摘要
背景:将种子细胞接种于三维支架材料上然后在生物反应器中进行三维培养是一种常见的体外组织工程培养手段,但是生物工程血管构建过程中的细胞增殖变化及代谢模式尚不清楚。目的:探究利用体外生物反应器进行生物血管组织构建过程中细胞耗氧等代谢变化及其原因。方法:以自主搭建的血管生物反应器系统为平台,牛血管壁平滑肌细胞为种子细胞,常规CO_(2)培养箱提供培养过程的外部气体环境。将种子细胞接种于管状多孔隙的聚乙醇酸支架材料上进行三维培养,全过程包括1周的静置期和7周脉动张应力刺激加载期。搭建一套非侵入式监测体系,采用光学溶解氧贴片法监测反应器中培养液溶解氧变化,并通过定期取样测定葡萄糖消耗量及乳酸生成量。采用CCK-8检测平滑肌细胞在聚乙醇酸三维支架材料上增殖情况,通过烟酰胺腺嘌呤二核苷酸的氧化态与还原态比值(NAD^(+)/NADH)了解培养早期阶段细胞增殖与代谢状况,采用RT-qPCR、Western blot方法检测增殖相关基因(Ki67)及糖酵解相关基因(GLUT-1、LDHA)的表达。结果与结论:(1)从细胞加注到静置期结束(第1周)培养液的溶解氧水平为(4.314±0.380)mg/L,张应力刺激加载后(后7周)溶解氧水平逐步稳定在(1.960±0.866)mg/L,两者有明显变化(P<0.05);(2)细胞培养液中乳酸生成量与葡萄糖消耗量的比值Y_(L/G)在加注细胞后快速升高,第5天最高值高于1,随后缓慢下降至0.5(静置期Y_(L/G)均值为0.89,加压期均值为0.57,P<0.05);(3)CCK-8检测显示A_(450)值在细胞加注之后逐渐增大,第5天达到最高值3.17,之后缓慢下降;同时发现Ki67 mRNA在培养第3天上调最显著,之后下降,Ki67蛋白在第3-5天的相对表达量较高;(4)细胞加注后第5-7天NAD~+/NADH明显升高,糖酵解相关基因(GLUT-1、LDHA)表达上调同步改变,前5 d相对表达量较高;(5)结果提示:利用血管生物反应器构建组织工程�
BACKGROUND:Seed cells are seeded on three-dimensional scaffold materials,and three-dimensional culture in bioreactors is a common in vitro tissue engineering culture method,but the changes in cell proliferation and metabolic patterns in bioengineered blood vessel construction are still unclear.OBJECTIVE:To explore the metabolic changes of cells such as oxygen consumption and their causes during the whole process of biological vascular tissue construction by in vitro bioreactor.METHODS:The self-built vascular bioreactor system was used as the platform;bovine vascular smooth muscle cells were used as the seed cells,and a conventional CO_(2) incubator provided the external gas environment for the cultivation process.Seed cells were seeded on a tubular porous polyglycolic acid scaffold material for three-dimensional culture,and the whole process included a one-week resting period and a seven-week pulsating tensile stress stimulation loading period.A non-invasive monitoring system was built,and the optical dissolved oxygen patch method was used to monitor the changes of dissolved oxygen in the culture solution in the reactor,and the glucose consumption and lactic acid production were measured by regular sampling.CCK-8 assay was used to determine the proliferation of smooth muscle cells on polyglycolic acid three-dimensional scaffold materials.Nicotinamide adenine dinucleotide oxidation state and reduction state ratio(NAD^(+)/NADH)was utilized to understand cell proliferation and metabolism in the early stage of culture.RT-qPCR and western blot assay were applied to detect the expression of proliferation-related genes(Ki67)and glycolysis-related genes(GLUT-1,LDHA).RESULTS AND CONCLUSION:(1)The dissolved oxygen level in the culture solution was(4.314±0.380)mg/L from the cell injection to the end of the resting period(the first week),and gradually stabilized at(1.960±0.866)mg/L after the tensile stress stimulation(the last seven weeks);the two had significant changes(P<0.05).(2)The ratio of glucose consumption to lacti
作者
梅静怡
刘江
肖聪
刘鹏
周浩浩
林展翼
Mei Jingyi;Liu Jiang;Xiao Cong;Liu Peng;Zhou Haohao;Lin Zhanyi(School of Biology and Biological Engineering,South China University of Technology,Guangzhou 510006,Guangdong Province,China;Guangdong Provincial People’s Hospital(Guangdong Academy of Medical Sciences),Southern Medical University,Guangzhou 510080,Guangdong Province,China;Ji Hua Institute of Biomedical Engineering Technology(Ji Hua Laboratory),Foshan 528200,Guangdong Province,China;School of Medicine,South China University of Technology,Guangzhou 510006,Guangdong Province,China)
出处
《中国组织工程研究》
CAS
北大核心
2024年第7期1043-1049,共7页
Chinese Journal of Tissue Engineering Research
基金
先进制造科学与技术广东省实验室(季华实验室)科研项目:动静脉造瘘血管移植体外工程化培养的装置研制(X210111TD210),项目负责人:林展翼。
关键词
生物反应器
组织工程
血管平滑肌细胞
聚乙醇酸
溶解氧
细胞增殖
有氧糖酵解
bioreactor
tissue engineering
vascular smooth muscle cell
polyglycolic acid
dissolved oxygen
cell proliferation
aerobic glycolysis
