骨髓间充质干细胞移植在小鼠急性肾损伤肾内的存活与分化情况研究

Survival and Differentiation of Transplanted Bone Marrow Mesenchymal Stem Cells in Kidneys of Acute Renal Injury Mice

为探讨急性肾损伤后,移植骨髓间充质干细胞(marrow mesenchymal stem cells,MSCs)在损伤肾内的存活能力及分化情况,通过复苏、扩增培养已完成鉴定、经慢病毒EGFP转染的MSCs,观察备用MSCs的EGFP(enhanced green fluorescent protein)表达情况,采用雄性C57BL/6J小鼠12只建立小鼠肾脏缺血再灌注损伤模型后(结扎双侧肾蒂40 min后开放血流并经尾静脉注射MSCs),分别于第1、3、7、14 d处死3只小鼠,采集小鼠左侧肾脏制作石蜡切片,倒置荧光显微镜下观察MSCs在肾内的存活及分化情况,定量分析每个时间点存活于肾内的数目的差异.结果表明复苏、扩增培养出的MSCs增殖活力旺盛,成功建立小鼠肾脏缺血再灌注损伤模型.移植的细胞随时间的延长,存活于肾脏内的数量显著减少(第14 d存活于肾内的细胞数量仅为移植后第1d的1/5),第1、3、7、14 d表达EGFP的MSCs主要分布于肾小球周围、肾脏小血管内壁、肾小管与肾小管之间的间质而肾小管内壁未见到表达EGFP的MSCs分布.这说明移植的MSCs在缺血再灌注损伤后肾脏内能够存活,但肾脏内的微环境限制了移植细胞的存活能力.在肾小管内壁未观测到表达EGFP的MSCs,提示MSCs对肾脏修复的途径不是直接向肾小管内皮细胞分化而另有其它途径.

To observe the survival and differentiation capacity of bone marrow mesenchymal stem cells（BM-SCs） transplanted in kidneys of acute renal injury mice.MSCs that had been transfected by enhanced green fluorescent protein（EGFP） were thawed and expanded,The expression of EGFP in MSCs was observed be-fore injection.Total 12 healthy male C57BL/6J mice were used to establish middle of ischemia and reperfu-sion injury model（After clamping bilateral renal pedicles and then reopening after 40 minutes.Meanwhile,mouse MSCs were injected through caudal veins into mice）.On day 1,day 3,day 7 and day 14 after the model establishment of the mode,three mice were killed,whose left kidneys were prepared for paraffin sec-tion.Observing the survival and differentiation of MSCs in the kidneys at different time points and quantita-tively analysing the number of survival MSCs at various time points after transplantation.The results showed that MSCs maintain biological activities of cells with vibrant proliferation and features after thawed and ex-panded.The modle of ischemia and reperfusion injury was established successfully.The number of survival MSCs decreased markedly as the extension of time,and day 1,day 3,day 7 and day 14 the MSCs which ex-pressing EGFP were detected in the visceral and parietal of the glomerulus,wall of small vessel,and mes-enchyma between renal tubule suggested that MSCs could survive in kidneys after ischemia and reperfusion injury,but the microenvironment in kidneys limit the survivability of MSCs,EGFP-positive cells were not detected in the lumens of renal tubule indicating that the recovery of renal function was not due to MSCs differentiate into renal tubular epithelial cells,there are other ways.