Effects of extracellular heat shock protein 70 on intestinal immune function of rats with severe scald injury
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摘要: 目的 了解严重烫伤大鼠肠道细胞外热休克蛋白70(eHSP70)与IL-2表达变化及两者间的关系,另体外观察eHSP70对严重烫伤大鼠肠道派伊尔结CD3+T淋巴细胞的影响。 方法 (1)取60只雄性SD大鼠,按随机数字表法分为正常对照组(仅麻醉)10只、烫伤组50只,烫伤组大鼠背部造成30%TBSAⅢ度烫伤。正常对照组于麻醉后即刻,烫伤组于伤后3、6、12、24、48 h,各取10只大鼠处死,取小肠组织,ELISA法检测eHSP70和IL-2的表达并进行相关分析。(2)另取2只雄性SD大鼠,同前致伤后12 h,分离小肠派伊尔结CD3+T淋巴细胞,用含体积分数10%FBS的RPMI 1640培养液培养,按随机数字表法分为空白对照组和5、10、20 μg/mL eHSP70组,每组6孔。空白对照组不行其他处理,后3组培养液中分别加入相应质量浓度重组大鼠eHSP70。培养48 h,流式细胞仪检测CD3+T淋巴细胞中Th1和Th2百分比及细胞凋亡率,ELISA法检测细胞培养上清液中IL-2和IL-10的表达。细胞实验均重复10次。对数据行单因素方差分析、Kruskal-Wallis秩和检验、SNK-
q 检验、Pearson相关分析。 结果 (1)与正常对照组的(1 278±135)、(48.6±4.9)ng/mg比较,烫伤组大鼠伤后3、6、12、24、48 h肠道eHSP70[(728±93)、(412±31)、(314±21)、(528±40)、(1 028±97)ng/mg]和IL-2[(38.6±2.3)、(32.3±1.0)、(25.3±3.6)、(33.9±4.1)、(44.3±2.6)ng/mg]水平均明显降低(q 值为3.48~5.32,P 值均小于0.05),均于伤后12 h达最低值。IL-2和eHSP70水平呈明显正相关(r =0.920,P <0.01)。(2)与空白对照组的(8.6±1.1)%、(3.75±0.45)%比较,5、10、20 μg/mL eHSP70组CD3+T淋巴细胞中Th1百分比明显增高[(11.3±2.1)%、(15.7±1.8)%、(10.8±1.5)%,q 值为2.97~4.57,P 值均小于0.05],Th2百分比明显降低[(2.39±0.38)%、(1.05±0.23)%、(2.67±0.26)%,q 值为2.48~4.32,P 值均小于0.05]。与空白对照组的(34.3±2.2)%、(254±16)pg/mL比较,5、10、20 μg/mL eHSP70组CD3+T淋巴细胞凋亡率明显降低[(26.1±2.6)%、(20.7±1.5)%、(31.5±2.4)%,q 值为3.47~4.95,P 值均小于0.05],细胞培养上清液中IL-2水平明显增高[(417±22)、(587±19)、(307±27)pg/mL,q 值为3.02~4.98,P 值均小于0.05]。4组CD3+T淋巴细胞培养上清液中IL-10水平相近(F =2.12,P >0.05)。 结论 大鼠严重烫伤后肠道中eHSP70和IL-2的表达均降低,且两者呈明显正相关。eHSP70可体外促进严重烫伤大鼠肠道派伊尔结中CD3+T淋巴细胞向Th1方向分化,明显降低其凋亡率,促进其释放IL-2。-
关键词:
- 烧伤 /
- 肠 /
- 免疫 /
- T淋巴细胞亚群 /
- 细胞外热休克蛋白70
Abstract: Objective To explore the change in the expression of extracellular heat shock protein 70 (eHSP70) and interleukin 2 (IL-2) and their correlation in intestine of rats with severe scald injury, and to observe the effects of eHSP70 on CD3+ T lymphocytes in Peyer's patch of intestine in rats with severe scald injury in vitro. Methods (1) Sixty male SD rats were divided into normal control group (NC,n =10, only anesthetized) and scald group (S,n =50) according to the random number table. Rats in scald group were inflicted with 30% total body surface area full-thickness scald on the back. Ten rats from group NC immediately after anesthetization and 10 rats from group S at post injury hour (PIH) 3, 6, 12, 24, 48 were sacrificed to harvest their small intestines. The expressions of eHSP70 and IL-2 were determined with enzyme-linked immunosorbent assay (ELISA), and their correlation was analyzed. (2) Another 2 male SD rats were inflicted with the same injury as above. At PIH 12, CD3+ T lymphocytes in Peyer's patch of small intestine were isolated and cultured with RPMI 1640 nutrient solution containing 10% fetal bovine serum. Cells were divided into blank control group (BC) and 5, 10, 20 μg/mL eHSP70 groups according to the random number table, with 6 wells in each group. Cells in group BC didn't receive any other treatment, while cells in the latter three groups were treated with corresponding mass concentration of recombinant rat eHSP70. After being cultured for 48 hours, the proportions of Th1 and Th2 in CD3+ T lymphocytes, and the apoptosis rate of CD3+ T lymphocytes were detected with flow cytometer, while the expressions of IL-2 and IL-10 in culture supernatant of cells were determined with ELISA. The cell experiments were repeated for 10 times. Data were processed with one-way analysis of variance, Kruskal-Wallis rank sum test, SNK-q test, and Pearson correlation analysis. Results (1) Compared with those in group NC [(1 278±135) and (48.6±4.9) ng/mg], the levels of eHSP70 [(728±93), (412±31), (314±21), (528±40), (1 028±97) ng/mg] and IL-2 [(38.6±2.3), (32.3±1.0), (25.3±3.6), (33.9±4.1), (44.3±2.6) ng/mg] in intestine of rats in group S obviously decreased at PIH 3, 6, 12, 24, 48 (withq values from 3.48 to 5.32,P values below 0.05), reaching the nadir both at PIH 12, with a significantly positive correlation between the level of IL-2 and the level of eHSP70 (r =0.920,P <0.01). (2) Compared with those in group BC [(8.6±1.1)% and (3.75±0.45)%], the proportion of Th1 obviously increased [(11.3±2.1)%, (15.7±1.8)%, (10.8±1.5)%, withq values from 2.97 to 4.57,P values below 0.05], while the proportion of Th2 obviously decreased [(2.39±0.38)%, (1.05±0.23)%, (2.67±0.26)%, withq values from 2.48 to 4.32,P values below 0.05] in CD3+ T lymphocytes of rats in 5, 10, 20 μg/mL eHSP70 groups. Compared with those in group BC [(34.3±2.2)% and (254±16) pg/mL], the apoptosis rate of CD3+ T lymphocytes obviously decreased [(26.1±2.6)%, (20.7±1.5)%, (31.5±2.4)%, withq values from 3.47 to 4.95,P values below 0.05], while the level of IL-2 obviously increased [(417±22), (587±19), (307±27) pg/mL, withq values from 3.02 to 4.98,P values below 0.05] in culture supernatant of CD3+ T lymphocytes of rats in 5, 10, 20 μg/mL eHSP70 groups. There was no significant difference in the level of IL-10 in culture supernatant of CD3+ T lymphocytes of rats among the four groups (F =2.12,P >0.05). Conclusions The expressions of eHSP70 and IL-2 in intestine of rats are decreased after severe scald, with a obviously positive correlation between them. eHSP70 can promote the differentiation of CD3+ T lymphocytes in Th1 orientation, decrease the apoptosis rate of the cells, and promote the release of IL-2 of cells in Peyer's patch of intestine in rats with severe scald injury in vitro.-
Key words:
- Burns /
- Intestines /
- Immunity /
- T-lymphocyte subsets /
- Extracellular heat shock protein 70
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