王浆主蛋白作为胎牛血清替代物培养中国仓鼠卵巢细胞CHO-K1的效果及作用机制
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摘要
用王浆主蛋白(major royal jelly proteins,MRJPs)部分代替胎牛血清(fetal bovine serum,FBS)培养中国仓鼠卵巢细胞(Chinese hamster ovary cell,CHO-K1),比较添加不同MRJPs/FBS(M/F)比例的培养基对细胞相对增殖率的影响。四唑盐比色法(methyl thiazolyl tetrazolium,MTT)结果显示:当M/F比例为50/50时对细胞的促增殖作用最显著;与完全培养基对照组(M/F 0/100)相比,培养48与72 h时的相对活细胞数分别上升了25.1%和13.6%。细胞成像仪分析表明,M/F 50/50培养基的细胞密度最高,其细胞直径显著大于完全培养基对照组。流式细胞仪结果显示,与完全培养基对照组相比,M/F 50/50培养基中的细胞增殖指数(proliferation index,PI)提高了19.4%,推测MRJPs的促细胞增殖作用可能与DNA、蛋白质及酶的合成有关。拉曼光谱检测结果显示,各处理组培养基的细胞光谱图之间没有显著差异,说明MRJPs在促进细胞增殖的同时维持了细胞的稳定性。以上结果证明,MRJPs可以部分替代FBS培养生物医药产业中应用广泛的CHO-K1细胞,降低细胞培养成本,具有产业化前景,并为蜂王浆深加工提供新的用途。
The major royal jelly proteins(MRJPs) as the alternative of fetal bovine serum(FBS) were used to culture Chinese hamster ovary cell line(CHO-K1). The effects of mixture media with different MRJPs/FBS(M/F) proportions on the proliferation of CHO-K1 cell were compared. The result of methyl thiazolyl tetrazolium(MTT) assay showed that the cells in the medium with M/F 50/50 possessed the highest relative proliferation activity. Compared to the CK(M/F 0/100), the relative cell proliferation activity in the medium with M/F 50/50 was increased by 25.1%(48 h) and 13.6%(72 h), respectively. Cell imager showed that the cell density and diameter in the medium with M/F 50/50 were significantly higher than those in the CK. The detection of cell cycle with flow cytometry showed that the cell proliferation index(PI) in the medium with M/F 50/50 was increased by19.4% compared to that in the CK, indicating that MRJPs might promote the synthesis of DNA, proteins and enzymes. The Raman spectrum of cells in the medium with M/F 50/50 was similar with those in the CK and bovine serum albumin(BSA) medium, demonstrating that the complex serum maintains the cell type stable. In conclusion, the results suggest that MRJPs could partially replace FBS in the cultivation of CHO-K1 cell line and possess industry prospect.
The major royal jelly proteins(MRJPs) as the alternative of fetal bovine serum(FBS) were used to culture Chinese hamster ovary cell line(CHO-K1). The effects of mixture media with different MRJPs/FBS(M/F) proportions on the proliferation of CHO-K1 cell were compared. The result of methyl thiazolyl tetrazolium(MTT) assay showed that the cells in the medium with M/F 50/50 possessed the highest relative proliferation activity. Compared to the CK(M/F 0/100), the relative cell proliferation activity in the medium with M/F 50/50 was increased by 25.1%(48 h) and 13.6%(72 h), respectively. Cell imager showed that the cell density and diameter in the medium with M/F 50/50 were significantly higher than those in the CK. The detection of cell cycle with flow cytometry showed that the cell proliferation index(PI) in the medium with M/F 50/50 was increased by19.4% compared to that in the CK, indicating that MRJPs might promote the synthesis of DNA, proteins and enzymes. The Raman spectrum of cells in the medium with M/F 50/50 was similar with those in the CK and bovine serum albumin(BSA) medium, demonstrating that the complex serum maintains the cell type stable. In conclusion, the results suggest that MRJPs could partially replace FBS in the cultivation of CHO-K1 cell line and possess industry prospect.
引文
[1]BUTTSTEDT A,MORITZ R F A,ERLER S.Origin and function of the major royal jelly proteins of the honeybee(Apis mellifera)as members of the yellow gene family.Biological Reviews,2014,89(2):255-269.
[2]SHEN L R,ZHANG W,JIN F,et al.Expression of recombinant Acc MRJP1 protein from royal jelly of Chinese honeybee in Pichia pastoris and its proliferation activity in an insect cell line.Journal of Agricultural and Food Chemistry,2010,58(16):9190-9197.
[3]KAMAKURA M.Royalactin induces queen differentiation in honeybees.Nature,2011,473:478-483.
[4]NAGAI T,INOUE R.Preparation and the functional properties of water extract and alkaline extract of royal jelly.Food Chemistry,2004,84(2):181-186.
[5]KAMAKURA M,SUENOBU N,FUKUSHIMA M.Fiftyseven-k Da protein in royal jelly enhances proliferation of primary cultured rat hepatocytes and increases albumin production in the absence of serum.Biochemical and Biophysical Research Communications,2001,282(4):865-874.
[6]SALAZAR-OLIVO L A,PAZ-GONZáLEZ V.Screening of biological activities present in honeybee(Apis mellifera)royal jelly.Toxicology in Vitro,2005,19(5):645-651.
[7]WATANABE K,SHINMOTO H,KOBORI M,et al.Stimulation of cell growth in the U-937 human myeloid cell line by honey royal jelly protein.Cytotechnology,1998,26(1):23-27.
[8]CHEN D,XIN X X,QIAN H C,et al.Evaluation of the major royal jelly proteins as an alternative to fetal bovine serum in culturing human cell lines.Journal of Zhejiang University:SCIENCE B,2016,17(6):476-483.
[9]于张颖,谌迪,王一然,等.王浆主蛋白(MRJPs)对张氏肝细胞的促增殖作用及其机制.浙江大学学报(农业与生命科学版),2015,41(1):7-14.YU Z Y,CHEN D,WANG Y R,et al.Effect of major royal jelly proteins(MRJPs)on proliferation activity of Chang’s liver cell line and their mechanism of action.Journal of Zhejiang University(Agriculture and Life Sciences),2015,41(1):7-14.(in Chinese with English abstract)
[10]沈立荣,张瓅文,丁美会,等.蜂王浆的营养保健功能及分子机制研究进展.中国农业科技导报,2009,11(4):41-47.SHEN L R,ZHANG L W,DING M H,et al.Research progress on nutritional and health-care functions and molecular mechanism of royal jelly.Journal of Agricultural Science and Technology,2009,11(4):41-47.(in Chinese with English abstract)
[11]LEE J H,KIM Y G,LEE G M.Effect of Bcl-x L overexpression on sialylation of Fc-fusion protein in recombinant Chinese hamster ovary cell cultures.Biotechnology Progress,2015,31(4):1133-1136.
[12]BALLEZ J S,MOLS J,BURTEAU C,et al.Plant protein hydrolysates support CHO-320 cells proliferation and recombinant IFN-γproduction in suspension and inside microcarriers in protein-free media.Cytotechnology,2004,44(3):103-114.
[13]BETANSKA K,CZOGALLA S,SPINDLER-BARTH M,et al.Influence of cell cycle on ecdysteroid receptor in CHO-K1 cells.Archives of Insect Biochemistry and Physiology,2009,72(3):142-153.
[14]JAYAPAL K P,WLASCHIN K F,HU W S,et al.Recombinant protein therapeutics from CHO cells:20 years and counting.Chemical Engineering Progress,2007,103(10):40-47.
[15]郑惠惠,江洪.CHO细胞表达系统研究进展.生物技术进展,2016,6(4):239-243.ZHENG H H,JIANG H.Progress of CHO expression system.Current Biotechnology,2016,6(4):239-243.(in Chinese with English abstract)
[16]BRADFORD M M.A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.Analytical Biochemistry,1976,72(1/2):248-254.
[17]BUSCHMAN H P,MARPLE E T,WACH M L,et al.In vivo determination of the molecular composition of artery wall by intravascular Raman spectroscopy.Analytical Chemistry,2000,72(16):3771-3775.
[18]SWAIN R J,JELL G,STEVENS M M.Non-invasive analysis of cell cycle dynamics in single living cells with Raman microspectroscopy.Journal of Cellular Biochemistry,2008,104(4):1427-1438.
[19]MOVASAGHI Z,REHMAN S,REHMAN I U.Raman spectroscopy of biological tissues.Applied Spectroscopy Reviews,2007,42(5):493-541.
[20]HAMADA K,FUJITA K,SMITH N I,et al.Raman microscopy for dynamic molecular imaging of living cells.Journal of Biomedical Optics,2008,13(4):044027.
[21]GSTRAUNTHALER G.Alternatives to the use of fetal bovine serum:Serum-free cell culture.Altex,2003,20(4):275-281.
[22]ELIOT M.Risks of virus transmission associated with animal sera or substitutes and methods of control.Developments in Biological Standardization,1999,99:9-16.
[23]SHAH G.Why do we still use serum in the production of biopharmaceuticals?Developments in Biological Standardization,1999,99:17-22.
[24]WESSMAN S J,LEVINGS R L.Benefits and risks due to animal serum used in cell culture production.Developments in Biological Standardization,1999,99:3-8.
[25]董慧君,曽宪卓,鲁菲.无血清培养CHO细胞的研究进展.科技视界,2015(32):71-72.DONG H J,ZENG X Z,LU F.Research progress of serumfree medium for CHO cells.Science&Technology Vision,2015(32):71-72.(in Chinese with English abstract)
[26]宗超,黄磊,蔡谨,等.无血清培养基中蛋白类补充因子重组生产研究进展.药物生物技术,2014,21(2):162-165.ZONG C,HUANG L,CAI J,et al.Research progress of bioproduction of recombinant protein supplements for serumfree medium formulation.Pharmaceutical Biotechnology,2014,21(2):162-165.(in Chinese with English abstract)
[27]KAMAKURA M.Signal transduction mechanism leading to enhanced proliferation of primary cultured adult rat hepatocytes treated with royal jelly 57-k Da protein.The Journal of Biochemistry,2002,132(6):911-919.
[28]KAMAKURA M,SAKAKI T.A hypopharyngeal gland protein of the worker honeybee Apis mellifera L.enhances proliferation of primary-cultured rat hepatocytes and suppresses apoptosis in the absence of serum.Protein Expression and Purification,2006,45(2):307-314.
[29]MATA?IN?,GAJGER I T,BEDRICA L.Cultivation of the EPC fish cell line in serum free media supplemented with honeybee royal jelly.Tier?rztliche Umschau,2008,63(8):446-448.
[30]HWANG E S,YOON G,KANG H T.A comparative analysis of the cell biology of senescence and aging.Cellular and Molecular Life Sciences,2009,66(15):2503-2524.
[31]李俊俊,闫明,吴锦涛,等.不同直径牙胚细胞增殖和骨向分化能力的评估.牙体牙髓牙周病学杂志,2014,24(12):687-691.LI J J,YAN M,WU J T,et al.Proliferation and osteogenic differentiation of tooth germ cells with different diameters.Chinese Journal of Conservative Dentistry,2014,24(12):687-691.(in Chinese with English abstract)
[32]WANG Y H,ZHENG H Y,QIN N L,et al.Involvement of ATP-sensitive potassium channels in proliferation and differentiation of rat preadipocytes.Acta Physiologica Sinica,2007,59(1):8-12.
[33]SCHNEIDER E L,BRAUNSCHWEIGER K,MITSUI Y.The effect of serum batch on the in vitro lifespans of cell cultures derived from old and young human donors.Experimental Cell Research,1978,115(1):47-52.
[34]BURGENER A,PATRICK M,COOMBS K,et al.The modification of a serum-free media formulation for the production of reovirus and the growth of Vero,MRC-5,MDCK and BHK cell lines//Animal Cell Technology:From Target to Market.Netherlands:Springer,2001:200-203.
[35]WISTROM C,VILLEPONTEAU B.Long-term growth of diploid human fibroblasts in low serum media.Experimental Gerontology,1990,25(2):97-105.
[36]YAN X L,DONG R X,ZHANG L,et al.Raman spectra of single cell from gastrointestinal cancer patients.World Journal of Gastroenterology,2005,11(21):3290-3292.
[37]LI Z F,CHEN Y,LI Y Z,et al.Raman microspectroscopy as a diagnostic tool to study single living nasopharyngeal carcinoma cell lines.Biochemistry and Cell Biology,2013,91(3):182-186.
[38]唐伟跃,王杰芳,徐平.胃癌组织拉曼光谱的研究.激光杂志,2004,25(1):82-83.TANG W Y,WANG J F,XU P.Research of stomach cancer tissue by Raman spectroscopy.Laser Journal,2004,25(1):82-83.(in Chinese with English abstract)
[39]杨文沛,姚辉璐,朱淼,等.单个肝癌细胞的拉曼光谱分析研究.激光与红外,2007,37(9):824-827.YANG W P,YAO H L,ZHU M,et al.Raman spectrums of mono-hepatocellular carcinoma.Laser&Infrared,2007,37(9):824-827.(in Chinese with English abstract)
[40]韦芳,岳惠芬,罗瑞琼,等.激光拉曼光谱系统无损鉴别大鼠原代胰岛α、β细胞.广西医科大学学报,2016,33(2):212-216.WEI F,YUE H F,LUO R Q,et al.A laser Raman spectroscopy system for non-destructive identification of rat primary pancreatic isletαandβcells.Journal of Guangxi Medical University,2016,33(2):212-216.(in Chinese with English abstract)
[41]MUSA M,NASIR N F M,THIRUMULU K P.Evaluation of royal jelly as an alternative to fetal bovine serum in cell culture using cell proliferation assays and live cell imaging.African Journal of Traditional Complementary and Alternative Medicines,2014,11(1):148-155.
[2]SHEN L R,ZHANG W,JIN F,et al.Expression of recombinant Acc MRJP1 protein from royal jelly of Chinese honeybee in Pichia pastoris and its proliferation activity in an insect cell line.Journal of Agricultural and Food Chemistry,2010,58(16):9190-9197.
[3]KAMAKURA M.Royalactin induces queen differentiation in honeybees.Nature,2011,473:478-483.
[4]NAGAI T,INOUE R.Preparation and the functional properties of water extract and alkaline extract of royal jelly.Food Chemistry,2004,84(2):181-186.
[5]KAMAKURA M,SUENOBU N,FUKUSHIMA M.Fiftyseven-k Da protein in royal jelly enhances proliferation of primary cultured rat hepatocytes and increases albumin production in the absence of serum.Biochemical and Biophysical Research Communications,2001,282(4):865-874.
[6]SALAZAR-OLIVO L A,PAZ-GONZáLEZ V.Screening of biological activities present in honeybee(Apis mellifera)royal jelly.Toxicology in Vitro,2005,19(5):645-651.
[7]WATANABE K,SHINMOTO H,KOBORI M,et al.Stimulation of cell growth in the U-937 human myeloid cell line by honey royal jelly protein.Cytotechnology,1998,26(1):23-27.
[8]CHEN D,XIN X X,QIAN H C,et al.Evaluation of the major royal jelly proteins as an alternative to fetal bovine serum in culturing human cell lines.Journal of Zhejiang University:SCIENCE B,2016,17(6):476-483.
[9]于张颖,谌迪,王一然,等.王浆主蛋白(MRJPs)对张氏肝细胞的促增殖作用及其机制.浙江大学学报(农业与生命科学版),2015,41(1):7-14.YU Z Y,CHEN D,WANG Y R,et al.Effect of major royal jelly proteins(MRJPs)on proliferation activity of Chang’s liver cell line and their mechanism of action.Journal of Zhejiang University(Agriculture and Life Sciences),2015,41(1):7-14.(in Chinese with English abstract)
[10]沈立荣,张瓅文,丁美会,等.蜂王浆的营养保健功能及分子机制研究进展.中国农业科技导报,2009,11(4):41-47.SHEN L R,ZHANG L W,DING M H,et al.Research progress on nutritional and health-care functions and molecular mechanism of royal jelly.Journal of Agricultural Science and Technology,2009,11(4):41-47.(in Chinese with English abstract)
[11]LEE J H,KIM Y G,LEE G M.Effect of Bcl-x L overexpression on sialylation of Fc-fusion protein in recombinant Chinese hamster ovary cell cultures.Biotechnology Progress,2015,31(4):1133-1136.
[12]BALLEZ J S,MOLS J,BURTEAU C,et al.Plant protein hydrolysates support CHO-320 cells proliferation and recombinant IFN-γproduction in suspension and inside microcarriers in protein-free media.Cytotechnology,2004,44(3):103-114.
[13]BETANSKA K,CZOGALLA S,SPINDLER-BARTH M,et al.Influence of cell cycle on ecdysteroid receptor in CHO-K1 cells.Archives of Insect Biochemistry and Physiology,2009,72(3):142-153.
[14]JAYAPAL K P,WLASCHIN K F,HU W S,et al.Recombinant protein therapeutics from CHO cells:20 years and counting.Chemical Engineering Progress,2007,103(10):40-47.
[15]郑惠惠,江洪.CHO细胞表达系统研究进展.生物技术进展,2016,6(4):239-243.ZHENG H H,JIANG H.Progress of CHO expression system.Current Biotechnology,2016,6(4):239-243.(in Chinese with English abstract)
[16]BRADFORD M M.A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.Analytical Biochemistry,1976,72(1/2):248-254.
[17]BUSCHMAN H P,MARPLE E T,WACH M L,et al.In vivo determination of the molecular composition of artery wall by intravascular Raman spectroscopy.Analytical Chemistry,2000,72(16):3771-3775.
[18]SWAIN R J,JELL G,STEVENS M M.Non-invasive analysis of cell cycle dynamics in single living cells with Raman microspectroscopy.Journal of Cellular Biochemistry,2008,104(4):1427-1438.
[19]MOVASAGHI Z,REHMAN S,REHMAN I U.Raman spectroscopy of biological tissues.Applied Spectroscopy Reviews,2007,42(5):493-541.
[20]HAMADA K,FUJITA K,SMITH N I,et al.Raman microscopy for dynamic molecular imaging of living cells.Journal of Biomedical Optics,2008,13(4):044027.
[21]GSTRAUNTHALER G.Alternatives to the use of fetal bovine serum:Serum-free cell culture.Altex,2003,20(4):275-281.
[22]ELIOT M.Risks of virus transmission associated with animal sera or substitutes and methods of control.Developments in Biological Standardization,1999,99:9-16.
[23]SHAH G.Why do we still use serum in the production of biopharmaceuticals?Developments in Biological Standardization,1999,99:17-22.
[24]WESSMAN S J,LEVINGS R L.Benefits and risks due to animal serum used in cell culture production.Developments in Biological Standardization,1999,99:3-8.
[25]董慧君,曽宪卓,鲁菲.无血清培养CHO细胞的研究进展.科技视界,2015(32):71-72.DONG H J,ZENG X Z,LU F.Research progress of serumfree medium for CHO cells.Science&Technology Vision,2015(32):71-72.(in Chinese with English abstract)
[26]宗超,黄磊,蔡谨,等.无血清培养基中蛋白类补充因子重组生产研究进展.药物生物技术,2014,21(2):162-165.ZONG C,HUANG L,CAI J,et al.Research progress of bioproduction of recombinant protein supplements for serumfree medium formulation.Pharmaceutical Biotechnology,2014,21(2):162-165.(in Chinese with English abstract)
[27]KAMAKURA M.Signal transduction mechanism leading to enhanced proliferation of primary cultured adult rat hepatocytes treated with royal jelly 57-k Da protein.The Journal of Biochemistry,2002,132(6):911-919.
[28]KAMAKURA M,SAKAKI T.A hypopharyngeal gland protein of the worker honeybee Apis mellifera L.enhances proliferation of primary-cultured rat hepatocytes and suppresses apoptosis in the absence of serum.Protein Expression and Purification,2006,45(2):307-314.
[29]MATA?IN?,GAJGER I T,BEDRICA L.Cultivation of the EPC fish cell line in serum free media supplemented with honeybee royal jelly.Tier?rztliche Umschau,2008,63(8):446-448.
[30]HWANG E S,YOON G,KANG H T.A comparative analysis of the cell biology of senescence and aging.Cellular and Molecular Life Sciences,2009,66(15):2503-2524.
[31]李俊俊,闫明,吴锦涛,等.不同直径牙胚细胞增殖和骨向分化能力的评估.牙体牙髓牙周病学杂志,2014,24(12):687-691.LI J J,YAN M,WU J T,et al.Proliferation and osteogenic differentiation of tooth germ cells with different diameters.Chinese Journal of Conservative Dentistry,2014,24(12):687-691.(in Chinese with English abstract)
[32]WANG Y H,ZHENG H Y,QIN N L,et al.Involvement of ATP-sensitive potassium channels in proliferation and differentiation of rat preadipocytes.Acta Physiologica Sinica,2007,59(1):8-12.
[33]SCHNEIDER E L,BRAUNSCHWEIGER K,MITSUI Y.The effect of serum batch on the in vitro lifespans of cell cultures derived from old and young human donors.Experimental Cell Research,1978,115(1):47-52.
[34]BURGENER A,PATRICK M,COOMBS K,et al.The modification of a serum-free media formulation for the production of reovirus and the growth of Vero,MRC-5,MDCK and BHK cell lines//Animal Cell Technology:From Target to Market.Netherlands:Springer,2001:200-203.
[35]WISTROM C,VILLEPONTEAU B.Long-term growth of diploid human fibroblasts in low serum media.Experimental Gerontology,1990,25(2):97-105.
[36]YAN X L,DONG R X,ZHANG L,et al.Raman spectra of single cell from gastrointestinal cancer patients.World Journal of Gastroenterology,2005,11(21):3290-3292.
[37]LI Z F,CHEN Y,LI Y Z,et al.Raman microspectroscopy as a diagnostic tool to study single living nasopharyngeal carcinoma cell lines.Biochemistry and Cell Biology,2013,91(3):182-186.
[38]唐伟跃,王杰芳,徐平.胃癌组织拉曼光谱的研究.激光杂志,2004,25(1):82-83.TANG W Y,WANG J F,XU P.Research of stomach cancer tissue by Raman spectroscopy.Laser Journal,2004,25(1):82-83.(in Chinese with English abstract)
[39]杨文沛,姚辉璐,朱淼,等.单个肝癌细胞的拉曼光谱分析研究.激光与红外,2007,37(9):824-827.YANG W P,YAO H L,ZHU M,et al.Raman spectrums of mono-hepatocellular carcinoma.Laser&Infrared,2007,37(9):824-827.(in Chinese with English abstract)
[40]韦芳,岳惠芬,罗瑞琼,等.激光拉曼光谱系统无损鉴别大鼠原代胰岛α、β细胞.广西医科大学学报,2016,33(2):212-216.WEI F,YUE H F,LUO R Q,et al.A laser Raman spectroscopy system for non-destructive identification of rat primary pancreatic isletαandβcells.Journal of Guangxi Medical University,2016,33(2):212-216.(in Chinese with English abstract)
[41]MUSA M,NASIR N F M,THIRUMULU K P.Evaluation of royal jelly as an alternative to fetal bovine serum in cell culture using cell proliferation assays and live cell imaging.African Journal of Traditional Complementary and Alternative Medicines,2014,11(1):148-155.