OBJECTIVE The FLT-3 ligand （fms-like tyrosine kinase receptor-3 ligand, FL） is a recently described growth factor affecting early hematopoietic progenitor cells. The FL plays a key role in the growth and differentia...OBJECTIVE The FLT-3 ligand （fms-like tyrosine kinase receptor-3 ligand, FL） is a recently described growth factor affecting early hematopoietic progenitor cells. The FL plays a key role in the growth and differentiation of primitive hematopoietic cells. To yield a high-level of recombinant human FL protein, a recembinant Pichia Pastoris （P. pastoris）strain was constructed. METHODS An artificial expression frame, with the same encoding protein sequence for the FL extracellular domain cDNA, was synthesized by using favored genetic codons of P. pastoris. P. pastoris strain KM71 cells were transformed with the endonuclease Bgl II linearized recombined plasmid, pPIC9K-FL. The plasmid then was linerized in the 5＇AOX1 site and integrated into the yeast KM71 genome. KM71 was transformed with pPIC9K plasmids as a control for the production of recombinant protein. Southern blotting and Northern blotting tests were used to screen the genotype of the recombined strain. Biological activity was demonstrated in vitro with culturing of CD34＋cells. RESULTS The recombinant human FL protein expressed into the yeast culture supertant was identified on the basis of its molecular weight and Western blotting analysis. Numerous bands were observed in the 10-100 kDa molecular mass range. SDS-PAGE showed that the expressed product, a 20 kDa protein, was secreted into the medium in the form of a soluble molecule. Western-blot analyses showed good antigenicity and specificity against polyclonal antibodies. A sharp band and a smeared band were observed at a molecular mass of approximately 20 kDa by Western blotting. The recombinant human FL protein was the major protein component observed in the culture supernatant. The highest yield （108 mg/L） was obtained when expression was induced with 0.5% methanol for 96 h. Deglycosylation with PNGase F resulted in a decrease in apparent molecular mass from 20 kDa to 18kDa forming three bands all of which were also detected by rabbit anti-FL antibodies, Culturing of CD34＋ cells in the presence of KM71pPIC9K-FL over 7 days increased 2.9 fold, while in the control group they increased only 1,5 fold. The biological assay showed that the expressed product could stimulate the proliferation of CD34＋ hematopoietic cells, CONCLUSION We demonstrated that human FL was secreted into the culture supernatant from P. pastoris, and that this yeast strain was a preferred host for recombinant human FL gene expression. This recombinant strain can provide a convenient process for pharmaceutical application.展开更多
Objective: To investigate the F1t3 receptor expression on the surface of malignant hematopoietic cells, the effect of TNFα and dexamethasone (DXM) on its expression and the responses of those cells to recombinant hum...Objective: To investigate the F1t3 receptor expression on the surface of malignant hematopoietic cells, the effect of TNFα and dexamethasone (DXM) on its expression and the responses of those cells to recombinant human F1t3 ligand (rhFL). Methods: Eighteen malignant hematopoietic cell lines were determined for the F1t3 receptor expression by flow cytometric analysis. The effect of rhFL on the proliferation of malignant hematopoietic cellsin vitro was measured using MTT assay. Results: The expressions of F1t3 receptor on the surface of Raji, Daudi, HL-60, 8266 and XG-6 cells were detected by flow cytometric analysis. Following incubation with 20 ng/ml TNFα for 24h, the number of F1t3 receptor positive cells decreased in Raji and 8266, increased in HL-60 and XG-6, and no difference in Daudi cells. After incubation with 10?6 mol/L DXM for 24h, the number of F1t3 receptor positive cells decreased in all the 5 F1t3 receptor positive cell lines. rhFL stimulated the proliferation of HL-60 and Raji cells. Conclusion: For most of the malignant hematopoietic cells, there was neither the expression of F1t3 receptor nor the response to rhFL. DXM may be useful to reduce the effect of FL on the proliferation of some F1t3 receptor positive malignant hematopoietic cells in vitro andin vivo.展开更多
In this study the three-dimensional (3-D) model of the ligand-binding domain (V106-P322) of human interleukin-6 receptor (hlL-6 R) was constructed by computer-guided ho-mology modeling technique using the crystal stru...In this study the three-dimensional (3-D) model of the ligand-binding domain (V106-P322) of human interleukin-6 receptor (hlL-6 R) was constructed by computer-guided ho-mology modeling technique using the crystal structure of the ligand-binding domain (K52-L251) of human growth hormone receptor (hGHR) as templet. Furthermore, the active binding region of the 3-D model of hlL-6R with the ligand (hlL-6) was predicted. In light of the structural characteristics of the active region, a hydrophobic pocket shielded by two hydrophilic residues (E115 and E505) of the region was identified by a combination of molecular modelling and the site-directed or double-site mutation of the twelve crucial residues in the ligand-binding domain of hIL-6R (V106-P322). We observed and analyzed the effects of these mutants on the spatial conformation of the pocket-like region of hlL-6 R. The results indicated that any site-directed mutation of the five Cys residues (four conservative Cys residues: Cyst 21, Cys132, Cys165, Cys176; near membrane Cys residue: Cys193) or each double-site mutation of the five residues in WSEWS motif of hIL-6R (V106-P322) makes the corresponding spatial conformation of the pocket region block the linkage between hlL-6 R and hlL-6. However, the influence of the site-directed mutation of Cys211 and Cys277 individually on the conformation of the pocket region benefits the interaction between hlL-6R and hlL-6. Our study suggests that the predicted hydrophobic pocket in the 3-D model of hIL-6R (V106-P322) is the critical molecular basis for the binding of hlL-6R with its ligand, and the active pocket may be used as a target for designing small hlL-6R-inhibiting molecules in our further study.展开更多
Several obstacles to the production,expansion and genetic modification of immunotherapeutic T cells in vitro have restricted the widespread use of T-cell immunotherapy.In the context of HSCT,delayed naïve T-cell ...Several obstacles to the production,expansion and genetic modification of immunotherapeutic T cells in vitro have restricted the widespread use of T-cell immunotherapy.In the context of HSCT,delayed naïve T-cell recovery contributes to poor outcomes.A novel approach to overcome the major limitations of both T-cell immunotherapy and HSCT would be to transplant human T-lymphoid progenitors(HTLPs),allowing reconstitution of a fully functional naïve T-cell pool in the patient thymus.However,it is challenging to produce HTLPs in the high numbers required to meet clinical needs.Here,we found that adding tumor necrosis factor alpha(TNFα)to a DL-4-based culture system led to the generation of a large number of nonmodified or genetically modified HTLPs possessing highly efficient in vitro and in vivo T-cell potential from either CB HSPCs or mPB HSPCs through accelerated T-cell differentiation and enhanced HTLP cell cycling and survival.This study provides a clinically suitable cell culture platform to generate high numbers of clinically potent nonmodified or genetically modified HTLPs for accelerating immune recovery after HSCT and for T-cell-based immunotherapy(including CAR T-cell therapy).展开更多
[目的]探究干扰素诱导T细胞α亚族/CXC趋化因子受体3(I-TAC/CXCR3)生物轴对甲状腺癌TPC-1细胞增殖、转移能力的影响。[方法]将体外培养的甲状腺癌细胞株TPC-1按处理方式不同分为对照组、I-TAC刺激组(100 ng/mL I-TAC)、I-TAC中和组(CXCR...[目的]探究干扰素诱导T细胞α亚族/CXC趋化因子受体3(I-TAC/CXCR3)生物轴对甲状腺癌TPC-1细胞增殖、转移能力的影响。[方法]将体外培养的甲状腺癌细胞株TPC-1按处理方式不同分为对照组、I-TAC刺激组(100 ng/mL I-TAC)、I-TAC中和组(CXCR3中和抗体+100 ng/mL I-TAC)、CXCR3抑制组(15μg/mL CXCR3抑制剂AMG 487),RT-PCR、CCK-8、流式细胞术、Transwell实验、划痕实验、Western Blot检测各组细胞CXCR3 mRNA表达、细胞增殖、凋亡、侵袭、转移及I-TAC、CXCR3蛋白表达。[结果]与对照组比较,I-TAC刺激组CXCR3 mRNA表达(0.52±0.06 vs 0.91±0.02)、细胞增殖水平(1.13±0.17 vs 1.98±0.14)、穿膜细胞数(37.05±7.14 vs 57.41±9.62)、迁移距离(142.7±20.5 vs 376.2±31.9)增加,凋亡水平(5.52±0.73 vs 3.07±0.52)降低,CXCR3抑制组CXCR3 mRNA表达、细胞增殖水平、穿膜细胞数、迁移距离降低,凋亡水平升高(P<0.05);与I-TAC刺激组比较,I-TAC中和组CXCR3 mRNA表达(0.91±0.02 vs 0.49±0.05)、细胞增殖水平(1.98±0.14 vs 1.67±0.19)、穿膜细胞数(57.41±9.62 vs 32.93±6.58)、迁移距离(376.2±31.9 vs 143.6±21.3)降低,凋亡水平升高(3.07±0.52 vs 5.44±0.82,P<0.05)。[结论]I-TAC/CXCR3生物轴激活可促进甲状腺癌细胞的增殖、侵袭和转移,抑制其凋亡,阻断I-TAC/CXCR3生物轴相互作用或许能成为治疗甲状腺癌的新方法。展开更多
文摘OBJECTIVE The FLT-3 ligand （fms-like tyrosine kinase receptor-3 ligand, FL） is a recently described growth factor affecting early hematopoietic progenitor cells. The FL plays a key role in the growth and differentiation of primitive hematopoietic cells. To yield a high-level of recombinant human FL protein, a recembinant Pichia Pastoris （P. pastoris）strain was constructed. METHODS An artificial expression frame, with the same encoding protein sequence for the FL extracellular domain cDNA, was synthesized by using favored genetic codons of P. pastoris. P. pastoris strain KM71 cells were transformed with the endonuclease Bgl II linearized recombined plasmid, pPIC9K-FL. The plasmid then was linerized in the 5＇AOX1 site and integrated into the yeast KM71 genome. KM71 was transformed with pPIC9K plasmids as a control for the production of recombinant protein. Southern blotting and Northern blotting tests were used to screen the genotype of the recombined strain. Biological activity was demonstrated in vitro with culturing of CD34＋cells. RESULTS The recombinant human FL protein expressed into the yeast culture supertant was identified on the basis of its molecular weight and Western blotting analysis. Numerous bands were observed in the 10-100 kDa molecular mass range. SDS-PAGE showed that the expressed product, a 20 kDa protein, was secreted into the medium in the form of a soluble molecule. Western-blot analyses showed good antigenicity and specificity against polyclonal antibodies. A sharp band and a smeared band were observed at a molecular mass of approximately 20 kDa by Western blotting. The recombinant human FL protein was the major protein component observed in the culture supernatant. The highest yield （108 mg/L） was obtained when expression was induced with 0.5% methanol for 96 h. Deglycosylation with PNGase F resulted in a decrease in apparent molecular mass from 20 kDa to 18kDa forming three bands all of which were also detected by rabbit anti-FL antibodies, Culturing of CD34＋ cells in the presence of KM71pPIC9K-FL over 7 days increased 2.9 fold, while in the control group they increased only 1,5 fold. The biological assay showed that the expressed product could stimulate the proliferation of CD34＋ hematopoietic cells, CONCLUSION We demonstrated that human FL was secreted into the culture supernatant from P. pastoris, and that this yeast strain was a preferred host for recombinant human FL gene expression. This recombinant strain can provide a convenient process for pharmaceutical application.
文摘Objective: To investigate the F1t3 receptor expression on the surface of malignant hematopoietic cells, the effect of TNFα and dexamethasone (DXM) on its expression and the responses of those cells to recombinant human F1t3 ligand (rhFL). Methods: Eighteen malignant hematopoietic cell lines were determined for the F1t3 receptor expression by flow cytometric analysis. The effect of rhFL on the proliferation of malignant hematopoietic cellsin vitro was measured using MTT assay. Results: The expressions of F1t3 receptor on the surface of Raji, Daudi, HL-60, 8266 and XG-6 cells were detected by flow cytometric analysis. Following incubation with 20 ng/ml TNFα for 24h, the number of F1t3 receptor positive cells decreased in Raji and 8266, increased in HL-60 and XG-6, and no difference in Daudi cells. After incubation with 10?6 mol/L DXM for 24h, the number of F1t3 receptor positive cells decreased in all the 5 F1t3 receptor positive cell lines. rhFL stimulated the proliferation of HL-60 and Raji cells. Conclusion: For most of the malignant hematopoietic cells, there was neither the expression of F1t3 receptor nor the response to rhFL. DXM may be useful to reduce the effect of FL on the proliferation of some F1t3 receptor positive malignant hematopoietic cells in vitro andin vivo.
文摘In this study the three-dimensional (3-D) model of the ligand-binding domain (V106-P322) of human interleukin-6 receptor (hlL-6 R) was constructed by computer-guided ho-mology modeling technique using the crystal structure of the ligand-binding domain (K52-L251) of human growth hormone receptor (hGHR) as templet. Furthermore, the active binding region of the 3-D model of hlL-6R with the ligand (hlL-6) was predicted. In light of the structural characteristics of the active region, a hydrophobic pocket shielded by two hydrophilic residues (E115 and E505) of the region was identified by a combination of molecular modelling and the site-directed or double-site mutation of the twelve crucial residues in the ligand-binding domain of hIL-6R (V106-P322). We observed and analyzed the effects of these mutants on the spatial conformation of the pocket-like region of hlL-6 R. The results indicated that any site-directed mutation of the five Cys residues (four conservative Cys residues: Cyst 21, Cys132, Cys165, Cys176; near membrane Cys residue: Cys193) or each double-site mutation of the five residues in WSEWS motif of hIL-6R (V106-P322) makes the corresponding spatial conformation of the pocket region block the linkage between hlL-6 R and hlL-6. However, the influence of the site-directed mutation of Cys211 and Cys277 individually on the conformation of the pocket region benefits the interaction between hlL-6R and hlL-6. Our study suggests that the predicted hydrophobic pocket in the 3-D model of hIL-6R (V106-P322) is the critical molecular basis for the binding of hlL-6R with its ligand, and the active pocket may be used as a target for designing small hlL-6R-inhibiting molecules in our further study.
基金supported by the French Institut National de la Sante et de la Recherche Medicale(INSERM)the European Union Seventh Framework Programme under grant agreements No 269037 and No 261387,the European Unionzs Horizon 2020 research and innovation programme under grant agreement No 666908+1 种基金state funding from the Agence Nationale de la Recherche under the"Investissement d'evenir"program(ANR-10-IAHU-01)the Paris Ile-de-France Region under the"DIM Th^rapie g^niquev initiative.K.M.was funded by the China Scholarship Council and the Fondation pour la Recherche Medicale.A.C.was funded by the French Institut National du Cancer.
文摘Several obstacles to the production,expansion and genetic modification of immunotherapeutic T cells in vitro have restricted the widespread use of T-cell immunotherapy.In the context of HSCT,delayed naïve T-cell recovery contributes to poor outcomes.A novel approach to overcome the major limitations of both T-cell immunotherapy and HSCT would be to transplant human T-lymphoid progenitors(HTLPs),allowing reconstitution of a fully functional naïve T-cell pool in the patient thymus.However,it is challenging to produce HTLPs in the high numbers required to meet clinical needs.Here,we found that adding tumor necrosis factor alpha(TNFα)to a DL-4-based culture system led to the generation of a large number of nonmodified or genetically modified HTLPs possessing highly efficient in vitro and in vivo T-cell potential from either CB HSPCs or mPB HSPCs through accelerated T-cell differentiation and enhanced HTLP cell cycling and survival.This study provides a clinically suitable cell culture platform to generate high numbers of clinically potent nonmodified or genetically modified HTLPs for accelerating immune recovery after HSCT and for T-cell-based immunotherapy(including CAR T-cell therapy).
文摘[目的]探究干扰素诱导T细胞α亚族/CXC趋化因子受体3(I-TAC/CXCR3)生物轴对甲状腺癌TPC-1细胞增殖、转移能力的影响。[方法]将体外培养的甲状腺癌细胞株TPC-1按处理方式不同分为对照组、I-TAC刺激组(100 ng/mL I-TAC)、I-TAC中和组(CXCR3中和抗体+100 ng/mL I-TAC)、CXCR3抑制组(15μg/mL CXCR3抑制剂AMG 487),RT-PCR、CCK-8、流式细胞术、Transwell实验、划痕实验、Western Blot检测各组细胞CXCR3 mRNA表达、细胞增殖、凋亡、侵袭、转移及I-TAC、CXCR3蛋白表达。[结果]与对照组比较,I-TAC刺激组CXCR3 mRNA表达(0.52±0.06 vs 0.91±0.02)、细胞增殖水平(1.13±0.17 vs 1.98±0.14)、穿膜细胞数(37.05±7.14 vs 57.41±9.62)、迁移距离(142.7±20.5 vs 376.2±31.9)增加,凋亡水平(5.52±0.73 vs 3.07±0.52)降低,CXCR3抑制组CXCR3 mRNA表达、细胞增殖水平、穿膜细胞数、迁移距离降低,凋亡水平升高(P<0.05);与I-TAC刺激组比较,I-TAC中和组CXCR3 mRNA表达(0.91±0.02 vs 0.49±0.05)、细胞增殖水平(1.98±0.14 vs 1.67±0.19)、穿膜细胞数(57.41±9.62 vs 32.93±6.58)、迁移距离(376.2±31.9 vs 143.6±21.3)降低,凋亡水平升高(3.07±0.52 vs 5.44±0.82,P<0.05)。[结论]I-TAC/CXCR3生物轴激活可促进甲状腺癌细胞的增殖、侵袭和转移,抑制其凋亡,阻断I-TAC/CXCR3生物轴相互作用或许能成为治疗甲状腺癌的新方法。
