Method for constructing milk cow blood milk barrier three-dimensional model in vitro

A three-dimensional model, technology of cows, applied in artificial cell constructs, biochemical equipment and methods, vascular endothelial cells, etc., can solve problems such as no 3D cultured blood-milk barrier model

Active Publication Date: 2018-08-14
NORTHEAST AGRICULTURAL UNIVERSITY
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AI Technical Summary

Problems solved by technology

The blood-milk barrier constructed by Guidry is composed of a single layer of cells, so far there is no 3D cultured blood-milk barrier model at home and abroad

Method used

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  • Method for constructing milk cow blood milk barrier three-dimensional model in vitro
  • Method for constructing milk cow blood milk barrier three-dimensional model in vitro
  • Method for constructing milk cow blood milk barrier three-dimensional model in vitro

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Embodiment Construction

[0041] as attached Figure 7 As shown, a method for constructing a three-dimensional model of the blood-milk barrier of dairy cows in vitro is characterized in that it comprises the following steps:

[0042] 1 Culture of cow mammary gland epithelial cells and fibroblasts

[0043] Aseptically cut out about 2g of breast tissue, put it in a sterile 50ml Erlenmeyer flask containing D-Hanks' solution, and bring it back to the laboratory in a warm box; put the breast tissue in a 50ml sterile beaker in a clean bench, Rinse 5 times with D-Hanks' solution containing 10× double antibody, and wash away the milk, blood and other impurities attached to the tissue; place the washed tissue in a 10ml sterile beaker, and cut it into small tissue pieces of 1mm3 with sterilized surgical scissors ;Transfer all the small tissue pieces to another sterile Erlenmeyer flask, add 20ml of digestion solution, seal the cap, place it in a shaker at 37°C, and shake and digest at 200r / min for 4h until no ob...

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Abstract

The invention provides a method for constructing a milk cow blood milk barrier three-dimensional model in vitro. The method comprises the steps of cultivation of mammary epithelial cells and fibroblast of a milk cow, cultivation of venous endothelial cells of the milk cow, immunofluorescent identification of the mammary epithelial cells and the venous endothelial cells, cocultivation of Transwell,preparation of a cocultivation medium by taking prolactin, insulin, an epidermal growth factor, cow growth hormone, a vascular endothelial growth factor and a fibroblast growth factor as hormone andcell factors related to growth of the mammary epithelial cells, the fibroblast and the endothelial cells; and finally the epithelial cells, the fibroblast and the endothelial cells are inoculated on atranswell chamber, and accordingly the milk cow blood milk barrier model is successfully constructed. The method can be used for researching a molecular mechanism by which nutrient substances, drugsand toxic substances enter the mammary tissue and the milk in real-time and effectively, not only fills in the gap both domestically and internationally, but also provides a research platform for research on the molecular mechanism by which lipid, vitamins, the drugs and the toxic substances pass through the blood milk barrier.

Description

technical field [0001] The invention relates to a method for constructing a three-dimensional model of a cow's blood-milk barrier in vitro, and belongs to the technical field of blood-milk barrier research. Background technique [0002] Traditional two-dimensional cell culture can only conduct molecular and genetic research on cells on a two-dimensional plane, so as to understand the impact of specific molecular and genetic changes on cell biological behavior. Although animal models can more accurately reflect the morphological characteristics, it is difficult to conduct large-scale molecular studies. The present invention constructs a three-dimensional blood-milk barrier model that combines the advantages of two-dimensional cell culture and animal models, and focuses on solving three types of cell inoculation density, culture time, three-dimensional combined medium type and Add the types and concentrations of cytokines that promote angiogenesis, and the concentrations of h...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071
CPCC12N5/0697C12N2501/11C12N2501/115C12N2501/165C12N2501/305C12N2501/315C12N2501/33C12N2502/095C12N2502/1323C12N2502/28C12N2533/54
Inventor 张娜张建涛迟新宇陈丹王伟华张艳莹赵锋
Owner NORTHEAST AGRICULTURAL UNIVERSITY
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