Preparation containing fibroblast exosome and application thereof

A technology of fibroblasts and exosomes, which can be used in medical preparations containing active ingredients, aerosol delivery, skin diseases, etc., and can solve the problems of undisclosed fibroblast exosomes

Inactive Publication Date: 2017-11-17
沃昕生物科技(深圳)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] However, fibroblast exosomes and preparations made of fibroblas

Method used

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  • Preparation containing fibroblast exosome and application thereof
  • Preparation containing fibroblast exosome and application thereof
  • Preparation containing fibroblast exosome and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Take fresh healthy umbilical cord and umbilical cord blood, collect the umbilical cord blood and centrifuge, take the upper serum and inactivate it for later use. After rinsing with PBS, remove the umbilical vessels with scissors and tweezers, peel off the Wahrenheit jelly tissue inside, and cut the resulting tissue to a size of 1mm3, add α-MEM culture medium and place at 37°C, 5% CO 2 Culture in an incubator with 10% inactivated umbilical cord serum in the culture medium. After 5-8 days of umbilical cord tissue culture, it can be seen that some cells crawled out from around the tissue block, showing a small spindle shape. After one week, the cells began to proliferate rapidly and formed cell colonies of various sizes. After the cells were full, use 0.25 % Trypsinization for passage. Such as figure 1 As shown, the human umbilical cord mesenchymal stem cells grow in the form of fibroblast-like adherence, and the cells are uniform in shape, spindle-shaped, and have a ce...

Embodiment 2

[0043] Take the UC-MSCs isolated and cultured at passages P3 and P6 respectively, and adjust the cell density to 1x10 6 / ml, respectively incubated with FITC-CD29, PE-CD31, FITC-CD40, PE-CD44, PC5-CD45, PC5-CD90, PE-CD105 and FITC-HLA-DR antibodies at 4°C for 30 min, washed 3 times with PBS , and then resuspended the cells in 200 μl PBS, and detected on the flow cytometer.

[0044] As shown in Table 1, UC-MSCs highly express CD29, CD44, CD90, and CD105, and have low or no expression of CD31, CD40, CD45, and HLA-DR, which meet the identification criteria of UC-MSCs.

[0045] Table 1 Identification of UC-MSC surface markers

[0046]

[0047]

Embodiment 3

[0049] Take P6 generation UC-MSC, adjust the cell density to 1x10 6 / ml, according to 2x10 per bottle 6 Cells were seeded into T175 cell culture flasks. After 24 hours, the medium was changed and divided into two groups, namely the experimental group and the control group. The control group was added with normal medium (α-MEM containing 10% inactivated cord blood serum by volume fraction), and the experimental group was added with conditioned medium (α-MEM containing 10% volume fraction of inactivated cord blood serum, 10ng / ml basic fibroblast growth factor, 1mmol / L vitamin C phosphate, 0.04mmo / L proline) for cell induction, and morphological observation of the cells after 3 days .

[0050] Such as figure 2 As shown, the cells in the experimental group formed a flattened spindle-shaped or star-shaped structure, which conformed to the morphological characteristics of fibroblasts.

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Abstract

The invention uses a method for inducing UC-MSC to fibroblasts. According to the method, after in-vitro induction amplification, the fibroblasts are collected to culture supernate, and exosome is obtained by a low temperature ultrafiltration concentration method; the exosome is used as a main active ingredient to prepare a spray. It is proved by experiments that the spray containing fibroblast exocrine can effectively promote the healing of a wound.

Description

technical field [0001] The present invention relates to fibroblast exosomes, a preparation method thereof, a preparation containing fibroblast exosomes and uses thereof. Background technique [0002] The skin is the largest organ of the human body, accounting for about 16% of the body weight. It contains about 1 / 3 of the circulating blood and about 1 / 4 of the water in the human body. It plays an important defense and protection function and is the first line of protection in the true sense of the human body. Wire. Under the action of internal and external injury factors of normal skin tissue, the integrity of the skin is destroyed, and the normal function of the skin is damaged, thus forming a skin wound. Mild trauma is limited to the epidermis of the skin, and in severe cases, the skin and subcutaneous tissue are broken, and wounds appear; severe trauma may have ruptures and fractures of muscles, tendons, and nerves. Wound healing is a complex process of restoring the fun...

Claims

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Application Information

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IPC IPC(8): A61K35/33A61K9/12A61P17/02A61K35/51
Inventor 李陶林词雄王旭林洁璇朱刚
Owner 沃昕生物科技(深圳)有限公司
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