Method for high content screening of therapeutic drugs for diabetes

A screening method and technology for therapeutic drugs, applied in biochemical equipment and methods, microbiological determination/testing, etc., can solve the problems of low success rate, high-content screening of cell structure and functional integrity without major progress, etc.

Active Publication Date: 2010-07-14
HANGZHOU DIANZI UNIV
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  • Description
  • Claims
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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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  • Method for high content screening of therapeutic drugs for diabetes
  • Method for high content screening of therapeutic drugs for diabetes
  • Method for high content screening of therapeutic drugs for diabetes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0073] Example 1 Seed cell culture

[0074] 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.

[0075] (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;

[0076] (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

[0084] Example 2 Isolation and culture of rat islets

[0085] (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.

[0086] (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.

[0087] (3) Quickly and completely rem...

Embodiment 3

[0090] Example 3 Multicellular system model construction process

[0091] (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.

[0092] (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 diabetes pathological model based on cell assembly technology and application thereof in high content drug screening. Adipose-derived stem cells and simulated extracellular matrix material are assembled to form a cell-containing three-dimensional structure with certain porosity; the inducing differentiation condition is controlled to enable the adipose-derived stem cells on the surface of the inner passage of the three-dimensional structure to differentiate into endothelial cells and to enable the adipose-derived stem cells inside the matrix material to differentiate into fat cells; and pancreas islet separated out from the body of an animal is directly assembled in the gaps of the three-dimensional structure. The system model is excited for a long time by high-concentration dextrose, fatty acid insulin, TNF or cytokine secretion of fat cells and the like so as to induce the system model to produce pathology and symptom of diabetes. The drug to be tested is put into a culture system, and by detecting the level of the relevant markers of the model under normal condition and pathological condition, the pharmacological activity of the drug to be tested can be analyzed and thereby high content screening can be carried out to the drug.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular, relates to a high-content drug screening method for diabetes drugs 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 low success rate (Bleicher KH, et al. Nat Rev Drug Discov .2003;2(5):369-78). The reason is that there is a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/02
Inventor 徐铭恩葛亚坤
Owner HANGZHOU DIANZI UNIV
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