Method for establishing osteoarthritis osteoclast-like cell model of mesenchymal stem cells after cartilage differentiation through two-step method

A technology of mesenchymal stem cells and osteoarthritis, which is applied in the field of establishing osteoarthritis-like cell models after cartilage-directed differentiation of mesenchymal stem cells by a two-step method, achieving the effects of less medical ethics, low cost, and no species difference

Active Publication Date: 2018-05-18
WUHAN UNIV
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  • Abstract
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Problems solved by technology

[0003] At present, stem cell-related research on osteoarthritis mainly focuses on stem cells as seed cells for cell transplantation after the occu...

Method used

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  • Method for establishing osteoarthritis osteoclast-like cell model of mesenchymal stem cells after cartilage differentiation through two-step method
  • Method for establishing osteoarthritis osteoclast-like cell model of mesenchymal stem cells after cartilage differentiation through two-step method
  • Method for establishing osteoarthritis osteoclast-like cell model of mesenchymal stem cells after cartilage differentiation through two-step method

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example 1

[0038] Human umbilical cord mesenchymal stem cells (Wharton`s Jelly-derived mesenchymal stem cells, WJ-MSCs) under high concentration of cortisol treatment have poor cartilage-oriented differentiation and susceptibility to osteoarthritis.

[0039] One-step and two-step method to establish osteoarthritis-susceptible cell model after cartilage-directed differentiation of WJ-MSCs ( figure 1 )

[0040] 1. Establishment of three-dimensional directed differentiation model of WJ-MSCs sodium alginate microspheres chondrocytes

[0041] Weigh 160 mg of alginate sodium alginate (Sigma, low-viscosity type), put it into a transparent and clean glass bottle with a volume greater than 20 mL, then add 10 mL of double-distilled water to the sterile operating table, and put a magnetic stirrer in the bottle; seal it, Mix well, and stir overnight with magnetic force until it turns into a uniform and transparent colloidal solution, and then sterilize in a pressure steam sterilizer at 120° C. for ...

example 2

[0093] Changes in mRNA expression of TGFβR1, TGFβR2 and downstream phenotype genes in normal control group and IUGR group neonatal umbilical cord mesenchymal stem cells (Wharton`s jelly-derived mesenchymal stem cells, WJ-MSCs).

[0094] 1. Umbilical cord mesenchymal stem cell culture for total RNA extraction

[0095] Neonatal umbilical cord specimens were collected from the Department of Obstetrics and Gynecology, Zhongnan Hospital of Wuhan University. Divided into two groups: normal control group and IUGR group (n≧8), total RNA was extracted after culturing mesenchymal stem cells.

[0096] Add 1ml Trizol to the homogenizer, after fully homogenized on ice, transfer to RNase-free 1.5ml EP tube; add 200μl chloroform for extraction, shake vigorously for 15s, let stand on ice for 10min; centrifuge at 12,000×rpm at 4°C for 15min; Carefully pipette 500 μl of the upper aqueous phase into a new RNase-free 1.5ml EP tube, add an equal volume of isopropanol, invert and mix to precipitat...

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Abstract

The invention discloses a method for establishing an osteoarthritis osteoclast-like cell model of mesenchymal stem cells after directional cartilage differentiation through a two-step method. The method comprises the steps that firstly, the mesenchymal stem cells are induced to be subjected to directional differentiation into articular cartilage cells, and then the articular cartilage cells are induced by adopting IL-1beta in-vitro induction to establish the osteoarthritis model. According to the method, the two-step method is used for establishing the osteoarthritis model after directional cartilage differentiation for the first time, and by exploring the cartilage cell morphology and expression change of phenotype genes aggrecan and Co12alpha1, the optimal use concentration and cell induction time of a solution are determined. The method is easy to operate and low in cost and can be applied to related studies such as cell quality evaluation of the mesenchymal stem cells after directional cartilage differentiation, early warning of easy infection of adult osteoarthritis, verification of osteoarthritis drug action targets and screening of drug toxicity/drug functions.

Description

technical field [0001] The invention belongs to the technical field of cell models, and relates to a two-step method for establishing an osteoarthritis-like cell model after cartilage-directed differentiation of mesenchymal stem cells. Background technique [0002] Osteoarthritis is a common and frequently-occurring disease among middle-aged and elderly people. It is a joint disease characterized by chronic degenerative changes in articular cartilage. It is the main cause of pain and disability in the elderly. Among people over 50 years old in the United States, the incidence of osteoarthritis ranks second, second only to cardiovascular diseases, accumulating about 21 million people, and the annual medical expenses for direct and indirect treatment of osteoarthritis are about 546 to 650 billion U.S. dollars [1]. The incidence of osteoarthritis in the total population of my country is about 4%, mainly concentrated in the elderly population, more than 50% of people over 65 ye...

Claims

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Application Information

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IPC IPC(8): C12N5/077
CPCC12N5/0655C12N2500/30C12N2500/32C12N2500/38C12N2501/2301C12N2502/137C12N2513/00C12N2533/74
Inventor 汪晖陈廖斌齐勇建杨旭何波
Owner WUHAN UNIV
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