干细胞外泌体减轻高糖腹膜透析液诱导的腹膜炎症

Stem cell-derived exosomes alleviated peritonitis induced by high glucose-based peritoneal dialysis dialysate

目的探讨间充质干细胞外泌体是否可减轻高糖腹膜透析液诱导的腹膜炎。方法选取雄性6~8周龄C57BL/6小鼠24只,体重20~25 g,随机分成4组:正常对照组、生理盐水组、4.25%含糖腹透液组、4.25%含糖腹透液+外泌体组。正常对照组不采取任何干预措施;生理盐水组每次腹腔内注射0.9%生理盐水0.1 ml/g;4.25%含糖腹透液组每次腹腔注射4.25%含糖腹透液0.1 ml/g;4.25%含糖腹透液+外泌体组每次腹腔注射4.25%含糖腹透液0.1 ml/g,并在第28天和35天腹腔注射外泌体200μg/kg。以上各组小鼠腹腔注射0.9%生理盐水或4.25%含糖腹透液每日2次,连续6周。6周后,评估小鼠腹膜转运及超滤功能;取壁层腹膜行PAS染色,评估腹膜增厚程度;免疫组化检测MPO、巨噬细胞标志F4/80、CD68,评估炎症细胞浸润情况;实时定量RT-PCR检测IL-6、IL-1β、TNF-α,评估炎症因子表达水平;免疫组化检测GSL-1,评估微血管及毛细血管新生程度。结果4.25%含糖腹透液组腹膜厚度显著高于正常对照组和生理盐水组(P<0.01)。与4.25%含糖腹透液组相比,4.25%含糖腹透液+外泌体组的腹膜增厚程度、腹膜间皮中性粒细胞和巨噬细胞数量、腹膜间皮新生血管数量、以及腹膜炎症因子(IL-6、IL-1β、TNF-α)的mRNA表达水平均明显下降(P<0.05)。与正常对照组、生理盐水组、4.25%含糖腹透液+外泌体组相比,4.25%含糖腹透液组小鼠的腹膜功能表现为葡萄糖平衡作用快、尿素氮清除力强,但超滤功能显著下降(P<0.05)。结论干细胞外泌体可有效减轻高糖腹膜透析液诱导的腹膜炎症水平,改善腹膜超滤功能。

Objective To explore whether mesenchymal stem cell-derived exosomes can reduce the peritonitis induced by high glucose-based peritoneal dialysate.Methods Twenty-four male C57BL/6 mice aged 6-8 weeks,weighing 20-25 g,were randomly divided into 4 groups:normal control group,normal saline group,4.25%glucose-containing peritoneal dialysate group,and 4.25%glucose-containing peritoneal dialysate plus exosomes group.The normal control group was not given any treatment.The normal saline group was given intraperitoneal injection of 0.9%normal saline(0.1 ml/g twice a day).The 4.25%glucose-containing peritoneal dialysate group was given intraperitoneal injection of 4.25%glucose-containing peritoneal dialysate(0.1 ml/g twice a day).The 4.25%glucose-containing peritoneal dialysate plus exosomes group was given intraperitoneal injection of both 4.25%glucose-containing peritoneal dialysate(0.1 ml/g twice a day)and exosomes(200μl/kg on the 28th and 35th days).After six weeks of the intraperitoneal injection,the peritoneal transport and ultrafiltration function of the mice were evaluated.The parietal peritoneum samples were taken for PAS staining to assess the degree of peritoneal thickening.Immunohistochemistry methods were applied to detect the levels of MPO,F4/80,and CD68 for the assessment of inflammatory cell infiltration.Real-time quantitative PCR was used to detect the expression of inflammatory factors as IL-6,IL-1β,and TNF-α.GSL-1 was detected by immunohistochemistry for evaluating the degree of angiogenesis of microvessels and capillaries.Results The peritoneal thickness of the 4.25%glucose-containing peritoneal dialysate group was significantly higher than that of the normal control group and the normal saline group(P<0.01).Compared with the 4.25%glucose-containing peritoneal dialysate group,the 4.25%glucose-containing peritoneal dialysate plus exosomes group showed lower levels in peritoneal thickness,the number of peritoneal mesothelial neutrophils and macrophages,the degree of peritoneal mesothelial neovascularization,and the mRNA expression of peritoneal inflammatory factors(IL-6,IL-1β,TNF-α)(P<0.05).Compared with the normal control group,the normal saline group,and the 4.25%glucose-containing peritoneal dialysate plus exosomes group,the 4.25%glucose-containing peritoneal dialysate group′s peritoneal function manifested a faster balance of glucose and stronger clearance of urea nitrogen,and a lower ultrafiltration function(P<0.05).Conclusion Stem cell-derived exosomes could significantly reduce the level of peritonitis induced by high glucose-based peritoneal dialysate,and improve the peritoneal ultrafiltration function.