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

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。

Objectives： To investigate the cell viability, proliferation and differentiation-related gene and pro-tein 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 ex-pression. 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 21 d. Collagen scaffolds showed great biocompatibility. There was no difference of LDH among groups at the 7thday（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 21 d culture, the m RNA 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 aggrecan. SOX9, type Ⅰ collagen, type Ⅱ collagen and aggrecan were up-regulated in COL-Ⅰ/Ⅱ and COL-Ⅱgroup, 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.