Construction of in vitro pathological model for metabolism syndrome and application of model in drug screening

A technology for metabolic syndrome and drugs, applied in the field of high-content drug screening, can solve the problems of high-content screening without major progress in cell structure and functional integrity, low success rate, etc.

Inactive Publication Date: 2010-07-07
HANGZHOU DIANZI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the field of drug research and development, due to the defect of low success rate of high-throughput screening technology, high-throughput screening urgently needs to be developed into high-content screening, but currently high-content screening has not made great progress in maintaining the integrity of cell structure and function

Method used

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  • Construction of in vitro pathological model for metabolism syndrome and application of model in drug screening
  • Construction of in vitro pathological model for metabolism syndrome and application of model in drug screening
  • Construction of in vitro pathological model for metabolism syndrome and application of model in drug screening

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Example 1 Seed cell culture

[0050] Adipose stem cells derived from rats are selected as the seed cells forming the basis of the system. Adipose stem cells are pluripotent stem cells that can differentiate into fat cells, osteoblasts, and muscle cells.

[0051] (1) Rats (100-150 g) were anesthetized by intraperitoneal injection of pentobarbital sodium, soaked in 70% ethanol for 10 minutes, aseptically took abdominal subcutaneous fat tissue, quickly put it into a sterile beaker, and transferred it to a clean table;

[0052] (2) After the above operations are completed, remove the connective tissue and blood clots on the adipose tissue with hemostatic forceps in the ultra-clean bench, and use D-Hank’s solution ((Ca 2+ , Mg 2+ -free Hank's, pH7.4): 8.175g NaCl, 0.403g KCl, 0.114g NaCl 2 HPO4, dissolved in 950mL double-distilled water, added HEPES (4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid, 4-hydroxyethylpiperazineethanesulfonic acid) to adjust the pH value, and...

Embodiment 2

[0060] Example 2 Isolation and Culture of Rat Pancreatic Islets

[0061] (1) SD rats were anesthetized by intramuscular injection of pentobarbital (30 mg / kg). Disinfection, laparotomy, pancreas and common bile duct were exposed, and a suture was threaded at the distal end of the common bile duct near the duodenum, and at the back of the common bile duct near the hilum of the liver, without ligation for now.

[0062] (2) Insert a 4.5-gauge needle (connected with a syringe equipped with pre-warmed digestion separation solution) antegrade to the far side of the middle section of the common bile duct, ligate the suture near the hepatic hilum of the common bile duct, and inject a small amount of digestion separation into the common bile duct After that, the distal end of the common bile duct was ligated with sutures, and 10-15ml of pre-warmed 5% collagenase solution was slowly retrogradely injected into the pancreatic duct to fully expand the pancreas.

[0063] (3) Quickly and com...

Embodiment 3

[0066] Example 3 Multicellular system model construction process

[0067] (1) Preparation of gelatin, sodium alginate, fibrinogen and cell blend material: dissolving gelatin in PBS buffer solution to form a 20% solution, dissolving sodium alginate in PBS buffer solution to form a 5% solution, Neutralize residual acetic acid with NaOH, adjust the pH value to 7.1, and perform batch sterilization in an oven at 70°C. Dissolve fibrinogen in DMEM medium under sterile conditions to form a 5% solution. Before use, the three materials were uniformly blended at a ratio of 1:1:1. Mix the material with cultured adipose stem cells to obtain a concentration of about 1×10 7 / mL adipose stem cell-composite blend material.

[0068] (2) The assembly of the energy metabolism system consists of two steps: system basic assembly and islet assembly. Inject the adipose-derived stem cell / composite material blend into a 1mL disposable syringe, and place it in a refrigerator at 4°C for more than 30 ...

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Abstract

The invention relates to a cell-assembly based pathological model for metabolism syndrome and the application thereof to high content drug screening. By assembling the extracellular matrix material of fat stem cells and stimulation cells, a cell-containing 3D structure with a certain porosity can be formed. By manipulating the inducing differentiation conditions, the fat stem cells on the surface of the internal channel of the 3D structure can be differentiated into endothelial cells, and the fat stem cell inside the matrix material differentiate into fat cells. The pancreatic islets separated from animals are directly arranged into the pores of the 3D structure to from a plurality of secretion regulation points. By providing the system models long time stimulus from energy metabolism regulation matters, such as glucose with high concentration, fatty acids insulin, TNF or fat cells secreting factors, etc; the fat cells, islet cells and endothelial cells can be induced to show pathological symptoms of metabolic syndrome. The invention also provides a method for applying the above mentioned pathological model of metabolic syndrome to drug screening.

Description

technical field [0001] The invention belongs to the field of biotechnology, and specifically relates to a new pathological model of metabolic syndrome constructed by cell assembly technology, a construction method and its application in high-content drug screening. Background of the invention [0002] The combination of life science, information science and manufacturing science is an important trend in the development of science and technology in the 21st century. The three-dimensional controlled assembly of cells based on advanced manufacturing theory and tissue engineering theory has expanded new theoretical and technical space for research fields such as tissue engineering and drug screening. In the field of drug research and development, drug screening and evaluation technologies such as high-throughput screening (High-Throughput Screening, HTS), which have been widely used by global drug research and development institutions since the 1990s, are facing the dilemma of l...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0775C12N5/071C12Q1/26C12Q1/02G01N33/53
Inventor 徐铭恩王小红葛亚坤
Owner HANGZHOU DIANZI UNIV
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