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目的探讨三氧化二砷(ATO)对MRL/lpr狼疮小鼠脾脏CD4+T细胞增殖以及IFN-γ和IL-4转录和翻译水平的影响。方法免疫磁珠分别分选15只MRL/lpr狼疮小鼠和15只C57BL正常对照小鼠脾脏CD4+T细胞,进而将CD4+T细胞随机分为空白组(RPMI1640),对照组(PHA20mg·L-1+IL-21000u·mL-1),ATO组[PHA20mg·L-1+IL-21000u·mL-1(48h)后+ATO1.0μmol·L-1],体外共培养至72h。采用细胞计数试剂盒(CCK-8)检测3组CD4+T细胞的增殖和抑制水平;酶联免疫吸附反应(ELISA)检测上清液中IFN-γ和IL-4水平;逆转录-聚合酶链反应(RT-PCR)检测上述细胞因子基因的转录水平。结果①分选后获得的CD4+T细胞纯度为(94.12±1.08)%,细胞存活率为(95.82±3.41)%;②MRL/lpr小鼠对照组CD4+T细胞的增殖水平(A值)(0.343±0.019)较空白组(0.054±0.008)高(P<0.001);却低于C57BL小鼠对照组的水平(0.375±0.018)(P<0.05);③MRL/lpr和C57BL小鼠ATO组CD4+T细胞增殖水平(A值)分别为(0.324±0.013)和(0.362±0.013),均低于对照组(P<0.05,P<0.05);④MRL/lpr小鼠对照组IFN-γ的表达水平[(562.287±60.345)ng·L-1]明显高于C57BL小鼠对照组[(253.208±59.434)ng·L-1](P<0.05);ATO对MRL/lpr小鼠IFN-γ的抑制程度大于C57BL小鼠(P<0.05);MRL/lpr小鼠对照组的IL-4表达水平[(41.332±8.039)ng·L-1]与ATO组[(23.046±3.253)ng·L-1]和C57BL小鼠对照组[(29.303±8.038)ng·L-1]比较,也具有明显的统计学差异(P<0.05和P<0.05);ATO对MRL/lpr小鼠IL-4的抑制程度高于C57BL小鼠(P<0.05);⑤在转录水平上,与C57BL小鼠相比,ATO对MRL/lpr小鼠IFN-γmRNA和IL-4mRNA的抑制程度更加明显(P<0.05和P<0.05);ATO对IFN-γ和IL-4表达水平的抑制和转录水平的抑制正相关,r=0.720(P<0.001)和r=0.557(P<0.01)。结论MRL/lpr小鼠存在CD4+T细胞的异常增殖,ATO能在不影响细胞存活率的浓度下抑制MRL/lpr小鼠CD4+T细胞的异常增殖活化,通过抑制其细胞因子IFN-γ和IL-4的mRNA分泌影响两者的表达,且对MRL/lpr小鼠抑制程度大于C57BL小鼠,表明ATO能抑制狼疮鼠的异常免疫功能,可进一步用于SLE的治疗。
Objective To investigate the effect of arsenic trioxide (ATO) on the proliferation of splenic CD4 + T cells and the transcription and translation of IFN-γ and IL-4 in MRL / lpr lupus mice. Methods Immunomagnetic beads were used to separate CD4 + T cells from 15 MRL / lpr lupus mice and 15 C57BL mice respectively. Then the CD4 + T cells were randomly divided into blank group (RPMI1640), control group (PHA 20 mg · L (+ AT-1 + IL-21000u · mL-1) and ATO group (ATO1.0μmol·L-1 after PHA20mg · L-1 + IL-21000u · mL-1 for 48h) The proliferation and inhibition of CD4 + T cells in three groups were detected by cell counting kit (CCK-8). The levels of IFN-γ and IL-4 in the supernatants were detected by enzyme linked immunosorbent assay (ELISA) Chain reaction (RT-PCR) was used to detect the transcription level of the above cytokine genes. Results ① The purity of CD4 + T cells obtained after sorting was (94.12 ± 1.08)% and the cell survival rate was (95.82 ± 3.41)%. ② The proliferation of CD4 + T cells in control group (A value) 0.343 ± 0.019) was significantly higher than that of blank control group (0.054 ± 0.008) (P <0.001), but lower than that of C57BL mice (0.375 ± 0.018) (P <0.05) (0.324 ± 0.013) and (0.362 ± 0.013), respectively, which were lower than those in the control group (P <0.05, P <0.05); ④The expression of IFN-γ in MRL / The level of [(562.287 ± 60.345) ng · L-1] was significantly higher in C57BL mice than that in C57BL mice [(253.208 ± 59.434) ng · L -1] (P <0.05). The level of IL-4 in MRL / lpr group was significantly higher than that in ATO group [(41.332 ± 8.039) ng · L -1] [(23.046 ± 3.253) ng · L- 1] and C57BL mice [(29.303 ± 8.038) ng · L-1], there was also a significant difference (P <0.05 and P <0.05) (P <0.05). (5) At the transcriptional level, the inhibitory effect of ATO on IFN-γ mRNA and IL-4 mRNA in MRL / lpr mice was more obvious than that in C57BL mice (P <0.05 and P <0.05); ATO had a positive correlation with the inhibition of IFN-|Ã and IL-4 expression and the inhibition of transcription, r = 0.720 (P <0.001) and r = 0.557 (P <0.01). Conclusions Abnormal proliferation of CD4 + T cells exists in MRL / lpr mice. ATO can inhibit the abnormal proliferation and activation of CD4 + T cells in MRL / lpr mice at concentrations that do not affect cell viability. By inhibiting IFN-γ and The mRNA secretion of IL-4 affected both the expression of MRL / lpr mouse and C57BL mice, indicating that ATO can inhibit the abnormal immune function in lupus mice and could be further used in the treatment of SLE.