血小板增强骨髓间充质干细胞促肿瘤转移作用

Platelets promote the enhanced tumor metastasis of bone marrow mesenchymal stem cells

目的观察血小板作用于人骨髓间充质干细胞(mesenchymal stem cells,MSCs)后对肿瘤细胞转移的作用。方法体外分离培养人骨髓MSCs及健康人外周血中的血小板;实验分为MSCs组、血小板+MSCs组及肿瘤细胞上清处理血小板+MSCs组(T-血小板+MSCs),分别收集3组培养24 h后的MSCs及培养上清(SGC-7901-CM及MSCs-CM);western blot检测其肿瘤相关成纤维母细胞(CAF)标志蛋白α-SMA及Vimentin的表达;Transwell实验检测骨髓MSCs的迁移能力;流式细胞术检测SGC-7901-CM及MSCs-CM共培养后血小板P选择素的表达水平;建立BALB/c裸鼠尾静脉注射SGC-7901胃癌细胞系转移模型,观察体内肿瘤转移情况。结果 SGC-7901-CM及MSCs-CM处理的血小板P选择素的表达水平[分别为(31.4±1.71)%、(21.37±1.00)%]明显高于对照组[(3.17±0.40)%],差异有统计学意义(t分别为27.85和29.18,P均<0.01);血小板+MSCs组及T-血小板+MSCs组α-SMA蛋白[分别为(0.79±0.08)、(0.90±0.06)]及Vimentin蛋白[分别为(0.88±0.01)、(0.96±0.04)]的表达水平均明显高于MSCs组[分别为(0.64±0.02)、(0.75±0.05)],差异有统计学意义(t分别为2.96和6.45,4.73和5.73,P均<0.01);血小板+MSCs组及T-血小板+MSCs组迁移细胞数[分别为(340.3±27.7)个、(424.3±17.6)个]明显高于MSCs组[(220.7±19.4)个],差异有统计学意义(t分别为6.14和13.48,P均<0.01);体内实验结果表明,血小板+MSCs组转移灶及T-血小板+MSCs组转移灶[分别为(4±2)个、(21±4)个]明显高于MSCs组[(0.33±0.06)个],差异有统计学意义(t分别为3.051和8.857,P均<0.01)。结论血小板能促进骨髓MSCs迁移,并能增强骨髓MSCs体内促进肿瘤细胞转移能力。

Objective To investigate the effect of human bone marrow mesenchymal stem cells（ BM-MSCs） stimulated by platelets in vitro on the metastasis of cancer cells. Methods The BM-MSCs were isolated and cultured in vitro and platelets from the peripheral blood of healthy persons were purified. The MSCs（ control）,platelets ＋ MSCs,and platelets treated with culture media（ CM） of SGC-7901 tumor cells ＋ MSCs（ T-platelets ＋ MSCs） were cultured,respectively,and the MSCs and supernatants（ MSCs-CM and SGC-7901-CM） were collected,respectively,after 24 hours. The expressions of markers of cancer-associated fibroblasts（ CAF）,such asα-SMA and Vimentin,were determined by Western-blotting. The immigration ability of BM-MSCs were analyzed by Transwell assay.The levels of P-selectin in platelets stimulated by MSCs-CM or SGC-7901-CM were detected with flow cytometry. The metastasis model of gastric cancer SGC-7901 cells was established in BALB/c nude mice by the injection of tail vein,and the tumor metastasis in vivo was observed. Results The expression levels of P-selectin in platelets stimulated by MSCs-CM（ [21. 37 ± 1. 00]%） or SGC-7901-CM（ [31. 4 ± 1. 71]% were significantly higher than that in the control（ [3. 17 ± 0. 40]%,t = 27. 85 and 29. 18,P〈0. 01）. The expression levels of α-SMA and Vimentin in platelets ＋ MSCs group（ 0. 79 ± 0. 08 and 0. 88 ± 0. 01） and T-platelets ＋ MSCs group（ 0. 90 ± 0. 06 and 0. 96 ± 0. 04） were significantly higher than that in the control（ 0. 64 ± 0. 02 and 0. 75 ± 0. 05,t = 2. 96 and 6. 45 forα-SMA,t = 4. 73 and 5. 73 for Vimentin,P〈0. 01）. The amounts of immigration cells in platelets ＋ MSCs group（ 340. 3 ± 27. 7）and T-platelets ＋ MSCs group（ 424. 3 ± 17. 6） were significantly higher than that in the control（ 220. 7 ± 19. 4,t = 6. 14 and 13. 48,P〈0. 01）. The in vivo experimental results showed that the metastatic foci in platelets ＋ MSCs group（ 4 ± 2） and T-platelets ＋ MSCs group（ 21 ± 4） were significantly higher than that in the control（ 0. 33 ± 0. 06,t = 3. 051 and 8. 857,P〈0. 01）. Conclusion Platelets promote the immigration and the enhanced tumor metastasis in vivo of BM-MSCs.