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A three-dimensional culture model of echinococcus in vitro and its application

An echinococcosis and model technology is applied in the field of in vitro three-dimensional culture models of hydatid cysts, which can solve the problems of loss of specific hepatocyte function and loss of gap junctions, and achieve the effect of maintaining the effect of good hepatocyte function.

Active Publication Date: 2017-04-12
LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The traditional hepatocyte culture is two-dimensional space culture under unnatural conditions, the gap junction will be lost within a few hours of culture, and the specific hepatocyte function will be lost rapidly after a week

Method used

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  • A three-dimensional culture model of echinococcus in vitro and its application
  • A three-dimensional culture model of echinococcus in vitro and its application
  • A three-dimensional culture model of echinococcus in vitro and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Example 1. Preparation and detection of echinococcus in vitro three-dimensional culture model

[0055] 1. Preparation of three-dimensional culture model of echinococcosis in vitro

[0056] (1) Preparation of mouse-derived primary hepatic parenchymal cells

[0057] According to the method reported in the literature (Seglen PO.Preparation of isolated rat liver cells.MethodsCell Biol, 1976,13:29-83.), the mouse-derived primary liver parenchymal cells were prepared, and the specific steps were as follows: Sodium perbital was injected into the abdominal cavity of SD rats for anesthesia; the rats were sterilized with 75% ethanol water solution by volume, the abdominal cavity was opened aseptically, the liver was exposed, and the hepatic portal vein and inferior vena cava were punctured and ligated respectively. Perfuse with calcium-free perfusion buffer pre-warmed at 37°C and saturated with oxygen for 10 minutes at a flow rate of 4-5 mL / min, and then perfuse with 5 mg / L coll...

Embodiment 2

[0073] Example 2. Research on the function of hepatic parenchymal cells in the three-dimensional culture model of echinococcosis in vitro

[0074] The function of the liver parenchymal cells of the hydatid in vitro three-dimensional culture model obtained in Example 1 was analyzed, including the following detection:

[0075] (1) Detection of albumin: The culture medium in the three-dimensional culture model of Echinococcus was collected regularly, centrifuged (100g, 5min), and the supernatant of the culture medium (without cell components) was taken, and the albumin ELISA Quantitation Kit (BethylLaboratories, Inc. product, the catalog number is E110-125) kit to detect the content of albumin secreted by hepatic parenchymal cells. For specific operations, refer to the kit instructions.

[0076] (2) 7-ethoxy-3-isophenoxazolone-deethylase (EROD) detection: regularly at 37 ° C, 5% CO 2 Add 7-ethoxyisophenoxazole (final concentration: 39.2 mmol / L) to the complete culture medium of...

Embodiment 3

[0082] Example 3, the comparison between the three-dimensional culture model of echinococcosis in vitro and the two-dimensional culture model of echinococcosis in vitro

[0083] 1. Detection of functional gene expression of hepatic parenchymal cells in three-dimensional culture model of Echinococcus in vitro

[0084] With the three-dimensional culture model of echinococcus prepared in step 1 in Example 1, the cells (i.e. hepatic parenchymal cells and bone marrow mesenchymal stem cells) cultured on the three-dimensional collagen scaffold were collected every 4 days from the third day of culture, and different The RNA of hepatic parenchymal cells and bone marrow mesenchymal stem cells collected at different time points was reverse-transcribed into cDNA. , G6P F and G6P R, α1-AT F and α1-AT R were used as primers for real-time quantitative PCR to detect the expression of functional genes AFP, G6P, and α1-AT in liver parenchymal cells, and GAPDH was used as an internal reference g...

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Abstract

The invention discloses an in-vitro three-dimensional cultivation model for hydatid cysts and application thereof. The model used for cultivating the hydatid cysts comprises a cultivating pond and a three-dimensional collagen scaffold arranged in the cultivating pond. The three-dimensional collagen scaffold is loaded with feeder cells. The feeder cells are primary parenchymal hepatic cells and bone mesenchymal stem cells. Bio-culture solutions are contained in the cultivating pond and are solutions used for cultivating the hydatid cysts and the feeder cells. The in-vitro three-dimensional cultivation model for the hydatid cysts can be used for researching the development process of the hydatid cysts; by means of the model, immunofluorescent staining observation, electron microscope detection and RT-PCR analysis can be performed on host cells and parasite tissue cells in the three-dimensional collagen scaffold, correlational researches on in-vitro long-time cultivation and development of the hydatid cysts are achieved, and compared with an existing cultivation model for the hydatid cysts, remarkable progress is made.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to an in vitro three-dimensional culture model of echinococcosis and its application. Background technique [0002] Multilocular echinococcosis, also known as alveolar echinococcosis or alveolar echinococcosis, is parasitic on animals and humans by the larvae of Echinococcus multilocularis in the genus Echinococcus multilocularis. A zoonotic parasitic disease caused by the body that seriously affects the health of humans and animals is one of the deadliest helminth-infected diseases for humans. It is called "worm cancer" because of the high mortality rate of patients. At present, research on the disease is mainly realized through experimental animal models and in vitro culture models of protoscoleiae. The animal model is an important method for the passage and conservation of Echinococcus in the laboratory. This model has been used for the study of host-parasite immunology and molecular ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/0775C12N5/071A01K67/033A61K35/56A61K35/12
CPCA01K67/033
Inventor 贾万忠李立陈冰肖志峰戴建武闫鸿斌娄忠子
Owner LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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