羊膜间充质干细胞对严重烧伤大鼠肾脏损伤的保护作用

Protective effects of amniotic mesenchymal stem cells on early kidney injury of rats induced by severe burns

目的探讨羊膜间充质干细胞(amniotil mesenehymal stem cells,AMSCs)对严重烧伤大鼠早期肾脏损害的作用及相关作用机制。方法体外分离培养大鼠AMSCs,流式细胞仪检测细胞表面分子标记物。36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、AMSCs治疗组。处理后48 h收集3组大鼠肾脏组织标本及腹主动脉血,ELISA法检测血肌酐、尿素氮水平,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)、肿瘤坏死因子α(tumor necrosis factor-α,TNF-α)、白细胞介素1β(IL-1β)、IL-10的m RNA表达水平。结果分离培养的AMSCs传至第三代经流式细胞仪检测,均表达CD29、CD44、CD90、CD105,阳性率分别为96.4%、89.8%、94.2%、98.3%,而CD34阳性率为2.1%。单纯烧伤组大鼠血清肌酐、尿素氮含量高于假伤组(P<0.05)。AMSCs治疗组大鼠肾脏组织中血肌酐和尿素氮含量均显著低于单纯烧伤组(P<0.05)。单纯烧伤组大鼠肾脏组织中TNF-α、caspase-3、IL-1β的m RNA表达量高于假伤组的(P<0.05)。AMSCs治疗组大鼠肾脏组织中IL-1β、caspase-3、TNF-α的m RNA表达量低于单纯烧伤组(P<0.05);AMSCs处理组大鼠肾脏组织中IL-10的m RNA表达量显著高于假伤组、单纯烧伤组(P<0.05)。结论 AMSCs可显著降低烧伤大鼠肌酐、尿素氮水平,减轻肾脏组织凋亡蛋白caspase-3的表达,降低炎症因子IL-1β和TNF-α表达、促进抑炎因子IL-10的表达,可能对严重烧伤大鼠肾脏损伤具有保护作用。

Objective To explore the effect and mechanism of amniotic mesenchymal stem cells on rats＇ early kidney injury induced by severe burns. Methods Fat tissue was isolated and cultured from rats, and trypsin-collagenase digestion method was used to culture rat AMSCs. The surface marker of AMSCs was assayed with flow cytometry. Thirty-six male SD rats were divided into sham group, burn group and AMSCs treatment group with the random number table. Rats were sacrificed and kidney tissue and abdominal aortic blood were collected after 48 hours. Serum creatinine and urea nitrogen were assayed with ELISA kit. Caspase-3, TNF-c~, IL-1 [3 and IL-10 mRNA levels were detected by fluorescence RT- PCR. Results AMSCs cultured in vitro were detected by flow cytometry after three passages. Positive rates of CD29, CD44, CD90, CD105 and CD34 were 96.4%, 89.8%, 94.2%, 98.3% and 2.1% respectively. Serum creatinine and urea nitrogen levels of burn group were higher than sham group （P〈0.05）. AMSCs treatment group had a lower level of serum creatinine and urea nitrogen than burn group （P〈O.05）. Caspase-3, TNF-a, and IL-1 β mRNA levels in the kidney of burn group were higher than in sham group （P〈0.05）. In AMSCs treatment group, the levels of caspase-3, TNF-a, and IL- 1β mRNA were all lower than in burn group （P〈0.05）. However, the IL-10 level was significantly higher than insham or burn group （P〈0.05）. Conelusion AMSCs can decrease serum creatinine and urea nitrogen apoptosis of kidney cells, inhibit the expression of such inflammatory factors as IL-1βand TNF-a the expression of the anti-inflammatory factor IL-10, thus having protective effects on early kidney induced by severe burns levels, reduce and promote injury of rats