王 瑾,陈其昕,陶轶卿,李方财.不同胶原支架对髓核间充质干细胞分化的影响[J].中国脊柱脊髓杂志,2015,(6):541-548. |
不同胶原支架对髓核间充质干细胞分化的影响 |
中文关键词: 椎间盘 髓核组织工程学 Ⅱ型胶原 细胞分化 |
中文摘要: |
【摘要】 目的:比较不同胶原支架中髓核间充质干细胞(NPMSCs)的细胞存活、增殖能力和分化相关基因及蛋白表达等方面的差异。方法:在体外构建Ⅰ型、Ⅰ/Ⅱ型混合和Ⅱ型胶原支架,观察其显微结构、孔隙率及降解特性。从健康雄性大鼠尾椎提取NPMSCs,分别采用细胞微球、Ⅰ型胶原、Ⅰ/Ⅱ型混合胶原、Ⅱ型胶原支架培养,其中细胞微球作为对照。通过乳酸脱氢酶(LDH)检测材料生物毒性,CCK-8测定细胞增殖,实时定量PCR和Western Blot测定SOX9、聚集蛋白聚糖、Ⅰ型胶原和Ⅱ型胶原的基因和蛋白表达水平,阿尔新蓝组织学染色检测硫酸蛋白聚糖的表达。结果:Ⅰ型、Ⅰ/Ⅱ型混合和Ⅱ型胶原支架孔隙率均为90%以上,构建21d后的降解率分别为(10.30±0.66)%、(9.87±0.71)%和(10.40±0.53)%。培养后7d细胞LDH检测结果Ⅰ型、Ⅰ/Ⅱ型混合和Ⅱ型胶原支架组分别为12.24±0.65、12.13±1.03、12.67±1.15,与对照组12.50±1.32比较无显著性差异(P>0.05)。胶原支架中培养5d及7d的细胞CCK-8检测结果(Ⅰ型胶原组为0.67±0.04、1.20±0.05,Ⅰ/Ⅱ型混合胶原组为0.62±0.05、1.20±0.07,Ⅱ型胶原组为0.69±0.02、1.34±0.04)明显高于对照组(0.53±0.03,1.02±0.02)(P<0.05)。培养21d后,三种胶原支架组与对照组比较,SOX9、Ⅰ型胶原、Ⅱ型胶原及聚集蛋白聚糖的基因表达均显著上升(P<0.05),其中Ⅱ型胶原支架组上述基因表达量最高,与Ⅰ型及Ⅰ/Ⅱ型混合胶原支架组有显著性差异(P<0.05);与对照组比较,Ⅰ型胶原支架组中仅Ⅰ型胶原及聚集蛋白聚糖的蛋白表达上升(P<0.05);Ⅰ/Ⅱ型混合及Ⅱ型胶原支架组中SOX9、Ⅰ型胶原、Ⅱ型胶原及聚集蛋白聚糖的蛋白表达有显著上升(P<0.05),其中Ⅱ型胶原支架组上述蛋白表达量最高,且与Ⅰ型及Ⅰ/Ⅱ型混合胶原支架组有显著性差异(P<0.05)。阿尔新蓝组织学染色检测硫酸蛋白聚糖在Ⅱ型胶原支架组表达明显高于其余各组。结论:Ⅰ型胶原、Ⅰ/Ⅱ型混合胶原、Ⅱ型胶原支架均促进NPMSC的分化,而Ⅱ型胶原支架促进NPMSC成髓核细胞分化作用尤为显著。Ⅱ型胶原是髓核组织工程学的理想生物支架材料。 |
Effect of different type of collagen scaffold for nucleus pulposusmesenchymal stem cell differentiation |
英文关键词:Intervertebral disc Nucleus pulposus tissue engineering Type Ⅱ collagen Differentiation |
英文摘要: |
【Abstract】 Objectives: To investigate the cell viability, proliferation and differentiation-related gene and protein expression of nucleus pulposus mesenchymal stem cells(NPMSCs) in different types of collagen scaffolds. Methods: Type Ⅰ, type Ⅰ/Ⅱ and type Ⅱ collagen scaffolds were formed in vitro, and microstructure, porosity and degradability were detected. NPMSCs isolated from coccygeal vertebra of healthy male rats were cultured as micromass or in type Ⅰ collagen(COL-Ⅰ), type Ⅰ/Ⅱ collagen(COL-Ⅰ/Ⅱ), type Ⅱ collagen scaffold(COL-Ⅱ), and micromass served as control. Cytotoxicity and cell proliferation were detected by lactate dehydrogenase(LDH) and CCK-8 methods respectively. Differentiation related gene and protein expressions were examined by real-time quantitative PCR and western Blotting respectively, including SOX9, aggrecan, type Ⅰ collagen and type Ⅱ collagen. Alcian blue staining was used to investigate sulfate proteoglycan expression. Results: The porosity of each of collagen scaffolds was measured by more than 90%, and the degradability of COL-Ⅰ, COL-Ⅰ/Ⅱ and COL-Ⅱ was detected as (10.30±0.66)%, (9.87±0.71)%, (10.40±0.53)% respectively in 21d. Collagen scaffolds showed great biocompatibility. There was no difference of LDH among groups at the 7th day(12.24±0.65, 12.13±1.03, 12.67±1.15 and 12.50±1.32, P>0.05). Collagen scaffolds enhanced proliferation of NPMSC after 5d and 7d culture(COL-Ⅰ 0.67±0.04, 1.20±0.05; COL-Ⅰ/Ⅱ 0.62±0.05, 1.20±0.07; COL-Ⅱ 0.69±0.02, 1.34±0.04) were much higher than those of control group(CTL 0.53±0.03, 1.02±0.02, P<0.05). After 21d culture, the mRNA expressions of SOX9, type Ⅰ collagen, type Ⅱ collagen and aggrecan were increased significantly in collagen scaffold groups compared to control group. Among those, COL-Ⅱ group was the highest one and there was significant differences compared to the other groups(P<0.05). Moreover, the expression of protein in COL-Ⅰ group was up-regulated in type Ⅰ collagen and aggre?鄄can. SOX9, type Ⅰ collagen, type Ⅱ collagen and aggrecan were up-regulated in COL-Ⅰ/Ⅱ and COL-Ⅱ group, compared to control group, COL-Ⅱ had the highest protein expression. Alcian blue staining also showed that sulfate proteoglycan synthesis was up-regulated in COL-Ⅱ group. Conclusions: All of type Ⅰ, type Ⅰ/Ⅱ and type Ⅱ collagen scaffolds can promote NPMCs′ differentiation towards nucleus pulposus cell type, and the effect of type Ⅱ collagen scaffolds is most significant. Type Ⅱ collagen is the ideal biological material for nucleus pulposus tissue engineering. |
投稿时间:2014-12-28 修订日期:2015-06-04 |
DOI: |
基金项目:国家自然科学基金项目(编号:81171756,81472114) |
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