雪旺细胞促进骨髓间充质干细胞增殖的实验研究

RESEARCH ON MARROW MESENCHYMAL STEM CELL PROLIFERATION BY COCULTURING WITH SCHWANN CELL

目的通过雪旺细胞(Schwann cell,SC)与骨髓间充质干细胞(marrow mesenchymal stem cells,MSCs)复合培养,观察SC对MSCs增殖的影响,为其在构建组织工程血管中应用提供实验依据。方法选用体重40gSD大鼠,用组织块培养法获取SC,用骨髓差速贴壁法获取MSCs;用免疫组织化学法分别对两种细胞进行鉴定。用Transwell培养板复合培养两种细胞,实验组于板上层接种MSCs,下层接种SC;同时上下两层均接种MSCs设为对照组。各组均选择第1、3、5和7天4个时间点进行检测,用氚标胸腺嘧啶核苷酸标记MSCs,液体闪烁仪进行细胞计数(counts per minute,CPM)。提取实验组SC、MSCs和对照组MSCs的细胞蛋白,分别对血管内皮细胞生长因子(vascular endothelial growth factor,VEGF)和其受体Flk-1,协同受体1(neuropilin-1,NRP-1)进行Westernblot检测。结果SC呈梭形,表达S-100蛋白,阳性率达90%。MSCs表达CD105和CD44,不表达CD34和CD45。Transwell复合培养:MSCs,第1、3、5和7天增殖数量分别为2411.00±270.84、3016.17±241.57、6570.83±2848.27、6375.83±1431.28,明显大于对照组2142.17±531.63、2603.33±389.64、2707.50±528.55、2389.00±908.01,差异有统计学意义(P<0.05),实验组CPM值于第5天最高。于复合培养第5天Westernblot显示:SC表达VEGF、Flk-1和NRP-1;VEGF、Flk-1、NRP-1在实验组MSCs中表达明显强于对照组。结论两种细胞体外复合培养后,SC在第1、3、5和7天均能显著增加MSCs的数量,并促进增殖,增殖高峰在第5天。与SC复合培养后,MSCs表达VEGF明显增加,其相应受体Flk-1、NRP-1表达上调,SC通过促进MSCs分泌VEGF增加参与了细胞的增殖过程。

Objective To evaluate the effect of Schwann cell （SC） on the proliferation of marrow mesenchymal stem cells （MSCs） and provide evidence for application of SC in construction of the tissue engineered vessels. Methods SC and MSCs were harvested from SD rats （weight 40 g）. SC were verified immunohistochemically by the S-100 staining, and MSCs were verified by CD 44, CD 105, CD 34 and CD 45. The 3rd passages of both the cells were cocuhured in the Transwell system and were amounted by the 3H-TDR integration technique at 1, 3, 5 and 7 days, respectively. The results were expressed by the CPM（counts per minute, CPM） values. However, MSCs on both the layers were served as the controls. The Western blot was performed to assess the expression of the vascular endothelial growth factor （VEGF）, its receptor Flk-1, and the associated receptor neuropilin 1 （NRP-1） in SC, the trial cells, and the controls. Results SC had a spindle shape in the flasks, and more than 90% of SC had a positive reaction for the S- 100 staining. MSCs expressed CD44 and CD105, and had a negative signal in CD 34 and CD 45. The CPM values of MSCs in the trial groups were 2 411.00±270.84,3 016.17±241.57,6 570.83±2 848.27 and 6 375.8±431.28 at 1, 3, 5 and 7 days, respectively. They were significantly higher in their values than the control group （2 142.17±531.63, 2 603.33±389.64,2 707.50±328.55,2 389.00±908.01）, especially at 5 days （P〈0.05）. The Western blot indicated that VEGF was expressed obviously in both the SC group and the cocuhured MSCs grou,p and was less visible in the control cells. The expressions of Flk-1 and NRP-1 in the cocultured MSCs were much stronger than in the controls. Conclusion SC can significantly promote the proliferation of MSCs when they are cocuhured. The peak time of the proliferation effect appeared at 5 days. This effect may be triggered by the up-regulation of VEGF in MSCs, which also leads to the upregulation of Flk-1 and NRP-1.