多房棘球绦虫severin基因过表达慢病毒载体的构建及对人正常肝细胞侵袭、迁移能力的影响
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摘要
目的构建多房棘球绦虫肌切蛋白(Em severin)基因过表达慢病毒载体,并分析其对人正常肝细胞L02侵袭、迁移能力的影响。方法扩增Em severin基因片段,并将其插入慢病毒载体pCDH-CMV-MCS-EF1-copGFP,构建重组慢病毒pCDH-Em severin。用pCDH-Em severin感染293T细胞,进行病毒包装并检测病毒滴度。以MOI=50的慢病毒感染人正常肝细胞L02,采用RT-qPCR和Western blot检测Em severin mRNA和蛋白表达水平,并通过Transwell和划痕试验检测过表达Em severin对L02细胞侵袭、迁移能力的影响。结果成功构建重组慢病毒pCDH-Em severin,感染293T细胞并进行包装后,获得滴度为1×10~8 TU/ml的pCDH-Em severin慢病毒。RT-qPCR和Western blot检测pCDH-Em severin感染组Em severin mRNA和蛋白表达水平显著高于空白组和空载病毒组(F值分别为98.731和86.074,P<0.05)。Transwell和划痕试验结果表明,过表达Em severin可增强人正常肝细胞L02的侵袭、迁移能力(F值分别为15.439和53.082,P<0.05)。结论成功构建重组慢病毒pCDH-Em severin,过表达Em severin可增强人正常肝细胞L02的侵袭、迁移能力,可为研究Em severin在泡球蚴的侵袭、转移中的作用及机制奠定了基础
Objective To construct a lentiviral vector overexpressing the severin gene of Echinococcus multilocularis(E. multilocularis severin) and to observe its effect on invasion and migration in L02 human liver cells. Methods The severin gene of E. multilocularis was amplified and inserted into the lentiviral vector pCDH-CMV-MCS-EF1-copGFP to construct the recombinant lentivirus pCDH-Em severin, which was transfected into 293 T cells for lentivirus packaging and determination of the viral titer. The recombinant lentivirus pCDH-Em severin was transfected into L02 human liver cells with a multiplicity of infection of 50. Levels of expression of mRNA and protein of E. multilocularis severin were detected using RT-qPCR and Western blotting, respectively. The effects of Em severin on invasion and migration in L02 human liver cells were identified using a Transwell assay and cell scratch test. Results The recombinant lentivirus pCDH-Em severin was successfully constructed, and 293 T cells were infected and packaged to obtain pCDH-Em severin lentivirus, with a titer of 1×10~8 TU/ml. The results of RT-qPCR and Western blotting indicated that the levels of expression of mRNA and protein of E. multilocularis severin in a group infected with the pCDH-Em severin lentivirus were significantly higher than those in the control group and the group infected with an empty lentivirus vector(F: 98.731 and 86.074, respectively, P<0.05). More importantly, results of a Transwell assay and cell scratch test indicated that overexpression of E. multilocularis severin increases invasion and migration in L02 human liver cells(F: 15.439 and 53.082, respectively, P<0.05). Conclusion The recombinant lentivirus pCDH-Em severin was successfully constructed, and overexpression of E. multilocularis severin increased invasion and migration in L02 human liver cells. These findings have laid the foundation for further study of the roles and mechanisms of severin in the process of invasion and migration by E. multilocularis.
Objective To construct a lentiviral vector overexpressing the severin gene of Echinococcus multilocularis(E. multilocularis severin) and to observe its effect on invasion and migration in L02 human liver cells. Methods The severin gene of E. multilocularis was amplified and inserted into the lentiviral vector pCDH-CMV-MCS-EF1-copGFP to construct the recombinant lentivirus pCDH-Em severin, which was transfected into 293 T cells for lentivirus packaging and determination of the viral titer. The recombinant lentivirus pCDH-Em severin was transfected into L02 human liver cells with a multiplicity of infection of 50. Levels of expression of mRNA and protein of E. multilocularis severin were detected using RT-qPCR and Western blotting, respectively. The effects of Em severin on invasion and migration in L02 human liver cells were identified using a Transwell assay and cell scratch test. Results The recombinant lentivirus pCDH-Em severin was successfully constructed, and 293 T cells were infected and packaged to obtain pCDH-Em severin lentivirus, with a titer of 1×10~8 TU/ml. The results of RT-qPCR and Western blotting indicated that the levels of expression of mRNA and protein of E. multilocularis severin in a group infected with the pCDH-Em severin lentivirus were significantly higher than those in the control group and the group infected with an empty lentivirus vector(F: 98.731 and 86.074, respectively, P<0.05). More importantly, results of a Transwell assay and cell scratch test indicated that overexpression of E. multilocularis severin increases invasion and migration in L02 human liver cells(F: 15.439 and 53.082, respectively, P<0.05). Conclusion The recombinant lentivirus pCDH-Em severin was successfully constructed, and overexpression of E. multilocularis severin increased invasion and migration in L02 human liver cells. These findings have laid the foundation for further study of the roles and mechanisms of severin in the process of invasion and migration by E. multilocularis.
引文
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[12] 刘军言, 刘佳佳, 吴益西, 等. 敲低肌切蛋白可抑制胃肠道间质瘤细胞的增殖能力[J]. 第三军医大学学报, 2016, 38(12): 1422-6.
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[16] 李瑞晓, 于垂恭, 张璟, 等. 慢病毒介导NDRG2基因过表达抑制人膀胱癌T24细胞的侵袭和迁移[J]. 中国肿瘤生物治疗杂志, 2013, 4(2): 414-8.
[17] 程凯, 孙浩杰, 张明智, 等. 慢病毒介导的调节因子XI过表达及其对胶质瘤细胞增殖的抑制作用[J]. 中南大学学报(医学版), 2016, 41(11): 1117-23.
[18] 任瑞芳, 赵君, 贵永堃, 等. 过表达BDNF基因重组慢病毒转染MSCs的实验研究[J]. 中国实用神经疾病杂志, 2018, 21(1): 1-6.
[19] 李慧, 惠玲, 李君红, 等. 慢病毒介导的CIB1过表达对人脑胶质瘤细胞增殖和周期影响[J]. 中华肿瘤防治杂志, 2017, 24(6): 357-61.
[20] 冯杰, 夏祥国, 陈礼刚, 等. HEPN1过表达慢病毒的构建及对人泌乳素型垂体瘤细胞的影响[J]. 东南大学学报(医学版), 2017, 36(3): 409-16.
[21] 常海平, 杨彩荣, 郑健. 人TPX2基因过表达慢病毒质粒的构建及其人宫颈癌细胞稳定表达株的筛选[J]. 中国生物制品学杂志, 2017, 30(6): 607-12.
[2] 张梦媛, 伍卫平, 官亚宜, 等. 我国棘球蚴病疾病负担分析[J]. 中国寄生虫学与寄生虫病杂志, 2018, 36(1): 15-9.
[3] Nazligul Y, Kucukazman M, Akbulut S. Role of chemotherapeutic agents in the management of cystic echinococcosis [J]. Int Surg, 2015, 100(1), 112-4.
[4] Musaev GK, Fatyanova AS, Levkin VV. Principles and modern trends in liver echinococcosis treatment [J]. Khirurqiia (Mosk), 2017, 12(1): 90-4.
[5] 李居怡, 陈灵, 邹吉利, 等. 棘球蚴病治疗的研究进展[J]. 医学综述, 2013, 19(16): 2941-43.
[6] 吴海福. Scinderin基因在结直肠癌肝转移中的相关功能研究[D]. 上海:复旦大学, 2010.
[7] 陈晓敏, 乐东海, 李克强, 等. Scinderin基因沉默对人胃癌细胞SGC-7901增殖、转移的影响[J]. 中国细胞生物学学报, 2014, 36(1): 26-32.
[8] 吴珊, 马晶晶, 姜旖, 等. 卵巢上皮性癌中肌切蛋白的表达及其临床意义[J]实用妇产科杂志, 2013, 29(7): 522-5.
[9] Miura N, Takemori N, Kikugawa T, et al. Adseverin: a novel cisplatin-resistant marker in the human bladder cancer cell line HT1376 identified by quantitative proteomic analysis[J]. Mol Oncol, 2012, 6(3): 311-22.
[10] Lueck A, Brown D, Kwiatkowski DJ. The actin-binding proteins adseverin and gelsolin are both highly expressed but differentially localized in kidney and intestine [J]. J Cell Sci, 1998, 111(24): 3633-43.
[11] 赖章超, 宋鑫. 肌切蛋白的结构与功能多样性[J]. 中国生物化学与分子生物学报, 2012, 28(7): 617-23.
[12] 刘军言, 刘佳佳, 吴益西, 等. 敲低肌切蛋白可抑制胃肠道间质瘤细胞的增殖能力[J]. 第三军医大学学报, 2016, 38(12): 1422-6.
[13] 华慧, 张波, 李向阳, 等. 华支睾吸虫脂肪酸结合蛋白CsFABP对巨噬细胞免疫功能的影响[J].中国病原生物学杂志, 2017, 12(11): 1058-61, 1065.
[14] 石梦辰, 陈庭金, 周丽娜, 等. 华支睾吸虫severin蛋白对肝癌细胞SMMC-7402增殖、迁移及侵袭作用的初步研究[J]. 热带医学杂志, 2016, 16(2): 127-30.
[15] 黄红, 张淑坤. 多房棘球蚴病的浸润和转移机制研究进展[J]. 中国人兽共患病学报, 2016, 32(7): 670-78.
[16] 李瑞晓, 于垂恭, 张璟, 等. 慢病毒介导NDRG2基因过表达抑制人膀胱癌T24细胞的侵袭和迁移[J]. 中国肿瘤生物治疗杂志, 2013, 4(2): 414-8.
[17] 程凯, 孙浩杰, 张明智, 等. 慢病毒介导的调节因子XI过表达及其对胶质瘤细胞增殖的抑制作用[J]. 中南大学学报(医学版), 2016, 41(11): 1117-23.
[18] 任瑞芳, 赵君, 贵永堃, 等. 过表达BDNF基因重组慢病毒转染MSCs的实验研究[J]. 中国实用神经疾病杂志, 2018, 21(1): 1-6.
[19] 李慧, 惠玲, 李君红, 等. 慢病毒介导的CIB1过表达对人脑胶质瘤细胞增殖和周期影响[J]. 中华肿瘤防治杂志, 2017, 24(6): 357-61.
[20] 冯杰, 夏祥国, 陈礼刚, 等. HEPN1过表达慢病毒的构建及对人泌乳素型垂体瘤细胞的影响[J]. 东南大学学报(医学版), 2017, 36(3): 409-16.
[21] 常海平, 杨彩荣, 郑健. 人TPX2基因过表达慢病毒质粒的构建及其人宫颈癌细胞稳定表达株的筛选[J]. 中国生物制品学杂志, 2017, 30(6): 607-12.