通过患者来源的原位异种移植建立慢性乙型肝炎病毒感染小鼠模型的方案。

Protocol for chronic hepatitis B virus infection mouse model development by patient-derived orthotopic xenografts.

机构信息

Central Research Institute for Epidemiology of the Federal Service for Supervision of Consumer Rights Protection and Human Welfare, Moscow, Russia.

出版信息

PLoS One. 2022 Feb 23;17(2):e0264266. doi: 10.1371/journal.pone.0264266. eCollection 2022.

DOI:10.1371/journal.pone.0264266

PMID:35196351

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC8865695/

Abstract

BACKGROUND

According to the World Health Organization, more than 250 million people worldwide are chronically infected with the hepatitis B virus, and almost 800.000 patients die annually of mediated liver disorders. Therefore, adequate biological test systems are needed that could fully simulate the course of chronic hepatitis B virus infection, including in patients with hepatocellular carcinoma.

METHODS

In this study, we will assess the effectiveness of existing protocols for isolation and cultivation of primary cells derived from patients with hepatocellular carcinoma in terms of the yield of viable cells and their ability to replicate the hepatitis B virus using isolation and cultivation methods for adhesive primary cells, flow cytometry and quantitative polymerase chain reaction. Another part of our study will be devoted to evaluating the effectiveness of hepatocellular carcinoma grafting methods to obtain patient-derived heterotopic and orthotopic xenograft mouse avatars using animal X-ray irradiation and surgery procedures and in vivo fluorescent signals visualization and measurements. Our study will be completed by histological methods.

DISCUSSION

This will be the first extensive comparative study of the main modern methods and protocols for isolation and cultivation primary hepatocellular carcinoma cells and tumor engraftment to the mice. All protocols will be optimized and characterized using the: (1) efficiency of the method for isolation cells from removed hepatocellular carcinoma in terms of their quantity and viability; (2) efficiency of the primary cell cultivation protocol in terms of the rate of monolayer formation and hepatitis B virus replication; (3) efficiency of the grafting method in terms of the growth rate and the possibility of hepatitis B virus persistence and replication in mice. The most effective methods will be recommended for use in translational biomedical research.

摘要

背景

据世界卫生组织统计，全球有超过 2.5 亿人慢性感染乙型肝炎病毒，每年几乎有 80 万人死于乙型肝炎病毒导致的肝脏疾病。因此，需要有足够的生物检测系统来模拟乙型肝炎病毒慢性感染的过程，包括在肝癌患者中。

方法

在本研究中，我们将根据分离和培养原发性肝癌细胞的现有方案的有效性，评估使用粘附性原代细胞分离和培养方法、流式细胞术和定量聚合酶链反应来分离和培养原发性肝癌细胞的有效性，评估其获得乙型肝炎病毒的能力。我们研究的另一部分将致力于评估肝癌移植方法的有效性，以获得患者来源的异质和原位异种移植小鼠模型，使用动物 X 射线照射和手术程序以及体内荧光信号可视化和测量。我们的研究将通过组织学方法完成。

讨论

这将是首次对原发性肝癌细胞分离和培养以及肿瘤移植到小鼠中的主要现代方法和方案进行广泛的比较研究。所有方案都将使用以下方法进行优化和特征描述：（1）从切除的肝癌中分离细胞的方法的效率，从细胞的数量和活力方面考虑；（2）原代细胞培养方案的效率，从单层形成和乙型肝炎病毒复制的速度方面考虑；（3）移植方法的效率，从增长率和乙型肝炎病毒在小鼠体内持续存在和复制的可能性方面考虑。将推荐最有效的方法用于转化医学研究。