种改良培养基的适用条件,为进一步细胞培养试验提供支持。试验结果表明,以L-15培养基为基础培养基,不同配方的培养效果差距较大,其中机械分离法可成功培养离体斑节对虾性腺组织,而酶解法与3号培养基结合效果较好;在培养过程中可见离体卵母细胞多呈悬浮生长,部分可见分裂相,离体精原细胞多呈贴壁生长,1号、2号培养基中的细胞可维持良好生长状态达一周以上;CCK测试结果表明,机械分离的性腺组织和酶解法分离的性腺细胞分别在1号、3号培养基中有更高的细胞活力,而2号培养基中的性腺组织及细胞在两种培养方法下的活力差别不大。进一步的荧光定量PCR检测了离体培养过程中MAPK信号通路相关基因的表达变化情况,结果表明,Ras、c-Mos、MEK1、ERK基因在卵母细胞中表达变化更显著,且c-Mos基因与离体细胞生长发育情况呈正相关。本试验系统性地筛选了适宜斑节对虾性腺组织、细胞的原代培养方法,为了解培养过程中的生理过程变化提供了相关参考数据。
The growth, cell activity and related-genes expression level were investigated in gonad tissues separated by mechanics and enzymes and primary cell culture of tiger shrimp Penaeus monodon cultured in three improved medium using CCK method and qPCR to determine the suitable conditions of three kinds of improved culture media and to provide support for further cell culture test. The results showed that the gonad tissues of the tiger shrimp were successfully cultured in the media based on L-15 by the mechanical separation method, with different effects. The gonadal tissue of tiger shrimp was successfully cultured by mechanical separation method and in the third type of medium by the enzymatic hydrolysis method in vitro. The growth of oocytes in suspension was mainly observed, some in the division phase, and the spermatogonia were growth as adherent cells in the first and second types of media at a good growth state for more than a week. However, the CCK counting test revealed that there were higher cell activities in the first and third types of media by the two methods compared with the second one. The changes in MAPK signaling pathway were also observed by q PCR, indicating that the key factors of MAPK signal pathway(Ras, c-Mos, MEK1, ERK) in oocyte were significantly higher than those in spermatogonium cells, especially, the c-Mos gene being positively correlated with the growth and development of the cells in vitro. The findings provide relative reference for understanding of the physiological process changes in the culture process.
The growth, cell activity and related-genes expression level were investigated in gonad tissues separated by mechanics and enzymes and primary cell culture of tiger shrimp Penaeus monodon cultured in three improved medium using CCK method and qPCR to determine the suitable conditions of three kinds of improved culture media and to provide support for further cell culture test. The results showed that the gonad tissues of the tiger shrimp were successfully cultured in the media based on L-15 by the mechanical separation method, with different effects. The gonadal tissue of tiger shrimp was successfully cultured by mechanical separation method and in the third type of medium by the enzymatic hydrolysis method in vitro. The growth of oocytes in suspension was mainly observed, some in the division phase, and the spermatogonia were growth as adherent cells in the first and second types of media at a good growth state for more than a week. However, the CCK counting test revealed that there were higher cell activities in the first and third types of media by the two methods compared with the second one. The changes in MAPK signaling pathway were also observed by q PCR, indicating that the key factors of MAPK signal pathway(Ras, c-Mos, MEK1, ERK) in oocyte were significantly higher than those in spermatogonium cells, especially, the c-Mos gene being positively correlated with the growth and development of the cells in vitro. The findings provide relative reference for understanding of the physiological process changes in the culture process.
引文
[1] Castaňon-Cervantes O, Lugo C, Aguilar M, et al. Photoperiodic induction on the growth rate and gonads maturation in the crayfish Procambarus clarkii during ontogeny [J]. Comparative Biochemistry & Physiology Part A Physiology,1995,110(2):139-146.
[2] Leelatanawit R, Sittikankeaw K, Yocawibun P, et al. Identification, characterization and expression of sex-related genes in testes of the giant tiger shrimp Penaeus monodon [J]. Comparative Biochemistry & Physiology Part A,2008,152(1):66-76.
[3] Zhao C, Fu M, Jiang S, et al. Molecular cloning and expression analysis of cyclin H from black tiger shrimp (Penaeus monodon) [J]. The Israeli Journal of Aquaculture - Bamidgeh,2015(67):1195.
[4] Jiang S, Qiu L, Zhou F, et al. Molecular cloning and expression analysis of a heat shock protein (Hsp90) gene from black tiger shrimp (Penaeus monodon)[J]. Molecular Biology Reports,2009,36(1):127-134.
[5] Jose S, Mohandas A, Philip E A. Primary hemocyte culture of Penaeus monodon as an in vitro model for white spot syndrome virus titration, viral and immune related gene expression and cytotoxicity assays [J]. Journal of Invertebrate Pathology,2010,105(3):312-321.
[6] Liu Q, Xu D, Jiang S, et al. Toll-receptor 9 gene in the black tiger shrimp (Penaeus monodon) induced the activation of the TLR-NF-κB signaling pathway [J]. Gene,2017(639):27-33.
[7] Chen S N, Chi S C, Kou G H, et al. Cell culture from tissues of grass prawn, Penaeus monodon [J]. Fish Pathology,1986,21(3):161-166.
[8] 胡珂,王立平,段爱梅.中国对虾的组织培养[J].水产学报,1991,15(4):328-331.
[9] Luedeman R A, Lightner D V. Development of an in vitro primary cell culture system from the penaeid shrimp, Penaeus stylirostris and Penaeus vannamei [J]. Aquaculture,1992,101(3/4):205-211.
[10] Hsu Y L, Yang Y H, Chen Y C, et al. Development of an in vitro subculture system for the oka organ (lymphoid tissue) of Penaeus monodon[J]. Aquaculture,1995,136(1/2):43-55.
[11] 王立平,宋晓玲,刘镁,等.中国对虾上皮样细胞培养研究[J].海洋水产研究,1997,18(1):16-20.
[12] 李国鹏.MAPK在斑马鱼卵母细胞第一次减数分裂恢复过程中的生理功能[D].上海:上海海洋大学,2011.
[13] Jose S, Jayesh P, Sudheer N S, et al. Lymphoid organ cell culture system from Penaeus monodon (Fabricius) as a platform for white spot syndrome virus and shrimp immune-related gene expression [J]. Journal of Fish Diseases,2012,35(5):321-334.
[14] 韩萍,杨丽诗,吴松,等.促性腺激素释放激素及多巴胺拮抗物地欧酮对斑节对虾卵巢组织发育的影响[J].南方水产科学,2015,11(2):50-56.
[15] 杨其彬,邱丽华,黄建华,等.切除单侧眼柄对不同体质量养殖斑节对虾卵巢发育及产卵的影响[J].水产科学,2016,35(5):516-521.
[16] 刘凯于,杨凯,余泽华,等.斑节对虾组织的原代培养[J].华中师范大学学报:自然科学版,1998,32(2):90-94.
[17] 高玮,黄灿华,兰萍章,等.斑节对虾肝胰腺和血淋巴组织细胞的体外培养[J].中山大学学报:自然科学版,2000,39(增刊):119-122.
[18] 黄建华,杨其彬,马之明,等.池塘养殖斑节对虾的生长、发育与性成熟[J].水产学报,2013,37(3):397-406.
[19] 黄建华,周发林,马之明,等.南海北部斑节对虾卵巢发育的形态及组织学观察[J].热带海洋学报,2006,25(3):47-52.
[20] 鄂征.组织培养和分子细胞学技术[M].北京:科学出版社,2001.
[21] 姜国建,樊廷俊,杨秀霞,等.对虾细胞培养现状与对策[J].水产科学,2009,28(1):47-49.
[22] 张岩,段虎,金松君,等.红螯螯虾XO-SG神经细胞的原代培养[J].水产学报,2016,40(10):1606-1612.
[23] Owens L, Smith J. Early attempts at production of prawn cell lines [J]. Methods in Cell Science,1999,21(4):207-212.
[24] 樊廷俊,汪小峰,史振平,等.碱性成纤维细胞生长因子与胰岛素样生长因子Ⅱ对中国对虾淋巴细胞培养物的协同诱导作用[J].海洋学报:中文版,2001,23(5):141-145.
[25] Crane M S J. Mutagensis and cell transformation in cell culture [J]. Methods in Cell Science,1999,21(4):245-253.
[26] Zhang W, Liu H T. MAPK signal pathways in the regulation of cell proliferation in mammalian cells [J]. Cell Res,2002,12(1):9-18.
[27] Yang L, Liu X, Huang J, et al. Molecular characterization and expression profile of MAP2K1ip1/MP1 gene from tiger shrimp, Penaeus monodon [J]. Molecular Biology Reports,2012,39(5):5811-5818.
[28] Mutter G L, Grills G S, Wolgemuth D J, et al. Evidence for the involvement of the proto-oncogene c-mos in mammalian meiotic maturation and possibly very early embryogenesis [J]. The EMBO Journal,1988,7(3):683-689.
[29] Newman B, Dai Y. Transcription of c-mos protooncogene in the pig involves both tissue-specific promoters and alternative polyadenylation sites [J]. Molecular Reproduction & Development, 1996, 44(3):275-288.
[30] 曹少锋. 精子发生/成熟和受精相关分子c-mos及ZP3-ppb的研究[D].上海:上海交通大学,2007.
[2] Leelatanawit R, Sittikankeaw K, Yocawibun P, et al. Identification, characterization and expression of sex-related genes in testes of the giant tiger shrimp Penaeus monodon [J]. Comparative Biochemistry & Physiology Part A,2008,152(1):66-76.
[3] Zhao C, Fu M, Jiang S, et al. Molecular cloning and expression analysis of cyclin H from black tiger shrimp (Penaeus monodon) [J]. The Israeli Journal of Aquaculture - Bamidgeh,2015(67):1195.
[4] Jiang S, Qiu L, Zhou F, et al. Molecular cloning and expression analysis of a heat shock protein (Hsp90) gene from black tiger shrimp (Penaeus monodon)[J]. Molecular Biology Reports,2009,36(1):127-134.
[5] Jose S, Mohandas A, Philip E A. Primary hemocyte culture of Penaeus monodon as an in vitro model for white spot syndrome virus titration, viral and immune related gene expression and cytotoxicity assays [J]. Journal of Invertebrate Pathology,2010,105(3):312-321.
[6] Liu Q, Xu D, Jiang S, et al. Toll-receptor 9 gene in the black tiger shrimp (Penaeus monodon) induced the activation of the TLR-NF-κB signaling pathway [J]. Gene,2017(639):27-33.
[7] Chen S N, Chi S C, Kou G H, et al. Cell culture from tissues of grass prawn, Penaeus monodon [J]. Fish Pathology,1986,21(3):161-166.
[8] 胡珂,王立平,段爱梅.中国对虾的组织培养[J].水产学报,1991,15(4):328-331.
[9] Luedeman R A, Lightner D V. Development of an in vitro primary cell culture system from the penaeid shrimp, Penaeus stylirostris and Penaeus vannamei [J]. Aquaculture,1992,101(3/4):205-211.
[10] Hsu Y L, Yang Y H, Chen Y C, et al. Development of an in vitro subculture system for the oka organ (lymphoid tissue) of Penaeus monodon[J]. Aquaculture,1995,136(1/2):43-55.
[11] 王立平,宋晓玲,刘镁,等.中国对虾上皮样细胞培养研究[J].海洋水产研究,1997,18(1):16-20.
[12] 李国鹏.MAPK在斑马鱼卵母细胞第一次减数分裂恢复过程中的生理功能[D].上海:上海海洋大学,2011.
[13] Jose S, Jayesh P, Sudheer N S, et al. Lymphoid organ cell culture system from Penaeus monodon (Fabricius) as a platform for white spot syndrome virus and shrimp immune-related gene expression [J]. Journal of Fish Diseases,2012,35(5):321-334.
[14] 韩萍,杨丽诗,吴松,等.促性腺激素释放激素及多巴胺拮抗物地欧酮对斑节对虾卵巢组织发育的影响[J].南方水产科学,2015,11(2):50-56.
[15] 杨其彬,邱丽华,黄建华,等.切除单侧眼柄对不同体质量养殖斑节对虾卵巢发育及产卵的影响[J].水产科学,2016,35(5):516-521.
[16] 刘凯于,杨凯,余泽华,等.斑节对虾组织的原代培养[J].华中师范大学学报:自然科学版,1998,32(2):90-94.
[17] 高玮,黄灿华,兰萍章,等.斑节对虾肝胰腺和血淋巴组织细胞的体外培养[J].中山大学学报:自然科学版,2000,39(增刊):119-122.
[18] 黄建华,杨其彬,马之明,等.池塘养殖斑节对虾的生长、发育与性成熟[J].水产学报,2013,37(3):397-406.
[19] 黄建华,周发林,马之明,等.南海北部斑节对虾卵巢发育的形态及组织学观察[J].热带海洋学报,2006,25(3):47-52.
[20] 鄂征.组织培养和分子细胞学技术[M].北京:科学出版社,2001.
[21] 姜国建,樊廷俊,杨秀霞,等.对虾细胞培养现状与对策[J].水产科学,2009,28(1):47-49.
[22] 张岩,段虎,金松君,等.红螯螯虾XO-SG神经细胞的原代培养[J].水产学报,2016,40(10):1606-1612.
[23] Owens L, Smith J. Early attempts at production of prawn cell lines [J]. Methods in Cell Science,1999,21(4):207-212.
[24] 樊廷俊,汪小峰,史振平,等.碱性成纤维细胞生长因子与胰岛素样生长因子Ⅱ对中国对虾淋巴细胞培养物的协同诱导作用[J].海洋学报:中文版,2001,23(5):141-145.
[25] Crane M S J. Mutagensis and cell transformation in cell culture [J]. Methods in Cell Science,1999,21(4):245-253.
[26] Zhang W, Liu H T. MAPK signal pathways in the regulation of cell proliferation in mammalian cells [J]. Cell Res,2002,12(1):9-18.
[27] Yang L, Liu X, Huang J, et al. Molecular characterization and expression profile of MAP2K1ip1/MP1 gene from tiger shrimp, Penaeus monodon [J]. Molecular Biology Reports,2012,39(5):5811-5818.
[28] Mutter G L, Grills G S, Wolgemuth D J, et al. Evidence for the involvement of the proto-oncogene c-mos in mammalian meiotic maturation and possibly very early embryogenesis [J]. The EMBO Journal,1988,7(3):683-689.
[29] Newman B, Dai Y. Transcription of c-mos protooncogene in the pig involves both tissue-specific promoters and alternative polyadenylation sites [J]. Molecular Reproduction & Development, 1996, 44(3):275-288.
[30] 曹少锋. 精子发生/成熟和受精相关分子c-mos及ZP3-ppb的研究[D].上海:上海交通大学,2007.