肝组织工程支架评价方法研究

Study on Evaluation Method of Scaffold for the Use of Liver Tissue Engineering

目的:以人肝细胞系HepG2作为种子细胞,建立一种细胞支架评价方法,为肝组织工程筛选合适支架。方法:将HepG2细胞接种在生物可降解聚合物支架——聚乳酸-乙醇酸共聚物(PLGA)、壳聚糖(3%CS)和丝素(2%SF)上,体外常规培养;采用四唑盐(MTT)比色法、HE染色和尿素氮检测试剂盒对接种在支架上HepG2细胞的生长、分布及功能情况进行检测。结果:培养在3种支架上的HepG2细胞均维持增殖状态,相比之下,丝素支架上的细胞增殖较快,而PLGA和壳聚糖支架上的细胞增殖相对缓慢;培养至第7d时,组织学检测显示丝素支架上的HepG2细胞分布均匀,数量最多;而在PLGA和壳聚糖支架上只能发现少量细胞;细胞功能检测显示丝素和PLGA支架上的尿素合成能力下降缓慢,而壳聚糖支架上的尿素合成能力快速下降。结论:3种支架均具有比较好的生物相容性;相比较而言,丝素更适合作为肝组织工程支架;该方法可用于批量筛选肝组织工程支架。

Objective To set up a method of scaffold evaluation using human cell line as seed cells and screen appropriate scaffold for live tissue engineering. Methods HepG2 cells were plated onto biodegradable polymer scaffolds： PLGA, 3% chitosan （3%CS） and 2% silk fibroin （2%SF）, and cultured in vitro. The growth, distribution and function of HepG2 cells in the scaffolds were evaluated using MTT assay, H.E. staining, and urea assay kit. Results HepG2 ceils plated on the three scaffolds maintained a proliferative state. In contrast, the cells on the 2%SF proliferated strongly, while the cells on the PLGA and chitin proliferated poorly. Histological examination showed that HepG2 cells distributed evenly on the 2%SF scaffold with a high amount, while few cells could be found on the PLGA and chitin at day 7. Cell function assay showed that HepG2 cells on the 2%SF and PLGA exhibited slower decrease of urea synthesis compared to HepG2 cells on the chitosan. Conclusion The three scaffolds have good biocompatibility. In contrast, 2%SF scaffold is more appropriate for liver tissue engineering. This method may be used for scale screening of scaffolds for liver tissue engineering.