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Primary mouse liver cancer model based on liver oval cell malignancy as well as establishment method and application of primary mouse liver cancer model

An established method and primary technology, applied in the direction of hepatocytes, chemical instruments and methods, cell culture active agents, etc., can solve problems such as the inability to accurately study the mechanism of specific genes

Active Publication Date: 2022-02-01
WEST CHINA HOSPITAL SICHUAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The present invention intends to provide a method for establishing a primary mouse liver cancer model based on the malignant transformation of hepatic oval cells, so as to solve the problem that the existing primary liver cancer mouse model cannot accurately study the role of specific genes in the development of liver cancer Technical questions about the mechanism of action

Method used

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  • Primary mouse liver cancer model based on liver oval cell malignancy as well as establishment method and application of primary mouse liver cancer model
  • Primary mouse liver cancer model based on liver oval cell malignancy as well as establishment method and application of primary mouse liver cancer model
  • Primary mouse liver cancer model based on liver oval cell malignancy as well as establishment method and application of primary mouse liver cancer model

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1: Establishment of mouse liver cancer cell lines

[0033] (1) Construction of retroviral vector and preparation of virus liquid

[0034]The original retroviral expression vector is MSCV-IRES-ZsGreen1. The MSCV-IRES-ZsGreen1 is transformed, and the cMYC gene is inserted to obtain the target plasmid. After the sequence is correct, a high-quality endotoxin-free target plasmid (ie, MSCV- CMYC-IRES-ZsGreen1). The cMYC gene is a known proto-oncogene in the prior art, which can promote cell division and proliferation. The detection of this gene is used as an auxiliary means for tumor screening, and can also be used in the establishment of tumor animal models to promote tumor formation. In this protocol, the accession number of the cMYC gene used is 110535641. This protocol takes cMYC as an example for illustration. In addition to the proto-oncogene, kRAS, P53, PTEN, KLF6 and P16 and other known proto-oncogenes can also be selected according to actual needs to constr...

Embodiment 2

[0040] Example 2: Establishment of mouse orthotopic liver cancer model

[0041] In this example, the cultured liver cancer cells were orthotopically transplanted into the liver of recipient mice by hepatic portal vein injection, and a mouse liver cancer model was successfully established. One day before the injection, C57BL / 6 mice were irradiated with X-rays (RS2000pro, Radsource, USA) at a dose of 5.5Gy (X-rays were irradiated at 320KV for 270s) for use the next day. The prepared malignant liver oval cells were digested and counted, resuspended in PBS, and prepared into 10 7 / mL cell suspension and put it on ice for later use. Mice were anesthetized with isoflurane, and their limbs were immobilized on an operating table. Routine povidone iodine disinfection, cutting along the midline of the abdomen to 2-3 cm below the xiphoid process of the mouse, exposing the abdominal cavity, exposing the hepatic portal vein with a wet cotton swab and gently provoking it, slowly injecting...

Embodiment 3

[0045] Example 3: Acquisition of primary liver oval cells

[0046] Eight-week-old male C57BL / 6 mice were fed with drinking water containing 0.1% DDC (3,5-diethoxycarbonyl-1,4-dihydro-collidine) for 2 weeks, and then the mice were sacrificed to test whether the DDC model was established success. Image 6 The liver pathology after the DDC mouse model was successfully established showed obvious cholestatic cholangitis. Immunohistochemical staining showed oval cells aggregated and proliferated around the portal vein in the portal area of ​​the liver and expressed CK19 and ALB.

[0047] The DDC model mice were anesthetized with isoflurane, opened the abdomen and exposed the hepatic portal vein, washed the mouse liver with 37°C preheated phosphate buffered saline (PBS), and then perfused with 0.01% type IV collagenase at a rate of 2 mL / min 10min. Then put the liver tissue into DMEM / F12 medium containing 0.01% collagenase IV, cut it into pieces, blow and blow to disperse the cells,...

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Abstract

The invention relates to the technical field of cancer animal model establishment, in particular to a primary mouse liver cancer model based on liver oval cell malignancy and an establishment method and an application thereof. The establishment method comprises the following steps: transforming liver oval cells by using a proto-oncogene to obtain liver cancer stem cells; performing in-situ transplantation on the liver cancer stem cells to the liver of a receptor mouse to obtain a primary mouse liver cancer model; wherein the acceptor mouse is a wild type mouse subjected to X-ray irradiation treatment. According to the scheme, aiming at the limitation of the existing primary liver cancer animal model, a novel primary model for simulating the whole occurrence and development natural process of the primary liver cancer is established, so that the effect and mechanism of a specific gene in the occurrence and development of the primary liver cancer can be accurately researched, and whether the gene can be used as an effective treatment target or not is verified. By utilizing the model in the scheme, potential new targets and new drugs for treating the primary liver cancer can be screened, and conditions are created for researching and developing novel drugs or treatment methods for treating the primary liver cancer.

Description

technical field [0001] The invention relates to the technical field of establishment of cancer animal models, in particular to a primary mouse liver cancer model based on the malignant transformation of hepatic oval cells and its establishment method and application. Background technique [0002] Animal models are one of the important tools for studying human disease mechanisms, identifying therapeutic targets, and evaluating treatment methods and drugs. Among them, the mouse model of primary liver cancer (hepatocellular carcinoma, HCC, also known as hepatocellular It plays an indispensable role in the study of the pathogenesis and development mechanism of primary liver cancer. Because of its simple genetic background, mice are very similar to humans in physiology, and the cost is lower, the cycle is shorter, and gene modification technology is easier to implement. Therefore, the mouse liver cancer model is an important tool for studying the mechanism of human liver cancer a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/867C12N5/09A01K67/027
CPCC12N15/86C07K14/82C12N5/067C12N5/0693A01K67/0271C12N2740/10043C12N2800/107C12N2510/04C12N2506/14C12N2501/11C12N2501/12C12N2501/235C12N2500/32A01K2227/105A01K2267/0331A01K2207/12
Inventor 何林烨
Owner WEST CHINA HOSPITAL SICHUAN UNIV
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