基于转录组测序分析甜菜夜蛾卵巢细胞离体培养成系进程
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摘要
【目的】本研究旨在分析甜菜夜蛾Spodoptera exigua蛹卵巢细胞建立细胞系的整个过程,探究细胞由体内到体外培养过程中其基因表达在转录水平的变化,为昆虫体外培养模型的建立提供理论基础。【方法】利用Illumina Hiseq测序平台对甜菜夜蛾蛹卵巢细胞离体培养过程中各阶段的细胞分别进行转录组测序,对获得注释的差异表达基因及其相关信号通路进行分析;通过荧光定量PCR对部分细胞周期相关基因(cycd和cdk4)、调控基因(cdc20,apc1,skp2和mad1)、增殖相关分子标志物(mcm4和pcna)在甜菜夜蛾卵巢细胞离体培养过程中的转录进行验证。【结果】甜菜夜蛾蛹卵巢细胞离体培养过程包括5个阶段:解剖获得离体的卵巢组织,卵巢组织贴壁培养后游离出原代细胞,细胞转化重新具备增殖能力,成功首次传代,以及能够连续传代15代以上建立细胞系。上述5个阶段的细胞经转录组测序、数据组装后共获得46 796条unigenes序列,组装得到序列长度完整性好;转录本unigenes序列拼接长度分布合理,样本碱基Q30均在94%以上。通过KEGG数据库获得注释的unigenes有1 473条,参与细胞过程紧密相关的20条信号通路,其中有92条unigenes在细胞周期信号通路中获得注释。聚类分析表明,在体内处于快速发育状态的卵巢细胞与同样处于增殖状态的细胞系基因表达模式非常接近。原代细胞由短暂停滞生长至成簇细胞的转化关键期,筛选到差异表达基因619个,cdk4在离体培养期表达量显著降低,cycd在细胞转化关键期之后表达量显著升高,cdc20,apc1,skp2和mad1在卵巢组织和细胞系的表达量显著高于原代细胞、转化关键期细胞和首次传代细胞的。从原代细胞至传代后,cycd的表达显著升高8.7倍,显著高于mcm4和pcna的变化水平。【结论】甜菜夜蛾卵巢细胞离体培养过程中5个阶段的细胞转录组测序获得的序列质量符合数据分析的基本要求。筛选获得了甜菜夜蛾蛹卵巢细胞经离体培养过程中的差异表达基因。原代细胞逆转增殖可能与cdk4,cycd,skp2和mad1等细胞周期调控基因表达有关。另外,cycd可作为原代细胞具备传代能力的标志物。
【Aim】 The aim of this study is to analyze the whole process of establishing cell lines from Spodoptera exigua pupal ovarian cells, and to explore the changes of gene expression at the transcriptional level from in vivo to in vitro culture, so as to provide a theoretical basis for the establishment of insect in vitro culture models. 【Methods】 In this study, the transcriptomes of various stages of S. exigua pupal ovarian cells during ex vivo culture were sequenced by using second-generation transcriptome sequencing technology Illumina Hiseq, and the differentially expressed genes and their related signaling pathways were analyzed. The expression of some cell cycle-related genes(cycd and cdk4), regulatory genes(cdc20, apc1, skp2 and mad1), and proliferation-related molecular markers(mcm4 and pcna) in different stages of S. exigua pupal ovarian cells during ex vivo culture were verified by real-time PCR. 【Results】 The process of ex vivo culture of S. exigua pupal ovarian cells includes five stages: anatomical obtaining of ex vivo ovarian tissues, primary cells isolated after adherent culture of ovarian tissues, transformed cells with re-proliferation ability, first generation cells capable of continuous passage, and cell lines capable of continuous passage for more than 15 generations. A total of 46 796 unigene sequences were obtained from the five stages of S. exigua pupal ovarian cells during ex vivo culture by RNA sequencing, with good sequence length integrity of the assembly. The splicing length distribution of unigene sequences was reasonable, and the Q30 of the sample base was above 94%. Based on the KEGG database, 1 473 unigenes were annotated to be involved in 20 signaling pathways closely related to cellular processes, and 92 unigenes in the cell cycle signaling pathway. Cluster analysis showed that the gene expression patterns of ovarian cells in a rapidly developing state in vivo were very close to that of cell lines which were also in a proliferative state. In addition, 619 differentially expressed genes were screened at the critical transformation stage of primary cells from transient stagnant growth to clustered cells. The expression of cdk4 significantly decreased ex vivo, and the expression level of cycd significantly increased after the critical stage of cell transformation. The expression levels of Cdc20, apc1, skp2, and mad1 were significantly higher in ovarian tissues and cell lines than in primary cells, key transformed cells, and first passage cells. From primary cells to passage, the expression level of cycd was significantly increased by 8.7-fold, which was significantly higher than those of mcm4 and pcna. 【Conclusion】 The sequence quality of five stages of S. exigua pupal ovarian cells during ex vivo culture by transcriptome sequencing met with the basic requirements for transcriptome data analysis. The differentially expressed genes in S. exigua pupal ovarian cells during ex vivo culture were obtained. The reverse cell proliferation of primary cells may be related to the expression of cell cycle regulatory genes such as cdk4, cycd, skp2 and mad1. In addition, cycd can be used as a marker for the ability of primary cells to pass through.
【Aim】 The aim of this study is to analyze the whole process of establishing cell lines from Spodoptera exigua pupal ovarian cells, and to explore the changes of gene expression at the transcriptional level from in vivo to in vitro culture, so as to provide a theoretical basis for the establishment of insect in vitro culture models. 【Methods】 In this study, the transcriptomes of various stages of S. exigua pupal ovarian cells during ex vivo culture were sequenced by using second-generation transcriptome sequencing technology Illumina Hiseq, and the differentially expressed genes and their related signaling pathways were analyzed. The expression of some cell cycle-related genes(cycd and cdk4), regulatory genes(cdc20, apc1, skp2 and mad1), and proliferation-related molecular markers(mcm4 and pcna) in different stages of S. exigua pupal ovarian cells during ex vivo culture were verified by real-time PCR. 【Results】 The process of ex vivo culture of S. exigua pupal ovarian cells includes five stages: anatomical obtaining of ex vivo ovarian tissues, primary cells isolated after adherent culture of ovarian tissues, transformed cells with re-proliferation ability, first generation cells capable of continuous passage, and cell lines capable of continuous passage for more than 15 generations. A total of 46 796 unigene sequences were obtained from the five stages of S. exigua pupal ovarian cells during ex vivo culture by RNA sequencing, with good sequence length integrity of the assembly. The splicing length distribution of unigene sequences was reasonable, and the Q30 of the sample base was above 94%. Based on the KEGG database, 1 473 unigenes were annotated to be involved in 20 signaling pathways closely related to cellular processes, and 92 unigenes in the cell cycle signaling pathway. Cluster analysis showed that the gene expression patterns of ovarian cells in a rapidly developing state in vivo were very close to that of cell lines which were also in a proliferative state. In addition, 619 differentially expressed genes were screened at the critical transformation stage of primary cells from transient stagnant growth to clustered cells. The expression of cdk4 significantly decreased ex vivo, and the expression level of cycd significantly increased after the critical stage of cell transformation. The expression levels of Cdc20, apc1, skp2, and mad1 were significantly higher in ovarian tissues and cell lines than in primary cells, key transformed cells, and first passage cells. From primary cells to passage, the expression level of cycd was significantly increased by 8.7-fold, which was significantly higher than those of mcm4 and pcna. 【Conclusion】 The sequence quality of five stages of S. exigua pupal ovarian cells during ex vivo culture by transcriptome sequencing met with the basic requirements for transcriptome data analysis. The differentially expressed genes in S. exigua pupal ovarian cells during ex vivo culture were obtained. The reverse cell proliferation of primary cells may be related to the expression of cell cycle regulatory genes such as cdk4, cycd, skp2 and mad1. In addition, cycd can be used as a marker for the ability of primary cells to pass through.
引文
Bashir T, Dorrello NV, Amador V, Guardavaccaro D, Pagano M, 2004. Control of the SCF (Skp2-Cks1) ubiquitin ligase by the APC/C (Cdh1) ubiquitin ligase. Nature, 428(6979): 190-193.
Chen QH, Lin J, Jinno S, Okayama H, 2003. Overexpression of Cdk6-cyclin D3 highly sensitizes cells to physical and chemical transformation. Oncogene, 22(7): 992-1001.
Du Z, Tong X, Ye X, 2013. Cyclin D1 promotes cell cycle progression through enhancing NDR1/2 kinase activity independent of cyclin-dependent kinase 4. J. Biol. Chem., 288(37): 26678-26687.
Fallon AM, 2008. Cytological properties of an Aedes albopictus mosquito cell line infected with Wolbachia strain wAlbB. In Vitro Cell. Dev. Biol. Anim., 44(5/6): 154-161.
Gong H, Zhu W, Zhang JH, Li X, Meng Q, Zhou GL, Wang ML, Wang H, Miao L, Qin QL, Zhang H, 2015. TTAGG-repeat telomeres and characterization of telomerase in the beet armyworm, Spodoptera exigua (Lepidoptera: Noctuidae). Insect Mol. Biol., 24(3): 358-367.
Hammoud ZT, Badve S, Saxena R, Kesler KA, Rieger K, Malkas LH, Hickey RJ, 2007. A novel biomarker for the detection of esophageal adenocarcinoma. J. Thorac. Cardiovasc. Surg., 133(1): 82-87.
Hardwick G, Murray AW, 1995. Mad1p, a phosphoprotein component of the spindle assembly checkpoint in budding yeast. J. Cell Biol., 131(3): 709-720.
Iwabuchi K, 2000. A continuous cell line derived from larval fat bodies of Thysanoplusia intermixta (Lepidoptera: Noctuidae). Appl. Entomol. Zool., 35(2): 245-249.
Izawa D, Pines J, 2015. The mitotic checkpoint complex binds a second CDC20 to inhibit active APC/C. Nature, 517(7536): 631-634.
Jia XP, Ye XQ, Liang LJ, Deng YM, Sun XB, She JM, 2014. Transcriptome characteristics of Paspalum vaginatum analyzed with Illumina sequencing technology. Acta Pratacul. Sin., 23(6): 242-252. [贾新平, 叶晓青, 梁丽建, 邓衍明, 孙晓波, 佘建明, 2014. 基于高通量测序的海滨雀稗转录组学研究. 草业学报, 23(6): 242-252]
Kikuchi J, Kinoshita I, Shimizu Y, Kikuchi E, Takeda K, Aburatani H, Oizumi S, Konishi J, Kaga K, Matsuno Y, Birrer MJ, Nishimura M, Dosaka-Akita H, 2011. Minichromosome maintenance (MCM) protein 4 as a marker for proliferation and its clinical and clinicopathological significance in non-small cell lung cancer. Lung Cancer, 72(2): 229-237.
Lewin B, Cassimeris L, Lingappa VR, Plopper G (translated by Sang JL, Lian ML et al.), 2009. Cell. Science Press, Beijing. 631-678. [卢因, 卡西梅里斯, 林加帕, 普洛泊(桑建利, 连慕兰等译), 2009. 细胞. 北京: 科学出版社. 631-678]
Li M, Shin YH, Hou L, Huang X, Wei Z, Klann E, Zhang P, 2008. The adaptor protein of the anaphase promoting complex Cdh1 is essential in maintaining replicative lifespan and in learning and memory. Nat. Cell Biol., 10(9): 1083-1089.
Liu SC, Klein-Szanto AJ, 2000. Markers of proliferation in normal and leukoplakic oral epithelia. Oral Oncol., 36: 145-151.
Malkas LH, Herbert BS, Abdel-Aziz W, Dobrolecki LE, Liu Y, Agarwal B, Hoelz D, Badve S, Schnaper L, Arnold RJ, Mechref Y, Novotny MV, Loehrer P, Goulet RJ, Hickey RJ, 2006. A cancer-associated PCNA expressed in breast cancer has implications as a potential biomarker. Proc. Natl. Acad. Sci. USA, 103(51): 19472-19477.
Mitsuhashi J, 2002. Invertebrate Tissue Culture Methods (Springer Lab Manual). Springer, Japan. 39.
Mukherjee P, Winter SL, Alexandrow MG, 2010. Cell cycle arrest by transforming growth factor beta1 near G1/S is mediated by acute abrogation of prereplication complex activation involving an Rb-MCM interaction. Mol. Cell Biol., 30(3): 845-856.
Smagghe G, Goodman CL, Stanley D, 2009. Insect cell culture and applications to research and pest management. In Vitro Cell. Dev. Biol. Anim., 45(3(4): 93-105.
Sobel RE, Sadar MD, 2005. Cell lines used in prostate cancer research: a compendium of old and new lines-part 2. J. Urol., 173(2): 360-372.
Wang XC, Tan HL, Chen Z, Meng LZ, Wang WB, Fan SC, 2015. Assembly and characterization of the transcriptome and development of SSR markers in Forsythia suspensa based on RNA-Seq technology. Sci. Sin. Vit., 45(3): 301-310. [王兴春, 谭河林, 陈钊, 孟令芝, 王文斌, 范圣此, 2015. 基于RNA-Seq技术的连翘转录组组装与分析及SSR分子标记的开发. 中国科学: 生命科学, 45(3): 301-310]
Wu CY, Chen YW, Lin CC, Hsu CL, Wang CH, Lo CF, 2012. A new cell line (NTU-SE) from pupal tissues of the beet armyworm, Spodoptera exigua (Lepidoptera: Noctuidae), is highly susceptible to S. exigua multiple nucleopolyhedrovirus (SeMNPV) and Autographa californica MNPV (AcMNPV). J. Invertebr. Pathol., 111(2): 143-151.
Yamashita YM, Nakaseko Y, Samejima I, Kumada K, Yamada H, Michaelson D, Yanagida M, 1996. 20S cyclosome complex formation and proteolytic activity inhibited by the Camp/PKA pathway. Nature, 384(6606): 276-279.
Zhang H, Meng Q, Tang P, Li X, Zhu W, Zhou GL, Shu RH, Zhang JH, Qin QL, 2014. Gene expression pattern of insect fat body cells from in vitro challenge to cell line establishment. In Vitro Cell. Dev. Biol. Anim., 50(10): 952-972.
Zhang H, Zhang YA, Qin QL, Wang YZ, Qu LJ, Li X, Miao L, Yin ZX, Zhang AJ, Wen FY, 2007. Advances in establishment of insect cell lines. Acta Entomol. Sin., 50(8): 834-839. [张寰, 张永安, 秦启联, 王玉珠, 曲良建, 李瑄, 苗麟, 殷珍仙, 张爱君, 温发园, 2007. 昆虫细胞系的培养和建立技术. 昆虫学报, 50(8): 834-839]
Zhang P, Qin WX, 2006. Functions of CDC20 in the cell division cycle. Chem. Life, 26(4): 297-299. [张萍, 覃文新, 2006. CDC20在细胞分裂周期中的作用. 生命的化学, 26(4): 297-299]
Chen QH, Lin J, Jinno S, Okayama H, 2003. Overexpression of Cdk6-cyclin D3 highly sensitizes cells to physical and chemical transformation. Oncogene, 22(7): 992-1001.
Du Z, Tong X, Ye X, 2013. Cyclin D1 promotes cell cycle progression through enhancing NDR1/2 kinase activity independent of cyclin-dependent kinase 4. J. Biol. Chem., 288(37): 26678-26687.
Fallon AM, 2008. Cytological properties of an Aedes albopictus mosquito cell line infected with Wolbachia strain wAlbB. In Vitro Cell. Dev. Biol. Anim., 44(5/6): 154-161.
Gong H, Zhu W, Zhang JH, Li X, Meng Q, Zhou GL, Wang ML, Wang H, Miao L, Qin QL, Zhang H, 2015. TTAGG-repeat telomeres and characterization of telomerase in the beet armyworm, Spodoptera exigua (Lepidoptera: Noctuidae). Insect Mol. Biol., 24(3): 358-367.
Hammoud ZT, Badve S, Saxena R, Kesler KA, Rieger K, Malkas LH, Hickey RJ, 2007. A novel biomarker for the detection of esophageal adenocarcinoma. J. Thorac. Cardiovasc. Surg., 133(1): 82-87.
Hardwick G, Murray AW, 1995. Mad1p, a phosphoprotein component of the spindle assembly checkpoint in budding yeast. J. Cell Biol., 131(3): 709-720.
Iwabuchi K, 2000. A continuous cell line derived from larval fat bodies of Thysanoplusia intermixta (Lepidoptera: Noctuidae). Appl. Entomol. Zool., 35(2): 245-249.
Izawa D, Pines J, 2015. The mitotic checkpoint complex binds a second CDC20 to inhibit active APC/C. Nature, 517(7536): 631-634.
Jia XP, Ye XQ, Liang LJ, Deng YM, Sun XB, She JM, 2014. Transcriptome characteristics of Paspalum vaginatum analyzed with Illumina sequencing technology. Acta Pratacul. Sin., 23(6): 242-252. [贾新平, 叶晓青, 梁丽建, 邓衍明, 孙晓波, 佘建明, 2014. 基于高通量测序的海滨雀稗转录组学研究. 草业学报, 23(6): 242-252]
Kikuchi J, Kinoshita I, Shimizu Y, Kikuchi E, Takeda K, Aburatani H, Oizumi S, Konishi J, Kaga K, Matsuno Y, Birrer MJ, Nishimura M, Dosaka-Akita H, 2011. Minichromosome maintenance (MCM) protein 4 as a marker for proliferation and its clinical and clinicopathological significance in non-small cell lung cancer. Lung Cancer, 72(2): 229-237.
Lewin B, Cassimeris L, Lingappa VR, Plopper G (translated by Sang JL, Lian ML et al.), 2009. Cell. Science Press, Beijing. 631-678. [卢因, 卡西梅里斯, 林加帕, 普洛泊(桑建利, 连慕兰等译), 2009. 细胞. 北京: 科学出版社. 631-678]
Li M, Shin YH, Hou L, Huang X, Wei Z, Klann E, Zhang P, 2008. The adaptor protein of the anaphase promoting complex Cdh1 is essential in maintaining replicative lifespan and in learning and memory. Nat. Cell Biol., 10(9): 1083-1089.
Liu SC, Klein-Szanto AJ, 2000. Markers of proliferation in normal and leukoplakic oral epithelia. Oral Oncol., 36: 145-151.
Malkas LH, Herbert BS, Abdel-Aziz W, Dobrolecki LE, Liu Y, Agarwal B, Hoelz D, Badve S, Schnaper L, Arnold RJ, Mechref Y, Novotny MV, Loehrer P, Goulet RJ, Hickey RJ, 2006. A cancer-associated PCNA expressed in breast cancer has implications as a potential biomarker. Proc. Natl. Acad. Sci. USA, 103(51): 19472-19477.
Mitsuhashi J, 2002. Invertebrate Tissue Culture Methods (Springer Lab Manual). Springer, Japan. 39.
Mukherjee P, Winter SL, Alexandrow MG, 2010. Cell cycle arrest by transforming growth factor beta1 near G1/S is mediated by acute abrogation of prereplication complex activation involving an Rb-MCM interaction. Mol. Cell Biol., 30(3): 845-856.
Smagghe G, Goodman CL, Stanley D, 2009. Insect cell culture and applications to research and pest management. In Vitro Cell. Dev. Biol. Anim., 45(3(4): 93-105.
Sobel RE, Sadar MD, 2005. Cell lines used in prostate cancer research: a compendium of old and new lines-part 2. J. Urol., 173(2): 360-372.
Wang XC, Tan HL, Chen Z, Meng LZ, Wang WB, Fan SC, 2015. Assembly and characterization of the transcriptome and development of SSR markers in Forsythia suspensa based on RNA-Seq technology. Sci. Sin. Vit., 45(3): 301-310. [王兴春, 谭河林, 陈钊, 孟令芝, 王文斌, 范圣此, 2015. 基于RNA-Seq技术的连翘转录组组装与分析及SSR分子标记的开发. 中国科学: 生命科学, 45(3): 301-310]
Wu CY, Chen YW, Lin CC, Hsu CL, Wang CH, Lo CF, 2012. A new cell line (NTU-SE) from pupal tissues of the beet armyworm, Spodoptera exigua (Lepidoptera: Noctuidae), is highly susceptible to S. exigua multiple nucleopolyhedrovirus (SeMNPV) and Autographa californica MNPV (AcMNPV). J. Invertebr. Pathol., 111(2): 143-151.
Yamashita YM, Nakaseko Y, Samejima I, Kumada K, Yamada H, Michaelson D, Yanagida M, 1996. 20S cyclosome complex formation and proteolytic activity inhibited by the Camp/PKA pathway. Nature, 384(6606): 276-279.
Zhang H, Meng Q, Tang P, Li X, Zhu W, Zhou GL, Shu RH, Zhang JH, Qin QL, 2014. Gene expression pattern of insect fat body cells from in vitro challenge to cell line establishment. In Vitro Cell. Dev. Biol. Anim., 50(10): 952-972.
Zhang H, Zhang YA, Qin QL, Wang YZ, Qu LJ, Li X, Miao L, Yin ZX, Zhang AJ, Wen FY, 2007. Advances in establishment of insect cell lines. Acta Entomol. Sin., 50(8): 834-839. [张寰, 张永安, 秦启联, 王玉珠, 曲良建, 李瑄, 苗麟, 殷珍仙, 张爱君, 温发园, 2007. 昆虫细胞系的培养和建立技术. 昆虫学报, 50(8): 834-839]
Zhang P, Qin WX, 2006. Functions of CDC20 in the cell division cycle. Chem. Life, 26(4): 297-299. [张萍, 覃文新, 2006. CDC20在细胞分裂周期中的作用. 生命的化学, 26(4): 297-299]