Ghrelin active agent for inducing stem cells differentiated to cartilage cells

A technology for inducing differentiation and chondrocytes, which is used in the treatment of osteoarthritis and cartilage damage. It can solve the problems of mixed cartilage subtypes, low expression of cartilage-related genes, and no different differentiation strategies, so as to improve efficiency and avoid cartilage. The effect of cell hypertrophy

Active Publication Date: 2019-03-26
PEKING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, as far as the current technical means are concerned, this method has the disadvantages of low induction efficiency, accelerated chondrocyte hypertrophy, low expression of chondrogenic-related genes in induced differentiated chondrocytes, mixed cartilage subtypes, and mostly growth plate cartilage rather than articular cartilage. And there are no different differentiation strategies for different stages of differentiation of mesenchymal stem cells into chondrocytes

Method used

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  • Ghrelin active agent for inducing stem cells differentiated to cartilage cells
  • Ghrelin active agent for inducing stem cells differentiated to cartilage cells
  • Ghrelin active agent for inducing stem cells differentiated to cartilage cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Isolation and Expansion of Rat Bone Marrow Mesenchymal Stem Cells

[0045] Bone marrow-derived rat bone marrow mesenchymal stem cells (BMSCs) were obtained from 6-week-old Sprague Dawley (SD) rats. After 6-week-old male SD rats were anesthetized to death with excessive chloral hydrate, the tibia and patella of the two hind legs were removed, and the whole bone marrow was used to adhere to the wall (Lin C W, Zhou S H. Journal of Clinical Rehabilitative Tissue Engineering Research, 2010,14(14):2508-2512), the bone marrow inside the bone was washed with PBS into the culture dish.

[0046] The isolated cells were washed with PBS, and then culture medium containing 90% DMEM (Dulbecco's modified Eagle's medium, Gibco), 10% fetal bovine serum (FBS, Gibco) and 0.1% penicillin / streptomycin (PS, Amresco) at 37°C, 5% CO 2 Monolayer cell culture was carried out in a humid incubator, and the medium was changed twice a week. When the confluency of the cultured cells reached 85%, th...

Embodiment 2

[0048] Effect of ghrelin on the proliferation of rat bone marrow mesenchymal stem cells

[0049] The rat bone marrow mesenchymal stem cells prepared in Example 1 were cultured with different concentrations of ghrelin (Contide Biotechnology (Beijing) Co., Ltd., 031-30) for 7 days, and the control group (Control group) was added with the same amount of medium, respectively. After 3 days and 7 days of culture, the stem cells of each group were collected and seeded in 96-well plates at a density of 3.5 x 10 3 cells / well, cultured in a DMEM medium incubator (37°C, 5% CO 2 ) 4 hours later, perform the stem cell proliferation test according to the instructions of the CCK-8 cell counting kit (CK04, Dojindo), add 10 μl of CCK-8 solution to each well of the 96-well plate, incubate at 37°C for 2 hours, and then use a microplate reader to detect Absorbance at 490nm wavelength, 4 wells in each group.

[0050] The result is as figure 1 As shown, after adding different concentrations of g...

Embodiment 3

[0052] Effects of Ghrelin on the Chondrogenic Differentiation of Rat Bone Marrow Mesenchymal Stem Cells Induced by TGF-β

[0053] The rat bone marrow mesenchymal stem cells prepared in Example 1 were made into a suspension with a serum-free chondrogenic differentiation induction medium, and the volume was 2.5×10 5 Each stem cell / tube was placed in a conical tube, centrifuged at 600g for 10min, and the collected agglomerates were placed in an incubator (37°C, 5% CO 2 ) were cultured with serum-free chondrogenic differentiation induction medium, and the medium was changed every 3 days. The serum-free chondrogenic differentiation induction medium contains 10 -7 High glucose DMEM medium of M dexamethasone, 50 μg / ml vitamin C, 1 mM sodium pyruvate, 4 mM proline and 1% v / v ITS+premix (BD Bioscience Inc., Franklin Lakes, NJ).

[0054] Grouping: Each group added TGF-β (10ng / ml human TGF-β3, Peprotech, 100-36E) and / or ghrelin (see Example 2) of different final concentrations to the m...

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Abstract

The invention provides a composition containing a ghrelin active agent and a method of using the composition to induce stem cells differentiated to cartilage cells. The invention specifically providesa method of using the composition to treat osteoarthritis and a method of using the composition to treat cartilage injury.

Description

technical field [0001] The present invention relates to a composition containing ghrelin active agent, and a method for using it to induce stem cells to differentiate into chondrocytes, specifically, a method for using it for treating osteoarthritis, a method for using it for treating cartilage damage, and corresponding uses. Background technique [0002] With the aging of the social population and the increase in the incidence of diseases such as osteoarthritis, cartilage tissue damage has become one of the most common clinical injuries. Articular cartilage has no blood vessels and nerves, relies on joint fluid for nutrition, and cannot repair itself once damaged. Whether it is through tissue engineering or regenerative medicine, efficiently obtaining a large number of chondrocytes is the core issue in the treatment of cartilage injuries. The chondrocytes of adult animals are terminally differentiated cells, their sources are limited, traumatic and difficult to obtain, poo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/077C07K14/575A61K38/22A61P19/02A61P19/08A61P29/00
CPCC12N5/0655C07K14/575C12N2506/1346C12N2506/45C12N2501/998C12N2501/15C12N2501/148C12N2501/155C12N2501/415C12N2501/13C12N2501/105C12N2501/113C12N2501/115C12N2501/119A61K38/00A61K38/22A61P19/02A61P19/08A61P29/00C07K14/47
Inventor 葛子钢樊丽彤
Owner PEKING UNIV
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