SDF-1/HGF融合蛋白介导的大鼠间充质干细胞对造血干细胞增殖和趋化作用的影响

Effect of SDF-1/HGF fusion protein-mediated BMSCs on proliferation and chemotaxis of HSCs

摘要:

目的：研究基质细胞衍生因子-1（stromal cell-derived factor-1，SDF-1）/肝细胞生长因子（hepatocyte growth factor，HGF）融合蛋白介导的骨髓间充质干细胞（bone marrow derived stroma cell，BMSCs）对造血干细胞（hematopoietic stem cell，HSC）增殖和趋化能力的影响，以期望通过联合移植的方式重建肝组织。方法：培养鉴定3~4周龄SD大鼠BMSCs及大鼠HSCs。荧光显微镜下观测重组腺病毒pAD-SDF-1-（GlySer）3-HGF-IRES-GFP感染BMSCs 1、3、7、14 d后的感染效率，同时ELISA检测SDF-1和HGF的表达。制备BMSCs细胞滋养层，HSCs种植于有滋养层的 24 孔板中，分为3组：HSCs加培养基组（A1组）；HSCs加BMSCs组（B1组）；HSCs加SDF/HGF融合蛋白介导的BMSCs组（C1组），检测共培养1、3、7、14 d后HSCs的增殖倍数。利用transwell培养体系，按实验要求分为3组：HSCs加细胞培养液组（A2组）；HSCs加BMSCs组（ B2组）； HSCs加SDF/HGF融合蛋白介导的BMSCs（C2组），测定HSCs的迁移指数。结果：重组腺病毒pAD-SDF-1-（GlySer）3-HGF-IRES-GFP感染BMSCs 1、3、7、14 d的感染效率分别为（25.28±3.72）%、（82.22±3.58）%、（64.85±4.48）%、（28.35±2.73）%，感染后第3天的感染效率最高（与第1、7、14天相比较，F=718.05，P＝0.000；F=66.80，P＝0.000；F=642.78，P＝0.000）。重组腺病毒pAD-SDF-1-（GlySer）3-HGF-IRES-GFP感染BMSCs 1、3、7、14 d后，SDF-1的表达分别为：（1.02±0.15） μg/ml，（4.51±0.52） μg/ml，（2.18±0.19） μg/ml，（1.22±0.21） μg/ml；HGF的表达分别为：（1.38±0.32） ug/ml，（5.01±0.43） μg/ml，（3.10±0.26） μg/ml，（1.63±0.27） μg/ml。感染后第3天SDF-1与第1、7、14天比较和HGF与第1、7、14天比较的表达明显增高（P均＝0.000）。BMSCs和HSCs共培养第3、7、14天，B1组、C1组中HSCs的扩增倍数明显高于A1组（P均＝0.000）。同时C1组中HSCs的扩增倍数明显高于B1组（P均＝0.000）。transwell显示B2、C2组的迁移指数明显高于A2组，同时C2组的迁移指数明显高于B2组。结论：SDF-1/HGF融合蛋白介导的BMSCs 可能在促进HSCs的增殖及趋化方面起到重要作用。

Abstract:

Objective：To investigate the effect of SDF-1/HGF fusion protein-mediated BMSCs on proliferation and chemotaxis of HSCs，then to rebuild liver tissue by combined transplantation. Methods：BMSCs and HSCs derived from SD rat at the age of 3-4 weeks were cultured and identified. After stably serial subcultivation，recombinated adenovirus pAD-SDF-1-（GlySer）3-HGF-IRES-GFP infected BMSCs，and the efficiency of infection was detected after infection for 1，3，7，14 d by fluorescence microscope. Meanwhile，the expressions of SDF-1 and HGF were assayed by ELISA. HSCs were planted in 24 well plates containing BMSCs cytotrophoblast. They were classified as HSCs plus cell culture fluid（group A1），HSCs plus BMSCs（group B1），HSCs plus SDF-1/HGF fusion protein-mediated BMSCs（group C1）. The proliferation index of HSCs after coculture for 1，3，7，14 d were investigated. In order to exam the immigration index of HSCs influenced by SDF-1/HGF fusion protein-mediated BMSCs，three experimental groups including HSCs plus cell culture fluid（group A2），HSCs plus BMSCs（group B2），HSCs plus SDF-1/HGF fusion protein-me－diated BMSCs（group C2） through transwell system were set. Results：After BMSCs being infected by recombinated adenovirus pAD-SDF-1-（GlySer）3-HGF-IRES-GFP for1，3，7，14 d，the efficiencies of infection were （25.28±3.72）%，82.22±3.58）%，（64.85±4.48）%，（28.35±2.73）%，respectively. And the efficiency of infection was the highest after infection for 3 d（com－pared with infection for 1，7，14 d，F=718.05，P＝0.000；F=66.80，P＝0.000；F=642.78，P＝0.000）. At the same time，after infection for 1，3，7，14 d，the expressions of SDF-1 were （1.02±0.15） μg/ml，（4.51±0.52） μg/ml，（2.13±0.19） μg/ml，（1.22±0.21） μg/ml，and the expressions of HGF were （1.38±0.32） μg/ml，（5.01±0.43） μg/ml，（3.10±0.26） μg/ml，（1.62±0.27） μg/ml. According to our data，after infection for 3 d，the expressions of SDF-1 and HGF were significantly increased compared with those after infection for 1，7，14 d（P＝0.000）. After coculture for 3，7，14 d，the proliferation of HSCs were significantly increased in group B1 and group C1（P＝0.000）compared with those of group A1. And the amplification of HSCs was obviously stronger in group C1 than in group B1（P＝0.000）. In transwell test，the immigration indexes of HSCs were prominently increased in group B2 and group C2 than in group A2. Meanwhile，the immigration index of HSCs in group C2 was remarkably higher than that in group B2. Conclusion：SDF-1/HGF fusion protein-mediated BMSCs might have an key role in promoting proliferation and chemotaxis of HSCs.