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

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.

Recent advances in different interactions between toll-like receptors and hepatitis B infection: a review

“…TLR response to HBV infection in many animal models has been well investigated ( 46 , 47 ). In animal models, TLR2 activation has been shown to expedite HBV clearance and improve HBV-specific T-cell responses ( 48 ).…”

Progress of Infection and Replication Systems of Hepatitis B Virus

“…Lastly, TK-NOG mice were developed by expressing herpes simplex virus thymidine kinase (HSV-tk) driven by mouse liver protein promoters. This enzyme phosphorylates nontoxic ganciclovir (GCV) into a highly toxic metabolite, effectively eliminating mouse hepatocytes . Among these models, uPA mice support HBV replication, facilitating antiviral testing, immunotherapy engineering, analysis of genotype-specific antigen expression, and investigations into innate immunity .…”

“…This enzyme phosphorylates nontoxic ganciclovir (GCV) into a highly toxic metabolite, effectively eliminating mouse hepatocytes. 45 Among these models, uPA mice support HBV replication, facilitating antiviral testing, immunotherapy engineering, analysis of genotype-specific antigen expression, and investigations into innate immunity. 7 However, due to hepatotoxicity or uPA gene overexpression, newborn mice exhibit hemolysis, chronic liver agenesis, or hypofibrinogenemia 4 days after delivery, and are prone to complement reaction after human hepatocyte transplantation, resulting in kidney function impairment and elevated mortality.…”

Human microbiomes in cancer development and therapy

“…It depends on the interaction among virus, host and environmental factors when HCV carriers developed into HCC. The HCV genome cannot be integrated into the host genome, but it can damage the host liver cells DNA through viral proteins and ROS producing by some certain carcinogenesis pathways, causing gene mutation and HCC occurrence 180 . It is possible for HBV carriers or HCV carriers to develop cancer due to some certain factors, such as sex, age, family history, viral load, infection time, coinfection with other virus (HDV, HIV, HBV/HDV), environmental factors, and so on 181 .…”