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Application of extracellular vesicles in preparation of preparation for regulating ovarian function

A technology of ovarian function and cells, applied in the application field of preparations, can solve the problems of time-consuming, high equipment requirements, and restrictions on the clinical transformation and application of exosome therapy

Pending Publication Date: 2022-02-22
EV CELL BIOTECH GUANGZHOU CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are still many problems in the current exosome-based cell vesicle therapy, mainly in the complex and time-consuming process of exosome extraction and purification, high requirements for equipment and reagents, and low yield of physiological exosomes etc. These defects limit the clinical transformation and application of exosome therapy

Method used

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  • Application of extracellular vesicles in preparation of preparation for regulating ovarian function
  • Application of extracellular vesicles in preparation of preparation for regulating ovarian function
  • Application of extracellular vesicles in preparation of preparation for regulating ovarian function

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0085] Example 1 Separation and culture of BMMSCs

[0086] Excessive CO based on the guidance of an animal ethics committee 2 The mice were sacrificed, and the tibial and femur were removed, and the muscles and connective tissue attached thereto were removed, and the dry tissue was further separated, exposed the bone marrow cavity, and extracted the volume fraction of 10% by 10 ml of sterile syringe. The PBS of the fetal bovine serum repeatedly rinsed the bone marrow cavity, 70 μm aperture cell mesh filtered, 500 g centrifugally 5 min, remove the cell precipitate at the bottom after the supernatant was removed, and the PBS was resuspended, and the final cell precipitate was collected by 5 min. The cells were subjected to flow sorting by CD34- and CD90 + for sorting standards, and bone marrow mesenchymal stem cells (BMMSCs) were selected. Finally, in DEX (-) culture solution and inoculated in 10 cm diameter cell culture dishes, 37 ° C, 5% CO 2 nourish. After 24 hours, the supernata...

Embodiment 2

[0093] Example 2 Preparation of IEV of BMMSC Source

[0094] Example 1 was cultured to the second generation MSCs (MSCs of the bone marrow), and the PBS washed 2 when the medium (DEX (+) culture solution) was continued in Example 1, PBS washed 2 The serum-free medium containing 500 nm STS was added (the medium was added to the medium in Example 1), and incubated for 16-24 h at 37 ° C, the cell supernatant was collected, and 800 g at 4 ° C centrifugation 10 In a minute, the supernatant was charged at 4 ° C for 10 minutes, and the supernatant was collected at 4 ° C for 30 minutes at 4 ° C, and the resulting precipitate was Iev. 500 μl of PBS resuspended, 16000 g at 4 ° C for 30 minutes, i.e., was obtained, i.e., Iev obtained.

[0095] Preparation route figure 2 Indicated.

Embodiment 3

[0099] Example 3 Analysis of IEVS

[0100] (1) Analysis of the quantification of IEVs and the analysis of membrane proteins

[0101] The IEVs obtained by the second embodiment were quantitatively analyzed by flow cytometry, and the measurement time points were firsth, fourth h, 8h, 16h and 24h, and the results showed 10 6 MSCs can produce 0.76 × 10 after induction to firsth, fourth, 8h, 16h and 24h, respectively 8 1.29 × 10 8 1.95 × 10 8 2.48 × 10 8 3.14 × 10 8 IEVS, it can be seen that after 24 hours, a single MSC can output 300 IEVs ( image 3 .

[0102] In addition, flow detection found that the particle diameter distribution of IEVs was concentrated below 1 μm, accounting for 94.97%.

[0103] Side-to-scattering light (SSC) analysis also shows that IEVS scattering light intensity is concentrated in 1 μm or less.

[0104] Further, the standardized small particle microspheres (0.2 μm, 0.5 μm, 1 μm) produced by Bangs Laboratories (0.2 μm, 0.5 μm, 1 μm) were analyzed, and the partic...

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Abstract

The invention discloses application of extracellular vesicles in preparation of a preparation for regulating ovarian functions. Compared with a wild type mouse, the MRL / lpr mouse shows ovarian enlargement, secondary follicles and mature follicles increasing, and is in an estrus period for a long time and other premature ovaries. After injection of the induced extracellular vesicles for treatment, the characterization of the MRL / lpr mouse can be recovered to a normal level, which indicates that the induced extracellular vesicles can improve the symptom of premature ovarian. Meanwhile, the invention also discloses an improvement effect of the induced extracellular vesicles on premature ovarian failure.

Description

Technical field [0001] The invention belongs to the field of biomedical, and specific to the application of cellular outer vesicles in preparations for preparing ovary function. Background technique [0002] Feminiological premature is due to premature production of ovarian follicles, although the actual age of women does not reach development age (before 8 years old), it will still enter adolescent development mode. Ovarian premature can result in premature development of follicles, excessive follicles. From hormone level, ovarian premature will lead to elevated estrogen, too high estrogen will lead to germoscopic, obesity, and excessive ovulation, and complications include uterine fibroids and endometriosis. [0003] Prematrue Ovarian Failure, POF refers to the early recession of women in the ovary function of the 40-year-old ovary, including some symptoms of ovarian function, such as menstrual cycle disorders, abnormal menstrual volume, reproductive road inflammation, will acc...

Claims

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Application Information

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IPC IPC(8): A61K35/28A61P15/08C12N5/0775
CPCA61K35/28A61P15/08C12N5/0662C12N2501/999
Inventor 傅钰寇晓星施松涛
Owner EV CELL BIOTECH GUANGZHOU CO LTD
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