金纳米粒子和碳纳米管对骨髓基质细胞成骨及成脂方向分化的影响
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摘要
近年来,纳米材料所具有的特殊物理性质,引起了人们的重视,科学家开始把它应用到疾病的防御、诊断和治疗等生物医学领域,特别是医学中的组织重建中。由于自身组织和器官是纳米级的,纳米材料的生物模拟特性和特殊的物理性质使之在进行组织重建时刺激细胞的生长,引导组织修复。尽管这是一个新的领域,但是已有很多科学家设计合成出了生物模拟材料包裹细胞(如干细胞、软骨细胞、骨细胞等)并应用于组织工程。然而,纳米材料的超小粒径使其能通过多种途径进入人体,如骨髓。当纳米粒子进入骨髓后,骨代谢平衡和骨组织功能是否会受到影响还需要我们进行深入的研究。因此,本文以骨髓基质细胞和成骨细胞为模型,采用四甲基偶氮唑蓝法、碱性磷酸酶比活性和油红O染色及定量测定等方法在细胞水平研究了金纳米粒子(Au NPs)和碳纳米管(CNTs)对小鼠骨髓基质细胞增殖、成骨、成脂分化和成骨细胞增殖及横向分化的影响,并用免疫印迹(Western blot)和实时定量聚合酶链式反应(Q-PCR)等方法对骨髓基质细胞成骨和成脂分化过程中相关重要蛋白和基因进行了研究。
研究结果表明：
1.细胞水平上金纳米粒子可以促进小鼠骨髓基质细胞、原代骨细胞向成骨方向分化,抑制其向成脂及横向成脂分化；
2.细胞水平上碳纳米管可以抑制小鼠骨髓基质细胞、原代骨细胞成骨成脂及横向成脂分化；
3.金纳米粒子上调成骨分化相关基因(Runx2、BMP-2、OCN、ALP等)的表达,下调成脂分化相关基因(PPARγ2、E/CBPα、E/CBPβ和E/CBPδ)的表达；
4.碳纳米管下调成骨成脂分化相关基因的表达；
5.金纳米粒子上调小鼠骨髓基质细胞、原代骨细胞成骨分化过程中Runx2和BMP-2蛋白的表达。
综合上述结果,金纳米粒子在促进成骨分化的同时抑制了成脂及成脂横向分化,而碳纳米管抑制了骨髓基质细胞成骨成脂的分化和原代成骨细胞成骨及横向成脂的分化。
Recently, due to the unique physical properies, nanomaterials have evoked a great amount of attention for improving diease prevention, diagnosis, treatment, and especially, tissue engineering. Since nature tissues or organs are nometer in dimension, the biomimetic features and excellent physiochemical properties of nanomaterials play a key role in stimulating cell growth as well as guide tissue regeneration. Even though it was a field in its infancy, currently, numerous researchers fabricate nanomaterials scaffolds encapsulating cells (such as stem cells, chondrocytes and osteoblasts, etc.) for tissue engineering applications.However, due to the ultra-small size, nanomaterials enter the body through variety ways, such as bone marrow. Whether the balance of bone metabolism and bone tissue function would be affected when nanoparticles entered bone marrow, still required further study. By using models of primary mouse bone marrow stromal cells (MSCs) and osteoblasts (OB) and methods of MTT, ALP activity assay, red O stain and assay, we study the effect of gold nanoparticle (Au NPs) and carbon nanotubes (CNTs) on osteogenic and adipogenic differentiation of MSCs and on adipogenic trans-differentiation of osteoblasts. Then we study the expression of osteogenic and adipogenic differentiation related genes by using western blot and real-time PCR.
And the results show that:
1.At cell level, Au NPs can promote osteogenic differentiation and inhibite adipogenic differentiation and adipogenic trans-differentiation of MSCs and OB;
2. At cell level, CNTs can inhibit osteogenic and adipogenic differentiaiton of MSCs;
3.Au NPs up-regulated the expression of osteogenic differentiation related genes (such as Runx2, BMP-2, OCN, ALP, etc.) and down-regulated the expression of adipogenic differentiation related genes (such as PPARγ2, E/CBPα, E/CBPβ和E/CBPδ);
4.CNTs down-regulated the expression of osteogenic and adipogenic differentiation genes of MSCs;
5.Au NPs up-regulated the expression of Runx2 and BMP-2 proteins, and down-regulated the expression of PPARy2 of MSCs.
In conclusion, Au NPs promoted the osteogenic differentiation and inhibited the adipogenic and trans-differentiation. While, CNTs inhibited the osteogenic and adipogenic differentiation of MSCs.
研究结果表明：
1.细胞水平上金纳米粒子可以促进小鼠骨髓基质细胞、原代骨细胞向成骨方向分化,抑制其向成脂及横向成脂分化；
2.细胞水平上碳纳米管可以抑制小鼠骨髓基质细胞、原代骨细胞成骨成脂及横向成脂分化；
3.金纳米粒子上调成骨分化相关基因(Runx2、BMP-2、OCN、ALP等)的表达,下调成脂分化相关基因(PPARγ2、E/CBPα、E/CBPβ和E/CBPδ)的表达；
4.碳纳米管下调成骨成脂分化相关基因的表达；
5.金纳米粒子上调小鼠骨髓基质细胞、原代骨细胞成骨分化过程中Runx2和BMP-2蛋白的表达。
综合上述结果,金纳米粒子在促进成骨分化的同时抑制了成脂及成脂横向分化,而碳纳米管抑制了骨髓基质细胞成骨成脂的分化和原代成骨细胞成骨及横向成脂的分化。
Recently, due to the unique physical properies, nanomaterials have evoked a great amount of attention for improving diease prevention, diagnosis, treatment, and especially, tissue engineering. Since nature tissues or organs are nometer in dimension, the biomimetic features and excellent physiochemical properties of nanomaterials play a key role in stimulating cell growth as well as guide tissue regeneration. Even though it was a field in its infancy, currently, numerous researchers fabricate nanomaterials scaffolds encapsulating cells (such as stem cells, chondrocytes and osteoblasts, etc.) for tissue engineering applications.However, due to the ultra-small size, nanomaterials enter the body through variety ways, such as bone marrow. Whether the balance of bone metabolism and bone tissue function would be affected when nanoparticles entered bone marrow, still required further study. By using models of primary mouse bone marrow stromal cells (MSCs) and osteoblasts (OB) and methods of MTT, ALP activity assay, red O stain and assay, we study the effect of gold nanoparticle (Au NPs) and carbon nanotubes (CNTs) on osteogenic and adipogenic differentiation of MSCs and on adipogenic trans-differentiation of osteoblasts. Then we study the expression of osteogenic and adipogenic differentiation related genes by using western blot and real-time PCR.
And the results show that:
1.At cell level, Au NPs can promote osteogenic differentiation and inhibite adipogenic differentiation and adipogenic trans-differentiation of MSCs and OB;
2. At cell level, CNTs can inhibit osteogenic and adipogenic differentiaiton of MSCs;
3.Au NPs up-regulated the expression of osteogenic differentiation related genes (such as Runx2, BMP-2, OCN, ALP, etc.) and down-regulated the expression of adipogenic differentiation related genes (such as PPARγ2, E/CBPα, E/CBPβ和E/CBPδ);
4.CNTs down-regulated the expression of osteogenic and adipogenic differentiation genes of MSCs;
5.Au NPs up-regulated the expression of Runx2 and BMP-2 proteins, and down-regulated the expression of PPARy2 of MSCs.
In conclusion, Au NPs promoted the osteogenic differentiation and inhibited the adipogenic and trans-differentiation. While, CNTs inhibited the osteogenic and adipogenic differentiation of MSCs.
引文
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