兔肝细胞体外分离及培养方法的实验研究  

A method in the isolation and culture of rabbit liver cells

作　　者：罗文[1] 周晓东[1] 巩萧音[1] 贺建国[1] 王莉[1] 秦海英[1] 

机构地区：[1]第四军医大学西京医院超声诊断科,西安710032

出　　处：《现代生物医学进展》2006年第10期5-7,共3页Progress in Modern Biomedicine

基　　金：国家自然科学基金面上项目(NO.30570489)

摘　　要：目的：研究体外兔肝细胞分离及培养方法,比较不同培养基条件下兔肝细胞培养过程。方法：采用非灌注胶原酶消化法分离兔肝细胞,分别采用RPIM 1640培养液(含10％新生牛血清),DMEM培养液(含10％新生牛血清),DMEM培养液(含10％胎牛血清)培养,计数法观察原代细胞增殖变化,MTT法观察传代细胞增殖情况。培养细胞采用PAS染色法鉴定,电镜观察细胞超微结构。结果：分离的肝细胞细胞活率大于85％；DMEM培养液(含10％胎牛血清)培养肝细胞生长状态较另两种培养液中的肝细胞强,DMEM培养液(含10％新生牛血清)中的细胞增殖能力较RPIM 1640培养液(含10％新生牛血清)高．具有统计学意义。PAS染色和透射电镜观察培养细胞胞质中有大量糖原颗粒。结论：本实验采用的分离方法可获得较纯的肝细胞,而且操作简便实用。DMEM培养液较RPIM 1640培养液更加适宜原代培养肝细胞生长,胎牛血清对培养肝细胞的生长促进作用明显高于新生牛血清。Objective： To explore a method for isolating and culturing rabbit liver cell in vitro and compare the cell growth in the different culture media. Methods： The rabbit liver cells were isolated by non - perfusion eollagenase digestion. The viability of cells was measured by typan blue staining. The liver cells were incubated respectively in three different media： Medium I （RPIM 1640 medium containing 10% new - born calf serum）, Medium II （DMEM medium containing 10 % new- born calf serum） and Medium 111（DMEM medium containing 10% fetal calf serum）, The growth of primary liver cells was measured by cell counting and MTr assay was used to assess the cell growth in the passage. The cell morphology was observed by the phase contrast microseope. The cultured livercells were identified by PAS （periodic acid- Schiff） staining and electron microscope. Results： The yield of hepatocytes was 0.8 - 1,0 x 106 from 69g liver tissue, The viability of the harvested primary hepatocytes was above 85 %, Numerous glycogen was detected in the cytoplasm of the cultured cells by PAS staining and electron microscope, The cultured cells in Medium II had more viability and proliferation ability than those in the other two medium, while those in Medium II was better than Medium I. Conclusions： The non- perfusion digestive method of collagenase can be used to isolate the rabbit liver cells, During the cell culture, DMEM medium containing 10% fetal calf serum was considered to promote cell growth better than the RPIM 1640 and DMEM containing 10% new- born calf serum,

关 键 词：兔 肝细胞 分离 培养 

分 类 号：Q813.11[生物学—生物工程]