骨髓间充质干细胞在重度烧伤大鼠心肌损伤中的保护作用

The protection effects of bone marrow stromal stem cells on rats early myocardial injury induced by severe burns

目的探讨骨髓间充质干细胞(BMSCs)对严重烫伤大鼠心肌的保护作用及作用机制。方法 (1)取10只健康雄性SD大鼠,采用全骨髓贴壁法体外分离大鼠BMSCs,流式细胞仪检测细胞表面分子标志物。(2)24只健康雄性SD大鼠,随机分为:对照组、模型组、治疗组。模型组及治疗组大鼠背部暴露于95℃热水浴18 s造成30%TBSAⅢ度烫伤,对照组暴露于37℃水浴18 s。水浴后即刻模型组及治疗组大鼠分别腹腔注射生理盐水10 mL(50 mL/kg),水浴后3 h模型组经尾静脉注射100μL生理盐水,治疗组经尾静脉注射100μL(2.5×107/mL)BMSCs。处理后48 h收集3组大鼠心脏组织标本及腹主动脉血,ELISA法检测血清肌酸激酶(CK)、乳酸脱氢酶(LDH)含量,实时荧光定量聚合酶链式反应(RT-PCR)法检测心脏组织中炎性细胞因子TNF-α、IL-1β、IL-10及凋亡相关分子caspase-3、Bcl-2、Bax的mRNA表达水平。结果流式细胞术检测提示,所培养的BMSCs表达CD44、CD90、CDl05的阳性率分别为96.8%、99.72%、95.93%,而CD34、CD45阳性率分别为1.42%、2.17%;模型组大鼠血清CK、LDH含量显著高于对照组(P<0.05)。治疗组CK、LDH含量显著低于模型组(P<0.05)。模型组大鼠心脏组织中caspase-3、Bax的mRNA表达量显著高于对照组(P<0.05)。治疗组大鼠心脏组织中caspase-3、Bax的m RNA表达量显著低于模型组(P<0.05)。模型组大鼠心脏组织中Bcl-2的mRNA表达量为显著低于对照组(P<0.05);治疗组大鼠心脏组织中Bcl-2的mRNA表达量显著高于对照组(P<0.05)。模型组大鼠心脏组织中TNF-α、IL-1β的mRNA表达量显著高于对照组(P<0.05)。治疗组大鼠心脏组织中IL-1β、TNF-α的m RNA表达量显著低于模型组(P<0.05)。模型组大鼠心脏组织中IL-10的mRNA表达量显著低于对照组(P<0.05);治疗组大鼠心脏组织中IL-10的mRNA表达量显著高于模型组(P<0.05)。结论 BMSCs可显著降低烧伤后大鼠CK、LDH水平,减轻心肌组织损伤,减少心肌细胞凋亡,降低炎性因子IL-1β和TNF-α表达、促进抑炎因子IL-10的表达,对严重烧伤大鼠心肌具有保护作用。

Objective To explore the effects and related mechanism of bone marrow stromal stem cells （BMSCs） on protecting rats myocardial injury induced by severe bums. Methods （1）Ten healthy male SD （Sprague - Dawley）rats were used to separate BMSCs through whole bone marrow adherent. The biological markers were assayed with flowcytometry. （2）Twenty-four healthy male SD rats were randomly divided into control group, model group and treatment group. Except for control group, abdominal skin of rats were exposed to 95℃ water for 18 seconds which led to the third degree, 30% TBSA burns while the rats in control group were exposed to 37℃ water for 18 seconds. Rats in burn model group and treatment group were given 10 mL（50 mL/kg） saline intraperitoneally immediately after burns. Three hours later,Saline （100 μL）or BMSCs （100 μL,2.5×107/mL） were given through caudal vein. Forty-eight later, rats were sacrificed and the myocardium and blood from aorta abdominalis were collected. The expression levelsof creatine kinase （CK）, lactic dehydrogenase （LDH） were detected by ELISA. The mRNA level of caspase-3, Bcl-2, Bax, TNF-α, IL-1β and IL-10 were detected by RT-PCR. Results The positive rates of CD44,CD90,CD105 were 96.8%, 99.72% and 95.93% separately in BMSCs. However, the positive rates of CD34 and CD45 were 1.42% and 2.17% separately. The expression level of CK and LDH in rats in model group were significantly higher than that in control group （P 〈 0.05）. The expression level of CK and LDH in treatment group were higher than those in control group （P 〈 0.05）. The mRNA level of caspase-3 and Bax in the myocardium in treatment group were significantly decreased compared with control group （P 〈 0.05）. The mRNA level of Bcl-2 in myocardium of treatment group was higher than that in control group （P 〈 0.05）. The mRNA levels of TNF-α and IL-1β in model group were higher than that in control group （P 〈 0.05）. However, in treatment group, these two were lower than in the model group （P 〈 0.05）. The mRNA level of IL-10 was significantly lower than that in control group （P 〈 0.05）. While in treatment group, the mRNA level of IL-10 was significantly higher than that in model group （P 〈 0.05）. Conclusion BMSCs could protect against myocardial injury induced by severe burns. The expression of CK and LDH decreased, indicating that the injury of myocardial decreased. During which, the inflammatory factors, IL-1β and TNF-α decreased and anti-inflammatory factor IL-10 increased.