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A method for chondrocyte proliferation based on wnt-5a/ror2 signaling pathway

A technology of chondrocytes and cells, which is applied in the field of cosmetic chondrocyte culture and regeneration preparations, can solve the problems of no ideal method, achieve the effect of reducing the content of use, simple operation, and promoting culture

Active Publication Date: 2022-04-12
肌因美生物基因工程(上海)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0011] The reduction or prevention of the immune response to cartilage transplantation has achieved certain results, but there is no ideal method, and most of them are intervened through purely immunological approaches

Method used

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  • A method for chondrocyte proliferation based on wnt-5a/ror2 signaling pathway
  • A method for chondrocyte proliferation based on wnt-5a/ror2 signaling pathway
  • A method for chondrocyte proliferation based on wnt-5a/ror2 signaling pathway

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] The configuration of the culture medium of embodiment 1 chondrocyte

[0045] To prepare media with different components, the types of media used are as follows:

[0046] Basal medium: DMEM medium, purchased from GIBCO.

[0047] Medium 1: basal medium + 10% fetal bovine serum.

[0048] Medium 2: basal medium + 6% fetal bovine serum + aloe-emodin, wherein, medium 2-1: basal medium + 6% fetal bovine serum + 40 μmol / L aloe-emodin; medium 2-2: basic Medium + 6% fetal bovine serum + 60 μmol / L aloe-emodin; medium 2-3: basal medium + 6% fetal bovine serum + 80 μmol / L aloe-emodin. .

[0049] Medium 3: basal medium + 6% fetal bovine serum + caffeic acid, wherein, medium 3-1: basal medium + 6% fetal bovine serum + 40 μmol / L caffeic acid; medium 3-2: basal medium + 6% fetal bovine serum + 60 μmol / L caffeic acid; medium 3-3: basal medium + 6% fetal bovine serum + 80 μmol / L caffeic acid.

[0050] Medium 4: basal medium + 6% fetal bovine serum + aloe-emodin + caffeic acid, wherei...

Embodiment 2

[0051] Preparation and cultivation of embodiment 2 chondrocytes

[0052] Preparation of chondrocytes: human articular cartilage tissue was taken, sterilized with 95% ethanol for 10 minutes, treated with 0.5% trypsin at 37°C for 2 hours, washed with phosphate buffer, and then digested with 0.08% type II collagenase; After the digested cells were diluted with medium 1, the cells were collected by centrifugation; the cells were centrifuged and washed with medium 1 to obtain primary human chondrocytes.

[0053] Cultivation of chondrocytes: the prepared medium 1 for chondrocytes was cultured in a carbon dioxide incubator, and the culture conditions were 37° C. and 5% carbon dioxide concentration. After culturing for 24 hours, the cells were examined by microscope observation. Observed human chondrocytes such as figure 1 shown.

Embodiment 3

[0054] Example 3 Different concentrations of cell proliferation preparations

[0055] Culture medium preparation: Prepare culture medium of different concentrations of cell culture and proliferation preparations, respectively as follows: medium 2 containing 40, 60 and 80 μmol / L aloe-emodin respectively, and medium 2 containing 40, 60 and 80 μmol / L caffeic acid respectively 3. Use Medium 1 as a control.

[0056] Cell proliferation experiment: Chondrocytes in the logarithmic growth phase were cultured in medium 1, medium 2 with different concentrations of aloe-emodin and medium 3 with different concentrations of caffeic acid, and cells in different groups were cultured at 1×10 5 Inoculate into a 96-well culture plate at a density of one per well, set 3 replicates in each group, and culture in a carbon dioxide incubator at 37°C and 5% carbon dioxide for 48 hours, add 10 μl of CCK-8 solution to each well, and continue to cultivate for 2 hours Then the OD value of the cells at 450...

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Abstract

The present invention provides a preparation and culture medium for cosmetic cell regeneration based on Wnt-5A / Ror2 signaling pathway, which can improve and maintain chondrocyte phenotype while improving chondrocyte proliferation ability and regeneration, and improve the application efficiency of chondrocytes in vivo. Stability and security. The cell culture preparation of the present invention can reduce the content of normal serum used in the basal medium, and significantly promote the cultivation and regeneration of chondrocytes, and solve the problems of complex components, difficult cultivation and slow growth rate of the culture medium used for cosmetic chondrocyte culture . The cell proliferation preparation of the present invention regulates the proliferation and regeneration of chondrocytes induced by IL-1β by inhibiting the Wnt-5A / Ror2 signaling pathway. Therefore, the preparation of the present invention can be used as a signal factor for the proliferation and regeneration of chondrocytes, etc. The cultured cells can be used as cosmetic raw materials for further application in cosmetic cartilage damage repair, etc. The method provided by the invention has the characteristics of simple operation and convenience, and is suitable for popularization and application.

Description

technical field [0001] The invention relates to the field of cell culture, in particular to a preparation for beautifying chondrocyte culture and regeneration and its application. Background technique [0002] Articular cartilage is composed of chondrocytes surrounded by a complex extracellular matrix produced by chondrocytes. The unique biochemical composition of this matrix provides a smooth, almost frictionless articulating surface for movement. Damage to cartilage due to trauma or diseases such as rheumatoid and osteoarthritis can lead to severe debilitating. The self-repair ability of cartilage injury is relatively weak, and clinical research has limited patient tissue materials, and because chondrocytes are terminally differentiated cells, their proliferation ability in vitro is limited and they are easy to dedifferentiate. Dedifferentiation refers to the loss of specific structural and functional changes of differentiated cells. A process with properties of undiffer...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/077A61L27/38
Inventor 杜剑波
Owner 肌因美生物基因工程(上海)有限公司