免疫调理联合骨髓间充质干细胞改善大鼠脓毒症所致肺损伤

Immunomodulation combined with bone marrow mesenchymal stem cells improves sepsis-induced acute lung injury

目的探讨蛋白酶抑制剂(乌司他丁)预处理后,骨髓间充质干细胞(BMSCs)改善脓毒症所致肺损伤的机制及治疗时机。方法采用盲肠末端置管持续引流(CASP)建立雄性SD大鼠脓毒症模型40只,随机分为5组:假手术组(A组)、脓毒症组(B组)、乌司他丁同时联合骨髓间充质干细胞(BMSCs)干预组(C组)、乌司他丁18 h后联合BMSCs干预组(D组)、乌司他丁66 h后联合BMSCs干预组(E组);采用苏木精-伊红(HE)染色,免疫组织化学染色及Western blot方法检测肺组织病理改变、肺泡表面活性蛋白C、E-钙黏蛋白的表达;采用生存曲线分析大鼠的生存率。结果各组大鼠生存率比较差异无统计学意义(P>0.05);各组大鼠肺组织病理评分:A组2.92±0.43、B组14.22±1.92、C组8.67±1.21、D组7.50±1.76、E组10.80±1.47,各治疗组大鼠肺组织病理损伤比脓毒症组轻,差异有统计学意义(P<0.05),E组肺组织损伤比C组、D组严重,差异有统计学意义(P<0.05);各组大鼠肺泡表面活性蛋白C表达灰度值:A组1.99±0.10、B组0.44±0.03、C组1.55±0.05、D组1.97±0.13、E组1.43±0.08,各治疗组大鼠肺泡表面活性蛋白C表达比脓毒症组高,差异有统计学意义(P<0.05);各治疗组大鼠肺泡上皮E-钙黏蛋白表达比脓毒症组高,差异有统计学意义(P<0.05),但各治疗组组间比较差异无统计学意义(P>0.05)。结论免疫调理后18 h内联合骨髓间充质干细胞治疗后大鼠脓毒症所致肺损伤程度最轻。可能机制是增加肺泡表面活性蛋白C的合成,抑制肺组织E-钙黏蛋白丢失,改善肺组织微环境,延缓脓毒症所致急性肺损伤肺纤维化过程。

Objective To explore the appropriate treatment opportunity and the possible mechanism of ulinastatin combined with bone marrow mesenchymal stem cells (BMSCs) improving sepsis-induced acute lung injury. Methods The cecum end catheter continuous drainage (CASP) was used to establish septic model in 40 male SD rats weighing 180-220 g. The animals were randomly divided into 5 groups (n=8 each group): control (A), septic group (B), ulinastatin combined with BMSCs treatment group at the same time (group C), BMSCs intervention 18 h after administration of ulinastatin (group D), BMSCs intervention 66 h after administration of ulinastatin (group E). The hematoxylin and eosin (HE) staining, immunohistochemistry and Western blotting were applied to examine the lung tissue pathologic changes, and alveolar surface activated protein C and E-calcium expression of mucin respectively. The survival curve was used to expore the survival rate. Results The survival rate among groups had no statistically significant difference (P>0.05). Pathological scores of rat lung tissues in groups A-E were 2.92±0.43, 14.22±1.92, 8.67±1.21, 7.50±1.76 and 10.80±1.47 respectively. The pathological injury of lung tissue in the intervention groups was milder than in group B (P<0.05), and that in group E was worse than in group C and group D (P<0.05). The gray scale values of protein expression of alveolar surface in groups A-E were 1.99±0.10, 0.44±0.03, 1.55±0.05, 1.97±0.13, and 1.43±0.08 respectively. The protein expression of alveolar surface activated protein C in the intervention group was significantly higher than in group B (P<0.05). The expression of E-calcium in mucin was significantly higher in the intervention groups than in group B, but there was no significant difference among the intervention groups (P>0.05). Conclusion After immunomodulation, the lung injury caused by sepsis in rats was the mildest within 18 h. The possible mechanism is contributed to the increases in the synthesis of alveolar surface active protein C, and inhibition of the loss of e-cadherin in lung tissue, which can improve the microenvironment of the lung tissue, and delay the lung fibrotic process of sepsis-induced acute lung injury.