儿童原发性肾病综合征尿源干细胞生物学特征研究

Biological characteristics of urine-derived stem cells from paediatrics patients with primary nephrotic syndrome

摘要:

目的：探讨正常儿童尿源干细胞（urine-derived stem cell，USCs）与原发性肾病综合征（primary nephrotic syndrome，PNS）患儿尿源干细胞（p-USCs）生物学特征差异。方法：收集正常儿童及PNS患儿清洁中段尿液，分离培养USCs及p-USCs，观察细胞形态差异；CCK-8检测细胞增殖能力；流式细胞术检测细胞凋亡及干细胞表面标志物（CD24、CD29、CD34、CD73、CD90、CD105、CD146）；免疫荧光及Western blot检测细胞肾系标志物（Pax2）表达；Western blot检测细胞自噬水平。结果：PNS患儿尿液中存在2种形态p-USCs：p-USCs Ⅰ 及p-USCs Ⅱ。p-USCs Ⅰ形态与正常USCs相似：米粒样、胞体较小、排列紧密。p-USCs Ⅱ胞体可见少量空泡，胞体较大，散在排列。USCs、p-USCs Ⅰ及p-USCs Ⅱ最大传代次数分别为11代、8代及5代。与USCs比较，p-USCs增殖能力减弱，且p-USCs Ⅱ减弱更加明显（F=217.300，P=0.000）；p-USCs细胞凋亡无明显差异；p-USCs干细胞表面标志物及周细胞表面标志物表达无统计学差异；p-USCs肾系标志物表达水平下降，且p-USCs Ⅱ下降更明显（F=26.930，P=0.001）；p-USCs自噬水平下降，差异有统计学意义（F=15.060，P=0.000）。结论：PNS患儿体内存在2种不同形态尿源干细胞：p-USCs Ⅰ及p-USCs Ⅱ。与USCs 相比，p-USCs形态有差异、自我更新能力降低、肾系标志物表达下降、自噬水平减弱。

Abstract:

Objective：To investigate and compare the biological characteristics of urine-derived stem cells（USCs） from paediatrics patients with primary nephrotic syndrom（p-USCs） and normal children. Methods：The clean midstream urine was collected from nor－mal children and paediatrics patients with simple primary nephrotic syndrome. USCs and p-USCs were cultured and their morphology features were observed by optical microscopy. The cell reproductive capacity was detected by CCK-8； apoptosis difference and stem cells’ surface markers were analyzed by flow cytometery（CD24，CD29，CD34，CD73，CD90，CD105，CD146）；kidney markers（Pax2） was detected by immunofluorescence and Western blot；autophagy levels was detected by Western blot. Results：Two types of cells were found in p-USCs：p-USCs Ⅰ and p-USCs Ⅱ. p-USCs Ⅰ had the same morphology with USCs：rice-like，small cell body and closely arranged. p-USCs Ⅱ had bigger cell body，small vacuoles and was dispersedly arranged. The largest passage of USCs，p-USCsⅠ and p-USCs Ⅱ were 11，8 and 5 respectively. Compared with those of USCs，the proliferation of p-USCs was decreased，p-USCs Ⅱ in particular（F=217.300，P=0.000）；no statistic difference in the apoptotic rate and expression of stem cell surface markers show was observed between USCs and p-USCs. Compared with those of USCs，the expression of Pax2 was decreased in p-USCs，but only p-USCs Ⅱ had statistic differences（F=26.930，P=0.001）. The autophagy level was lower in p-USCs than in USCs（F=15.060，P=0.000）. Conclusion：There were two types of p-USCs in peadiatric patients with simple primary nephrotic syndrome，namely p-USCs Ⅰ and p-USCs Ⅱ. Compared with USCs，the p-USCs has different mor－phological features，lower proliferation，Pax2 expression and au－tophagy level.