摘要
目的提供适用于日本血吸虫感染小鼠模型活化后肝星状细胞分离的新方法,并确定最适宜的密度分离液浓度。方法构建日本血吸虫感染BALB/c小鼠模型,对小鼠模型分别进行经下腔静脉逆行灌注和门静脉顺行灌注,充分完全消化小鼠肝脏,获得肝脏细胞悬液。利用Optiprep密度梯度离心法逐步纯化获得活化的原代肝星状细胞。通过流式分选法和Real-time PCR验证实验结果,统计分析通过不同浓度分离液密度获得的肝星状细胞数量和纯度。结果当分离液密度分别为13.5%、15.0%、16.5%和18.0%时,富集到的肝星状细胞数量依次升高,肝星状细胞的纯度分别可达56.1%、90.3%、75.9%和71.1%。比较流式分选法得到的细胞与本实验得到的原代肝星状细胞纯度,二者间差异无统计学意义(P>0.05)。结论改良后的实验方法可在日本血吸虫感染小鼠模型中高产量地分离出活化的高纯度原代肝星状细胞,且更加经济便捷、实验结果稳定。
Objective A new method for isolating hepatic stellate cells after activation of a mouse model of Schistosoma japonicum infection was provided,and the optimal density of the separation solution was determined.Methods A BALB/c mouse model of Schistosoma japonicum infection was constructed,and the mouse models were perfused inferiorly via the inferior vena cava and the portal vein by antegrade perfusion to fully digest the mouse liver and obtain liver cell suspension.Optiprep density gradient centrifugation was used to gradually obtain activated primary hepatic stellate cells,and the number and purity of the hepatic stellate cells were counted.The experimental results were verified by flow cytometry and Real-time PCR,and the number and relative purity of hepatic stellate cells obtained from different concentrations of separation solution were statistically analyzed.Results When the density of separation solution was 13.5%,15.0%,16.5%and 18.0%,the number of enriched HSCs increased successively,and the purity of HSCs could reach 56.1%,90.3%,75.9%and 71.1%,respectively.Comparing the purity of the cells obtained by flow sorting with the primary hepatic stellate cells obtained in this experiment,there was no significant difference between the two(P>0.05).Conclusion The result showed that this improved method of isolation for primary HSCs could provide high-purity and high-yield,which was convenient and consistant.
作者
张亦舒
宋秋月
汤文燕
张蓓蓓
宋兰桂
刘嘉华
雷俊霞
吴忠道
赵玉敏
ZHANG Yi⁃shu;SONG Qiu⁃yue;TANG Wen⁃yan;ZHANG Bei⁃bei;SONG Lan⁃gui;LIU Jia⁃hua;LEI Jun⁃xia;WU Zhong⁃dao;ZHAO Yu⁃min(Guilin Medical University,Guilin,Guangxi 541000;Zhongshan School of Medicine,Sun Yat⁃sen University,Guangzhou,Guangdong 510080;The First Affiliated Hospital,Sun Yat⁃sen University,Guangzhou,Guangdong 510080;Xuzhou Medical University,Xuzhou,Jiangsu 221000;The Eighth Affiliated Hospital,Sun Yat⁃sen University,Shenzhen,Guangdong 518000,China)
出处
《热带医学杂志》
CAS
2020年第3期319-322,349,F0003,共6页
Journal of Tropical Medicine
基金
国家自然科学基金(81960376)。
