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Dental pulp stem cell exosome as well as preparation method and application thereof

A technology of dental pulp stem cells and exosomes, which is applied in the field of cell exosome preparation and can solve problems such as limited effect

Pending Publication Date: 2021-08-31
STOMATOLOGICAL HOSPITAL OF CHONGQING MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The present invention intends to provide a method for preparing exosomes from dental pulp stem cells to solve the technical problem of limited therapeutic effect of exosomes obtained by prior art methods

Method used

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  • Dental pulp stem cell exosome as well as preparation method and application thereof
  • Dental pulp stem cell exosome as well as preparation method and application thereof
  • Dental pulp stem cell exosome as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1: Three-dimensional and two-dimensional culture of dental pulp mesenchymal cells (DPSCs)

[0043] The source of DPSCs: collect healthy premolars extracted due to orthodontics, cut off the teeth from the enamel-cementum junction, take out the pulp in the ultra-clean bench, isolate and culture DPSCs by improved enzyme digestion method, and take the third generation of cells after subculture. The following experiments (in actual operation, P3-P6 generations can be selected, and ideal results can be obtained). The DPSCs used in this protocol are taken from the healthy premolars of normal adults. The protocol only involves the cultivation of DPSCs and the acquisition of exosomes, and does not involve the process of dental surgery when collecting premolars. More specific methods for obtaining DPSCs are as follows:

[0044] The healthy premolars extracted due to orthodontics in the oral and maxillofacial surgery clinic were collected, placed in α-MEM medium containin...

Embodiment 2

[0050] Example 2: Extraction and identification of exosomes

[0051] Exosomes were extracted using the exoEasy Maxi Kit kit. Thaw the collected supernatant at 4°C, concentrate it with an ultrafiltration tube, add XBP and XWP in sequence according to the kit instructions, centrifuge, discard the filtrate, and finally add XE to elute and resuspend, which is the exosome solution. The exosomes were extracted from the supernatants of the three-dimensional culture and the two-dimensional culture, and the obtained three-dimensional culture exosomes and two-dimensional culture exosomes were identified.

[0052] Exosome morphology observed by transmission electron microscope:

[0053] Take 20ul of freshly extracted DPSC-Exo suspension under three-dimensional culture and two-dimensional culture conditions and drop it on the copper grid, add 10μl of 2% uranyl acetate on the copper grid for 1min after 10 minutes, wash, dry at room temperature, put the sample on the machine, and transmit ...

Embodiment 3

[0058] Example 3: miRNA analysis of exosomes

[0059] The miRNA high-throughput sequencing of the two-dimensional and three-dimensional cultured exosomes in this experiment was completed in cooperation with Wuhan Huada Medical Laboratory Co., Ltd., and BGISEQ-500 was used for sample sequencing. The exosomal protein concentration was corrected by the BCA method, followed by library construction. The specific process of library construction is: 1) total RNA isolation, ligation of 5' and 3' ends; 2) cDNA synthesis after adapter removal; 3) cDNA amplification, separation of target fragments; 4) library quantification and pooling circularization; 5) Quality inspection, on-machine sequencing. Statistical analysis is performed on the original data obtained by sequencing to obtain credible target sequences for standby analysis by removing joints, removing low quality, and removing contamination.

[0060] Using the Dr.Tom platform under BGI, the miRNA expression profile was statistic...

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Abstract

The invention relates to the technical field of cell exosome preparation, in particular to a dental pulp stem cell exosome and a preparation method and application thereof. The preparation method of the dental pulp stem cell exosome sequentially comprises the following steps: mixing a dental pulp mesenchymal cell suspension and a low-melting-point agarose solution to obtain a mixture, then inoculating the mixture into a culture container, and carrying out pre-culture and induction culture to obtain a supernatant containing the exosome; and extracting the exosome in the supernate. The preparation method of the scheme solves the technical problem that the treatment effect of the exosome obtained by the existing technical method is limited. In the exosome obtained by the scheme, the expression quantity of miRNAs such as miR-302a / b / c / d family and miR-24-3p is remarkably improved, and the exosome obtained by using the three-dimensional cultured dental pulp mesenchymal cells has a relatively remarkable treatment application potential.

Description

technical field [0001] The invention relates to the technical field of cell exosome preparation, in particular to a dental pulp stem cell exosome and its preparation method and application. Background technique [0002] Dental pulp stromal cells (DPSCs), isolated from dental pulp tissue, are commonly used dental-derived mesenchymal cells and have been used in the repair and regeneration of various tissues. DPSCs are derived from neural crest and have high proliferative potential for self-renewal and multilineage differentiation. Exosomes (Exosomes, Exo) are small vesicles formed by cells through the invagination of lysosomal particles, which contain rich active substances, such as proteins, lipids, mRNA, miRNA, etc., and can exchange information and substances between cells. function in exchange. Recent studies have found that exosomes from DPSCs (DPSC-Exo) have a variety of therapeutic effects, and some applications have been reported in the field of regenerative medicine...

Claims

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Application Information

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IPC IPC(8): C12N5/0775A61L27/38
CPCC12N5/0664A61L27/3834A61L27/3895C12N2513/00C12N2509/00C12N2533/76A61L2300/412A61L2430/40
Inventor 杨德琴窦磊艾晓青
Owner STOMATOLOGICAL HOSPITAL OF CHONGQING MEDICAL UNIV
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