CD40 siRNA对 MRL/Lpr狼疮小鼠炎症反应的影响

doi:10.3969/j.issn.1671-167X.2016.05.004

摘要/Abstract

摘要：

目的：观察CD40的小干扰RNA（small interfering RNA, siRNA）对系统性红斑狼疮（systemic lupus erythematosus, SLE）动物模型MRL/Lpr小鼠细胞因子IFNγ、IL17、IL-4和抗双链DNA(anti-double strand DNA,anti-ds-DNA)抗体表达水平的影响，探讨CD40 siRNA治疗MRL/Lpr小鼠的可能性。方法：16只MRL/Lpr小鼠随机分为对照组、空载体组、CD40-siRNA1组及CD40-siRNA2组，每组4只，将成功构建的CD40 siRNA表达载体尾静脉注射MRL/Lpr小鼠，对照组和空载体组小鼠分别注射等剂量、等比例的磷酸盐缓冲液（PBS）和pGFP-V-RS空载体，每隔1天1次，共6次,14 d处死小鼠，称其脾的质量，组织切片在荧光显微镜下观察小鼠脾是否有CD40 siRNA的表达，注射前及注射后第2、5、8、11和14 天小鼠尾部采血，ELISA法检测4组小鼠血清中IFN-γ、IL-17、IL-4和anti-dsDNA抗体的表达水平变化， RT-PCR法检测小鼠脾组织中CD40 mRNA的表达水平，免疫组织化学法检测小鼠脾组织中CD40蛋白的表达水平。结果：注射后的siRNA表达载体可以在小鼠脾中表达；注射CD40 siRNA组的小鼠脾质量减轻，由对照组（141.88±7.81） mg和空载体组（153.10±7.60） mg降低至CD40-siRNA1组（78.85 ±5.61） mg和CD40-siRNA2组（80.25±4.07） mg，注射前4组小鼠血清中IFN-γ、IL-17、IL-4和anti-dsDNA抗体的（质量）浓度比较，差异无统计学意义；而注射后第2、5和8天 CD40-siRNA1组和CD40-siRNA2组MRL/Lpr小鼠血清中IFN-γ、IL-17和（质量）浓度明显下降，CD40-siRNA1组的IFN-γ的（质量）浓度依次为（118.74±10.32） ng/L（pg/mL）、（115.24±8.26） ng/L、（113.71±5.02） ng/L ，CD40-siRNA2组依次为（117.83±6.83） ng/L 、（114.07±0.97） ng/L、（112.67±9.66） ng/L； CD40-siRNA1组的IL-17的(质量)浓度依次为（7.05±0.41） ng/L、（6.34±0.76） ng/L、（5.83±0.43） ng/L，CD40-siRNA2组依次为（7.07±0.22） ng/L、（6.35±0.49） ng/L、（6.12±0.80） ng/L；CD40-siRNA1组的anti-dsDNA的浓度依次为（7.51±0.29） ng/L、（6.74±0.45） ng/L、（6.32±0.39） ng/L；CD40-siRNA2组依次为（8.19±0.38） ng/L、（7.14±0.50） ng/L、（6.48±0.29） ng/L。IL-4的（质量）浓度明显升高，CD40-si-RNA1组IL-4的（质量）浓度依次为（26.51±1.81） ng/L、（27.80±1.72） ng/L、（28.08±2.21） ng/L，CD40-siRNA2组依次为（26.28±2.03） ng/L、（28.15±2.95） ng/L、（28.37±1.71） ng/L。 CD40-siRNA1组和CD40-siRNA2组与对照组和空载体组比较差异有统计学意义（P<0.05），注射后第11和14天 CD40siRNA1组和CD40-siRNA2组中这4个指标的（质量）浓度逐渐恢复至对照组和空载体组的（质量）浓度水平，IFN-γ、 IL-17、IL-4的（质量）浓度4组小鼠比较，差异无统计学意义，注射后第11天 CD40-siRNA1组和CD40-siRNA2组anti-dsDNA抗体的水平尽管与对照组和空载体组比较有所提高，4组比较差异有统计学意义（P<0.05），而注射后第14天 anti-dsDNA抗体的水平4组小鼠比较，差异无统计学意义。注射后第14天 CD40-siRNA1组和CD40-siRNA2组 MRL/Lpr小鼠脾组织中CD40 mRNA和蛋白的表达水平明显低于对照组和空载体组，差异有统计学意义（P<0.05）。结论： MRL/Lpr小鼠注射CD40 siRNA载体后，血清中IFN-γ、IL17和anti-dsDNA抗体水平降低，IL-4的浓度升高，CD40 mRNA和蛋白的表达水平下降，说明CD40 siRNA抑制其mRNA和蛋白后，MRL/Lpr小鼠炎症反应减轻，疾病活动度下降，提示其对SLE动物模型MRL/Lpr小鼠有治疗作用。

关键词: 抗原, CD40, RNA, 小分子干扰, 红斑狼疮, 系统性, 细胞因子类, 小鼠, 近交MRL Lpr

Abstract:

Objective：To observe the effect of CD40 siRNA on expression of IFN-γ, IL-17, IL-4 and anti-dsDNA antibody of systemic lupus erythematosus (SLE) animal model MRL/Lpr mice and to discuss its therapy on MRL/Lpr mice. Methods: In the study, 16 female MRL/Lpr mice were randomly divided into control group (n=4), empty vector group (n=4), CD40siRNA1 group (n=4) and CD40-siRNA2 group (n=4). The vectors expressing siRNA against CD40 were injected by tail veil into MRL/Lpr mice, while MRL/Lpr mice in control group and empty vector group were injected with the same dose of PBS and pGFP-V-RS vector respectively. The injection was given six times and every one day. The mice were sacrificed 14 d after injection, and the spleen tissue was weighed. The pGFP-V-RS was labeled by green fluorescent protein（GFP) and the tissue sections were observed whether siRNA expressed in the spleen. The expression levels of IFN-γ, IL-17, IL-4 and anti-dsDNA antibody in the sera were detected by ELISA method on the 1st day before the first time and the 2nd, 5th, 8th, 11th, and 14th days after last injection, and the expression levels of CD40 mRNA in spleen tissue of MRL/Lpr mice were detected by RT-PCR and the expression levels of CD40 protein in spleen tissue of MRL/Lpr mice were detected by immunohistochemistry method. Results: The expression vector of CD40-siRNA could express in the spleen of MRL/Lpr. The spleens in CD40-siRNA1 group ［(78.85 ±5.61) mg］ and CD40-siRNA2 group ［(80.25±4.07) mg］ were lower than those in control ［(141.88±7.81) mg］ and empty vector group ［(153.10±7.60) mg］. The levels of IL-17, IFN-γ and anti-dsDNA antibody were lower and the levels of IL-4 was higher in CD40-siRNA1 group and CD40-siRNA2 group on the 2nd, 5th and 8th days after last injection than on the 1st day before the first time (P<0.05). The levels of IFN-γ in CD40-siRNA1 group were (118.74±10.32) ng/L, (115.24±8.26) ng/L and (113.71±5.02) ng/L in turn, the levels of IFNγ in CD40siRNA2 group were (117.83±6.83) ng/L, (114.07±0.97) ng/L and (112.67±9.66) ng/L in turn. The levels of IL-17 in CD40-siRNA1 group were (7.05±0.41) ng/L, (6.34±0.76) ng/L and (5.83±0.43) ng/L in turn, the levels of IL-17 in CD40-siRNA2 group were (7.07±0.22) ng/L, (6.35±0.49) ng/L and (6.12±0.80) ng/L in turn. The levels of anti-dsDNA antibody in CD40-siRNA1 group were (7.51±0.29) ng/L, (6.74±0.45) ng/L and (6.32±0.39) ng/L in turn, the levels of anti-dsDNA antibody in CD40-siRNA2 group were (8.19±0.38) ng/L, (7.14±0.50) ng/L and (6.48±0.29) ng/L in turn. The levels of IL-4 in CD40siRNA1 group were (26.51±1.81)ng/L (27.80±1.72) ng/L and (28.08±2.21) ng/L in turn, the level of IL-4 in CD40-siRNA2 group were (26.28±2.03) ng/L, (28.15±2.95) ng/L and (28.37±1.71) ng/L in turn. The expression levels of IL-17 and IFN-γ antibody increased gradually and the levels of IL-4 decreased gradually in CD40-siRNA1 group and CD40-siRNA2 group on the 11th and 14th days after last injection, then reached to the levels of control group and empty vector group (P>0.05). Though the levels of anti-dsDNA antibody in CD40-siRNA1 group and CD40-siRNA2 group on the 11th day was higher than on the 8th day, there was more significance than those in control group and empty vector group (P<0.05). There was no significance between the 4 groups on the 14th day. The levels of CD40 mRNA and protein were lower in CD40-siRNA1 group and CD40-siRNA2 group than in control group and empty vector group on the 14th day after last injection (P<0.05). Conclusion: CD-40 si-RNA can reduce the concentration of IL-17, IFN-γ and of anti-dsDNA antibody in serum, and at the same time, it can elevate the concentration of IL-4 and suppress CD40 mRNA and protein of spleen in MRL/Lpr. Meanwhile after suppressing CD40 mRNA and protein, it can reduce inflammatory response of the mice and the disease activity of MRL/Lpr, suggesting that CD-40 siRNA has therapy effect on SLE.

Key words: Antigens, CD40, RNA, small interfering, Lupus erythematosus, systemic, Cytokines, Mice, inbred MRL Lpr

中图分类号:

王志华，张伟，张艳清，庞春艳，王永福. CD40 siRNA对 MRL/Lpr狼疮小鼠炎症反应的影响[J]. 北京大学学报(医学版), 2016, 48(5): 771-776.

WANG Zhi-hua, ZHANG Wei, ZHANG Yan-qing, PANG Chun-yan,WANG Yong-fu. Effect of CD40 siRNA on inflammatory response of MRL/Lpr mice[J]. Journal of Peking University(Health Sciences), 2016, 48(5): 771-776.

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