تاثیر سرم جنین گاوی بر رشد سلول‌های خونی لاروهایHelicoverpa armigera، Anagasta kuehniella و Arge ochropus در محیط کشت سلولی400 Ex-cell

نوع مقاله: مقاله پژوهشی

نویسندگان

1 گروه گیاه‌پزشکی، دانشگاه گیلان، رشت، ایران

2 گروه ژنتیک و زیست‌شناسی ملکولی، دانشگاه علوم پزشکی اصفهان، ایران

10.22124/iprj.2019.3769

چکیده

استفاده از رده­های سلولی حشرات برای تولید آفت­کش­های زیستی، پروتئین­های نوترکیب یا تحویل ژن، در حال گسترش است. لذا تولید رده­های سلولی جدید به غنی­تر کردن این فن­آوری کمک می­کند، بنابراین هدف از تحقیق حاضر، تهیه چندین کشت اولیه از سلول­های خونی لاروهای کرم غوزه پنبه Helicoverpa armigera (Hubner)، شب­پره مدیترانه­ای آرد Anagasta kuehniella (Zeller) و زنبور برگ­خوار رز Arge ochropus (Gmelin) می­باشد. سلول­های خونی در محیط کشت EX-cell 400 حاوی غلظت­های مختلف 0، 5، 10، 15 و 20 درصد سرم جنینی گاوی در دمای 28 درجه سلسیوس نگه­داری شدند. غلظت­های سرم جنینی بر اساس پژوهش­های پیشین کشت سلولی حشرات انتخاب شدند. نتایج نشان داد که زمانی که سرم جنینی گاوی از محیط کشت حذف شد، هیچ رشد سلولی مشاهده نشد. بیشینه رشد سلول­های خونی لارو کرم غوزه در محیط کشت حاوی 15 درصد سرم جنینی گاوی مشاهده شد. با افزایش سرم جنینی گاوی به 20 درصد، افزایشی در رشد سلولی مشاهده نشد. بیشینه رشد سلول­های خونی لارو شب­پره مدیترانه­ای آرد در محیط کشت با 10 درصد سرم جنینی گاوی مشاهده شد. رشد این سلول­ها در سرم جنینی گاوی 15 و 20 درصد به­طور معنی­داری کاهش یافت. با افزایش غلظت سرم جنینی گاوی، رشد سلول­های خونی A. ochropus نیز افزایش یافت. در نتیجه، پژوهش حاضر، حداقل غلظت سرم جنینی گاوی مورد نیاز برای بیشینه رشد سلول­های حشرات را در محیط کشت سلولی نشان داد.

کلیدواژه‌ها


عنوان مقاله [English]

Effect of fetal bovine serum on the hemocytes growth of Helicoverpa armigera, Anagasta kuehniella and Arge ochropus in Ex-cell 400 culture

نویسندگان [English]

  • B. Valizadeh 1
  • J. Jalali Sendi 1
  • R. Salehi 2
1 Department of Plant Protection, Faculty of Agricultural Sciences, University of Guilan, Rasht, Iran,
2 Department of Genetics and Molecular Biology, Medical University of Isfahan, Isfahan, Iran
چکیده [English]

The use of insect cell lines for producing bio-pesticides, recombinant protein expression or gene delivery is increasing. Hence, the establishment of new cell lines will help to enrich this technology. Therefore, the purpose of this research is preparation of several primary cultures initiated from hemocytes of Helicoverpa armigera (Hubner),Anagasta kuehniella (Zeller) and Arge ochropus (Gmelin). The cultures were incubated in EX-cell 400 medium supplemented with 0, 5, 10, 15 and 20% concentrations of fetal bovine serum (FBS) at 28ºC. The concentrations of FBS were chosen on the basis of previous research. Results showed no growth in any of the cell lines when FBS was omitted from the medium. Maximal growth of H. armigerahemocytes was obtained with 15% FBS. There was no further increase in growth as FBS concentration was increased to 20%, Maximal growth of A. kuehniella hemocytes was obtained at 10% FBS. The growth of these cells was significantly lower at 15 and 20% FBS. As the fetal bovine serum concentration increased, A. ochropus hemocytes growth increased.  In conclusion, the current investigation demonstrated the lowest concentration of FBS required for maximal growth of insect cells in vitro.

کلیدواژه‌ها [English]

  • Arge ochropus
  • Anagasta kuehniella
  • Helicoverpa armigera
  • Fetal bovine serum
  • Insect cell culture
Aghili, Z. and Zarkesh, H. 2016. Investigating the effects of fetal bovine serum on morphology, cell viability and production of recombinant human growth hormone in comparison to serum-free medium in CHO cells. New Cellular and Molecular Biotechnology Journal 6(24): 65-71. (In Farsi)

Arif, B. and Pavlik, L. 2012. Insect cell culture: Virus replication and applications in biotechnology. Journal of Invertebrate Pathology 112: 136-141.

Baghban, A., Jalali Sendi, J. and Zibaee, A. 2015. The effect of zinc (Zn2+) on cellular immune system of American cotton bollworm, Helicoverpa armigera Hübner (Lep.: Noctuidae). Plant Pest Research 5(3): 71-84. (In Farsi)

Chaeychomsri, S. Chaeychomsri W. Ikeda M. and Kobayashi M. 2017. Susceptibility of a Cloned Cell Line from Helicoverpa armigera to Homologous Nucleopolyhedrovirus. Journal of Advanced Agricultural Technologies 4: 3.

Davis, J. M. 1994. Basic cell culture A Practical Approach. Oxford University Press.

Drugmand, J. C., Schneider Y. J. and Agathos, S. N. 2012. Insect cells as factories for biomanufacturing. Biotechnology Advances 30: 1140–1157.

Evans, C. J., Liu, T. and Banerjee, U. 2014. Drosophila hematopoiesis: Markers and methods for molecular genetic analysis. Methods 68: 242-251.

Fan, J. 2016. Hemocytometer cell count and trypan blue cell viability (4th ed). Bowdish Lab, McMaster University Hamilton, ON, Canada.

Ferruzza, S., Rossi, C., Sambuy, Y. and Scarino, M. L. 2013. Serum reduced and serum free media for differentiation of Caco2 cells. Altext 30:159-168.

Ghasemi, V., Moharramipour, S. and Sendi, J. J. 2013. Circulating hemocytes of Mediterranean flour moth, Ephestia kuehniella Zell. (Lep.: Pyralidae) and their response to thermal stress. Invertebrate Survival Journal 10: 128-140.

Gómez, D. L. M., Belaich, M. N., Rodríguez, V. A. and Ghiringhelli, P. D. 2018. Effects of fetal bovine serum deprivation cultures on the production of Anticarsia gemmatalis multinucleopolyhedrovirus. Contributions on Biotechnology 10: 68.

Harrison, R. L., Herniou, E. A., Jehle, J. A., Theilmann, D. A., Burand, J. P., Becnel, J. J., Krell, P. J., van Oers, M. M., Mowery, J. D., Bauchan, G. R. and Lefkowitz, E. J. 2018. ICTV virus taxonomy profile: Baculoviridae. Journal of General Virology 99(9): 1185-1186.

He, F., Yang, Y., Xiao, Y., Peng, R., Yao, H., Li, X., Peng, J., Hong, H., Liu, K. and Li,J. 2015. Establishment and characterization of a novel cell line from midgut tissue of Helicoverpa armigera (Lepidoptera: Noctuidae). In Vitro Cellular and Developmental Biology - Animal 51(6): 562-571.

Khosravi, R., Sendi, J. J., Brayner, F. A., Alves, L. C. and Feitosa, A. P. S. 2015. Hemocytes of the Rose Sawfly Arge ochropus (Gmelin) (Hymenoptera: Argidae).Neotropical Entomology 45(1): 58-65.

Lemaitre, B. and Hoffmann, J. A. 2007. The host defense of Drosophila melanogaster. Annual Review of Immunology 25: 697-743.

Liu, C. H. and Chang, T. Y. 2006. Rational development of serum free medium for Chinese hamster ovary cells. Process Biochemistry 41: 2314–2319.

Lua, L. H. L., Pedrini, M. R. S., Reid, S., Robertson, A. and Tribe, D. E. 2002. Phenotypic and genotypic analysis of Helicoverpa armigera nucleopolyhedrovirus serially passaged in cell culture. Journal of General Virology 83: 945–955.

Lynn, D., Stephen, E. and Ferkovich, M. 2004. New cell lines from Ephestia kuehniella: characterization and susceptibility to baculoviruses. Journal of Insect Science 4(9): 1-5.

Mangalika, P. R., Kawamoto, T., Takahashi-Nakaquchi, A. and Iwabuchi, K. 2010. Characterization of cell clusters in larval hemolymph of the cabbage armyworm Mamestra brassicae and their role in maintenance of hemocyte populations. Journal of Insect Physiology 56(3): 314-323.

Matindoost, L., Jalali Sendi, J., Soleiman-Jahi, H., Etebari K. and Rahbarizade, F. 2008. In vitro establishment of embryonic primary cultures of silkworm, Bombyx mori (Lep.: Bombycidae). Journal of Entomological Society of Iran 38(2): 173-186. (In Farsi)

Maranga, L., Coroadinha, A. S. and Carrondo, M. J. T. 2002. Insect cell culture medium supplementation with fetal bovine serum and bovine serum albumin: Effects on Baculovirus adsorption and infection kinetics. Biotechnology Progress 18: 855−861.

Merten, O. W., Kierulff, J. V., Castignolles, N. and Perrin, P. 1994. Evaluation of the new serum free medium (MDSS2) for the production of different biological: use of various cell lines. Cytotechnology 14: 47-59.

Merten, O. W., Kallel, H. and Manuguerra, J. C. 1999. The new medium MDSS2N, free of any animal protein supports cell growth and production of various viruses. Cytotechnology 30: 191-201.

Mitsuhashi, J. 2002. Invertebrate tissue culture methods. Springer-Verlag, Tokyo, p. 446.

Mitsuhashi, J. and Shozawa, A. 1985. Continuous cell lines from larval hemocytes of the cabbage armyworm, Mamestra brassicae. Development, Growth and Differentiation 27(5): 599-606.

Pedrini, M. R. S., Reid, S., Nielsen, L. and Chan, L. C. L. 2011. Kinetic characterization of the group II Helicoverpa armigera nucleopolyhedrovirus propagated in suspension cell cultures: implications for development of a biopesticides production process. Biotechnological Progress 27: 614–624.

Ranga Rao, C., Kumar, S., Sireesha, K. and Lava, P. 2015. Role of nucleopolyhedroviruses (NPVs) in the management of lepidopteran pests. In Asia G. V. (Eds). International Publishing Switzerland, Switzerland, Europe. pp. 43.

Reid, S., Chan, L. C. L. and Van Oers, M. M. 2014. Production of entomopathoginic viruses. In: Morales-Ramos, J. A., Rojas, M. G. and Shapiro-Ilan D. I. (Eds.). Mass production of beneficial organisms invertebrates and entomopathogens. Elsevier, Amsterdam, Netherland, Europe. ISBN 978-0-12-391453-8.

Rezaei, M., Zarkesh Esfahani, S. H. and Gharagozloo, M. 2013. The effect of different media composition and temperatures on the production of recombinant human growth hormone by CHO cells. Research in Pharmaceutical Sciences 8: 211-217.

Shorey, H. and Hale, R. 1965. Mass-rearing of the larvae of nine noctuid species on a simple artificial medium. Journal of Economic Entomology 58: 522–524.

Sohi, S. S. and Smith, C. 1970. Effect of fetal bovine serum on the growth and survival of insect cell cultures. Canadian Journal of Zoology 48: 427-432.

Stanley, D. and Kim, Y. 2014. Eicosanoid signaling in insects: from discovery to plant protection. Critical Reviews in Plant Sciences 33: 1. 342-349.

Van der Valk, J., Brunner, D., De Smet, K., Fex Svenningsen, A., Honegger, P., Knudsen, L. E., Lindl, T., Noraberg, J., Price, A., Scarino, M. L. and Gstraunthaler, G. 2010. Optimization of chemically defined cell culture media replacing fetal bovine serum in mammalian in vitro methods. Toxicology in Vitro 24:1053-1063.

Wagner, G. R. and Hirschey, M. D. 2014. Nonenzymatic protein acylation as a carbon stress regulated by sirtuin deacylases. Molecola Cell 54(1): 5-16.

Wen, F., Caputo, G. and Hooey, S. h. 2015. Establishment of a cell line from the ash and privet borer beetle Tylonotus bimaculatus Haldeman and assessment of its sensitivity to diacylhydrazine insecticides. In Vitro Cellular and Developmental Biology Animal 42: 683–707.

Yasunaga-Aoki, G., Imanishi, S., Iiyama K. and Kawarabata, T. 2004. Establishment of phagocytic cell lines from larval hemocytes of the beet armyworm, Spodoptera exigua. In Vitro Cellular and Developmental Biology- Animal 40: 183-186.

Yazdanian, M. 2000. Evaluating the amount of growth and fecundity of mill moth, Ephestia kuehniella Zeller on different media prepared from flour. M. Sc. Thesis, University of Tabriz, 125 pp. (In Farsi)

Yoon, S. K. and Hong, J. K. 2006. Adaptation of Chinese hamster ovary cells to low culture temperature: cell growth and recombinant protein production. Journal of Biotechnology 122: 463-472.

Yuanyuan, M., Wu, W., Chen, H., Liu, Q., Jia, D., Mao, Q., Chen, Q., Wu, Z. and Taiyun, W. 2013. An insect cell line derived from the small brown plant hopper supports replication of rice stripe virus, a tenuivirus. Journal of General Virology 94: 1421–1425.