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A kind of separation, culture method and application of testicular mesenchymal stem cells

A separation method and testicular interstitial technology, applied in the field of separation and culture of testicular interstitial stem cells, can solve the problems of unclear cell type, low amount of obtained cells, and lack of cell identification standards.

Active Publication Date: 2017-09-15
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the transplantation of testicular mesenchymal stem cells for the treatment of LOH provides a good prospect for clinical application, the current method has many disadvantages such as the small amount of cells obtained, the type of cells is not clear, and the lack of cell identification standards, which obviously restricts clinical application and needs further in-depth study. Research
There are no research reports on the isolation and identification of testicular mesenchymal stem cells using CD51 as a marker

Method used

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  • A kind of separation, culture method and application of testicular mesenchymal stem cells
  • A kind of separation, culture method and application of testicular mesenchymal stem cells
  • A kind of separation, culture method and application of testicular mesenchymal stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1: Isolation of CD51-positive cells in the interstitium of mouse adult testis

[0026] In this example, testicular mesenchymal stem cells were isolated from mouse adult testes.

[0027] The separation method is as follows:

[0028] a. Take out the male C57BL / 6 male mice with healthy reproductive system 7 days after birth, kill them by cervical dislocation, open the scrotum under aseptic conditions, take out the bilateral testes, remove the capsule and blood vessels under a dissecting microscope, and use Wash repeatedly with 1×PBS containing 1% double antibody. Then blow gently with a straw and a gun tip to disperse the interstitial tissue and the fine tubules as much as possible, and cut the tissue part with small scissors. Then put in 1mg / ml type IV collagenase solution containing a small amount of DNase enzyme, incubate at 37°C and 5% CO2 for 15 minutes, add a large amount of 1×PBS containing 0.5% BSA to terminate the digestion of collagenase, and centrifuge...

Embodiment 2

[0031] Example 2: Identification of self-renewal and proliferation abilities of CD51-positive testicular mesenchymal stem cells

[0032] In this example, on the basis of Example 1, the self-renewal and proliferation abilities of the obtained CD51-positive testicular mesenchymal stem cells were identified.

[0033] a. Identification of self-renewal ability of CD51-positive testicular stromal stem cells:

[0034] 1) Single CD51-positive cells sorted from mouse adult testes were placed in each single well of a 6-well plate for culture. The components of the culture solution include 1nM dexamethasone, 1ng / ml LIF (leukemia inhibitory factor), 5mg / liter insulin (insulin), 5mg / litertransferrin (transferrin), 5ug / litersodium selenite (selenite) added to DMEM-F12 medium ITS (insulin-transferrin-sodium selenite), 5% chicken embryo extract, 0.1mM β-mercaptoethanol, 1% non-essential amino acids, 1% N2 (N2 additive), 2% B27 (B27 serum-free additive) , 20ng / ml bFGF (basic fibroblast growt...

Embodiment 3

[0042] Example 3: Observing the role of CD51-positive testicular mesenchymal stem cells in tissue repair in vivo

[0043] Establishment of rat EDS model:

[0044] The ability of stem cells to regenerate damaged tissues in the body is an important attribute. Therefore, we investigated whether CD51-positive Leydig stem cells could promote Leydig cell recovery in a rat model of Leydig cell loss. Previous studies have shown that the cytotoxin ethane dimethylesulfonate (EDS) may deplete Leydig cells after 4 days of treatment. We selected three groups of adult rats, which were normal group, model group and cell group. Rats in model group and cell group were inoculated with EDS, 1x10 after 4 days 6 CD51-positive Leydig stem cells were injected into the cell group mice. Serum testosterone concentrations were measured on day 10 after transplantation. The result is as Figure 7 As shown, CD51-positive Leydig stem cells significantly promoted the secretion of testosterone in the te...

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Abstract

The invention discloses a method for separating, culturing and using testicular mesenchymal stem cells. The method provided by the invention includes: using a 7-day or 2-month-old mouse to separate the testis, remove the capsule and blood vessels, and expose the seminiferous tubules Digest with collagenase IV, filter the digested cell suspension; use CD51 cell-specific markers, sort by flow cytometry, obtain single cells, and collect CD51-expressing cells, thereby isolating the CD51-positive Leydigm testis stem cell. The present invention provides Leydig stem cells isolated according to the above method, as well as self-renewal and proliferation cells with multi-lineage differentiation potential obtained by further culturing in the culture medium, and provides the use of these cells for the preparation of low testosterone levels Use of the composition resulting in a related disease.

Description

technical field [0001] The invention relates to the technical field of stem cells and tissue engineering, in particular to a method for separating and culturing testicular mesenchymal stem cells and its application. Background technique [0002] With the acceleration of global aging, various aging-related diseases have attracted increasing attention. Among them, the incidence rate of diseases such as delayed hypogonadism (LOH) and senile androgen deficiency caused by primary or secondary Leydig cell dysfunction is as high as more than 20% in men over 50 years old. It not only affects Quality of life can also easily lead to the occurrence of major diseases such as cardiovascular disease and mental disease. Testosterone replacement therapy is currently the main treatment. It is estimated that the market size of testosterone products in the United States was approximately US$49 million in 1997, and exceeded US$300 million in 2002. Statistics show that 20% of men over the age...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/0775A61K35/52A61P5/26
Inventor 项鹏姜美花蔡炳臧志军汪建成
Owner SUN YAT SEN UNIV
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