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Dental pulp stem cell conditioned medium and preparation method and application thereof

A technique of dental pulp stem cells and conditioned medium is applied to the conditioned medium of human dental pulp stem cells and preparation thereof. Expensive, the concentration does not reach the protective effect, the secretion content is not high, etc., to improve the vitality of genioglossus muscle cells, the treatment effect is excellent, and the effect of reducing oxidative stress is achieved.

Active Publication Date: 2020-05-19
上海市口腔病防治院
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, at present, the nutrients in the conditioned medium obtained by the traditional preparation method have been partially consumed by the mesenchymal stem cells, the content of the secretion is not high, and the concentration cannot meet the requirements of the protective effect, and then used in cell experiments will attenuate the original Effect
Moreover, the existing technology for extracting conditioned medium secretion is expensive and has a long cycle

Method used

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  • Dental pulp stem cell conditioned medium and preparation method and application thereof
  • Dental pulp stem cell conditioned medium and preparation method and application thereof
  • Dental pulp stem cell conditioned medium and preparation method and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0033] A preparation method for dental pulp stem cell conditioned medium, comprising the steps of:

[0034] (1) Isolation and culture: normal adults (18-25 years old) were collected from wisdom teeth extracted or impacted due to orthodontic reduction. All methods were implemented in accordance with relevant ethical guidelines and regulations, and the patients gave informed consent. Pre-cooled phosphate-buffered saline (PBS) with a volume concentration of 5% double-antibody, and then sent it to a laboratory biosafety cabinet within 1 hour, rinsed repeatedly with PBS with a volume concentration of 1% double-antibody for 10 times, and then rinsed after cleaning the root surface of the blade 3-5 times, place the jaws of the vise on the enamel-cementum junction to cut off the tooth, gently separate the pulp with a pulp-pulling needle, and then cut it to 1mm with ophthalmic scissors 3size, and then digested in type I collagenase and Dispase enzyme (volume ratio 1:1) at 37°C for 45mi...

Embodiment 2

[0038] An application of improving hypoxia-induced genioglossus muscle cell injury by using the dental pulp stem cell conditioned medium obtained in Example 1, specifically comprising the following steps:

[0039] (1) Isolation and culture of genioglossus myoblasts

[0040] The genioglossus tissue was isolated from male 3-4 week-old C57BL / 6 mice and placed in a petri dish, washed with PBS repeatedly for 10 times, and the specimen was cut into 1mm with ophthalmic scissors 3 After the tissue block was removed, transfer it to a centrifuge tube; add type I collagenase, digest at 37°C for 30min, centrifuge at 1000r / min for 3min, remove the supernatant; add 0.05% trypsin, digest at 37°C for 30min; add serum to stop, 1000r / min, centrifuge for 3min; add 10% FBS, 1% double antibody DMEM culture medium to resuspend; transfer the cell suspension to a culture dish, put it in 37℃, 5%CO 2 After 30 minutes of attachment in the incubator, the supernatant was transferred to a new culture dish...

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Abstract

The invention discloses a preparation method of a dental pulp stem cell conditioned medium. The method comprises the following steps: cutting dental pulp tissue, separating dental pulp stem cells, culturing, digesting and passaging, continuously culturing the dental pulp stem cells of the third generation to the fifth generation, discarding a culture medium when the cells reach 70-80% confluence rate, replacing with a serum-free culture medium after cleaning, continuously culturing, collecting the culture medium, centrifuging, taking supernate, filtering the supernate with a filter, filling the supernate into an ultrafiltration centrifuge tube, and centrifugally concentrating to obtain the dental pulp stem cells. The preparation method can obtain the dental pulp stem cell conditioned medium with high concentration and high purity, has advantages of short time, simple operation, low price and low pollution, and greatly improves efficiency of the experiment. The invention also disclosesapplication of the dental pulp stem cell conditioned medium in improving damage of genioglossal muscle cells induced by hypoxia. The prepared dental pulp stem cell conditioned medium can effectively improve activity of genioglossal muscle cells and reduce generation of intracellular and mitochondrial oxidative stress.

Description

technical field [0001] The invention belongs to the field of biomedicine, and in particular relates to a human dental pulp stem cell conditioned medium and a preparation method thereof, as well as the application of the conditioned medium in improving hypoxia-induced genioglossus muscle cell damage. Background technique [0002] The genioglossus belongs to the skeletal muscle and is the main muscle that pulls the tongue forward. It is called the "upper airway safety muscle". Its dysfunction is closely related to the pathogenesis of obstructive sleep apnea-hypopnea syndrome (OSA) . During frequent apnea in OSA patients, the compensatory contraction of the genioglossus muscle is enhanced under long-term hypoxia, and continuous compensatory contraction can easily lead to muscle fatigue, which further aggravates the degree of upper airway collapse, forming a vicious circle and affecting muscles. structure and function. [0003] Dental pulp stem cells are a type of mesenchymal ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0775C12N5/077
CPCC12N5/0664C12N5/0658
Inventor 张维华刘月华於丽明韩欣欣卢芸朱露莹邓佳佳
Owner 上海市口腔病防治院
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