腺病毒介导的“睡美人”转座子与IL-10共表达对NOD小鼠Th17/Treg细胞平衡的影响

Effect of co-expression of IL-10 and sleeping beauty transposon on balance of Th17/Treg cells in splenocytes of NOD mice

目的:观察腺病毒介导的"睡美人"(SB)转座子与白细胞介素10(IL-10)共表达对非肥胖糖尿病(NOD)小鼠脾细胞中Th17/Treg细胞平衡的影响,阐明IL-10对NOD小鼠的可能治疗机制。方法:提取C57BL6小鼠的脾细胞并培养,将脾细胞分为对照组、空载体组和治疗组。对照组脾细胞不做任何处理,空载体组将不含IL-10基因而有转座子序列的腺病毒载体和不含转座酶的腺病毒载体共同感染小鼠脾细胞,治疗组将含有IL-10基因和转座子序列的腺病毒载体以及含有转座酶的腺病毒载体共同感染小鼠脾细胞。感染48h后RTPCR法检测各组小鼠脾细胞中IL-10mRNA表达水平。感染48h后将上述3组处理的脾细胞分别种植到对照组、空载体组和治疗组NOD小鼠右后腿的腘窝皮下,每组6只小鼠,每周注射1次,共6次。注射结束后4周处死小鼠,ELISA法检测各组NOD小鼠血清中IL-10表达水平;流式细胞术检测各组NOD小鼠脾细胞中CD4+IL-10+、CD4+IFN-γ+、Th17和Treg细胞的比例。结果:与对照组和空载体组比较,治疗组C57BL6小鼠脾细胞中IL-10mRNA表达水平明显升高(F=72.71,P<0.05),NOD小鼠血清中IL-10表达水平明显升高(F=8.89,P<0.05),NOD小鼠脾细胞中CD4+IL-10+细胞和Treg细胞比例明显升高(F=72.09,P<0.05;F=12.98,P<0.05);但治疗组小鼠脾细胞中Th17细胞比例明显下降(F=6.39,P<0.05);NOD小鼠脾细胞中CD4+IFN-γ+细胞比例在对照组、空载体组和治疗组之间比较差异无统计学意义(F=2.72,P>0.05)。结论:腺病毒介导的SB转座子与IL-10共表达后可以升高NOD小鼠血清中IL-10表达水平;同时可明显升高NOD小鼠脾细胞中CD4+IL-10+细胞比例,降低Th17细胞比例,升高Treg细胞比例,调控Th1/Th2和Th17/Treg的平衡,对NOD小鼠起到治疗作用。

Objective:To investigate the effect of co-expression of sleeping beauty(SB)transposon and IL-10 mediated by adenovirus on the balance of Th17/Treg cells in the splenocytes of the non-obese diabetes(NOD)mice,and to illuminate the possible mechanism of IL-10 in the treatment of NOD mice.Methods:The splenocytes were extracted from spleen of the C57BL6 mice and cultured.The splenocytes were divided into control group,empty vector group and therapy group.The cells in control group didn’t receive any treatment,the cells in empty vector group were co-infected with the adenovirus vector without IL-10 gene containing transposon sequence and the adenovirus vector without transposase,and the cells in therapy cells were co-infected with the adenovirus vector containing IL-10 gene and transposon sequence and the adenovirus vector with transposase.After 48 h of infection,the expression leves of IL-10 mRNA in the splenocytes of the mice in various groups were assessed by RT-PCR.The cells of above three groups were subcutaneously injected into popliteal space in right hind leg of the NOD mice in control group,empty vector group and therapy group;there were six mice in each group;once a week and six times.The mice were killed 4 weeks after injection,the expression levels of IL-10 in serum of the NOD mice in various groups were assessed by ELISA,and the proportions of CD4+IL-10+,CD4+IFN-γ+,Th17 and Treg cells in the splenocytes were detected by flow cytometry.Results:Compared with control group and empty vector group,the expression level of IL-10 mRNA in splenocytes of the C57BL6 mice in therapy group was increased(F=72.71,P<0.05);the expression level of IL-10 in serum of the NOD mice was increased(F=8.89,P<0.05);the proportions of CD4+IL-10+cells and Treg cells were significantly increased(F=72.09,P<0.05;F=12.98,P<0.05);the proportion of Th17 cells was decreased(F=6.39,P<0.05).The proportion of CD4+IFN-γ+cells had no significant differences between various groups(F=2.72,P>0.05).Conclusion:The co-expression of SB transposon and IL-10 can significantly increase the IL-10 level in serum of the NOD mice,increase the proportion of CD4+IL-10+cells,decrease the proportion of Th17 cells and increase the proportion of Treg cells in the splenocytes,which can regulate the balance of Th1/Th2 and Th17/Treg cells and play a therapeutic effect in the NOD mice.