低氧环境对组织工程血管移植物胶原蛋白含量的提升作用

doi:10.12141/j.issn.1000-565X.240137

华南理工大学学报(自然科学版) ›› 2025, Vol. 53 ›› Issue (1): 129-135.doi: 10.12141/j.issn.1000-565X.240137

低氧环境对组织工程血管移植物胶原蛋白含量的提升作用

林展翼¹^,²^,³, 肖聪¹, 许健宜¹, 刘青¹, 孙盱衡¹, 方丽君¹

^1.华南理工大学医学院，广东广州 510006
^2.广东省人民医院（广东省医学科学院）心内科，广东广州 510080
^3.季华实验室，广东佛山 528200

收稿日期:2024-03-25 出版日期:2025-01-25 发布日期:2025-01-02
作者简介:林展翼（1965—），男，博士，教授，主要从事组织工程和生物医学工程研究。E-mail： linzhanyi@hotmail.com
基金资助:
广东省科技计划项目(2023B110009);季华实验室项目(X210111TD210)

The Effect of Hypoxic Environment on the Enhancement of Collagen Content in Tissue-Engineered Vascular Grafts

LIN Zhanyi¹^,²^,³, XIAO Cong¹, XU Jianyi¹, LIU Qin¹, SUN Xuheng¹, FANG Lijun¹

^1.School of Medicine，South China University of Technology，Guangzhou 510006，Guangdong，China
^2.Department of Cardiology，Guangdong Provincial People’s Hospital （Guangdong Academy of Medical Sciences），Guangzhou 510080，Guangdong，China
^3.Ji Hua Laboratory，Foshan 528200，Guangdong，China

Received:2024-03-25 Online:2025-01-25 Published:2025-01-02
About author:林展翼（1965—），男，博士，教授，主要从事组织工程和生物医学工程研究。E-mail： linzhanyi@hotmail.com
Supported by:
the Guangdong S & T Programme(2023B110009);the Program of Ji Hua Laboratory(X210111TD210)

摘要/Abstract

摘要：

在血管组织工程领域，目前的主要技术方向集中于利用血管平滑肌细胞（VSMCs）在体外增殖和分泌富含胶原蛋白的细胞外基质（ECM），并且施加特定力学刺激重构出具有所需力学性能的生物材料。合适的氧气含量对于体外细胞三维培养至关重要，但在组织工程血管移植物（TEVG）应用方面还缺乏足够研究。为此通过设计不同的氧气含量培养环境，对VSMCs增殖活性检测以确定细胞培养的氧气含量最佳值；进而，在常规氧含量和低氧含量下，对胶原蛋白的基因表达和蛋白分泌情况进行检测，并通过羟脯氨酸检测法对沉积在细胞层中的总胶原含量进行定量；最后，在聚乙醇酸（PGA）支架材料上接种VSMCs进行三维培养形成TEVG，通过组织切片染色和总胶原定量来观察低氧环境下TEVG的培养效果。结果显示：7%氧气条件下VSMCs细胞活性增长最快；低氧环境下VSMCs的Ⅰ型胶原蛋白（Col Ⅰ）和Ⅲ型胶原蛋白（Col Ⅲ）基因表达有所上调，在第5至第9天可以检测到更多胶原蛋白分泌到细胞培养基中，尤其是Col Ⅲ，并且第11天细胞层ECM中总胶原蛋白的含量为常氧组的3.1倍；7%氧气条件可促进PGA支架上VSMCs三维培养过程的胶原沉积，与常氧组相比总胶原含量提高至2.09倍，并形成更致密的胶原纤维。结果表明，选择低氧环境可以促进组织工程血管移植物胶原蛋白的含量，可为进一步优化TEVG的体外培养条件提供依据。

关键词: 血管平滑肌细胞, 低氧环境, 组织工程血管移植物, 胶原蛋白

Abstract:

In the field of vascular tissue engineering, current technological advancements are focused on the proli-feration and secretion of collagen-rich extracellular matrix (ECM) by vascular smooth muscle cells (VSMCs) in vitro, coupled with specific mechanical stimuli to reconstruct biomaterials with desired mechanical properties. The optimal oxygen concentration is crucial for three-dimensional cell culture in vitro. However, there is a lack of sufficient research regarding its application in tissue-engineered vascular graft (TEVG). To this end, this study designed varying oxygen concentration environments to determine the optimal value for VSMCs culture by assessing cell proliferation activity. Furthermore, collagen gene expression and protein secretion were measured under normoxic and hypoxic conditions, along with quantification of total collagen content deposited in the cell layer using hydroxyproline assay. Subsequently, VSMCs were seeded on polyglycolic acid (PGA) scaffolds for three-dimensional culturing to form TEVG, and the effects of hypoxic conditions on TEVG cultivation were observed through histological staining and total collagen quantification. The results indicate that VSMC cell activity increased most rapidly at 7% oxygen concentration; under hypoxic conditions, an upregulation of type I (Col I) and type Ⅲ (Col Ⅲ) collagen gene expression was observed, with an increased secretion of collagen into the culture medium between days 5 and 9, particularly Col Ⅲ. Furthermore, the total collagen content on the 11th day demonstrates a 3.1-fold increase relative to the normoxic group. The 7% oxygen concentration facilitated collagen deposition during the three-dimensional culture of VSMCs on PGA scaffolds, resulting in a 2.09-fold increase in total collagen content compared to the normoxic group. Additionally, the formation of more dense collagen fibrils was observed. These findings indicate that the utilization of a hypoxic environment can enhance the collagen content in TEVG, thereby providing a foundation for the further optimization of in vitro culture conditions for TEVG.

Key words: vascular smooth muscle cell, hypoxia environment, tissue-engineered vascular graft, collagen

中图分类号:

Q813.1

林展翼, 肖聪, 许健宜, 刘青, 孙盱衡, 方丽君. 低氧环境对组织工程血管移植物胶原蛋白含量的提升作用[J]. 华南理工大学学报(自然科学版), 2025, 53(1): 129-135.

LIN Zhanyi, XIAO Cong, XU Jianyi, LIU Qin, SUN Xuheng, FANG Lijun. The Effect of Hypoxic Environment on the Enhancement of Collagen Content in Tissue-Engineered Vascular Grafts[J]. Journal of South China University of Technology(Natural Science Edition), 2025, 53(1): 129-135.

图/表 5

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基因名称	引物
Col ⅠA1	正向：GCTACTACCGGGCTGATGAT
Col ⅠA1	反向：TTCTCCGCTCTTCCAGTCAG
Col ⅢA1	正向：CTATGGGCATCAAAGGACATCG
Col ⅢA1	反向：CAGATCCTCTTTCACCTCTGTT
β-actin	正向：GCATGGAATCCTGCGGCATTCACGAA
β-actin	反向：TGGCGTAGAGGTCCTTGCGGATGTC

低氧环境对组织工程血管移植物胶原蛋白含量的提升作用

The Effect of Hypoxic Environment on the Enhancement of Collagen Content in Tissue-Engineered Vascular Grafts

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