The continued development of new cell culture technology is essential for the future growth and application of insect cell and baculovirus biotechnology. The use of cell lines for academic research and for commercial ...The continued development of new cell culture technology is essential for the future growth and application of insect cell and baculovirus biotechnology. The use of cell lines for academic research and for commercial applications is currently dominated by two cell lines; the Spodoptera frugiperda line, SF21 (and its clonal isolate, SF9), and the Trichoplusia ni line, BTI 5B1-4, commercially known as High Five cells. The long perceived prediction that the immense potential application of the baculovirus-insect cell system, as a tool in cell and molecular biology, agriculture, and animal health, has been achieved. The versatility and recent applications of this popular expression system has been demonstrated by both academia and industry and it is clear that this cell-based system has been widely accepted for biotechnological applications. Numerous small to midsize startup biotechnology companies in North America and the Europe are currently using the baculovirus-insect cell technology to produce custom recombinant proteins for research and commercial applications. The recent breakthroughs using the baculovirus-insect cell-based system for the development of several commercial products that will impact animal and human health will further enhance interest in this technology by pharma. Clearly, future progress in novel cell and engineering advances will lead to fundamental scientific discoveries and serve to enhance the utility and applications of this baculovirus-insect cell system.展开更多
Recombinant Escherichia coli BL21 is used to produce human-like collagen. The key constituents of media are optimized using response surface methodology (RSM). Before thermal induction, the highest biomass production ...Recombinant Escherichia coli BL21 is used to produce human-like collagen. The key constituents of media are optimized using response surface methodology (RSM). Before thermal induction, the highest biomass production and the lowest production of some hazardous by-products, especially acetic acid, were obtained in the media containing 0.085 mol·L-1 glucose and 0.019 mol·L-1 nitrogen (carbon-nitrogen ratio, 4.47:1). After thermal induction, when the concentrations of glucose and nitrogen in the media were 0.065 mol·L-1 and 0.017 mol·L-1 , respectively (carbon-nitrogen ratio, 3.82:1), the productivity of human-like collagen per cell was the highest while that of acetic acid was the lowest. The extended analysis showed that the production of lactic acid and propionic acid increased while that of some intermediate acids of the tricarboxylic acid cycle decreased if the dose of glucose increased.展开更多
Recombinant E. coli BL 21 was cultivated in high cell density to produce human-like collagen. The effects of the feeding of nitrogen source, controlled by an auto on/off-feeding mode with two different cycles of 0.5mi...Recombinant E. coli BL 21 was cultivated in high cell density to produce human-like collagen. The effects of the feeding of nitrogen source, controlled by an auto on/off-feeding mode with two different cycles of 0.5min and 4min intervals, oxygen-enrichment methods and inducement strength on the cell yield and human-like collagen production were investigated. The studies showed that nitrogen source feeding in fast cycle could result in higher human-like collagen production than that in slow cycle; and the feedback regulation of glucose, increase of the pressure of fermentation bioreactor, and supply of oxygen-enriched air could all increase cell yield and human-like collagen production. The effects of inducement strength on protein expression were found important. When OD600 reached 90-100, the cultivation temperature was increased to 42℃ to begin induction for 2-3 h, and then shifted to 39℃ for 5-6h induction, the cell density and human-like collagen production could reach 96g·L-1 [DCW (dry cell mass)] and 19.8% (g·g-1 DCW) respectively.展开更多
Background:Tooth is vital not only for a good smile,but also good health.Yet,we lose tooth regularly due to accidents or diseases.An ideal solution to this problem is to regenerate tooth with patients’own cells.Here ...Background:Tooth is vital not only for a good smile,but also good health.Yet,we lose tooth regularly due to accidents or diseases.An ideal solution to this problem is to regenerate tooth with patients’own cells.Here we describe the generation of tooth-like structures from integration-free human urine induced pluripotent stem cells(ifhU-iPSCs).Results:We first differentiated ifhU-iPSCs to epithelial sheets,which were then recombined with E14.5 mouse dental mesenchymes.Tooth-like structures were recovered from these recombinants in 3 weeks with success rate up to 30%for 8 different iPSC lines,comparable to H1 hESC.We further detected that ifhU-iPSC derived epithelial sheets differentiated into enamel-secreting ameloblasts in the tooth-like structures,possessing physical properties such as elastic modulus and hardness found in the regular human tooth.Conclusion:Our results demonstrate that ifhU-iPSCs can be used to regenerate patient specific dental tissues or even tooth for further drug screening or regenerative therapies.展开更多
This study assessed and compared the immunogenicity of various immunization strategies in mice using combinations of re- combinant DNA （pCCMp24） and recombinant attenuated vaccinia virus Tian Tan （rddVTT_ccMpe4）. ...This study assessed and compared the immunogenicity of various immunization strategies in mice using combinations of re- combinant DNA （pCCMp24） and recombinant attenuated vaccinia virus Tian Tan （rddVTT_ccMpe4）. Intramuscular immuniza- tion was performed on days 0 （prime） and 21 （boost）. The immunogenicity of the vaccine schedules was determined by meas- uring human immunodeficiency virus （HIV）-specific binding antibody levels and cytokine （interleukin-2 and interleukin-4） concentrations in peripheral blood, analyzing lymphocyte proliferation capacity against HIV epitopes and CD4~/CD8＋cell ratio, and monitoring interferon-gamma levels at different times post-immunization. The results showed that pCCMp24, rddVTT.ccMp24 and their prime-boost immunization induced humoral and cellular immune responses. The pCCMp24/ rddVTT.ccMp24 immunization strategy increased CD8＋ T cells and induced more IFN-7-secreting cells compared with sin- gle-shot rDNA. The prime-boost immunization strategy also induced the generation of cellular immunological memory to HIV epitope peptides. These results demonstrated that prime-boost immunization with rDNA and rddVTT_ccMp24 had a tendency to induce greater cellular immune response than single-shot vaccinations, especially IFN-7 response, providing a basis for further studies.展开更多
Extracellular superoxide dismutase(EcSOD)protects tissues from oxidative stress,and thus is considered as a therapeutic agent for many diseases such as atherosclerosis,hypertension,and cancer.However,cost-effective pr...Extracellular superoxide dismutase(EcSOD)protects tissues from oxidative stress,and thus is considered as a therapeutic agent for many diseases such as atherosclerosis,hypertension,and cancer.However,cost-effective production of bioactive recombinant human EcSOD(rhEcSOD)remains a challenge.Herein,we developed an efficient strategy for producing active rhEcSOD by transgenic silkworms.rhEcSOD was successfully synthesized as homodimers and homotetramers in the middle silk gland and spun into the cocoons with a concentration of 9.48±0.21 mg/g.Purification of rhEcSOD from the cocoons could be conveniently achieved with a purity of 99.50%and a yield of 3.5±0.5 mg/g.Additionally,N-glycosylation at the only site of N89 in rhEcSOD with 10 types were identified.The purified rhEcSOD gained the potent enzymatic activity of 4162±293 U/mg after Cu/Zn ions incorporation.More importantly,rhEcSOD was capable of penetrating and accumulating in the nuclei of cells to maintain cell morphology and attenuate ultraviolet B-induced cell apoptosis by eliminating reactive oxygen species and inhibiting the C-Jun N-terminal kinase signaling pathway.These results demonstrated that the transgenic silkworm could successfully produce rhEcSOD with enzymatic and biological activities for biomedical applications.展开更多
BACKGROUND Mesenchymal stromal cells(MSCs)are multipotent cell populations obtained from fetal and adult tissues.They share some characteristics with limb bud mesodermal cells such as differentiation potential into os...BACKGROUND Mesenchymal stromal cells(MSCs)are multipotent cell populations obtained from fetal and adult tissues.They share some characteristics with limb bud mesodermal cells such as differentiation potential into osteogenic,chondrogenic,and tenogenic lineages and an embryonic mesodermal origin.Although MSCs differentiate into skeletal-related lineages in vitro,they have not been shown to selforganize into complex skeletal structures or connective tissues,as in the limb.In this work,we demonstrate that the expression of molecular markers to commit MSCs to skeletal lineages is not sufficient to generate skeletal elements in vivo.AIM To evaluate the potential of MSCs to differentiate into skeletal lineages and generate complex skeletal structures using the recombinant limb(RL)system.METHODS We used the experimental system of RLs from dissociated-reaggregated human placenta(PL)and umbilical cord blood(UCB)MSCs.After being harvested and reaggregated in a pellet,cultured cells were introduced into an ectodermal cover obtained from an early chicken limb bud.Next,this filled ectoderm was grafted into the back of a donor chick embryo.Under these conditions,the cells received and responded to the ectoderm’s embryonic signals in a spatiotemporal manner to differentiate and pattern into skeletal elements.Their response to differentiation and morphogenetic signals was evaluated by quantitative poly-merase chain reaction,histology,immunofluorescence,scanning electron microscopy,and in situ hybridization.RESULTS We found that human PL-MSCs and UCB-MSCs constituting the RLs expressed chondrogenic,osteogenic,and tenogenic molecular markers while differentially committing into limb lineages but could not generate complex structures in vivo.MSCs-RL from PL or UCB were committed early to chondrogenic lineage.Nevertheless,the UCB-RL osteogenic commitment was favored,although preferentially to a tenogenic cell fate.These findings suggest that the commitment of MSCs to differentiate into skeletal lineages differs according to the s展开更多
文摘The continued development of new cell culture technology is essential for the future growth and application of insect cell and baculovirus biotechnology. The use of cell lines for academic research and for commercial applications is currently dominated by two cell lines; the Spodoptera frugiperda line, SF21 (and its clonal isolate, SF9), and the Trichoplusia ni line, BTI 5B1-4, commercially known as High Five cells. The long perceived prediction that the immense potential application of the baculovirus-insect cell system, as a tool in cell and molecular biology, agriculture, and animal health, has been achieved. The versatility and recent applications of this popular expression system has been demonstrated by both academia and industry and it is clear that this cell-based system has been widely accepted for biotechnological applications. Numerous small to midsize startup biotechnology companies in North America and the Europe are currently using the baculovirus-insect cell technology to produce custom recombinant proteins for research and commercial applications. The recent breakthroughs using the baculovirus-insect cell-based system for the development of several commercial products that will impact animal and human health will further enhance interest in this technology by pharma. Clearly, future progress in novel cell and engineering advances will lead to fundamental scientific discoveries and serve to enhance the utility and applications of this baculovirus-insect cell system.
基金Supported by the National High Technology Research and Development Program of China （2006AA02Z246 2007AA03Z456） the National Natural Science Foundation of China （20776119 21076169）+4 种基金 Xi’an Research and Development Program（CX0735） the Scientific Research Program of Shaanxi Provincial Department of Education China （07JK417 07JC16 JG08181） the Natural Science Foundation of Shaanxi Province （2010JQ2012） the Specialized Research Fund for the Doctoral Program of Higher Education of China （20096101120023 20096101110014） Shaanxi Key Subject Program China
文摘Recombinant Escherichia coli BL21 is used to produce human-like collagen. The key constituents of media are optimized using response surface methodology (RSM). Before thermal induction, the highest biomass production and the lowest production of some hazardous by-products, especially acetic acid, were obtained in the media containing 0.085 mol·L-1 glucose and 0.019 mol·L-1 nitrogen (carbon-nitrogen ratio, 4.47:1). After thermal induction, when the concentrations of glucose and nitrogen in the media were 0.065 mol·L-1 and 0.017 mol·L-1 , respectively (carbon-nitrogen ratio, 3.82:1), the productivity of human-like collagen per cell was the highest while that of acetic acid was the lowest. The extended analysis showed that the production of lactic acid and propionic acid increased while that of some intermediate acids of the tricarboxylic acid cycle decreased if the dose of glucose increased.
基金Supported by the National Science and Technology Key Funds (2003DA901A32)the National Nature Science Foundation (No. 20476085).
文摘Recombinant E. coli BL 21 was cultivated in high cell density to produce human-like collagen. The effects of the feeding of nitrogen source, controlled by an auto on/off-feeding mode with two different cycles of 0.5min and 4min intervals, oxygen-enrichment methods and inducement strength on the cell yield and human-like collagen production were investigated. The studies showed that nitrogen source feeding in fast cycle could result in higher human-like collagen production than that in slow cycle; and the feedback regulation of glucose, increase of the pressure of fermentation bioreactor, and supply of oxygen-enriched air could all increase cell yield and human-like collagen production. The effects of inducement strength on protein expression were found important. When OD600 reached 90-100, the cultivation temperature was increased to 42℃ to begin induction for 2-3 h, and then shifted to 39℃ for 5-6h induction, the cell density and human-like collagen production could reach 96g·L-1 [DCW (dry cell mass)] and 19.8% (g·g-1 DCW) respectively.
基金We thank Prof.Yanding Zhang and Prof.Dajiang Qin for valuable suggestions and all staffs working for the South Stem Cell Bank.This work was supported by the grants from Ministry of Science and Technology 973 Program(2010CB944800,2011CB965200)National Natural Science Foundation of China(31000402)+3 种基金the“Strategic Priority Research Program”of the Chinese Academy of Sciences(XDA01020401,XDA01020202)863 Program(2011AA020109)Ministry of Science and Technology International Technology Cooperation Program(2012DFH30050)Open Project of Key Laboratory of Regenerative Biology,Chinese Academy of Sciences(KLRB201217).
文摘Background:Tooth is vital not only for a good smile,but also good health.Yet,we lose tooth regularly due to accidents or diseases.An ideal solution to this problem is to regenerate tooth with patients’own cells.Here we describe the generation of tooth-like structures from integration-free human urine induced pluripotent stem cells(ifhU-iPSCs).Results:We first differentiated ifhU-iPSCs to epithelial sheets,which were then recombined with E14.5 mouse dental mesenchymes.Tooth-like structures were recovered from these recombinants in 3 weeks with success rate up to 30%for 8 different iPSC lines,comparable to H1 hESC.We further detected that ifhU-iPSC derived epithelial sheets differentiated into enamel-secreting ameloblasts in the tooth-like structures,possessing physical properties such as elastic modulus and hardness found in the regular human tooth.Conclusion:Our results demonstrate that ifhU-iPSCs can be used to regenerate patient specific dental tissues or even tooth for further drug screening or regenerative therapies.
基金supported by the National Natural Science Foundation of China (81001342)the National Basic Research Program of China (2011CB512110)the National Mega Project on Major Infectious Diseases Prevention (2012ZX10001005-006)
文摘This study assessed and compared the immunogenicity of various immunization strategies in mice using combinations of re- combinant DNA （pCCMp24） and recombinant attenuated vaccinia virus Tian Tan （rddVTT_ccMpe4）. Intramuscular immuniza- tion was performed on days 0 （prime） and 21 （boost）. The immunogenicity of the vaccine schedules was determined by meas- uring human immunodeficiency virus （HIV）-specific binding antibody levels and cytokine （interleukin-2 and interleukin-4） concentrations in peripheral blood, analyzing lymphocyte proliferation capacity against HIV epitopes and CD4~/CD8＋cell ratio, and monitoring interferon-gamma levels at different times post-immunization. The results showed that pCCMp24, rddVTT.ccMp24 and their prime-boost immunization induced humoral and cellular immune responses. The pCCMp24/ rddVTT.ccMp24 immunization strategy increased CD8＋ T cells and induced more IFN-7-secreting cells compared with sin- gle-shot rDNA. The prime-boost immunization strategy also induced the generation of cellular immunological memory to HIV epitope peptides. These results demonstrated that prime-boost immunization with rDNA and rddVTT_ccMp24 had a tendency to induce greater cellular immune response than single-shot vaccinations, especially IFN-7 response, providing a basis for further studies.
基金supported by the National Key Research and Development Program of China(2022YFD1201600)Chongqing Science and Technology Commission(cstc2020jcyj-cxttX0001,CSTB2022TIAD-KPX0083)Yongchuan District Science and Technology Commission(2022ycyfjg20002).
文摘Extracellular superoxide dismutase(EcSOD)protects tissues from oxidative stress,and thus is considered as a therapeutic agent for many diseases such as atherosclerosis,hypertension,and cancer.However,cost-effective production of bioactive recombinant human EcSOD(rhEcSOD)remains a challenge.Herein,we developed an efficient strategy for producing active rhEcSOD by transgenic silkworms.rhEcSOD was successfully synthesized as homodimers and homotetramers in the middle silk gland and spun into the cocoons with a concentration of 9.48±0.21 mg/g.Purification of rhEcSOD from the cocoons could be conveniently achieved with a purity of 99.50%and a yield of 3.5±0.5 mg/g.Additionally,N-glycosylation at the only site of N89 in rhEcSOD with 10 types were identified.The purified rhEcSOD gained the potent enzymatic activity of 4162±293 U/mg after Cu/Zn ions incorporation.More importantly,rhEcSOD was capable of penetrating and accumulating in the nuclei of cells to maintain cell morphology and attenuate ultraviolet B-induced cell apoptosis by eliminating reactive oxygen species and inhibiting the C-Jun N-terminal kinase signaling pathway.These results demonstrated that the transgenic silkworm could successfully produce rhEcSOD with enzymatic and biological activities for biomedical applications.
基金Supported by the Dirección General de Asuntos del Personal Académico(DGAPA)-Universidad Nacional Autónoma de México,No.IN211117Consejo Nacional de Ciencia y Tecnología(CONACyT),No.1887 CONACyT-Fronteras de la Ciencia awarded to Chimal-Monroy J+1 种基金García-García RD and Garay-Pacheco E received an undergraduate scholarshipMarin-Llera JC a postdoctoral fellowship from the Consejo Nacional de Ciencia y Tecnología,No.CONACyT-Fronteras de la Ciencia-1887.
文摘BACKGROUND Mesenchymal stromal cells(MSCs)are multipotent cell populations obtained from fetal and adult tissues.They share some characteristics with limb bud mesodermal cells such as differentiation potential into osteogenic,chondrogenic,and tenogenic lineages and an embryonic mesodermal origin.Although MSCs differentiate into skeletal-related lineages in vitro,they have not been shown to selforganize into complex skeletal structures or connective tissues,as in the limb.In this work,we demonstrate that the expression of molecular markers to commit MSCs to skeletal lineages is not sufficient to generate skeletal elements in vivo.AIM To evaluate the potential of MSCs to differentiate into skeletal lineages and generate complex skeletal structures using the recombinant limb(RL)system.METHODS We used the experimental system of RLs from dissociated-reaggregated human placenta(PL)and umbilical cord blood(UCB)MSCs.After being harvested and reaggregated in a pellet,cultured cells were introduced into an ectodermal cover obtained from an early chicken limb bud.Next,this filled ectoderm was grafted into the back of a donor chick embryo.Under these conditions,the cells received and responded to the ectoderm’s embryonic signals in a spatiotemporal manner to differentiate and pattern into skeletal elements.Their response to differentiation and morphogenetic signals was evaluated by quantitative poly-merase chain reaction,histology,immunofluorescence,scanning electron microscopy,and in situ hybridization.RESULTS We found that human PL-MSCs and UCB-MSCs constituting the RLs expressed chondrogenic,osteogenic,and tenogenic molecular markers while differentially committing into limb lineages but could not generate complex structures in vivo.MSCs-RL from PL or UCB were committed early to chondrogenic lineage.Nevertheless,the UCB-RL osteogenic commitment was favored,although preferentially to a tenogenic cell fate.These findings suggest that the commitment of MSCs to differentiate into skeletal lineages differs according to the s
