细粒棘球绦虫重组亲肌肉抗原ZW-15免疫小鼠LncRNA 031520表达的研究  

Study on the expression of LncRNA 031520 after immunization with recombinant Eg myophilin ZW-15 of Echinococcus granulosus

作　　者：杨继辉 杨松昊 王婵 徐士梅 朱明星[1,3] 赵巍 YANG Ji-hui;YANG Song-hao;WANG Chan;XU Shi-mei;ZHU Ming-xing;ZHAO Wei(Medical Science and Technology Research Center,Ningxia Medical University,Yinchuan 750004,China;School of Basic Medicine,Ningxia Medical University,Yinchuan 750004,China)

机构地区：[1]宁夏医科大学医学科学技术研究中心,宁夏银川750004 [2]宁夏医科大学基础医学院 [3]宁夏常见传染病防治重点实验室

出　　处：《中国病原生物学杂志》2021年第6期671-675,共5页Journal of Pathogen Biology

基　　金：宁夏自然科学基金项目(No.NZ17209);宁夏高等学校科学研究项目(No.NGY2017110);宁夏重点研发计划重大(重点)项目(No.2018BEG02003)。

摘　　要：目的探讨长链非编码RNA031520(LncRNA 031520)分子在细粒棘球绦虫重组亲肌肉抗原ZW-15诱导小鼠免疫保护的表达分析。方法36只雌性BALB/c小鼠分为PBS对照组、弗氏佐剂组和ZW-15免疫组,12只/组。PBS对照组、弗氏佐剂组分别皮下多点注射PBS溶液和完全/不完全弗氏佐剂100μl/只,ZW-15免疫组注射重组抗原100μl/只(10μg/只)。弗氏佐剂组和ZW-15免疫组初次免疫使用完全弗氏佐剂,加强免疫使用不完全弗氏佐剂。共免疫3次,初次免疫后的2次加强免疫间隔时间为两周。免疫小鼠经麻醉取血,分离血清。采用ELISA法检测特异IgG、IgG1、IgG2a、IgG2b抗体水平。无菌取脾脏,分离淋巴细胞采用流式细胞术分选CD4^(+)T淋巴细胞、CD8^(+)T淋巴细胞及B淋巴细胞群;利用qRT-PCR检测CD4^(+)T淋巴细胞、CD8^(+)T淋巴细胞、B淋巴细胞及总淋巴细胞中LncRNA 031520及内参GAPDH的表达;利用流式细胞术检测脾脏CD4^(+)T淋巴细胞中IFN-γ、IL-4的表达。使用SPSS 17.0统计软件和Graphpad Prism 8.0绘图软件对实验数据进行统计分析。结果小鼠经亲肌肉重组抗原ZW-15免疫后血清特异IgG、IgG1、IgG2a及IgG2b抗体水平均升高,且均显著高于PBS组、弗氏佐剂对照组,差异均有统计学意义(P<0.001)。LncRNA 031520在ZW-15免疫组CD4^(+)T淋巴细胞、总淋巴细胞中相对表达量显著高于PBS对照组和弗氏佐剂对照组(P<0.01),在ZW-15免疫组B淋巴细胞及CD8^(+)T淋巴细胞中相对表达量与PBS对照组及弗氏佐剂对照组相比差异均无统计学意义(均P>0.05)。免疫组小鼠脾脏淋巴细胞Th1亚群标志分子IFN-γ表达量为(19.07±3.44)%,显著高于PBS对照组(8.03±2.67)%和弗氏佐剂对照组(9.37±1.25)%(P<0.05),Th2亚群标志分子IL-4表达量为(0.79±0.02)%,显著高于PBS对照组(0.44±0.13)%和弗氏佐剂对照组(0.50±0.15)%(P<0.05)。结论LncRNA 031520在细粒棘球绦虫重组亲肌肉抗原ZW-15免疫小鼠淋巴细胞中表达上调,可能Objective To preliminarily analyze the expression of long non-coding RNA 031520(LncRNA 031520)molecule in Echinococcus granulosus recombinant Eg myophilin ZW-15 to induce immune protection in mice.Methods Thirty-six female BALB/c mice were divided into a PBS control group,a Freund’s adjuvant group,and a ZW-15 immunization group.Twelve mice/group were injected subcutaneously at multiple points.The PBS control group and Freund’s adjuvant group were injected with a PBS solution or Freund’s adjuvant,100μl/mouse.The ZW-15 immune group was injected with recombinant antigen,100μl/mouse(10μg/mouse).The initial immunization group and the ZW-15 immunization group received Freund’s complete adjuvant,and the booster group received Freund’s incomplete adjuvant.Three rounds of immunization were performed,with an interval of two weeks between the initial immunization and the booster.Serum was collected,and the spleen was removed aseptically.Lymphocytes were separated using a lymphocyte separation solution.ELISA was used to detect the expression of IgG,IgG1,IgG2 a,and IgG2 b antibodies in serum.Flow cytometry was used to sort CD4^(+)T cells,CD8^(+)T cells,and B lymphocytes.qRT-PCR was used to detect the expression of LncRNA 031520 in CD4^(+)T lymphocytes,CD8^(+)T lymphocytes,B lymphocytes,and total lymphocytes.Flow cytometry was used to detect the expression of IFN-γand IL-4 in CD4^(+)T lymphocytes of the spleen.The statistical software SPSS 17.0 and the graphing software Graphpad Prism 8.0 were used to statistically analyze and display results.Results After the mice were immunized with the recombinant Eg myophilin ZW-15,the titer of specific IgG,IgG1,IgG2 a,and IgG2 b antibodies was significantly higher than that in the PBS group and Freund’s adjuvant control group,and the differences were significant(P<0.01).The relative expression of LncRNA 031520 in the total lymphocytes and CD4^(+)T lymphocytes from the ZW-15 immunization group was significantly higher than that in the PBS control group and Freund’s adjuva

关 键 词：亲肌肉抗原ZW-15 长链非编码RNA CD4+T淋巴细胞 免疫保护 

分 类 号：R383.33[医药卫生—医学寄生虫学]