目的制备小鼠抗人B7-1(CD80)单克隆抗体(mAb),研究其对天然表达B7-1的恶性肿瘤细胞体外生长和迁移的影响。方法以人B7-1基因转染的L929-B7-1细胞免疫BALB/c小鼠,采用B淋巴细胞杂交瘤技术制备mAb,并通过流式细胞术检测mAb对肿瘤细胞膜型B...目的制备小鼠抗人B7-1(CD80)单克隆抗体(mAb),研究其对天然表达B7-1的恶性肿瘤细胞体外生长和迁移的影响。方法以人B7-1基因转染的L929-B7-1细胞免疫BALB/c小鼠,采用B淋巴细胞杂交瘤技术制备mAb,并通过流式细胞术检测mAb对肿瘤细胞膜型B7-1的识别。以天然表达B7-1的Daudi细胞、Raji细胞及8266细胞为观察对象,分别加入(5、10、20、40)μg/m L B7-1 mAb处理,采用噻唑蓝(MTT)法检测mAb对肿瘤细胞体外增殖的影响,TranswellTM法分析mAb对肿瘤细胞体外迁移的影响,流式细胞术分析mAb对肿瘤细胞凋亡的诱导作用。结果成功制备1株能稳定分泌小鼠抗人B7-1 mAb的杂交瘤,命名为5G10。经流式细胞术分析,该抗体与Daudi、Raji、8266、U266、BEL-7402及MCF-7肿瘤细胞的阳性结合率分别为(96.30±2.12)%、(95.70±1.79)%、(96.80±2.48)%、(23.20±2.35)%、(1.68±0.35)%、(0.55±0.04)%;与对照组相比,20μg/m L 5G10 mAb明显抑制Daudi细胞、Raji细胞及8266细胞的增殖,肿瘤细胞迁移率明显降低,细胞凋亡率明显增加。结论小鼠抗人B7-1 mAb能特异性识别和结合不同肿瘤细胞膜表面的B7-1,并能显著抑制天然表达B7-1的肿瘤细胞的体外生长、迁移并促进其凋亡。展开更多
Objective: To construct the human B7. 1 (CD80) eukaryocytic expressing vector and its expression on HL60 cells to investigate the immunotherapeutic and immunoprotective effects of B7. 1 molecule on acute leukemia. Met...Objective: To construct the human B7. 1 (CD80) eukaryocytic expressing vector and its expression on HL60 cells to investigate the immunotherapeutic and immunoprotective effects of B7. 1 molecule on acute leukemia. Methods: ①B7. 1 gene was subcloned from the cloning vector using PCR. Both of tlie PCR products and eukaryocytic expressing vector pHook were digested with Apa I , Sal 1 and linked using T4 DNA ligase. Tlie ligased products were transduced into DH5a. B7. 1 gene containing clones were selected by digestion with Apa I and Sal I which were further conformed by sequencing. ②HL60 cells were transformed by B7. 1 with lipofectamine and detected by FACS. ③Tumor formation, mice survival time and the immunotherapeutic and immunoprotective effects by immunization with B7. 1+ cells were evaluated. Results: ①The PCR products were about 620 bp. Six clones were all digested by Apa I and Sal I to produce 620 bp gene fragment which was in tlie same way as 67. 1. It demonstrated t/iat t/ie recombinant vector had been constructed successfully. Further sequencing confirmed the validity of the construction. There was no nucleotide mutation. ② B7. 1 was availably expressed on HL60 cells. ③ B7. 1+ HL60 cells delayed the growth of tumor and prolonged the survival time of leukemic mice, distinctively. So did tlie immunoprotection of B7. 1 + HL60 cells. Conclusion: The human B7. 1(CD80) eukaryocytic expressing vector can be successfully constructed by molecular cloned methods and can be stably and availably expressed on tlie membrane of B7. 1 negative acute myelocytic leukemia (AML) cell line HL60. Furthermore, the costimulatory molecular vaccine has significant effection of immunotherapy and immunoprotection and gives the rationale for clinical application.展开更多
The CD80 （B7-1） costimulatory molecule is expressed on the surface of B cells and its expression is transcriptionally upregulated upon stimuli. To identify the region of murine CD80 promoter that direct cell type sp...The CD80 （B7-1） costimulatory molecule is expressed on the surface of B cells and its expression is transcriptionally upregulated upon stimuli. To identify the region of murine CD80 promoter that direct cell type specific gene expression, four promoters construct of CD80 gene were generated with DNA fragments fused to the GFP reporter gene. In the present study, significant promoter activity was detected with all four promoter constructs only in the murine B lymphocyte. Further, the CD80 promoter region extending from -3,005 bp to ＋273 bp in relation to the previously reported transcription start site, was identified as tissue specific region. Interestingly, the shortest 700 bp （-427/＋273） of promoter fragment was sufficient to direct the CD80 gene expression. The level of CD80 expression was also found to be modulated by exogenous stimuli in B lymphocyte. Additionally, it was demonstrated that the CD80 gene expression is regulated at the level of transcription where the inducible CD80 gene transcript was detected in B lymphocyte with increasing time.展开更多
文摘目的制备小鼠抗人B7-1(CD80)单克隆抗体(mAb),研究其对天然表达B7-1的恶性肿瘤细胞体外生长和迁移的影响。方法以人B7-1基因转染的L929-B7-1细胞免疫BALB/c小鼠,采用B淋巴细胞杂交瘤技术制备mAb,并通过流式细胞术检测mAb对肿瘤细胞膜型B7-1的识别。以天然表达B7-1的Daudi细胞、Raji细胞及8266细胞为观察对象,分别加入(5、10、20、40)μg/m L B7-1 mAb处理,采用噻唑蓝(MTT)法检测mAb对肿瘤细胞体外增殖的影响,TranswellTM法分析mAb对肿瘤细胞体外迁移的影响,流式细胞术分析mAb对肿瘤细胞凋亡的诱导作用。结果成功制备1株能稳定分泌小鼠抗人B7-1 mAb的杂交瘤,命名为5G10。经流式细胞术分析,该抗体与Daudi、Raji、8266、U266、BEL-7402及MCF-7肿瘤细胞的阳性结合率分别为(96.30±2.12)%、(95.70±1.79)%、(96.80±2.48)%、(23.20±2.35)%、(1.68±0.35)%、(0.55±0.04)%;与对照组相比,20μg/m L 5G10 mAb明显抑制Daudi细胞、Raji细胞及8266细胞的增殖,肿瘤细胞迁移率明显降低,细胞凋亡率明显增加。结论小鼠抗人B7-1 mAb能特异性识别和结合不同肿瘤细胞膜表面的B7-1,并能显著抑制天然表达B7-1的肿瘤细胞的体外生长、迁移并促进其凋亡。
基金Supported by Grant from the Key Clinical Department Development Item of China,Health Ministry(20012131)
文摘Objective: To construct the human B7. 1 (CD80) eukaryocytic expressing vector and its expression on HL60 cells to investigate the immunotherapeutic and immunoprotective effects of B7. 1 molecule on acute leukemia. Methods: ①B7. 1 gene was subcloned from the cloning vector using PCR. Both of tlie PCR products and eukaryocytic expressing vector pHook were digested with Apa I , Sal 1 and linked using T4 DNA ligase. Tlie ligased products were transduced into DH5a. B7. 1 gene containing clones were selected by digestion with Apa I and Sal I which were further conformed by sequencing. ②HL60 cells were transformed by B7. 1 with lipofectamine and detected by FACS. ③Tumor formation, mice survival time and the immunotherapeutic and immunoprotective effects by immunization with B7. 1+ cells were evaluated. Results: ①The PCR products were about 620 bp. Six clones were all digested by Apa I and Sal I to produce 620 bp gene fragment which was in tlie same way as 67. 1. It demonstrated t/iat t/ie recombinant vector had been constructed successfully. Further sequencing confirmed the validity of the construction. There was no nucleotide mutation. ② B7. 1 was availably expressed on HL60 cells. ③ B7. 1+ HL60 cells delayed the growth of tumor and prolonged the survival time of leukemic mice, distinctively. So did tlie immunoprotection of B7. 1 + HL60 cells. Conclusion: The human B7. 1(CD80) eukaryocytic expressing vector can be successfully constructed by molecular cloned methods and can be stably and availably expressed on tlie membrane of B7. 1 negative acute myelocytic leukemia (AML) cell line HL60. Furthermore, the costimulatory molecular vaccine has significant effection of immunotherapy and immunoprotection and gives the rationale for clinical application.
文摘The CD80 （B7-1） costimulatory molecule is expressed on the surface of B cells and its expression is transcriptionally upregulated upon stimuli. To identify the region of murine CD80 promoter that direct cell type specific gene expression, four promoters construct of CD80 gene were generated with DNA fragments fused to the GFP reporter gene. In the present study, significant promoter activity was detected with all four promoter constructs only in the murine B lymphocyte. Further, the CD80 promoter region extending from -3,005 bp to ＋273 bp in relation to the previously reported transcription start site, was identified as tissue specific region. Interestingly, the shortest 700 bp （-427/＋273） of promoter fragment was sufficient to direct the CD80 gene expression. The level of CD80 expression was also found to be modulated by exogenous stimuli in B lymphocyte. Additionally, it was demonstrated that the CD80 gene expression is regulated at the level of transcription where the inducible CD80 gene transcript was detected in B lymphocyte with increasing time.
