三维多孔微载体支持下人肝细胞L-02的培养

CULTURING HUMAN HEPATOCYT L-02 BY THREE-DIMENSIONAL POROUS CHITOSAN MICROCARRIER

目的:对壳聚糖三维多孔微载体培养的人肝细胞L-02进行定时的形态学观察、细胞功能和代谢活性检测。方法:本研究以自制的壳聚糖三维多孔微载体样本为支架来培养人肝细胞L-02,用细胞计数、倒置相差生物显微镜和扫描电子显微镜对其进行定时的形态学观察,并进行定时的细胞功能检测(包括测定谷草转氨酶,谷丙转氨酶和乳酸脱氢酶水平)和代谢活性检测(包括检测培养基中白蛋白、尿素、葡萄糖含量)。结果:两组培养的细胞数量均呈现前3d增长,在第3d细胞数量达到最高值;而且实验组3个样本培养的细胞数与对照组无微载体培养的细胞数量相比,均始终明显高于后者,两者差异具有统计学意义(P<0.05),而实验组各样本培养的细胞之间无明显差异(P>0.05);在倒置相差生物显微镜下进行动态观察,可看到前3d微载体表面黏附生长的肝细胞则逐渐增多,在第3d时可见大部分微载体表面有许多肝细胞黏附成团,总的存活率均在90%以上,且肝细胞保持着良好的形态学结构;扫描电子显微镜下,在微载体表面、切面和内部均可看到有许多球状肝细胞紧密黏附。实验组和对照组测定的AST、ALT和LDH含量在前3d持续下降,第3d降到最低,而从第4dAST、ALT、LDH含量开始重新反弹上升,但ALB、UREA和GLUCOSE含量在前3d持续上升,第3d升到最高值,而从第4d ALB、UREA和GLUCOSE含量开始逐渐下降,实验组每天测定上述各项含量始终明显高于对照组,表明两组各项含量的差异具有统计学意义(P<0.05),提示实验组中的人肝细胞L-02代谢活性较对照组增强;而实验组中微载体样本1、2、3之间测定的各项含量无明显差异,表明实验组3个样本间各项含量的差异无统计学意义(P<0.05)。结论:本研究以自制的壳聚糖三维多孔微载体作为一种支架,在体外三维环境下可以进行高密度细胞培养,为肝细胞移植、生物人工肝研究中进一步以微载体黏附培养足够数量、功能良好的肝细胞提供了一定的理论和实验依据。

Objective：To observe the morphological changes and detect the function and metabolic activity of human hepatocyte L-02 cultured by the three-dimensional porous chitosan microcarrier at each time.Methods：To observe the morphological changes by using cell count,inverted microscope,scanning electron microscope and detect the function（includes the aspartate aminotransferase,alanine aminotransferase and lactate dehydrogenase level）and metabolic activity（includes the albumin,urea,glucose content）of human hepatocyte L-02 cultured by the three-dimensional porous chitosan microcarrier at each time.Results：The quantity of the two groups cultured cell has always increased in the former three days and reached the highest value at the third day.The cell quantity of the experiment group was obviously higher than the compare group and it has significant difference（P 0.05）.But it has no significant difference in the experiment group（P0.05）.The development observation results under inverted microscope as follow：the quantity of these cell adhered to microcarrier has increased gradually in the experiment group,there were lots of hepatocyte adhered to ball in the surface of a greater part of microcarrier,the total survive rate of hepatocyte was more than 90% and hepatocyte kept a good morphological structure.Under scanning electron microscope,lots of hepatocyte adhered intensely each other in the surface,section,inside of microcarrier.In two groups,the concentration of aspartate aminotransferase（AST）,alanine aminotransferase（ALT）,lactate dehydrogenase（LDH）,albumin（ALB） secretion,the level of urea synthesis and glucose consumption dropped in the former three days,reached the lowest value at the third day and began to increase at the fourth day.But the concentration of ALB、UREA and GLUCOSE increased in the former three days,reached the highest value at the third day and began to drop at the fourth day.These concentration of the experimental group was obviously higher than them of the compare group（P0.05）.It indicated that these different concentration of the two groups has significance in statistics and metabolic activity of human hepatocyte L-02 in the experimental group more higher than then of the compare group.But it has no significant difference in the experiment group（P0.05） and indicated that these different concentration has no significance in statistics.Conclusoin：This study cultured successfully human hepatocyte L-02 by using the three-dimensional porous chitosan microcarrier as a support in three-dimensional environment of vitro.It provides a theoretical basis and experimental evidence for culturing adequate numbers and well-functioning hepatocyte in the hepatocyte transplantation and BAL study.