不同胶原支架对髓核间充质干细胞分化的影响

Effect of different type of collagen scaffold for nucleus pulposusmesenchymal stem cell differentiation

中文关键词:  椎间盘  髓核组织工程学  Ⅱ型胶原  细胞分化

英文关键词:Intervertebral disc  Nucleus pulposus tissue engineering  Type Ⅱ collagen  Differentiation

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。

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.