脂肪间充质干细胞对严重烧伤大鼠早期心肌损伤的保护作用

Protective effects of adipose derived mesenchymal stem cells on early myocardial injury in rats after severe burns

目的探讨脂肪间充质干细胞(ADSC)对严重烧伤大鼠早期心肌损害的作用及相关作用机制。方法 (1)取6只健康雄性SD大鼠双侧腹股沟脂肪组织,采用胶原酶消化法培养大鼠ADSC,流式细胞仪检测细胞表面分子标记物,并进行成骨、成脂诱导分化鉴定。(2)18只健康雄性SD大鼠,按照随机数字表法分为假伤组、单纯烧伤组、ADSC治疗组(每组各6只大鼠)。单纯烧伤组、ADSC治疗组大鼠背部于95℃热水浴15 s,制成30%总体表面积Ⅲ度烧伤,伤后即刻2组大鼠分别腹腔注射0.9%氯化钠10 m L(50 m L/kg)抗休克,3 h后经尾静脉注射0.9%氯化钠溶液和ADSC;假伤组大鼠仅背部于37℃水浴18 s模拟烧伤过程。均于处理后48 h收集3组大鼠心肌组织标本及腹主动脉血,酶联免疫吸附没定法检测血清肌酸激酶(CK)、乳酸脱氢酶(LDH)含量,实时荧光定量RT-PCR法检测组织中半胱氨酸天冬氨酸特异性蛋白酶-3(caspase-3)、肿瘤坏死因α(TNF-α)、白细胞介素(IL-1β)、IL-10的mRNA表达水平。对数据行单因素方差分析、LSD-t检验。结果 (1)分离培养的ADSC传至第三代时形态规则,经流式细胞仪检测,所培养的ADSC均表达CD29、CD90、CD105,阳性率分别为88.6%、99.7%、92.8%,而CD3l、CD34、CD45阳性率分别为4.4%、4.7%、3.3%;经诱导培养后可向成骨成脂分化。(2)单纯烧伤组大鼠血清CK、LDH含量分别为(1963.72±642.33)、(2732.45±215.13)U/L,高于假伤组的(347.77±72.33)、(825.36±94.26)U/L(t值分别为12.322、17.244,P值均小于0.05)。ADSC处理组CK、LDH含量(1163.09±135.47)、(2069.24±152.46)U/L低于单纯烧伤组(t值分别为-8.757、-6.556,P值均小于0.05)。(3)单纯烧伤组大鼠心肌组织中caspase-3、TNF-α、IL-1β的mRNA表达量分别为2.68±0.43、2.22±0.31、2.57±0.19,高于假伤组的1.00±0.05、1.00±0.09、1.00±0.08、(t值分别为13.242、8.651、14.332,P值均小于0.05)。ADSC处理组大鼠心肌组织中caspase-3、IL-1β、TNF-α的mRNA表达量1.41±0.08、1.37±0.05、1.12±0.04低于单纯烧伤组(t值分别为-11.257、-7.990、15.248,P值均小于0.05)。单纯烧伤组大鼠心肌组织中IL-10的mRNA表达量为0.43±0.02,低于假伤组的1.00±0.05(t=8.768,P<0.05);ADSC处理组大鼠心肌组织中IL-10的mRNA表达量为0.61±0.03,高于假伤组(t=11.446,P<0.05)。结论 ADSC可显著降低CK、LDH水平,减轻心肌组织损伤,减少心肌细胞凋亡,降低炎症因子IL-1β和TNF-α表达、促进抑炎因子IL-10的表达,对严重烧伤大鼠心脏损伤具有保护作用。

Objective To explore the effect of adipose derived mesenchymal stem cells on early myocardial damage in rats after severe burns and related mechanism. Method （1） Bilateral inguinal fat tissue was taken from 6 healthy male SD rats through eollagenase digestion. The flow cytometry instrument was used to detect the cell surface molecular to identify the osteogenesis and adipogenesis. （2） Eighteen healthy male SD rats, according to the random nmnber table method were divided into shame injury group （control）, burn group, ADSC-treatment group. The burn group and the ADSC-treatment group rats back were bathed 15 s at 95 ℃ hot water, caused a 30% TBSA Ⅲ degree burn injury. Then 10 mL （50 mL/kg） saline was injected into abdominal cavity of the rats respectively to avoid shock. Then 3 hours later, saline or ADSC were injected through tail vein. The shame injury group rats back bath in 37 ℃ water 18 s. fourty eight hours later, heart tissue specimens and blood from abdominal aortic were collected from all mice. ELISA was used to detect serum creatine kinase （CK）, lactate dehydrogenase （LDH） levels, Tissue eysteine aspartie acid proteinase 3 （ caspase -3） , TNF-α, IL- 1β, IL- 10 mRNA levels were detected by RT- PCR. Data were detected by one-way analysis of variance and LSD-t test. Results （1） ADSC were cultured to the third generation, the morphology of ADSC is fine. The positive rate of CD29, CD90 and CD105 is 88.6%, 99.7% and 92.8% respectively, while the positive rate of CD31, CD34 and CD45 is 4.4%, 4.7% and 3.3% respectively. The osteogenesis was obvious. （2） In burn group, the content of serum CK and LDH was （1963. 72±642. 33）, （2732. 45±215. 13） U/L respectively, which was significantly higher than that in the shame injury group （ 347.77±72.33 ） , （ 825.36±94.26） U/L （ t = 12. 322, 17. 244, P 〈 0.05）. CK and LDH levels of ADSC treatment group were （1163.09±135.47）U/L and （2069.24±152.46 ） U/L respectively, which were significantly lower that1 that in the shame burn group （ t = - 8. 757, - 6.556, respectively, P 〈 O. 05）. （3） In the burn group rat heart tissue, the mRNA levels of caspase-3, TNF-α, IL-1β were 2.68±0.43, 2.22±0.31, 2.57±0. 19, which were significantly higher than those in the shame injury group （1.00±0.05, 1.00±0.09, 1.00±0.09） （t = 13. 242, 8.651, 14. 332, respectively, P 〈 0.05）. In the ADSC group rat heart tissue, the mRNA levels of easpase- 3, IL-1β and TNF-α （1.41±0.08, 1.37 ±0.05, 1.12±0.04） were significantly lower than those in the burn group （ t = - 11. 257, - 7. 990, 15. 248, respectively, P 〈 0.05）. In the burn group, the mRNA level of IL- 10 （ 0.43±0.02 ） is significantly lower than that in the shame injury group （ 1. 00±0.05 ） （ t = 8. 768, P 〈 0.05 ） ; In ADSC treatment group, the mRNA level of IL- 10 （0.61±0. 03） is significantly higher than that in the false injury group （t = 11. 446, P 〈 0.05 ）. Conclusion ADSC can significantly reduce the expression of CK, LDH, IL-Iβ and TNF-α and prevent myocardial tissue injury at well as myocardial cell apoptosis. ADSC could increase the expression of inflammation suppression factor IL-10, which has a protective effect on severe burn rats＇ myocardial.