骨髓间充质干细胞调控肝星状细胞RhoA、p27的表达
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摘要
目的研究体外大鼠骨髓间充质干细胞(MSCs)对肝星状细胞(HSCs)RhoA信号因子及其细胞周期调控因子p27表达的影响,探讨MSCs调控HSCs细胞周期G1/S转换机制。
方法贴壁筛选法培养、纯化SD大鼠MSCs,传代至第4代使用;大鼠肝星状细胞(HSC-T6)系及纤维原细胞系冻融后传代使用。应用6孔塑料细胞培养盒,每孔使用半透膜(transwell insert)建立上下双层细胞共培养体系,常规培养。实验分3组:空白对照组;阴性对照组;MSCs实验组.用WST-8法对肝星状细胞增殖率进行测定;流式细胞仪检测细胞周期;RT-PCR、Western blot检测MSCs与HSCs共培养后HSCs内RhoA,p27 mRNA和蛋白的表达。
结果(1) HSCs与MSCs共培养24h后,HSCs表现明显增殖抑制(P<0.01),且呈现时间依赖性.MSCs试验组与空白对照组、阴性对照组比较均有显著性差异(P<0.01;P<0.01)。(2)共培养12h后,MSCs可阻滞HSCs由G0/G1期向S期转换,使G0/G1期细胞增多,S期细胞减少,与空白对照组、阴性对照组比较差异均有统计学意义(P<0.01)。(3)共培养12h后,MSCs实验组RhoA mRNA表达与空白对照组、阴性对照组比较均有统计学差异(P<0.05;P<0.01),随时间的延长呈递减趋势;共培养后,MSCs实验组p27 mRNA表达与空白对照组、阴性对照组比较均无统计学差异。(4)共培养12h后,MSCs实验组RhoA蛋白表达与空白对照组、阴性对照组比较均有统计学差异(P<0.01,P<0.01),随时间的延长呈递减趋势;共培养12h后, MSCs实验组p27蛋白与空白对照组、阴性对照组比较均有统计学差异(P<0.01;P<0.01),随时间的延长呈递增趋势。(5)相关性分析显示: RhoA与p27 mRNA的表达无明显相关( r=-0.105);RhoA与p27蛋白的表达呈显著负相关( r=-0.943, P<0.01)。
结论骨髓间充质干细胞抑制肝星状细胞增殖的机制可能是通过RhoA-p27通路调控HSCs细胞周期改变;RhoA活性的下调可能是引起HSCs内p27蛋白表达增加的原因。
OBJECTIVE To investigate the effects of Bone marrow mesenchymal stem cells (MSCs) on the mRNA and protein expression of RhoA and p27 in hepatic stellate cells (HSCs) by co-cultured, and explore the underlying mechanism of cell cycle regulation of MSCs on the HSCs.
METHODS MSCs were isolated from bone marrow in rats and grown, propagated in culture flask. HSCs and fiberblast cells were recoveried and activated morphologically. An indirect coculture system between MSCs/fiberblast cells and HSCs were established using Transwell membrane systems(24mm diameter, 0.4μm pore size). Three groups were divided randomly:①HSCs control group②fiberblast control group③MSCs group. The inhibitory rate of HSCs proliferation with MSCs coculture were tested by the method of WST-8 and cell cycle was determined by flow cytometry, the mRNA and protein expressions of RhoA, p27 in HSCs were determined by reverse transcription-polymerase chain reaction(RT- PCR)and Western blot respectinvely.
RESULTS The inhibitory rate of HSCs proliferation with MSCs coculture were significant higher than that of HSCs control group and Fiberoblast control group at times 24h,48h and 72h(P < 0.01). Flow cytometry showed that MSCs blocked the HSCs from G0/G1 period convert to the S phase as compared with the control group, and the percentage of G0/G1 phase cells becomed large and the S phase cells small (P<0.01). Furthermore, the mRNA expression of RhoA in MSCs group were significantly reduced at time 12h(P<0.01)as compared with the controls, and the same effects existed at the following times; but p27 expression in MSCs group was not changed during the whole cocultrue process. Finally, the protein expression of RhoA in MSCs group were significantly reduced at time 12h(P<0.01),and the same effects were taken place at the following times; whereas p27 expression in MSCs group was increased at time 12h(P<0.05), and significantly increased at time 24h and the following times(P<0.01). There were not relationship between the mRNA expression of RhoA and p27(r=0.105);However, there were negative relationship between the protein expression of RhoA and p27(r=-0.943, P<0.01).
CONCLUSIONS The mechanism of MSCs inhibit the proliferation of HSCs may modulate the cell cycle process of HSCs through regulating the RhoA-p27 pathway. The underlying reason for the increased expression of p27 protein may be the down-regulated RhoA activity inhibited by MSCs.
方法贴壁筛选法培养、纯化SD大鼠MSCs,传代至第4代使用;大鼠肝星状细胞(HSC-T6)系及纤维原细胞系冻融后传代使用。应用6孔塑料细胞培养盒,每孔使用半透膜(transwell insert)建立上下双层细胞共培养体系,常规培养。实验分3组:空白对照组;阴性对照组;MSCs实验组.用WST-8法对肝星状细胞增殖率进行测定;流式细胞仪检测细胞周期;RT-PCR、Western blot检测MSCs与HSCs共培养后HSCs内RhoA,p27 mRNA和蛋白的表达。
结果(1) HSCs与MSCs共培养24h后,HSCs表现明显增殖抑制(P<0.01),且呈现时间依赖性.MSCs试验组与空白对照组、阴性对照组比较均有显著性差异(P<0.01;P<0.01)。(2)共培养12h后,MSCs可阻滞HSCs由G0/G1期向S期转换,使G0/G1期细胞增多,S期细胞减少,与空白对照组、阴性对照组比较差异均有统计学意义(P<0.01)。(3)共培养12h后,MSCs实验组RhoA mRNA表达与空白对照组、阴性对照组比较均有统计学差异(P<0.05;P<0.01),随时间的延长呈递减趋势;共培养后,MSCs实验组p27 mRNA表达与空白对照组、阴性对照组比较均无统计学差异。(4)共培养12h后,MSCs实验组RhoA蛋白表达与空白对照组、阴性对照组比较均有统计学差异(P<0.01,P<0.01),随时间的延长呈递减趋势;共培养12h后, MSCs实验组p27蛋白与空白对照组、阴性对照组比较均有统计学差异(P<0.01;P<0.01),随时间的延长呈递增趋势。(5)相关性分析显示: RhoA与p27 mRNA的表达无明显相关( r=-0.105);RhoA与p27蛋白的表达呈显著负相关( r=-0.943, P<0.01)。
结论骨髓间充质干细胞抑制肝星状细胞增殖的机制可能是通过RhoA-p27通路调控HSCs细胞周期改变;RhoA活性的下调可能是引起HSCs内p27蛋白表达增加的原因。
OBJECTIVE To investigate the effects of Bone marrow mesenchymal stem cells (MSCs) on the mRNA and protein expression of RhoA and p27 in hepatic stellate cells (HSCs) by co-cultured, and explore the underlying mechanism of cell cycle regulation of MSCs on the HSCs.
METHODS MSCs were isolated from bone marrow in rats and grown, propagated in culture flask. HSCs and fiberblast cells were recoveried and activated morphologically. An indirect coculture system between MSCs/fiberblast cells and HSCs were established using Transwell membrane systems(24mm diameter, 0.4μm pore size). Three groups were divided randomly:①HSCs control group②fiberblast control group③MSCs group. The inhibitory rate of HSCs proliferation with MSCs coculture were tested by the method of WST-8 and cell cycle was determined by flow cytometry, the mRNA and protein expressions of RhoA, p27 in HSCs were determined by reverse transcription-polymerase chain reaction(RT- PCR)and Western blot respectinvely.
RESULTS The inhibitory rate of HSCs proliferation with MSCs coculture were significant higher than that of HSCs control group and Fiberoblast control group at times 24h,48h and 72h(P < 0.01). Flow cytometry showed that MSCs blocked the HSCs from G0/G1 period convert to the S phase as compared with the control group, and the percentage of G0/G1 phase cells becomed large and the S phase cells small (P<0.01). Furthermore, the mRNA expression of RhoA in MSCs group were significantly reduced at time 12h(P<0.01)as compared with the controls, and the same effects existed at the following times; but p27 expression in MSCs group was not changed during the whole cocultrue process. Finally, the protein expression of RhoA in MSCs group were significantly reduced at time 12h(P<0.01),and the same effects were taken place at the following times; whereas p27 expression in MSCs group was increased at time 12h(P<0.05), and significantly increased at time 24h and the following times(P<0.01). There were not relationship between the mRNA expression of RhoA and p27(r=0.105);However, there were negative relationship between the protein expression of RhoA and p27(r=-0.943, P<0.01).
CONCLUSIONS The mechanism of MSCs inhibit the proliferation of HSCs may modulate the cell cycle process of HSCs through regulating the RhoA-p27 pathway. The underlying reason for the increased expression of p27 protein may be the down-regulated RhoA activity inhibited by MSCs.
引文
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36王小中,冯文莉,刘兴,等.SKP2反义寡核苷酸对K562细胞生长和增殖的影响.癌症,2003,22(9):948-952.
37 Hengst L, and Reed SI. Translation contral of p27kip1 accumulation during the cell cycle . Science, 1996,271: 1861–1864.
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