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Brown adipocyte-derived exosome as well as preparation method and application thereof

A brown fat and exosome technology, applied in the field of molecular biology, can solve problems such as local infection, local tissue embolism, and local bleeding, and achieve the effects of less side effects, improved pain symptoms, and pain relief.

Inactive Publication Date: 2022-05-20
INST OF ZOOLOGY CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, most of the existing methods of stem cell treatment for arthritis are to inject stem cells in situ at the patient's site, but the injection method is an invasive treatment, which will inevitably have certain risks, including local bleeding, hematoma, etc., especially when the aseptic technique is improper , may cause local infection
In addition, if the injection is improper, it may also cause local tissue embolism, leading to local tissue necrosis, etc.

Method used

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  • Brown adipocyte-derived exosome as well as preparation method and application thereof
  • Brown adipocyte-derived exosome as well as preparation method and application thereof
  • Brown adipocyte-derived exosome as well as preparation method and application thereof

Examples

Experimental program
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preparation example Construction

[0025] Aiming at the many defects of the arthritis treatment methods in the prior art, the present invention provides exosomes derived from brown adipocytes transdifferentiated from human skin fibroblasts, which can be exosomes by externally applying the exosomes to painful areas of arthritis-related diseases. The exosome method is used to treat arthritis-related diseases, and a preparation method of the exosome is provided.

[0026] The inventors were surprised to find that although HEF cells are terminally differentiated cells that have lost their pluripotent stemness, when they are transformed into cells that stably co-express PPARγ and CEBPβ, they can have the ability to differentiate into brown adipocytes potential, and the inventors also found that using the exosomes secreted by the brown adipocytes transdifferentiated from the human skin fibroblasts can treat arthritis-related diseases, and has a good therapeutic effect. Based on this, the present invention provides exo...

Embodiment 1

[0031] Example 1: Transformation of HEF cells (human skin fibroblasts) into precursor cells with the potential to differentiate into brown adipocytes

[0032] This embodiment aims to transform HEF cells into precursor cells with the potential to differentiate into brown adipocytes, and to identify the characteristics of the precursor cells, specifically including the following steps.

[0033] 1.1. Plasmid construction

[0034] The human source PPARγ cDNA sequence (its nucleotide sequence is shown in SEQ ID NO: 1) and the human source CEBPβ ​​cDNA sequence (its nucleotide sequence is shown in SEQ ID NO: 2) were respectively cloned into pCDH-EF1-MCS - From the T2A-Gfp vector (commercially available) and the pCDH-EF1-MCS-T2A-Puro vector (commercially available), a PPARγ viral expression vector and a CEBPβ ​​viral expression vector were obtained, respectively.

[0035] 1.2. Lentiviral packaging

[0036] Cultivate HEK 293T cells (the complete medium for cell culture is complete m...

Embodiment 2

[0044] Example 2: Preparation of exosomes derived from mature brown adipocytes transdifferentiated from HEF cells

[0045] This example aims to induce the HEF cells that stably co-express PPARγ and CEBPβ ​​obtained in Example 1 and have the potential to differentiate into brown adipocytes to transdifferentiate into mature brown adipocytes, and use the mature brown adipocytes to prepare exosomes, Specifically include the following steps.

[0046] (2.1) HEF cells stably co-expressing PPARγ and CEBPβ ​​obtained in Example 1 were placed in brown adipocyte induction differentiation medium (shown in Table 1, the specific formula used in this example is: 98% by volume High glucose DMEM, 2% by volume fetal bovine serum, 33 μM biotin, 0.5 μM insulin, 17 μM pantothenic acid, 0.10 μM DEX, 2 μM T3, 0.55 mM IBMX and 0.03 mM indomethacin) for 6 days.

[0047] (2.2) The differentiated cells induced in step (2.1) were washed 3 times with PBS, and 20 mL of phenol red-free DMEM basal medium w...

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Abstract

The invention discloses a brown adipocyte-derived exosome as well as a preparation method and application thereof, and belongs to the technical field of molecular biology. The preparation method of the exosome provided by the invention comprises the following steps: constructing human skin fibroblasts co-expressing PPAR gamma and CEBP beta to obtain brown adipose precursor cells; carrying out induced differentiation culture on the brown adipose precursor cells by using the brown adipose cell induced differentiation culture medium to obtain brown adipose cells; and enabling the brown adipocytes to secrete exosomes to obtain the exosomes from the brown adipocytes. The exosome provided by the invention can be used for treating the pain symptom of arthritis in an external application mode, so that the exosome can be used for preparing medicines for treating diseases related to arthritis.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and in particular relates to an exosome derived from brown adipocytes transdifferentiated from human skin fibroblasts (HEF cells) and a preparation method and application thereof. Background technique [0002] Arthritis symptoms include inflammation in one or more joints. The main symptoms are joint pain, stiffness, limited movement, hot and red joint skin, muscle weakness and muscle atrophy, etc. The etiology is not completely clear, but known and multifactorial Related, including congenital genetic factors, acquired environmental factors, age, gender, obesity, joint damage, joint overuse, etc. [0003] According to the latest epidemiological research, there are at least 60 million arthritis patients in my country, and the number of patients is gradually increasing. Arthritis not only affects the quality of life of patients, but also imposes a great burden on society and national prod...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N15/12C12N15/867A61K35/35A61P19/02A61P29/00
CPCC12N5/0653C12N15/86C07K14/47A61K35/35A61P19/02A61P29/00C12N2510/00C12N2500/38C12N2501/33C12N2501/395C12N2501/999C12N2740/15043C07K14/515C12N5/0656C12N2800/107C12N2501/39C12N2500/40Y02A50/30
Inventor 金万洙叶荣财
Owner INST OF ZOOLOGY CHINESE ACAD OF SCI
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