羟化重组人源Ⅲ型胶原蛋白对血管平滑肌细胞功能的影响

Influences of hydroxylated recombinant human type III collagen on the functional characteristics of vascular smooth muscle cells

摘要:

为了探讨羟化修饰的重组人源Ⅲ型胶原蛋白对血管平滑肌细胞(vascular smooth muscle cells, VSMCs)增殖、迁移等生物学行为的影响，明确其生物相容性及促血管生成潜力，本研究采用CCK-8法检测原代培养的VSMCs活力，利用细胞周期试剂盒分析不同浓度羟化修饰的重组人源Ⅲ型胶原蛋白对细胞增殖的影响，通过随机迁移实验和伤口愈合实验评估其对细胞迁移能力的作用，并结合蛋白质-蛋白质相互作用(protein-protein interaction, PPI)网络筛选与VSMCs功能相关的关键基因，进一步利用实时荧光定量PCR对筛选结果进行验证。CCK-8实验结果表明，羟化修饰的重组人源Ⅲ型胶原蛋白在不同浓度下均可提高VSMCs细胞活力，在0.1 mg/mL浓度下最为显著，表现出良好的生物相容性；迁移实验结果显示，0.1 mg/mL的羟化修饰重组人源Ⅲ型胶原蛋白对细胞迁移的促进作用最为显著，其24 h伤口愈合率达到74.93%，显著高于未羟化胶原蛋白(1206)各浓度处理组及空白对照组；细胞周期分析表明，经羟化修饰的胶原蛋白处理后，VSMCs处于S期和M期的比例较未羟化组显著增加；通过PPI网络筛选出MMP-3和MMP-13这2个关键基因，二者均属基质金属蛋白酶家族，在血管生物学过程中发挥重要作用；实时荧光定量PCR结果进一步证实，MMP-3和MMP-13在VSMCs中的表达水平均显著上调，表明羟化修饰的重组人源Ⅲ型胶原蛋白可促进血管生成相关基因的表达。羟化的重组人源Ⅲ型胶原蛋白能够上调血管生成相关基因MMP-3和MMP-13的表达，有效促进VSMCs的增殖与迁移，且无明显细胞毒性，研究结果为将该蛋白作为人工血管细胞外基质组分提供了理论依据。

Abstract:

This study was designed to investigate the effects of hydroxylated recombinant human type III collagen on the proliferation and migration of vascular smooth muscle cells (VSMCs). The viability of primarily cultured VSMCs was examined by the CCK-8 assay. A cell cycle assay kit was used to evaluate the effect of hydroxylated recombinant human type III collagen at different concentrations on cell proliferation. Cell migration was evaluated via random migration and wound healing assays. A protein-protein interaction (PPI) network was built to screen the key genes associated with functions of VSMCs, and the screening results were further validated by RT-qPCR. The CCK-8 assay indicated that hydroxylated recombinant human type III collagen at various concentrations increased the viability of VSMCs, demonstrating good biocompatibility. Migration assays showed that hydroxylated recombinant human type III collagen at a concentration of 0.1 mg/mL most significantly promoted cell migration, with a 24 h wound healing rate of 74.93%, which was significantly higher than those of the control group and the groups treated with non-hydroxylated collagen at all concentrations. Cell cycle analysis revealed that treatment with hydroxylated collagen significantly increased the proportion of VSMCs in the S and M phases compared with the non-hydroxylated collagen group. Two key genes, MMP-3 and MMP-13, both belonging to the matrix metalloproteinase (MMP) family which plays a crucial role in vascular biology, were screened from the PPI network. RT-qPCR results further confirmed that the expression levels of MMP-3 and MMP-13 in VSMCs were significantly up-regulated, indicating that hydroxylated recombinant human type III collagen promoted the expression of angiogenesis-related genes. In conclusion, hydroxylated recombinant human type III collagen can up-regulate the expression of MMP-3 and MMP-13, effectively promote the proliferation and migration of VSMCs, and shows no significant cytotoxicity. This provides a theoretical basis for the potential application of hydroxylated recombinant human type III collagen as an extracellular matrix component in artificial vascular grafts.