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CN-119530127-B - Construction method of natural lipid deposition model of liver organoids of laying hens

CN119530127BCN 119530127 BCN119530127 BCN 119530127BCN-119530127-B

Abstract

The invention relates to the technical field of cell culture, and discloses a construction method of a natural lipid deposition model of liver organoids of laying hens, which verifies that liver tissue lipid synthesis and transport related functions of 1-day-old laying hens are more active, and the isolated liver organoids and liver primary cells show the same trend and have more lipid deposition, so that the organoids cultured by the liver primary cells of the 1-day-old laying hens are selected as a natural in-vitro lipid deposition model. In order to solve the technical problem that the endogenous lipid deposition model of the laying hen is difficult to build, the invention successfully builds the natural lipid deposition model of the liver organoid of the laying hen from the primary liver cells by separating the primary liver cells of the laying hen at 1 day old and improving the organoid inoculation and culture technology, reduces the research cost, has higher lipid metabolism activity, and provides an ideal in-vitro model for the research of liver lipid metabolism and related disease mechanisms of the laying hen.

Inventors

  • YANG XIAOJUN
  • WANG YIBIN
  • HOU QIHANG
  • LIU RUI
  • LIU YANLI

Assignees

  • 西北农林科技大学

Dates

Publication Date
20260508
Application Date
20241104

Claims (8)

  1. 1. The method for constructing the natural lipid deposition model of the liver organoids of the laying hens is characterized by comprising the following steps of: (1) Taking the liver of the laying hen, removing the membranous and the gall bladder, transferring the liver to precooled PBS containing 2% of green streptomycin, and cleaning for 2-3 times until the PBS is clear; (2) Placing the liver tissue of the laying hen obtained by the treatment in the step (1) into a centrifuge tube, adding 500 mu L of PBS containing 2% of green streptomycin, and shearing into liver tissue fragments; (3) Transferring the liver tissue fragments obtained in the step (2) into a new centrifuge tube, naturally settling and cleaning the liver tissue fragments for 2-3 times by using PBS containing 2% of green streptomycin, centrifuging at 4 ℃ and 700rpm for 3min, and collecting the liver tissue fragments; (4) Transferring the liver tissue fragments obtained in the step (3) to a centrifuge tube containing 10mL of liver dissociation liquid, digesting for 30min in a 37 ℃ water bath, taking out the centrifuge tube every 10min, and shaking up and down for 30s with force, and after digestion, shaking up and down for 30s with force, and collecting digestion liquid with liver cells; (5) Filtering the digestive juice with the liver cells collected in the step (4) by using a 70 mu m cell filter screen, transferring the filtrate to a new centrifuge tube, centrifuging at 4 ℃ and 1500rpm for 5min, collecting liver cell sediment, and removing the digestive juice; (6) Resuspension the hepatocyte sediment obtained in step (5) with 7mL ADVANCED DMEM/F12 containing 1% of green streptomycin, centrifuging at 4 ℃ and 1500rpm for 5min, collecting new hepatocyte sediment, repeating for 2-3 times until washing to obtain washed hepatocytes; (7) Re-suspending the cleaned liver cells obtained in the step (6) by using a growth medium, wherein the concentration of the cell suspension is about 600,000 cells/mL, adding precooled matrigel, fully and uniformly mixing, and inoculating 50 mu L to 24-hole culture plate culture hole centers into each hole; (8) Placing the culture plate prepared in the step (7) into a cell culture box, standing for 30min, adding 500 mu L of HepatiCultTM organoid growth medium containing 1% of streptomycin into each hole after matrix gel is solidified, and placing into the cell culture box; (9) And (5) changing liquid for the liver organoids of the laying hens every 3d to obtain a natural lipid deposition model of the liver organoids of the laying hens.
  2. 2. The method for constructing a natural lipid deposition model of a liver organoid of a laying hen according to claim 1, wherein, The layer liver in the step (1) adopts 1-day-old layer liver.
  3. 3. The method for constructing a natural lipid deposition model of a liver organoid of a laying hen according to claim 1, wherein, In the step (4), the liver dissociation liquid comprises Collagenase Type IV of 125 mu L/mL, dispese of 125 mu L/mL, ADVANCED DMEM/F-12 of 750 mu L/mL, HEPES of 15 mu L/mL and a mixture of 10 mu L/mL of the green streptomycin.
  4. 4. The method for constructing a natural lipid deposition model of a liver organoid of a laying hen according to claim 1, wherein, The size of the tissue fragments in the step (2) is 0.5mm 3 .
  5. 5. The method for constructing a natural lipid deposition model of a liver organoid of a laying hen according to claim 1, wherein, In the step (7), the 24-well culture plate is preheated for 30min in a 37 ℃ cell culture incubator before inoculation.
  6. 6. The method for constructing a natural lipid deposition model of a liver organoid of a laying hen according to claim 1, wherein, The ratio of the growth medium to the matrigel in the step (7) is 1:2.
  7. 7. The method for constructing a natural lipid deposition model of a liver organoid of a laying hen according to claim 1, wherein, The number of the liver organoids of the laying hens cultured in the step (7) is 10,000/hole.
  8. 8. The method for constructing a natural lipid deposition model of a liver organoid of a laying hen according to claim 1, wherein, The temperature of the incubator in the step (8) is 37 ℃, and the content of CO 2 is 5%.

Description

Construction method of natural lipid deposition model of liver organoids of laying hens Technical Field The invention relates to the technical field of cell culture, in particular to a method for constructing a natural lipid deposition model of a liver organoid of a laying hen. Background In addition to humans and mammals, the incidence of fatty liver hemorrhagic syndrome of laying hens is up to 30%, and the sick laying hens are often accompanied with obesity, reduced laying rate, accumulation of abdominal or subcutaneous fat, and even death caused by internal hemorrhage, which causes serious economic loss to poultry industry. In order to explore the molecular mechanism of fatty liver and reduce its morbidity and mortality, it is highly desirable to build a suitable representative model for subsequent studies. Currently there are three types of cell sources for liver organoids, including stem cells, cancer cell lines and primary cells. Organoids derived from primary hepatoma cells exhibit histological morphology similar to that of in vivo cancers, and most cancer cell lines can carry genetic abnormalities, primarily for use in mimicking primary hepatoma and colon cancer liver metastasis. Since liver primary cells do not require extensive reprogramming and differentiation, it is more convenient and economical for rapid and high throughput pharmacological studies. Compared to the stem cells and the organoids derived from cancer cell lines, the primary hepatocyte-derived organoids retain more functions than donor livers, carry 10-fold fewer base substitutions in their genes than induced pluripotent stem cells, and retain donor-specific characteristics. Research shows that the organ tissue from primary liver cell shows higher physiological relevance in medicine metabolism and toxicity test, and has gene expression spectrum similar to that of original liver tissue and complex liver functions, such as bile synthesis, lipid metabolism, etc. This makes them an important tool for studying liver diseases and developing new drugs. In recent years, researchers have established some in vitro models that adequately reproduce the characteristics of the liver. Traditional two-dimensional hepatocyte culture techniques are relatively simple and cost-effective and can be used to explore cellular molecular mechanisms and drug efficacy, but two-dimensional cells often have difficulty reproducing the structural, functional and physiological properties of cells in vivo. Culturing hepatocytes in conventional culture dishes forces the cells to grow on the plastic surface of the culture dish, can affect important metabolic pathways thereof, lead to cytoskeletal changes, and lose cell polarity and enzymatic activity within 2-3 days. The three-dimensional liver organoid culture technology overcomes some limitations of two-dimensional culture, not only retains the operability of the experiment, but also maintains higher physiological relevance. The microenvironment created in the three-dimensional culture more closely approximates the in vivo environment, mimicking cytokine gradients and nutrient diffusion functions, cell polarization, cell-cell and cell-environment interactions, and the like. Compared with two-dimensional cell culture, the modeling effect of fatty liver, hepatic fibrosis, hepatocellular carcinoma and other diseases in a three-dimensional in-vitro model is better. However, at present, the method for establishing the lipid deposition model of the laying hen is complex, and the traditional method for externally adding oleic acid or palmitic acid has the defects of great effect difference, poor stability, cytotoxicity and difficulty in simulating endogenous lipid deposition. Disclosure of Invention Aiming at the defects existing in the prior art, the invention aims to solve the technical problem of difficulty in establishing a layer chicken lipid deposition model, and aims to provide a method for establishing a layer chicken liver organoid natural lipid deposition model, which verifies that liver tissue lipid synthesis and transport related functions of 1-day-old layer chicken are more active, isolated liver organoids and liver primary cells show the same trend, and liver has lipid deposition. Therefore, the natural lipid deposition model of the liver organoids of the laying hens, which is derived from the primary liver cells, is successfully constructed by selecting the organoids cultured by the primary liver cells of the laying hens of 1 day old as a natural in-vitro lipid deposition model, separating the primary liver cells of the laying hens of 1 day old, and improving the organoid inoculation and culture technology. In order to solve the technical problems, the technical scheme provided by the invention is as follows: The method for constructing the natural lipid deposition model of the liver organoids of the laying hens comprises the following steps: (1) Taking the liver of the laying hen, removing the membranous an