鼠抗人CD86单克隆抗体的制备及对肿瘤细胞生长与迁移的影响
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摘要
目的:制备鼠抗人CD86分子的单克隆抗体(m Ab),分析其对天然表达CD86分子的恶性肿瘤细胞株的生长与迁移的影响。方法:采用小鼠腹水诱生法制备CD86 m Ab,Protein A亲和层析法纯化抗体。采用流式细胞术(FCM)检测抗体对细胞膜型CD86分子的识别。以Raji、Daudi和8266细胞为观察对象,加入CD86 m Ab共同孵育,用FCM检测抗体对细胞凋亡的诱导作用,MTT检测抗体对细胞增殖的影响,Transwell~(TM)研究抗体对细胞迁移的影响。结果:本实验获得的CD86 m Ab能很好地识别细胞膜型分子,其与Raji、Daudi和8266细胞的结合率分别为96. 5%、99. 0%和83. 9%。FCM结果显示,共同孵育24 h,当抗体浓度为20μg/ml时,上述细胞的凋亡率分别为16%、18%及14%,均明显高于同型对照组(P<0. 05)。MTT结果显示,共同孵育48 h,20μg/ml抗体对细胞的增殖己可产生抑制作用,40μg/ml抗体对上述细胞增殖的抑制效果更明显(P<0. 05),抑制率依次为23%、25%及26%。TranswellTM结果显示,共同孵育48 h,20μg/ml抗体组的Raji、Daudi和8266细胞的迁移率分别为26%、23%和24%,均明显低于同型对照组(P<0. 05)。结论:本实验制备的抗体能很好地识别细胞膜型分子,其对表达CD86分子的肿瘤细胞的增殖及迁移具有抑制作用,同时可诱导肿瘤细胞发生凋亡,提示该抗体对表达相应抗原分子的肿瘤具有一定的抗瘤效应。
Objective: To prepare monoclonal antibodies( m Ab) against murine anti-human CD86 molecules and analyze their effects on the growth and migration of malignant tumor cell lines that naturally express CD86 molecules. Methods: CD86 m Ab was prepared by mouse ascites induced method. CD86 m Ab was purified by Protein A affinity chromatography. Flow cytometry( FCM) was used to detect antibody recognition of cell membrane type CD86 molecules. Raji,Daudi and 8266 cells were used as observation objects,CD86 m Ab was added to co-incubate. FCM was used to detect the apoptotic cells by antibody induction,MTT was used to detect the effect of antibodies on cell proliferation,and TranswellTMwas used to study the effect of antibodies on cell migration. Results: The CD86 m Ab obtained in this experiment was able to identify the cell membrane molecules well. The binding rates to Raji,Daudi and 8266 cells were 96. 5%,99. 0% and 83. 9% respectively. FCM showed that when the cells were incubated together for 24 hours,apoptosis rate of cells was 16%,18% and 14% at the antibody concentration of 20 μg/ml,which was significantly higher than that of the isotype control group( P<0. 05). MTT results showed that 20 μg/ml antibody could inhibit the proliferation of cells after 48 hours of incubation,the inhibition effect of 40 μg/ml antibody on the above cells was more obvious( P<0. 05),and the inhibition rate was 23%,25% and 26%in order. TranswellTMresults showed that the migration rates of Raji,Daudi and 8266 cells in the 20 μg/ml antibody group were 26%,23% and 24% respectively,which were significantly lower than those in the isotype control group( P<0. 05). Conclusion: The antibody prepared in this experiment can well recognize the cell membrane type molecules,which has an inhibitory effect on the proliferation and migration of tumor cells expressing CD86 molecule,and can induce apoptosis of tumor cells. It is suggested that the antibody has a certain anti-tumor effect on the tumor expressing the corresponding antigen molecule.
Objective: To prepare monoclonal antibodies( m Ab) against murine anti-human CD86 molecules and analyze their effects on the growth and migration of malignant tumor cell lines that naturally express CD86 molecules. Methods: CD86 m Ab was prepared by mouse ascites induced method. CD86 m Ab was purified by Protein A affinity chromatography. Flow cytometry( FCM) was used to detect antibody recognition of cell membrane type CD86 molecules. Raji,Daudi and 8266 cells were used as observation objects,CD86 m Ab was added to co-incubate. FCM was used to detect the apoptotic cells by antibody induction,MTT was used to detect the effect of antibodies on cell proliferation,and TranswellTMwas used to study the effect of antibodies on cell migration. Results: The CD86 m Ab obtained in this experiment was able to identify the cell membrane molecules well. The binding rates to Raji,Daudi and 8266 cells were 96. 5%,99. 0% and 83. 9% respectively. FCM showed that when the cells were incubated together for 24 hours,apoptosis rate of cells was 16%,18% and 14% at the antibody concentration of 20 μg/ml,which was significantly higher than that of the isotype control group( P<0. 05). MTT results showed that 20 μg/ml antibody could inhibit the proliferation of cells after 48 hours of incubation,the inhibition effect of 40 μg/ml antibody on the above cells was more obvious( P<0. 05),and the inhibition rate was 23%,25% and 26%in order. TranswellTMresults showed that the migration rates of Raji,Daudi and 8266 cells in the 20 μg/ml antibody group were 26%,23% and 24% respectively,which were significantly lower than those in the isotype control group( P<0. 05). Conclusion: The antibody prepared in this experiment can well recognize the cell membrane type molecules,which has an inhibitory effect on the proliferation and migration of tumor cells expressing CD86 molecule,and can induce apoptosis of tumor cells. It is suggested that the antibody has a certain anti-tumor effect on the tumor expressing the corresponding antigen molecule.
引文
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[9]Qian H,Chen Y,Huang T,et al.Combined application of embelin and tumor necrosis factor-related apoptosis-inducing ligand inhibits proliferation and invasion in osteosarcoma cells via caspase-induced apoptosis[J].Oncol Lett,2018,15(5):6931-6970.
[10]Skau CT,Fischer RS,Gurel P,et al.FMN2 makes perinuclear actin to protect nuclei during confined migration and promote metastasis[J].Cell,2018,173(2):529.
[11]Wang TJ,Liu HT,Lai YH,et al.Honokiol,a polyphenol natural compound,attenuates cisplatin-induced acute cytotoxicity in renal epithelial cells through cellular oxidative stress and cytoskeleton modulations[J].Front Pharmacol,2018,9(24):357-362.
[2]Agadjanyan MG,Kim JJ,Trivedi N,et al.CD86(B7-2)can function to drive MHC-restricted antigen-specific CTL responses in vivo[J].J Immunol,1999,162(6):3417-3443.
[3]Barker E,Bossart KN,Fujimura SH,et al.The role of CD80 and CD86 in enhancing CD8(+)cell suppression of HIV replication[J].Cell Immunol,1999,196(2):95-103.
[4]Abodllah ZR,Jalilian N,Sahraian MA,et al.Polymorphisms of RPS6KB1 and CD86 associates with susceptibility to multiple sclerosis in Iranian population[J].Neurol Res,2017,39(3):217-239.
[5]安萌,王志强,朱华亭,等.抗人CD86单链抗体的基因克隆、表达及结合活性分析[J].细胞与分子免疫学杂志,2013,29(2):178-181.An M,Wang ZQ,Zhu HT,et al.Gene cloning and expression and binding activity analysis of anti-human CD86 ScFv[J].Chin J Cell Mol Immunol,2013,29(2):178-181.
[6]刘玉华,孙杰,胡玲玲,等.抗人CD86嵌合抗体对B淋巴瘤细胞株Raji生长抑制及杀伤作用研究[J].中国免疫学杂志,2009,25(6):513-516.Liu YH,Sun J,Hu LL,et al.Anti-human CD86 chimeric antibody inhibits growth of B lymphoma cell line Raji and killing effect study[J].Chin J Immunol,2009,25(6):513-516.
[7]Plumas J,Chaperot L,Jacob MC,et al.Malignant B lymphocytes from non-hodgkin's lymphoma induce allogeneic proliferative and cytotoxic T cell responses in primary mixed lymphocyte cultures:an important role of co-stimulatory molecules CD80(B7-1)and CD86(B7-2)in stimulation by tumor cells[J].Eur J Immunol,1995,25(12):3332-3372.
[8]陶然,石云杰,孙中文,等.鼠抗人B7-2单克隆抗体的研制及生物学特性研究[J].现代免疫学,2006,26(3):202-206.Tao R,Shi YJ,Sun ZW,et al.Preparation and biological characteristics of mouse anti-human B7-2 monoclonal antibody[J].Cur Immunol,2006,26(3):202-206.
[9]Qian H,Chen Y,Huang T,et al.Combined application of embelin and tumor necrosis factor-related apoptosis-inducing ligand inhibits proliferation and invasion in osteosarcoma cells via caspase-induced apoptosis[J].Oncol Lett,2018,15(5):6931-6970.
[10]Skau CT,Fischer RS,Gurel P,et al.FMN2 makes perinuclear actin to protect nuclei during confined migration and promote metastasis[J].Cell,2018,173(2):529.
[11]Wang TJ,Liu HT,Lai YH,et al.Honokiol,a polyphenol natural compound,attenuates cisplatin-induced acute cytotoxicity in renal epithelial cells through cellular oxidative stress and cytoskeleton modulations[J].Front Pharmacol,2018,9(24):357-362.
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