小鼠骨髓间充质干细胞向平滑肌样细胞分化过程中myocardin的表达
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摘要
背景:平滑肌细胞(SMC)的过度增生是血管病变发生发展中的重要环节。长久以来的观点认为动脉粥样硬化(AS)斑块中的SMC源于中膜SMC的迁移和增生。虽然对于中膜SMC的迁移和增殖的分子机制做了大量的研究工作,但是至今仍没有建立有效的阻止闭塞性血管重建的治疗方法。然而近年来有报道,骨髓细胞参与了血管疾病的发生,发展。研究发现骨髓细胞可能具有分化成血管祖细胞的潜能,这种祖细胞迁移至受损的血管,分化成平滑肌细胞或内皮细胞,参与血管的修复,重塑和病变的形成。这些发现为探索血管疾病的新疗法提供了基础。但是骨髓细胞向平滑肌方向分化的分子机制仍不清楚。然而令人兴奋的是近年来研究者发现了一个关键的促平滑肌分化因子-myocardin。目前对于myocardin的研究仍处于初步阶段。
目地:探讨血小板源性生长因子-BB(PDGF-BB)及高浓度的胎牛血清(20%FBS)体外诱导骨髓间充质干细胞(bone marrow mesenchymal stem cell,BMSC)定向分化为平滑肌细胞的可行性及myocardin在此过程中的表达变化。
方法:采用全骨髓贴壁分离和消化控制相结合分离小鼠BMSC,然后用PDGF-BB(50μg/L)联合高浓度胎牛血清(20%FBS)诱导BMSC,分别于诱导前及诱导4天、8天后用免疫细胞化学法检测细胞α-SMA,SM-MHC,myocardin的表达,同时在倒置相差显微镜下观察细胞的形态学变化。
结果:从单个细胞的形态上看,诱导前BMSC呈梭形,分支状或多边形,诱导后的细胞体积略增加,仍以梭形细胞为主,但是诱导8天时细胞密集处出现束状,漩涡状排列,与SMC相似。细胞免疫组化显示:诱导前及诱导4天、8天时,α-SMA,SM-MHC,myocardin三种蛋白的表达逐渐增强,平均灰度值逐渐降低,不同时间点组组之间统计学比较有显著性差异(P<0.05)。
结论: PDGF-BB联合高浓度的胎牛血清(20%FBS)可有效诱导BMSC向平滑肌样细胞分化。myocardin在此分化过程中起重要作用。
Background Exuberant accumulation of smooth muscle cells (SMC) plays a principal role in the pathogenesis of vascular diseases. The long-standing dogma has been that SMC in atherosclerotic lesions are derived primarily from preexisting medial SMC. Although much effort has been devoted to understanding the molecular pathways regulating migration and proliferation of medial SMC, no effective therapy to prevent occlusive vascular remodeling has been established. It was recently reported that bone marrow cells substantially contribute to the pathogenesis of vascular diseases. It was suggested that bone marrow cells may have the potential to give rise to vascular progenitor cells that home in on the damaged vessels and differentiate them into smooth muscle cells or endothelial cells, thereby contributing to vascular repair, remodeling, and lesion formation. The present findings may provide the basis for the development of new therapeutic strategies for vascular diseases. But the molecular mechanism of bone marrow cells differentiate into SMC is not unclear. However, one exciting advance for the field of SMC differentiation in recent years was the discovery of myocardin, a key transcription factor for SMC differentiation. At present, we have not known a lot about myocardin.
Objective To investigate the feasibility of differentiation of bone marrow mesenchymal stem cells(BMSC) into SMC in vitro under PDGF-BB(50μg/L) and high concentrations of FBS(20%), and the positive effect of myocardin on BMSC differentiating into SMC.
Methods Mouse BMSC were isolated and purifed from bone marrow by means of adhesion seperation plus digestion control, then PDGF-BB(50μg/L) and FBS(20%)were added to the BMSC. And the cells were cultuted for 4 and 8 days respectively. Immunohistochemistry assay and image analysis were used to value the expression extent ofα-SMA, SM-MHC and myocardin. Meanwhile, cell morphology was observed by inverted phase contrast microscope.
Results In morphology, before induction, BMSC were spind-shaped, branch-shaped or polygonal. After 4、8 days induction, most cell were spind-shaped, no big changes with before. But cells were fascl and whirlpool-shaped, similar to SMC. Intensities of the three proteines immunostaining significantly increased after 4 and 8 days. And Test of One-way ANOVA showed that there was statistically significance in different times(p<0.05).
Conclusions BMSC could be induced to differentiate into SM-like cells through adding PDGF-BB(50μg/L) and FBS(20%) with BMSC in vitro. Myocardin plays an important role in this process.
目地:探讨血小板源性生长因子-BB(PDGF-BB)及高浓度的胎牛血清(20%FBS)体外诱导骨髓间充质干细胞(bone marrow mesenchymal stem cell,BMSC)定向分化为平滑肌细胞的可行性及myocardin在此过程中的表达变化。
方法:采用全骨髓贴壁分离和消化控制相结合分离小鼠BMSC,然后用PDGF-BB(50μg/L)联合高浓度胎牛血清(20%FBS)诱导BMSC,分别于诱导前及诱导4天、8天后用免疫细胞化学法检测细胞α-SMA,SM-MHC,myocardin的表达,同时在倒置相差显微镜下观察细胞的形态学变化。
结果:从单个细胞的形态上看,诱导前BMSC呈梭形,分支状或多边形,诱导后的细胞体积略增加,仍以梭形细胞为主,但是诱导8天时细胞密集处出现束状,漩涡状排列,与SMC相似。细胞免疫组化显示:诱导前及诱导4天、8天时,α-SMA,SM-MHC,myocardin三种蛋白的表达逐渐增强,平均灰度值逐渐降低,不同时间点组组之间统计学比较有显著性差异(P<0.05)。
结论: PDGF-BB联合高浓度的胎牛血清(20%FBS)可有效诱导BMSC向平滑肌样细胞分化。myocardin在此分化过程中起重要作用。
Background Exuberant accumulation of smooth muscle cells (SMC) plays a principal role in the pathogenesis of vascular diseases. The long-standing dogma has been that SMC in atherosclerotic lesions are derived primarily from preexisting medial SMC. Although much effort has been devoted to understanding the molecular pathways regulating migration and proliferation of medial SMC, no effective therapy to prevent occlusive vascular remodeling has been established. It was recently reported that bone marrow cells substantially contribute to the pathogenesis of vascular diseases. It was suggested that bone marrow cells may have the potential to give rise to vascular progenitor cells that home in on the damaged vessels and differentiate them into smooth muscle cells or endothelial cells, thereby contributing to vascular repair, remodeling, and lesion formation. The present findings may provide the basis for the development of new therapeutic strategies for vascular diseases. But the molecular mechanism of bone marrow cells differentiate into SMC is not unclear. However, one exciting advance for the field of SMC differentiation in recent years was the discovery of myocardin, a key transcription factor for SMC differentiation. At present, we have not known a lot about myocardin.
Objective To investigate the feasibility of differentiation of bone marrow mesenchymal stem cells(BMSC) into SMC in vitro under PDGF-BB(50μg/L) and high concentrations of FBS(20%), and the positive effect of myocardin on BMSC differentiating into SMC.
Methods Mouse BMSC were isolated and purifed from bone marrow by means of adhesion seperation plus digestion control, then PDGF-BB(50μg/L) and FBS(20%)were added to the BMSC. And the cells were cultuted for 4 and 8 days respectively. Immunohistochemistry assay and image analysis were used to value the expression extent ofα-SMA, SM-MHC and myocardin. Meanwhile, cell morphology was observed by inverted phase contrast microscope.
Results In morphology, before induction, BMSC were spind-shaped, branch-shaped or polygonal. After 4、8 days induction, most cell were spind-shaped, no big changes with before. But cells were fascl and whirlpool-shaped, similar to SMC. Intensities of the three proteines immunostaining significantly increased after 4 and 8 days. And Test of One-way ANOVA showed that there was statistically significance in different times(p<0.05).
Conclusions BMSC could be induced to differentiate into SM-like cells through adding PDGF-BB(50μg/L) and FBS(20%) with BMSC in vitro. Myocardin plays an important role in this process.
引文
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