CHO-dhfr细胞改造的研究
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摘要
CHO-dhfr~-细胞表达体系是重要的药用蛋白表达体系,但其在培养过程中存在着一系列的问题,为了使其具有更适应大规模培养的特性,我们利用Flp定点整合系统,通过在CHO-dhfr~-细胞中稳定整合调控基因对CHO-dhfr~-细胞进行了的改造。首先在CHO-dhfr~-细胞中定点整合并过表达人抗凋亡基因Bc1-2,获得的重组细胞对诱导凋亡因素的耐受能力增强,凋亡水平降低,增加了对无血清和高氨离子浓度不利培养条件。乳酸是细胞的重要代谢产物,其积累可明显影响细胞的生长和对外源蛋白的表达。为了减少细胞内乳酸的积累,选择了四个CHO-dhfr~-细胞LDH-A基因中RNAi位点,设计了可转录产生相应shRNA片段的DNA序列,克隆于表达载体pAV/U6的下游。通过将shRNA表达载体在CHO-dhfr~-细胞中稳定整合使之持续表达针对CHO-dhfr细胞LDH-A基因的shRNA,在建立的四株重组细胞中,筛选到一株具有较低的乳酸脱氢酶的mRNA的细胞株,获得的重组细胞株CHO-shRNA能降低乳酸的产生和在培养基中的积累,使细胞的凋亡水平降低,提高了细胞贴壁培养密度11.4%。我们的第三方面工作是实现对细胞增殖进行调控,因为细胞在培养过程中的过度增殖不利于外源蛋白的产生,当细胞达到最佳密度后,生长的停滞将有利于提高外源蛋白的生产。为了实现对细胞生长调控蛋白P27表达的调控以实现细胞生长的停滞,利用T-REx~(TM)(四环素诱导)诱导表达系统,通过两步重组来达到建立稳定整合并可诱导表达p27基因的重组细胞株的目的,首先筛选了高表达TetR的重组细胞株,在此基础上,定点整合了受TetO_2调控之下的p27基因,以实现对p27基因的诱导表达和对细胞周期的控制,在p27诱导表达的情况下,有效的抑制细胞周期停滞于G1期,通过对EGFP检测,表明在细胞生长停滞条件下,细胞对外源蛋白的表达水平可提高70%。
CHO-dhfr~- cell is the most important host cell for biopharmaceutical protein, however, there are some problems still existing in CHO-dhfr~- cell culturing.In order to make CHO-dhfr~- cell more suitable for culturing on a large scale, we modified the cell by steady integrating the anti-apoptosis gene Bcl-2 in genome of CHO-dhfr~- used the Flp target integrating system.Firstly, human anti-apoptosis gene Bcl-2 was target integrated into CHO-dhfr~- cell and overexpressed. The viability of the reconstructing cell increased to face the inductive apoptosis factors such as culturing in serum-free and in high-concentration ammonia ion.As accumulation of Lactic acid which is a kind of side products can obviously influence the growth of the cell. In order to reduce lactate formation in the cell culture by genetically manipulation of the pathway of lactate synthesis, 4 RNAi targets were selected in the LDH-A gene of CHO-dhfr~-cell. Four DNA sequence to produce corresponding shRNA is designed and cloned in the lower position of the expressing vector pAV/U6. shRNA toward LDH-A gene of CHO-dhfr~- can be expressed continuously by steady integrating expressing vector within genome of CHO-dhfr~- cell. One cell line was screened out from the 4 reconstructing cells lines, the result of analysis showed that the LDH-A mRNA , the accumulation of lactate , cell apoptosis in medium all decreased, the density of cell culturing clinging to walls increased by 11. 4%.The third part of the job was to control the cell proliferation bioprocesses. Because the over proliferation of the cell in culturing process was unfavorable for the heterologous protein production, growth stagnation is
CHO-dhfr~- cell is the most important host cell for biopharmaceutical protein, however, there are some problems still existing in CHO-dhfr~- cell culturing.In order to make CHO-dhfr~- cell more suitable for culturing on a large scale, we modified the cell by steady integrating the anti-apoptosis gene Bcl-2 in genome of CHO-dhfr~- used the Flp target integrating system.Firstly, human anti-apoptosis gene Bcl-2 was target integrated into CHO-dhfr~- cell and overexpressed. The viability of the reconstructing cell increased to face the inductive apoptosis factors such as culturing in serum-free and in high-concentration ammonia ion.As accumulation of Lactic acid which is a kind of side products can obviously influence the growth of the cell. In order to reduce lactate formation in the cell culture by genetically manipulation of the pathway of lactate synthesis, 4 RNAi targets were selected in the LDH-A gene of CHO-dhfr~-cell. Four DNA sequence to produce corresponding shRNA is designed and cloned in the lower position of the expressing vector pAV/U6. shRNA toward LDH-A gene of CHO-dhfr~- can be expressed continuously by steady integrating expressing vector within genome of CHO-dhfr~- cell. One cell line was screened out from the 4 reconstructing cells lines, the result of analysis showed that the LDH-A mRNA , the accumulation of lactate , cell apoptosis in medium all decreased, the density of cell culturing clinging to walls increased by 11. 4%.The third part of the job was to control the cell proliferation bioprocesses. Because the over proliferation of the cell in culturing process was unfavorable for the heterologous protein production, growth stagnation is
引文
1.胡显文,陈惠鹏,汤仲明,马清钧.生物制药的现状和未来(一):历史与与现实市场.中国生物工程杂志.2004,24(12):95-101
2. Florian M Wurm. Production of recombinant protein therapeutics in cultivated mammalian cells. Nature Biotechnology. 2004,22(4): 1393-1398
3. HS Conradt, M Nimtz, KE Dittmar,et al. Expression of human interleukin-2 in recombinant baby hamster kidney, Ltk-, and Chinese hamster ovary cells. Structure of O-linked carbohydrate chains and their location within the polypeptide. J. Biol. Chem. 1989,264: 17368-17373.
4.申烨华,耿信笃.CHO细胞表达系统研究新进展.生物工程进展.2000,20(94):23-25
5.刘文军,杨芙蓉,阮力,等.三种病毒启动子在中国仓鼠卵巢(CHO)细胞中表达活性的比较.病毒学报.1997,13(2):164-168
6. Kaufman R J,Sharp P A,Latt S A,et al. Evolution of chromosomal regions containing transfected and amplified dihydrofolate reductase sequences. Mol Cell Biol. 1983,3(4):699-711
7. Kimura R,Miller WM. Effects of elevated pCO2 and Por osmolality on the growth and recombinant tPA production of CHO cells. Biotechnol Bioeng. 1996,52:152-160
8. Gray DR, Kashyap ML, Gozzip CG,et al. CO_2 in large scale and high density CHO cell perfusion culture. Cytotech.1996,22:65～78
9. Jan DCH,Petch DA,Huzel N,et al.The effect of dissolved oxygen on the metabolic profile of a murine hybridoma grown in serum free medium in continuous culture. Biotechnol Bioeng. 1997,54 (2):153
10.胡显文,肖成祖,高丽华,等.血清浓度及溶氧对培养rCHO细胞生产尿激酶原的影响.生物技术通讯.2001,12(3):198-201
11. Senger RS, Karim MN. Effect of shear stress on intrinsic CHO culture state and glycosylation of recombinant tissue type plasminogen activator protein. Biotechnol Prog.2003,19(4): 1199-1209
12.孙丽萍,吴海珍,张惠展.第三代基因工程--代谢工程.药物生物技术,2000,7(2):71-76
13. Fussenegger M, Michael J. Metabolic engineering Ⅱ.eukaryotic systems. Biotechnol Bioeng.2002,79:509-531
14. Kim NS, Lee GM. Overexpression of bcl-2 inhibits sodium butyrate-induced apoptosis in Chinese hamster ovary cell line expressing a chimeric antibody. Biotechnol Bioeng.2001,71: 184-193
15. Kim YH, Kitayama A, Takahashi M, et al. Establishment of an apoptosis-supressible, cell-cycle arrestable cell line and its application for enhancing protein production of serum free or-supplement culture. Biotechnol Prog.1998,14:807-833
16. Mastrangelo A J, Marie J.Hardwick, Part Ⅰ. Bcl-2 and Bcl-xL Limit Apoptosis upon Infection with Alphavirus Vectors. Biotechnol Bioeng.2000.67(5):545-554
17. Nicholas H. Simpson,Anne E. Prevention of Hybridoma Cell Death by bcl-2 During Suboptimal Culture Conditions. Biotechnol Bioeng. 1997,54(5):1-16
18. Meents H, Enenkei B, Eppenberger H M.et al.Impact of coexpression and coamplification of sicam and antiapoptosis determinants bcl-2/bcl-xl on productivity, cell survival, and mitochondria number in CHO-DG44 grown in suspension and serum-free media. Biotechnol Bioeng. 2002,80(6), 706-716
19. Figueroa B,l Sauerwald T M,. Mastrangelo A J,et al.Comparison of bcl-2 to a bcl-2 deletion mutant for mammalian cells exposed to culture insults Biotechnol Bioeng. 2001, 73,(3):211-222
20. Figueroa B J, Sulin Chen,Oyler G A,et al.Aven and bcl-xl enhance protection against apoptosis for mammalian cells exposed to various culture conditions. Biotechnol Bioeng.2004 85(6):589-600
21. Kim YH, Kitayama A, Takahashi M, et al. Establishment of an apoptosis-supressible, cell-cycle arrestable cell line and its application for enhancing protein production of serum free or-supplement culture. Biotechnol Prog. 1998(14):807-833
22. Renner WA, Lee KH, Hatzimanikatis V, Vailey JE, Eppenberger HM. Recombinant cyclin E expression activates proliferation and obviates surface attachment of Chinese hamster ovary (CHO) cells in protein-free medium. Biotechnol Bioeng.1995,47:476-482.
23. Lee KH, Sburlati AR, Renner W et al. Deregulated expression of cloned transcription factor E2F-1 in Chinese hamster ovary cells shifts protein patterns and activates growth in protein-free medium. Biotechnol Bioeng. 1996,50:273-279.
24. Mazur X, Fussenegger M, Renner WA et al. Higher productivity of growth-arrested Chinese hamster ovary cells expressing thecyclin-dependent kinase inhibitor p27.Biotechnol Prog. 1998,14:705-713.
25. Fussenegger M, Schlatter S, Daetwyler D et al. Controlled proliferation by multigene metabolic engineering enhances the productivity of Chinese hamster ovary cells. Nat Biotechnol. 1998, 16:468-472.
26. Minn AJ, Boise LH, Thompson CB. Expression of BCL-XL and loss of p53 can cooperate to overcome a cell-cycle checkpoint induced by mitotic spindle damage. Genes Dev. 1996,10:2621-2631.
27. Minch SL, Kallio PT, Bailey JE. Tissue plasminogen activator coexpressed in Chinese hamster ovary cells with _(2,6)-sialytransferasecontainsNeuAc_(2,6)Gal_(1,4)G1c-N-AcR linkages. Biotechnol Prog. 1995,11:348-351.
28. Uman'a P, Jean-Mairet J, Moudry R et al. Engineered glycoforms of an antineuroplastoma IgG1 with optimized antibody-dependent cellular cytotoxic activity. Nat Biotechnol, 1999,17:176-180.
29. Fux C, Fussenegger M. Improved dual-regulated expression systems for independent control of two different transgenes. From target to market. The Netherland: Kluwer Academic Publishers.2001,129-132.
30. Fux C, Moser S, Schlatter S et al.Streptogramin-and tetracycline-responsive dual-regulated expression of p27 Kipl sense and antisense enables positive and negative growth control of Chinese hamster ovary cells. Nucleic Acids Res.2001,29:3-7
31. Lai DZ, Fu L, Weng SJ, et al. Blocking caspase-3 activity with a U6 SnRNA promoter driven ribozyme enhances survivability of CHO cells cultured in low serum medium and production of interferon-β. Biotechnol Bioeng.2004,85 (1) 20-28
32.来大志,付玲,陈薇等.用于生产重组蛋白药物的抗凋亡CHO宿主细胞株的建立.生物工程学报 2003,19(4):745-749
33 Goswami J. Sinskey H. Steller, et al Apoptosis in Batch Cultures of Chinese Hamster Ovary Cells. Biotechnol Bioeng. 1999,62,(6):632-640
34 Moore A, Donahue CJ, Hooley J, Stocks DL,et al. Apoptosis in CHO cell batch cultures: examination by flow cytometry. Cytotech.1995.17:1-11.
35 James A. Zanghi, Martin Fussenegger, et al.Serum Protects Protein-Free Competent Chinese Hamster Ovary Cells Against Apoptosis Induced by Nutrient Deprivation in Batch Culture. Biotechnol Bioeng.1999, 64(1):108-119
36 Mastrangelo,AJ,Hardwick JM, Betenbaugh MJ.Cytotechnology. 1996,22:169-178
37 Simpson N H,Milner A E,Al-Rubeai. M Biotechnol Bioeng. 1997,54:1-16
38 Alison J, Mastrangelo, Michael Betengauth. Overcoming apoptosis: new methods for improve protein-expression system. Tibtech. 1998,16:88-95
39 林其谁.线粒体与细胞凋亡.生物化学与生物物理学报.1999,31(2)116-123
40 刘志刚,林建波,许凌峰等.新型CHO-dhfr~-细胞定点整合和表达系统的建立.军事医学科学院院刊.2004,28(6):501-504
41 Kluck RM,Bossy-Wetzal E,Green DR,Newmeyer DD. The release ofcytochrome c from mitochondria: A primary site for bcl-2 regulation of apoptosis. Science. 1997,275:1132-1136
42 James A. Zanghi, Martin Fussenegger, James E. Bailey.Serum protects protein-free competent chinese hamster ovary cells against apoptosis induced by Nutrient Deprivation in Batch Culture. Biotechnol Bioeng. 1999,64(1):108-119
43 B.T.Tey R.P, Singh.Piredda, M.Piredda et al. Influence of Bcl-2 on cell death during the cultivation of a chinese hamster ovary cell line expressing a chimeric antibody. Biotechnol Bioeng.2000,68:33-43
44 Singh RP, AL-rubeai M. apoptosis and bioprocess technology. Adv Biochem Eng Biotechnol. 1998.62:167-184
45 孙祥明,张元兴.乳酸对重组CHO细胞生长代谢及EPO表达的影响.化工学报.2002,(53)10:1034-1039
46 龚伟,方学平,刘燕.用填充床生物反应器连续灌流培养CHO细胞生产HbsAg.中国生物工程杂志.2004,24(7):82-84
47 李玉,姚伟,张岩锐,等.重组CHO细胞培养过程中代谢产物对细胞分泌HBsAg的影响.药物生 物技术.2002,9(4):209-211
48 Chang YHD, Grodzinsky AJ, Wang DIC. Nutrient enrichment and in-situ removal of ammonium and lactate through electrical means for suspension hybridoma cultures. Biotechnol Bioeng. 1995,47:319-326.
49 Omasa T, Higashiyama K, Shioya S et al. Effects of lactateconcentration on hybridoma culture in lactate-controlled fed-batch operation. Biotechnol Bioeng. 1992,39:556-564.
50 Glacken MW, Fleischaker RJ, Sinskey AJ. Reduction of waste product excretion via nutrient control: Possible strategies for maximizing product and cell yields on serum in cultures of mammalian cells. Biotechnol Bioeng. 1986.28:1376-1389.
51 Xie L, Wang DIC. Fed-batch cultivation of animal cells using different medium design concepts and feeding strategies. Biotechnol Bioeng. 1994.43:1175-1189.
52 Baumgart, E., Fahimi, D., Stich, A.et al. L-Lactate dehydrogenase A4- and A3B isoforms are bona fide peroxisomal enzymes in rat liver: Evidence for involvement in intraperoxisomal NADH reoxidation. J. Biol. Chem. 1996, 271:3846-3855.
53 Keqin Chen, 1 Qian Liu,Liangzhi Xie et al, Engineering of a Mammalian Cell Line for Reduction of Lactate Formation and High Monoclonal Antibody Production.2001, 72(1):55-61
54 Dae-won Jeong, Tae Soo Kim, Joon Won Lee et al. Blocking of Acidosis-Mediated Apoptosis by a Reduction of Lactate Dehydrogenase Activity through Antisense mRNA Expression, Biochemical and Biophysical Research Communications.2001,289:1141-1149
55 Dae-won Jeong, Tae Soo Kim,Joon Won Lee.et al. Blocking of acidosis-mediated apoptosis by a reduction of lactate dehydrogenase activity through antisense mRNA expression. Biochemical And Biophysical Research Communications.2001,289(5),1141-1149
56 Cynthia P.P,Paul D G,Ira W et al.Effective expression of small interfering rna in human cell. Nat Biotechnol. 2002,20:505-508
57 Patrick J P, Amy AC, Emily B, et al. Short hairpin RNAs(shRNAs) induce sequence-specific silencing in mammalian cell. Genes dev. 2002.16:948-958
58 Cocket MI, Bebbington CR, Yarranton GT. High level expression oftissue inhibitor of
2. Florian M Wurm. Production of recombinant protein therapeutics in cultivated mammalian cells. Nature Biotechnology. 2004,22(4): 1393-1398
3. HS Conradt, M Nimtz, KE Dittmar,et al. Expression of human interleukin-2 in recombinant baby hamster kidney, Ltk-, and Chinese hamster ovary cells. Structure of O-linked carbohydrate chains and their location within the polypeptide. J. Biol. Chem. 1989,264: 17368-17373.
4.申烨华,耿信笃.CHO细胞表达系统研究新进展.生物工程进展.2000,20(94):23-25
5.刘文军,杨芙蓉,阮力,等.三种病毒启动子在中国仓鼠卵巢(CHO)细胞中表达活性的比较.病毒学报.1997,13(2):164-168
6. Kaufman R J,Sharp P A,Latt S A,et al. Evolution of chromosomal regions containing transfected and amplified dihydrofolate reductase sequences. Mol Cell Biol. 1983,3(4):699-711
7. Kimura R,Miller WM. Effects of elevated pCO2 and Por osmolality on the growth and recombinant tPA production of CHO cells. Biotechnol Bioeng. 1996,52:152-160
8. Gray DR, Kashyap ML, Gozzip CG,et al. CO_2 in large scale and high density CHO cell perfusion culture. Cytotech.1996,22:65～78
9. Jan DCH,Petch DA,Huzel N,et al.The effect of dissolved oxygen on the metabolic profile of a murine hybridoma grown in serum free medium in continuous culture. Biotechnol Bioeng. 1997,54 (2):153
10.胡显文,肖成祖,高丽华,等.血清浓度及溶氧对培养rCHO细胞生产尿激酶原的影响.生物技术通讯.2001,12(3):198-201
11. Senger RS, Karim MN. Effect of shear stress on intrinsic CHO culture state and glycosylation of recombinant tissue type plasminogen activator protein. Biotechnol Prog.2003,19(4): 1199-1209
12.孙丽萍,吴海珍,张惠展.第三代基因工程--代谢工程.药物生物技术,2000,7(2):71-76
13. Fussenegger M, Michael J. Metabolic engineering Ⅱ.eukaryotic systems. Biotechnol Bioeng.2002,79:509-531
14. Kim NS, Lee GM. Overexpression of bcl-2 inhibits sodium butyrate-induced apoptosis in Chinese hamster ovary cell line expressing a chimeric antibody. Biotechnol Bioeng.2001,71: 184-193
15. Kim YH, Kitayama A, Takahashi M, et al. Establishment of an apoptosis-supressible, cell-cycle arrestable cell line and its application for enhancing protein production of serum free or-supplement culture. Biotechnol Prog.1998,14:807-833
16. Mastrangelo A J, Marie J.Hardwick, Part Ⅰ. Bcl-2 and Bcl-xL Limit Apoptosis upon Infection with Alphavirus Vectors. Biotechnol Bioeng.2000.67(5):545-554
17. Nicholas H. Simpson,Anne E. Prevention of Hybridoma Cell Death by bcl-2 During Suboptimal Culture Conditions. Biotechnol Bioeng. 1997,54(5):1-16
18. Meents H, Enenkei B, Eppenberger H M.et al.Impact of coexpression and coamplification of sicam and antiapoptosis determinants bcl-2/bcl-xl on productivity, cell survival, and mitochondria number in CHO-DG44 grown in suspension and serum-free media. Biotechnol Bioeng. 2002,80(6), 706-716
19. Figueroa B,l Sauerwald T M,. Mastrangelo A J,et al.Comparison of bcl-2 to a bcl-2 deletion mutant for mammalian cells exposed to culture insults Biotechnol Bioeng. 2001, 73,(3):211-222
20. Figueroa B J, Sulin Chen,Oyler G A,et al.Aven and bcl-xl enhance protection against apoptosis for mammalian cells exposed to various culture conditions. Biotechnol Bioeng.2004 85(6):589-600
21. Kim YH, Kitayama A, Takahashi M, et al. Establishment of an apoptosis-supressible, cell-cycle arrestable cell line and its application for enhancing protein production of serum free or-supplement culture. Biotechnol Prog. 1998(14):807-833
22. Renner WA, Lee KH, Hatzimanikatis V, Vailey JE, Eppenberger HM. Recombinant cyclin E expression activates proliferation and obviates surface attachment of Chinese hamster ovary (CHO) cells in protein-free medium. Biotechnol Bioeng.1995,47:476-482.
23. Lee KH, Sburlati AR, Renner W et al. Deregulated expression of cloned transcription factor E2F-1 in Chinese hamster ovary cells shifts protein patterns and activates growth in protein-free medium. Biotechnol Bioeng. 1996,50:273-279.
24. Mazur X, Fussenegger M, Renner WA et al. Higher productivity of growth-arrested Chinese hamster ovary cells expressing thecyclin-dependent kinase inhibitor p27.Biotechnol Prog. 1998,14:705-713.
25. Fussenegger M, Schlatter S, Daetwyler D et al. Controlled proliferation by multigene metabolic engineering enhances the productivity of Chinese hamster ovary cells. Nat Biotechnol. 1998, 16:468-472.
26. Minn AJ, Boise LH, Thompson CB. Expression of BCL-XL and loss of p53 can cooperate to overcome a cell-cycle checkpoint induced by mitotic spindle damage. Genes Dev. 1996,10:2621-2631.
27. Minch SL, Kallio PT, Bailey JE. Tissue plasminogen activator coexpressed in Chinese hamster ovary cells with _(2,6)-sialytransferasecontainsNeuAc_(2,6)Gal_(1,4)G1c-N-AcR linkages. Biotechnol Prog. 1995,11:348-351.
28. Uman'a P, Jean-Mairet J, Moudry R et al. Engineered glycoforms of an antineuroplastoma IgG1 with optimized antibody-dependent cellular cytotoxic activity. Nat Biotechnol, 1999,17:176-180.
29. Fux C, Fussenegger M. Improved dual-regulated expression systems for independent control of two different transgenes. From target to market. The Netherland: Kluwer Academic Publishers.2001,129-132.
30. Fux C, Moser S, Schlatter S et al.Streptogramin-and tetracycline-responsive dual-regulated expression of p27 Kipl sense and antisense enables positive and negative growth control of Chinese hamster ovary cells. Nucleic Acids Res.2001,29:3-7
31. Lai DZ, Fu L, Weng SJ, et al. Blocking caspase-3 activity with a U6 SnRNA promoter driven ribozyme enhances survivability of CHO cells cultured in low serum medium and production of interferon-β. Biotechnol Bioeng.2004,85 (1) 20-28
32.来大志,付玲,陈薇等.用于生产重组蛋白药物的抗凋亡CHO宿主细胞株的建立.生物工程学报 2003,19(4):745-749
33 Goswami J. Sinskey H. Steller, et al Apoptosis in Batch Cultures of Chinese Hamster Ovary Cells. Biotechnol Bioeng. 1999,62,(6):632-640
34 Moore A, Donahue CJ, Hooley J, Stocks DL,et al. Apoptosis in CHO cell batch cultures: examination by flow cytometry. Cytotech.1995.17:1-11.
35 James A. Zanghi, Martin Fussenegger, et al.Serum Protects Protein-Free Competent Chinese Hamster Ovary Cells Against Apoptosis Induced by Nutrient Deprivation in Batch Culture. Biotechnol Bioeng.1999, 64(1):108-119
36 Mastrangelo,AJ,Hardwick JM, Betenbaugh MJ.Cytotechnology. 1996,22:169-178
37 Simpson N H,Milner A E,Al-Rubeai. M Biotechnol Bioeng. 1997,54:1-16
38 Alison J, Mastrangelo, Michael Betengauth. Overcoming apoptosis: new methods for improve protein-expression system. Tibtech. 1998,16:88-95
39 林其谁.线粒体与细胞凋亡.生物化学与生物物理学报.1999,31(2)116-123
40 刘志刚,林建波,许凌峰等.新型CHO-dhfr~-细胞定点整合和表达系统的建立.军事医学科学院院刊.2004,28(6):501-504
41 Kluck RM,Bossy-Wetzal E,Green DR,Newmeyer DD. The release ofcytochrome c from mitochondria: A primary site for bcl-2 regulation of apoptosis. Science. 1997,275:1132-1136
42 James A. Zanghi, Martin Fussenegger, James E. Bailey.Serum protects protein-free competent chinese hamster ovary cells against apoptosis induced by Nutrient Deprivation in Batch Culture. Biotechnol Bioeng. 1999,64(1):108-119
43 B.T.Tey R.P, Singh.Piredda, M.Piredda et al. Influence of Bcl-2 on cell death during the cultivation of a chinese hamster ovary cell line expressing a chimeric antibody. Biotechnol Bioeng.2000,68:33-43
44 Singh RP, AL-rubeai M. apoptosis and bioprocess technology. Adv Biochem Eng Biotechnol. 1998.62:167-184
45 孙祥明,张元兴.乳酸对重组CHO细胞生长代谢及EPO表达的影响.化工学报.2002,(53)10:1034-1039
46 龚伟,方学平,刘燕.用填充床生物反应器连续灌流培养CHO细胞生产HbsAg.中国生物工程杂志.2004,24(7):82-84
47 李玉,姚伟,张岩锐,等.重组CHO细胞培养过程中代谢产物对细胞分泌HBsAg的影响.药物生 物技术.2002,9(4):209-211
48 Chang YHD, Grodzinsky AJ, Wang DIC. Nutrient enrichment and in-situ removal of ammonium and lactate through electrical means for suspension hybridoma cultures. Biotechnol Bioeng. 1995,47:319-326.
49 Omasa T, Higashiyama K, Shioya S et al. Effects of lactateconcentration on hybridoma culture in lactate-controlled fed-batch operation. Biotechnol Bioeng. 1992,39:556-564.
50 Glacken MW, Fleischaker RJ, Sinskey AJ. Reduction of waste product excretion via nutrient control: Possible strategies for maximizing product and cell yields on serum in cultures of mammalian cells. Biotechnol Bioeng. 1986.28:1376-1389.
51 Xie L, Wang DIC. Fed-batch cultivation of animal cells using different medium design concepts and feeding strategies. Biotechnol Bioeng. 1994.43:1175-1189.
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