慢病毒介导沉默PD-L1基因在乳腺癌细胞中的表达

doi:10.13560/j.cnki.biotech.bull.1985.2017-0181

摘要/Abstract

摘要： 构建含细胞程序性死亡因子配体1（PD-L1）基因的慢病毒载体三质粒体系,并检测其在乳腺癌MDA-MB-231细胞中的表达情况。pMAGic 7.1慢病毒表达载体与质粒△8.91和pVSVG用聚乙烯亚胺（PEI）共转293T包装病毒,感染并筛选获得MDA-MB-231稳转细胞系,检测PD-L1表达情况。（1）MDA-MB-231在3种乳腺癌细胞中PD-L1表达量最高；（2）三质粒转染293T细胞48 h后荧光强度增强,达到90%以上；用病毒感染MDA-MB-231细胞,48 h获得20%左右荧光强度；（3）经筛选的MDA-MB-231稳转细胞系在mRNA水平和蛋白水平低表达PD-L1,其中由pMAGic-sh1包装的病毒干扰能力最强；（4）分别加入病毒后,干扰组细胞增殖能力下降,pMAGic-sh1干扰组细胞在加入病毒7 d后生长速度约为阴性对照的64.77%；（5）混合淋巴实验显示,sh-1干扰组的肿瘤抑制率为35.8%,是正常组的3.06倍,且干扰组的T淋巴细胞活性显著增强。成功通过PEI转染试剂建立了三质粒慢病毒包装体系,该体系获得的慢病毒可有效干扰乳腺癌MDA-MB-231细胞中PD-L1的表达量,所得的干扰细胞株细胞增殖能力下降,可增强T细胞增殖活性及肿瘤细胞杀伤活性。

关键词: 乳腺癌, 细胞程序性死亡因子配体1基因, 慢病毒载体, 聚乙烯亚胺, 共转染

Abstract: This study aims at constructing three-plasmid system of the lentiviral vector carrying the programmed death ligant 1（PD-L1）gene and investigating the expression of PD-L1 in human breast carcinoma cell MDA-MB-231. A pMAGic 7.1 recombinant lentiviral expression vector,plasmid △8.91 and pVSVG were co-transfected into 293T cells for packaging virus,using polyethylenimine（PEI）as transfected solution. The lentiviral products were added to infect and extract stably-expressed MDA-MB-231 cells,and finally the PD-L1 expression of mRNA and protein levels were detected. The results showed as followings：（1）The PD-L1 expression in MDA-Mb-231 was the highest,compared with other two breast cancer cells. （2）At 48 h after co-transfecting three plasmids,the fluorescence intensity reached over 90%. Moreover,the fluorescence intensity reached almost 20% at 48 h after using virus infecting MDA-MB-231 cells. （3）There were low PD-L1 expressions of mRNA and protein levels in the stably-expressed MDA-MB-231 cell lines,and the pMAGic-sh1 had the best effect on it among the three plasmids. （4）After adding viruses,the cell proliferation declined in interfering groups,and the cell proliferation rate in the pMAGic-sh1 interfering groups was 64.77% at 7 days of adding virus compared with the negative group. （5）Mixed lymphatic experiments showed that the tumor inhibition rate of sh-1 group was 35.8%,which was 3.06 times of that in normal group,and the T lymphocyte proliferation rate significantly enhanced. In conclusion,the three-plasmid system of lentiviral vector containing PD-L1 gene using PEI reagent is successfully established. The study demonstrates that the PD-L1 expression of mRNA and protein is lower through the virus infecting system,and interfering groups have lower cell proliferation rate and they enhance the T lymphocyte proliferation rates and its ability to kill tumor cells.

Key words: breast cancer, programmed death factor ligant 1 gene, lentiviral vector, PEI, co-transfected

欧阳婧,孙园园,唐泽民,唐政山,李放军,杨寅柯. 慢病毒介导沉默PD-L1基因在乳腺癌细胞中的表达[J]. 生物技术通报, 2017, 33(9): 259-266.

OUYANG Jing,SUN Yuan-yuan,TANG Ze-min,TANG Zheng-shan,LI Fang-jun,YANG Yin-ke. Low Expression of PD-L1 Gene in Human Breast Carcinoma Cell Mediated by Lentiviral Vector[J]. Biotechnology Bulletin, 2017, 33(9): 259-266.

参考文献

[1] Ferlay J, Steliarova-Foucher E, Lortet-Tieulent J, et al. Cancer incidence and mortality patterns in Europe：estimates for 40 countries in 2012[J] . Eur J Cancer, 2013, 49（6）：1374-1403.
[2] Mauricio Z, Baptista D, et al. Prognostic significance of PD-L1 and PD-L2 in breast cancer[J] . Human Pathlogy, 2016, 47（1）：78-84.
[3] Li B, VanRoey M, Wang C, et al. Anti- programmed death-1 synergizes with granulocyte macrophage colony-stimulating factor-secreting tumor cell immunotherapy providing therapeutic benefit to mice with established tumors[J] . Clin Cancer Res, 2009, 15（5）：1623-1634.
[4] Cimino-Mathews A, Thompson E, Taube JM, et al. PD-L1（B7-H1）expression and the immune tumor microenvironment in primary and metastatic breast carcinomas[J] . Human Pathology, 2016, 47（1）：52-63.
[5] Dai S, Jia R, Zhang X, et al. The PD-1/PD-Ls pathway and autoimmune diseases[J] . Cellular Immunology, 2014, 290（1）：72-79.
[6] Gianchecchi E, Delfino DV, Fierabracci A. Recent insights into the role of the PD-1/PD-L1 pathway in immunological tolerance and autoimmunity[J] . Autoimmunity Reviews, 2013, 12（11）：1091-1100.
[7] Iwai Y, Ishida M, Tanaka Y, et al. Involvement of PD-L1 on tumor cells in the escape from host immune system and tumor immunotherapy by PD- L1 blockade[J] . Proc Natl Acad Sci USA, 2002, 99（19）：12293-12297.
[8] Dong H, Strome SE, Salomao DR, et al. Tumor-associated B7-H1 promotes T-cell apotosis：a potential mechanism of immune evasion[J] . Nat Med, 2008, 8（8）：793-800.
[9] Dulos J, Carven GJ, van Boxtel SJ, et al. PD-1 blockade augments Th1 and Th17 and suppresses Th2 responses in peripheral blood from patients with prostate and advanced melanoma cancer[J] . J Immunother, 2012, 35（2）：169-178.
[10] Marzec M, Zhang Q, Goradia A, et al. Oncogenic kinase NPM/ALK induces through STAT3 expression of immunosuppressive protein CD274（PD-L1, B7-H1）[J] . Proc Natl Acad Sci USA, 2008, 105（52）：20852-20857.
[11] Tuchscherer M, Kanitz E, Otten W, et al. Effects of prenatal stress on cellular and humoral immune responses in neonatal pigs[J] . Veterinary Immunology and Immunopathology, 2002, 86（3）：195-203.
[12] Okudaira K, Hokari R, Tsuzuki Y, et al. Blockade of B7-H1 or B7-DC induces an anti-tumor effect in a mouse pancreatic cancer model[J] . Int J Oncol, 2009, 35（4）：741-749.
[13] Ghebeh H, Mohammed S, Al-Omair A, et al. The B7-H1（PD-L1）T lymphocyte-inhibitory molecule is expressed in breast cancer patients with infiltrating ductal carcinoma：correlation with important high-risk prognostic factors[J] . Neoplasia, 2006, 8（3）：190-198.
[14] D’Addio F, Riella LV, Mfarrej BG, et al. The link between the PD-L1 costimulatory pathway and Th17 in fetomaternal tolerance[J] . J Immunol, 2011, 187（9）：4530-4541.
[15] Liu H, Bakthavatsalam R, Meng Z, et al. PD-L1 signal on liver dendritic cells is critical for Foxp3+CD4+CD25 + Treg and liver tolerance induction in mice[J] . Transplant Proc, 2013, 45（5）：1853-1855.
[16] Franceschini D, Paroli M, Francavilla V, et al. PD-L1 negatively regulates CD4+ CD25+Foxp3+Tregs by limiting STAT-5 phosphorylation in patients chronically infected with HCV[J] . J Clin Invest, 2009, 119（3）：551-564.
[17] Beswick EJ, Pinchuk IV, Das S, et al. Expression of the programmed death ligand 1, B7-H1, on gastric epithelial cells after Helicobacter pylori exposure promotes development of CD4+CD25+Foxp3+ regulatory T cells[J] . Infect Immune, 2007, 75（9）：4334-4341.
[18] Sumimoto H, Kawakami Y. Lentiviral vector-mediated RNAi and its use for cancer research[J] . Future Oncol, 2007, 3（6）：655-664.
[19] Katayama K, Koichiro W, Miyoshi H, et al. RNAinterfering approach forclarifying the PPAR pathway using lentiviral vectors expressing short hairpin RNA[J] . FEBS Letters, 2004, 560（3）：178-182.
[20] 李跃萍, 宋丽萍, 邱曙东, 等. 慢病毒载体在肿瘤基因治疗中的应用[J] . 现代肿瘤医学, 2006, 14（12）：1615-1617.
[21] Naldini L, Blomer U, Gallay P, Ory D, Mulligan R, et al. In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector[J] . Science, 1996, 272（5259）：263-267.
[22] 罗望, 张泓, 许淼, 顾林, 等. 慢病毒——基因转移的潜在新载体[J] . 江苏药学与临床研究, 2006, 14（6）：366-371.