Cheng Xiao-Xia, Yang Qiao-Yan, Qi Yong-Li, Liu Zhi-Zhen, Liu Dan, He Sheng, Yang Li-Hong, Xie Jun
Department of Biochemistry and Molecular Biology, Shanxi Medical University, Taiyuan, Shanxi 030001, China.
The First Affiliated Hospital of Shanxi Medical University, Taiyuan, Shanxi 030001, China.
Chronic Dis Transl Med. 2019 Mar 19;5(1):53-63. doi: 10.1016/j.cdtm.2019.02.002. eCollection 2019 Mar.
The aim of this study was to investigate the effect and possible mechanism of action of roof plate-specific spondin1 (Rspo1) in the apoptosis of rat bone marrow mesenchymal stem cells (BMSCs).
Osteogenic and adipogenic differentiation of BMSCs was identified by Alizarin Red and Oil Red O staining, respectively. BMSC surface markers (cluster of differentiation 29 [CD29], CD90, and CD45) were detected using flow cytometry. BMSCs were transfected with an adenoviral vector encoding (BMSCs-Rspo1 group). The expression levels of gene and Rspo1 protein in the BMSCs-Rspo1 group and the two control groups (untransfected BMSCs group and BMSCs-green fluorescent protein [GFP] group) were analyzed and compared by quantitative polymerase chain reaction and Western blot. The occurrence of apoptosis in the three groups was detected by flow cytometry and acridine orange-ethidium bromide (AO-EB) double dyeing. The activity of the Wnt/β-catenin signaling pathway was evaluated by measuring the expression levels of the key proteins of the pathway (β-catenin, c-Jun N-terminal kinase [JNK], and phospho-JNK).
Osteogenic and adipogenic differentiation was confirmed in cultured BMSCs by the positive expression of CD29 and CD90 and the negative expression of CD45. Significantly increased expression levels of Rspo1 protein in the BMSCs-Rspo1 group compared to those in the BMSCs (0.60 ± 0.05 . 0.13 ± 0.02; =95.007, =0.001) and BMSCs-GFP groups (0.60 ± 0.05 . 0.10 ± 0.02; =104.842, =0.001) were observed. The apoptotic rate was significantly lower in the BMSCs-Rspo1 group compared with those in the BMSCs group ([24.06 ± 2.37]% . [40.87 ± 2.82]%; = 49.872, = 0.002) and the BMSCs-GFP group ([24.06 ± 2.37]% . [42.34 ± 0.26]%; = 62.358, = 0.001). In addition, compared to the BMSCs group, the protein expression levels of β-catenin (2.67 ± 0.19 . 1.14 ± 0.14; = -9.217, = 0.000) and JNK (1.87 ± 0.17 . 0.61 ± 0.07; = -22.289, = 0.000) were increased in the BMSCs-Rspo1 group. Compared to the BMSCs-GFP group, the protein expression levels of β-catenin (2.67 ± 0.19 . 1.44 ± 0.14; = -5.692, = 0.000) and JNK (1.87 ± 0.17 . 0.53 ± 0.06; = -10.589, = 0.000) were also upregulated in the BMSCs-Rspo1 group. Moreover, the protein expression levels of phospho-JNK were increased in the BMSCs-Rspo1 group compared to those in the BMSCs group (1.89 ± 0.10 . 0.63 ± 0.09; = -8.975, = 0.001) and the BMSCs-GFP group (1.89 ± 0.10 . 0.69 ± 0.08; = -9.483, = 0.001).
The Wnt/β-catenin pathway could play a vital role in the Rspo1-mediated inhibition of apoptosis in BMSCs.
本研究旨在探讨顶板特异性促分泌素1（Rspo1）对大鼠骨髓间充质干细胞（BMSCs）凋亡的影响及其可能的作用机制。
分别采用茜素红和油红O染色鉴定BMSCs的成骨和成脂分化。使用流式细胞术检测BMSC表面标志物（分化簇29 [CD29]、CD90和CD45）。用编码Rspo1的腺病毒载体转染BMSCs（BMSCs-Rspo1组）。通过定量聚合酶链反应和蛋白质印迹法分析并比较BMSCs-Rspo1组与两个对照组（未转染BMSCs组和BMSCs-绿色荧光蛋白[GFP]组）中Rspo1基因和蛋白的表达水平。采用流式细胞术和吖啶橙-溴化乙锭（AO-EB）双染法检测三组细胞凋亡的发生情况。通过检测该信号通路关键蛋白（β-连环蛋白、c-Jun氨基末端激酶[JNK]和磷酸化JNK）的表达水平来评估Wnt/β-连环蛋白信号通路的活性。
通过CD29和CD90的阳性表达以及CD45的阴性表达证实培养的BMSCs具有成骨和成脂分化能力。与BMSCs组（0.60±0.05比0.13±0.02；t = 95.007，P = 0.001）和BMSCs-GFP组（0.60±０.05比0.10±0.02；t = 104.842，P = 0.０01）相比，BMSCs-Rspo1组中Rspo1蛋白表达水平显著升高。与BMSCs组（[24.06±2.37]%比[40.87±2.82]%；t = 49.872，P = 0.002）和BMSCs-GFP组（[24.06±2.37]%比[42.34±0.26]%；t =６2.358，P = 0.001）相比，BMSCs-Rspo1组的凋亡率显著降低。此外，与BMSCs组相比，BMSCs-Rspo1组中β-连环蛋白（2.6７±0.19比1.14±0.14；t = -9.217，P = 0.000）和JNK（1.87±0.17比0.61±0.07；t = -22.289，P = 0.000）的蛋白表达水平升高。与BMSCs-GFP组相比，BMSCs-Rspo1组中β-连环蛋白（2.67±0.19比1.44±0.14；t = -5.692，P = 0.000）和JNK（1.87±0.17比0.53±0.06；t = -10.589，P = 0.000）的蛋白表达水平也上调。此外，与BMSCs组（1.89±0.10比0.63±0.09；t = -8.975，P = 0.001）和BMSCs-GFP组（1.89±0.10比0.69±0.08；t = -9.483，P = 0.001）相比,BMSCs-Rspo1组中磷酸化JNK的蛋白表达水平升高。
Wnt/β-连环蛋白通路可能在Rspo1介导的抑制BMSCs凋亡中起关键作用。
