西罗莫司对创伤小鼠脾脏树突状细胞诱导T淋巴细胞应答能力的影响

目的 了解西罗莫司对创伤小鼠脾脏树突状细胞(DC)诱导异源性T淋巴细胞应答能力的体外调节作用.方法 将24只BALB/c小鼠按随机数字表法分为对照组与创伤组,每组12只.将创伤组麻醉后造成失血合并闭合性骨折,对照组仅麻醉不致伤,24 h后分离2组小鼠脾脏DC.将2组DC分为西罗莫司阴性对照组和创伤组(不用西罗莫司处理),以及西罗莫司阳性对照组和创伤组(用10μg/L西罗莫司处理6 h).检测各组DC自噬活性(用荧光强度值表示)及DC介导的混合淋巴细胞反应(MLR)变化(用吸光度值表示).流式细胞仪检测细胞表面主要组织相容性复合物(MHC)Ⅱ与共刺激分子CD40、CD80、CD86表达.用ELISA法检测LPS刺激后DC中IL-12p40、IL-12p70和IL-10的水平.对数据进行单因素方差分析.结果 (1)西罗莫司阴性创伤组小鼠脾脏DC自噬活性(荧光强度值为13±2)及其介导的MLR强度均较西罗莫司阴性对照组(荧光强度值为22±6)明显减弱(F=212.836,P<0.05).与西罗莫司阴性对照组和创伤组比较,西罗莫司阳性对照组和创伤组自噬活性(45±8、44±8)均明显增强(F=212.836,P<0.05或P<0.01).西罗莫司阳性创伤组MLR强度较西罗莫司阴性创伤组明显增强(F值分别为101.426、86.533,P值均小于0.05).(2)西罗莫司阴性创伤组小鼠脾脏DC表面的MHCⅡ(60±9)%]及CD40(4±1)%]表达较西罗莫司阴性对照组(85±6)%、(8±1)%]明显降低(F值分别为37.918、40.426,P值均小于0.05),西罗莫司阳性创伤组MHCⅡ表达(78±7)%]较西罗莫司阴性创伤组明显提高(F=37.918,P<0.05).(3)两罗莫司阴性创伤组小鼠脾脏DC的IL-12p40、IL-12p70表达水平(120±13)、(10±3)pg/mL]较西罗莫司阴性对照组(200±25)、(20±6)pg/mL]明显降低(F值分别为218.646、310.253,P值均小于0.05);与西罗莫司阴性对照组和创伤组比较,西罗莫司阳性对照组和创伤组IL-12p40(560±34)、(540±29)pg/mL]、IL-12p70(55±8)、(60±11)pg/mL]表达水平均明显提高(F值分别为218.646、310.253,P值均小于0.01),IL-10表达水平降低(F=246.108,P<0.01).结论 西罗莫司在体外可部分改善创伤小鼠DC功能,并提高其诱导T淋巴细胞的应答能力.

Effect of sirolimus on capacity of splenic dendritic cells from traumatized mice in inducing T cell responses ex vivo

Objective To study the ex vivo effect of sirolimus on capacity of splenic dendritic cells ( DC ) from traumatized mice in inducing T cell responses. Methods Twenty-four BALB/c mice were divided into control group and trauma group according to the random number table, with 12 mice in each group. Mice in trauma group were bled followed by closed femor fracture after anaesthesia, while mice in control group were only anaesthetized without injury. Twenty-four hours later DC were isolated from spleens and divided into 4 subgroups: sirolimus devoid control (trauma) groups consisted of cells from control (trauma) groups, without sirolimus treatment] and sirolimus treated control (trauma) groups consisted of cells from control (trauma) groups, treated with 10 μg/L sirolimus for 6 hours]. Then their autophagic activity, DC-induced mixed lymphocyte reaction (MLR) were measured and recorded as fluorescence intensity (FI) value and absorbance value respectively. The expression of major histocompatibility complex class (MHC) Ⅱ and costimulatory molecules CD40, CD80, and CD86 on DC surface were measured with flow cytometry. IL-12p40, IL-12p70 and IL-10 levels in lipopolysaccharide-stimulated DC supernatants were determined by ELISA. Data were processed with one-way analysis of variance. Results (1) Compared with those of sirolimus devoid control group ( FI value = 22 ± 6) , DC autophagic activity ( FI value = 13 ± 2) and DC-induced MLR in mice from sirolimus devoid trauma group were significantly weakened ( F =212.836, P < 0.05). Compared with those of sirolimus devoid control (trauma) groups, DC autophagic activity in mice from sirolimus treated control ( trauma) groups ( FI = 45 ± 8, 44 ± 8 respectively) were significantly strengthened ( F =212.836, P <0. 05 or P < 0. 01). MLR in mice from sirolimus treated trauma group was stronger than that from sirolimus devoid trauma group ( with F value respectively 101. 426, 86. 533 , P values all below 0.05). (2) Compared with those of sirolimus devoid control group MHC Ⅱ (85 ± 6) % , CD40 (8 ±1)% ], the expressions of MHC II (60±9)%] and CD40 (4 ± 1 )% ] on DC surface from sirolimus devoid trauma group were significantly reduced ( with F value respectively 37.918, 40.426, P values all below 0. 05 ). The expression of MHC Ⅱ from sirolimus treated trauma group (78 ±7)%] was higher than that from sirolimus devoid trauma group ( F =37.918, P <0. 05). (3) IL-12p40, IL-12p70 secretion by DC from sirolimus devoid trauma group ( 120 ± 13) , (10 ±3) pg/mL] were significantly reduced as compared with those from sirolimus devoid control group (200 ± 25) , (20 ± 6) pg/mL, with F value respectively 218. 646, 310. 253 , P values all below 0. 05 ]. Compared with those from sirolimus devoid control (trauma) groups, IL-12p40 (560 ±34) , (540 ±29) pg/mL] , IL-12p70 (55 ±8) , (60 ± 11 ) pg/mL] secretion by DC from sirolimus treated control (trauma) groups were obviously enhanced (with F value respectively 218.646, 310.253 , P values all below 0. 01) , while IL-10 secretion levels were significantly decreased ( F =246. 108, P <0.01). Conclusions Sirolimus can partially ameliorate DC functions ex vivo in traumatized mice, and further enhance the capacity of DC in inducing T cell responses.