Department of Cardiovascular Surgery, Renji Hospital, Shanghai Jiaotong University School of Medicine, Shanghai 200127, PR China.
Int J Mol Med. 2013 Oct;32(4):805-12. doi: 10.3892/ijmm.2013.1445. Epub 2013 Jul 16.
The aim of this study was to investigate the expression of nesprin‑1 protein and its effects on rat bone marrow mesenchymal stem cells (MSCs). MSCs were cultured in DMEM and surface-associated antigens of MSCs were detected by flow cytometry. The protein expression of nesprin-1 was detected by immunofluorescence and western blot analysis. A lentiviral vector expressing small interfering RNA (siRNA) targeting nesprin-1 was constructed (LV-siNesprin-1) and the MSCs were subsequently transfected with this vector. Another group of MSCs was transfected with the LV-GFP vector and another group of untransfected cells was used as the controls (normal group). The protein expression level of nesprin-1 in the 3 groups of MSCs (LV-siNesprin-1, LV-GFP and normal group) was measured by western blot analysis. Cell proliferation was assessed by MTT assay, and the cell cycle and apoptosis were detected by flow cytometry. DAPI was used to stain the nucleus of the MSCs. The MSCs appeared spindle-shaped with irregular processes and were positive for CD90, CD29 and negative for CD45. Nesprin-1 protein was found in the nuclear membrane. The protein expression of nesprin-1 in the LV-siNesprin-1 group was lower than that in the LV-GFP (P=0.03) and normal group (P=0.028); this difference was significant (P<0.05). The cell proliferation of the MSCs transfected with LV-siNesprin-1 was reduced; the apoptotic rate was higher in the LV-siNesprin-1 group compared with the other 2 groups (LV-GFP and normal group) (P=0.032, P=0.025, respectively; P<0.05). The changes in the morphology of the nucleus in the LV-siNesprin-1 group included fusion and fragmentation. In conclusion, the data presented in this study indicate that nesprin-1 regulates the proliferation and apoptosis of MSCs; our results are consistent with those from previous studies. Thus, nesprin-1 protein plays an important role in the proliferation and apoptosis of MSCs.
本研究旨在探讨核膜蛋白 nesprin-1 的表达及其对大鼠骨髓间充质干细胞（MSCs）的影响。将 MSCs 培养于 DMEM 中，并通过流式细胞术检测 MSCs 的表面相关抗原。通过免疫荧光和 Western blot 分析检测 nesprin-1 的蛋白表达。构建了表达靶向 nesprin-1 的小干扰 RNA（siRNA）的慢病毒载体（LV-siNesprin-1），并将该载体转染 MSCs。将另一组 MSCs 转染 LV-GFP 载体，另一组未转染的细胞作为对照（正常组）。通过 Western blot 分析测量 3 组 MSCs（LV-siNesprin-1、LV-GFP 和正常组）中 nesprin-1 的蛋白表达水平。通过 MTT 测定评估细胞增殖，通过流式细胞术检测细胞周期和凋亡。用 DAPI 染色 MSCs 的细胞核。MSCs 呈纺锤形，有不规则突起，CD90、CD29 阳性，CD45 阴性。核膜中存在 nesprin-1 蛋白。LV-siNesprin-1 组 nesprin-1 的蛋白表达低于 LV-GFP 组（P=0.03）和正常组（P=0.028）；差异具有统计学意义（P<0.05）。转染 LV-siNesprin-1 的 MSCs 增殖减少；LV-siNesprin-1 组的凋亡率高于其他 2 组（LV-GFP 和正常组）（P=0.032，P=0.025；P<0.05）。LV-siNesprin-1 组细胞核形态变化包括融合和碎片化。综上所述，本研究的数据表明 nesprin-1 调节 MSCs 的增殖和凋亡；我们的结果与之前的研究一致。因此，nesprin-1 蛋白在 MSCs 的增殖和凋亡中发挥重要作用。
