LncRNA 017418及细胞因子在细粒棘球蚴rP29免疫小鼠中的表达分析

LncRNA 017418 and cytokine expression in mice immunized with Echinococcus granulosus rP29

目的初步探讨长链非编码RNA 017418(lncRNA 017418)和细胞因子在细粒棘球蚴重组蛋白P29(rP29)诱导小鼠产生免疫反应中的表达。方法重组融合表达菌株pET28a-P29/BL21与LB液体培养基混合孵育,加入异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达,十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)检测纯化rP29。36只雌性BALB/c小鼠随机分为对照组、佐剂组和免疫组,每组12只,免疫组将纯化的10μg rP29与等体积弗氏完全佐剂混合乳化后,腹部皮下3点注射,总剂量100μl/鼠,佐剂组注射等体积弗氏完全佐剂,对照组不作处理;首次免疫后2周,相同剂量加强免疫1次,加强免疫采用弗氏不完全佐剂。加强免疫后2周,无菌环境下制备脾细胞悬液,分离淋巴细胞;流式细胞术分选淋巴细胞亚群CD4^+T、CD8^+T和B淋巴细胞;实时荧光定量PCR(qRT-PCR)检测lncRNA 017418、Notch1、白细胞介素-2(IL-2)、γ干扰素(IFN-γ)、IL-10在淋巴细胞中的表达量;采用SPSS 17.0统计学软件和GraphPad Prism6.0软件对实验结果进行分析。结果经IPTG诱导表达、亲和层析法纯化,rP29蛋白相对分子质量(Mr)约为31000。qRT-PCR检测lncRNA 017418在脾淋巴细胞中的相对表达量,免疫组为2.25±0.10,高于对照组(0.81±0.05)和佐剂组(0.99±0.13)(P<0.01)。流式细胞术分析的CD4^+T、CD8^+T和B淋巴细胞所占比例分别为:对照组22.0%、9.2%和59.8%,佐剂组22.8%、7.6%和60.7%,免疫组22.1%、9.7%和60.4%。LncRNA 017418在免疫组CD4^+T淋巴细胞中的相对表达量为1.49±0.03,高于对照组(0.97±0.02)和佐剂组(1.06±0.10)(P<0.01),LncRNA 017418在免疫组CD8^+T细胞中的相对表达量为1.87±0.12,与对照组(2.06±0.14)和佐剂组(2.00±0.09)相比,差异无统计学意义(P>0.05),LncRNA 017418在免疫组B淋巴细胞中的相对表达量为1.03±0.03,与对照组(0.97±0.07)和佐剂组(1.08±0.12)相比,差异无统计学意义(P>0.05)。Notch1在免疫组CD4+T淋巴细胞中的相对表达量为1.92±0.04,高于对照组(1.10±0.10)和佐剂组(1.20±0.08)(P<0.01),IL-2的相对表达量为1.45±0.07,高于对照组(1.07±0.08)和佐剂组(1.09±0.11)(P<0.05);IFN-γ的相对表达量为1.51±0.11,高于对照组(0.92±0.05)和佐剂组(1.03±0.14)(P<0.01、0.05);IL-10的相对表达量为0.52±0.01,低于对照组(0.95±0.04)和佐剂组(1.05±0.10)(P<0.01);对照组和佐剂组比较,差异均无统计学意义(P>0.05)。Pearson相关系数分析lncRNA 017418与Notch1、IL-2、IFN-γ之间存在正相关关系(r=0.8244、0.5902、0.8415,均P<0.01),与IL-10之间存在负相关关系(r=-0.4432,P<0.05)。结论LncRNA 017418在细粒棘球蚴rP29诱导的小鼠中表达上调,Notch1、IL-2、IFN-γ在CD4+T淋巴细胞中表达上调,IL-10在CD4+T淋巴细胞中表达下调。

Objective To preliminarily investigate the expression of long-chain non-coding RNA 017418(lncRNA 017418)and cytokines during immune response of mice induced by recombinant protein P29(rP29).Methods The recombinant fusion expression strain of Escherichia coli pET28a-P29/BL21 was incubated with LB liquid medium,to which isopropyl-β-D-thioglycoside(IPTG)was added to induce protein expression.The purified recombinant protein rP29 was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE).Thirty-six female BALB/c mice were randomly divided into the control group,adjuvant group and immunization group(12 mice each).To immunization group mice,10μg purified rP29 was mixed with an equal volume of Freund’s complete adjuvant forming emulsion,and injected subcutaneously at three points at abdomen at a total volume of 100μl/mouse.The adjuvant group was injected with an equal volume of Freund’s complete adjuvant alone,while the control group received no treatment.Two weeks after the first immunization,the same volume of booster with incomplete Freund’s adjuvant was given once.Two weeks after boosting,spleen cell suspensions were prepared in sterile condition and lymphocytes were separated.Flow cytometry was performed to assay the lymphocyte subsets CD4^+T,CD8^+T and B lymphocytes.The expression of lncRNA 017418,Notch1,interleukin-2(IL-2),interferon gamma(IFN-γ)and IL-10 in lymphocytes was detected by real-time fluorescence quantitative PCR(qRT-PCR).Data were analyzed with SPSS 17.0 and GraphPad Prism 6.0 softwares.Results Produced by IPTG induction and affinity chromatography purification,the purified r P29 showed a relative molecular weight of^31000.qRT-PCR revealed that the relative expression of lncRNA 017418 in spleen lymphocytes in the immunization group was 2.25±0.10,which was significantly higher than those in the control(0.81±0.05)and adjuvant groups(0.99±0.13)(P<0.01).The proportions of CD4^+T,CD8^+T and B lymphocytes found by flow cytometry were 22.0%,9.2%and 59.8%in the control group;22.8%,7.6%and 60.7%in the adjuvant group;and 22.1%,9.7%and 60.4%in the immunization group,respectively.The relative expression of lncRNA 017418 in CD4^+T lymphocyte in the immunization group was 1.49±0.03,significantly higher than those in the control group(0.97±0.02)and the adjuvant group(1.06±0.10)(P<0.01);those in the CD8^+T cells in the three groups were 1.87±0.12,2.06±0.14,and 2.00±0.09,respectively(P>0.05);and those in the B cells were 1.03±0.03,0.97±0.07,and 1.08±0.12,respectively(P>0.05).The relative expression of Notch1 in the CD4^+T lymphocytes was 1.92±0.04 in the i mmunization group,higher than those in the control group(1.10±0.10)and adjuvant group(1.20±0.08)(P<0.01).The relative expression of IL-2 was 1.45±0.07 in the immunization group,higher than those in the control group(1.07±0.08)and adjuvant group(1.09±0.118)(P<0.01).The relative expression of IFN-γwas 1.51±0.11 in the immunization group,higher than those in the control group(0.92±0.05)and adjuvant group(1.03±0.14)(P<0.01,P<0.05).The relative expression of IL-10 was 0.52±0.01 in the immunization group,lower than those in the control group(0.95±0.04)and adjuvant group(1.05±0.10)(P<0.01).There is no statistically significant difference found between the control group and the adjuvant group(P>0.05).Pearson correlation coefficient analysis showed that lncRNA 017418 was positively correlated with Notch1,IL-2,and IFN-γ(r=0.8244,0.5902,0.8415;P<0.01),and negatively correlated with IL-10(r=-0.4432;P<0.05).Conclusion LncRNA 017418 expression is up-regulated in rP29-immunized mice;the expression of Notch1,IL-2 and IFN-γare up-regulated in CD4^+T lymphocytes;and IL-10 expression is down-regulated in CD4^+T lymphocyte.