A method for isolating, culturing and using testicular mesenchymal stem cells expressing nestin

A technology for Leydig and Nestin, which is applied in the field of separation and culture of Leydig stem cells, can solve the problems of restricting clinical application, obtaining a small amount of cells, and unclear cell types.

Active Publication Date: 2017-09-01
SUN YAT SEN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

These studies suggest that testicular mesenchymal stem cells in rats can be cultured and expanded in vitro, but 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. further in-depth research
At present, there are no relevant research reports on the separation and identification of testicular mesenchymal stem cells using nestin as a marker

Method used

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  • A method for isolating, culturing and using testicular mesenchymal stem cells expressing nestin
  • A method for isolating, culturing and using testicular mesenchymal stem cells expressing nestin
  • A method for isolating, culturing and using testicular mesenchymal stem cells expressing nestin

Examples

Experimental program
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Effect test

Embodiment 1

[0029] Example 1: Isolation of Nestin-positive Leydig cells from mouse adults

[0030] In this example, testicular mesenchymal stem cells expressing Nestin were isolated from mouse adult testis.

[0031] Selection of Nestin-GFP mice: The transgenic mice used in this experiment were from the specific marker Nestin-GFP transgenic mouse model (Yamaguchi M, Saito H, Suzuki M, Mori K. Visualization of neurogenesis in the central nervous system using nestin promoter-GFP transgenic mice. Neuroreport 2000.11:1991-1996). However, other Nestin-GFP mice can also be used, and their common characteristics are: a plasmid that expresses GFP driven by a Nestin promoter, and the promoter region including the upstream translation site is 2.5kb long and contains the second exon The enhancer region from 3' to the 5' of the third intron is 1.8kb long, and the polyadenylation site is connected with enhanced GFP (EGFP) cDNA.

[0032] The present invention utilizes the research of the animal model ...

Embodiment 2

[0037] Example 2: Identification of nestin-positive testicular mesenchymal stem cells self-renewal and proliferation ability

[0038] In this example, on the basis of Example 1, the self-renewal and proliferation abilities of the obtained nestin-positive testicular mesenchymal stem cells were identified.

[0039] a. Identification of the self-renewal ability of nestin-positive testicular stromal stem cells:

[0040] Single nestin-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 medium include ITS added with 1nM dexamethasone, 1ng / ml LIF, 5mg / literinsulin, 5mg / litertransferrin, 5ug / liter sodium selenite in DMEM-F12 medium, 5% chicken embryo extract, 0.1mM β-mercaptoethanol, 1% non-essential amino acids, 1% N2, 2% B27 (Gibco), 20ng / ml bFGF, 20ng / ml EGF, 20ng / ml PDGFBB, 20ng / ml OSM. In the above medium, the sorted primary cells grow adherently. Passage after 7 days, form spherical growth....

Embodiment 3

[0047] Example 3: Observing the role of nestin-positive testicular stromal stem cells in tissue repair in vivo

[0048] a. Establishment of rat EDS model:

[0049]The ability of stem cells to regenerate damaged tissues in the body is an important feature. Therefore, we investigated whether nestin-positive Leydig stem cells could promote functional recovery of Leydig cells 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 the model group and the cell group were inoculated with EDS, and 4 days later, PKH26-labeled 1x10 6 Nestin-positive Leydig stem cells were infused into the testicular parenchyma. Serum testosterone concentrations were measured on day 10 of transplantation. Figure 8 showed that nestin-positive testicular mesenchymal stem cell transplantation...

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Abstract

The invention discloses a method for isolating, culturing and using testicular mesenchymal stem cells expressing nestin. The method of the invention includes: providing a nestin-green fluorescent protein transgenic mouse with C57BL / 6 as the genetic background. Rat model; feed the mice to the age of 1 week or more, separate the testis, remove the capsule and blood vessels, expose the seminiferous tubules and digest with collagenase IV, filter the digested cell suspension; sort by flow cytometry to obtain For a single cell, cells expressing green fluorescent protein whose fluorescence intensity is 10 times or more than that of the negative control cells are collected, thereby isolating the testicular mesenchymal stem cells expressing nestin. The present invention provides Leydig cells derived from testicular stem / progenitor cells isolated according to the above method, as well as self-renewing and proliferating cells obtained by further culturing in culture medium, and provides the use of these cells for the preparation of diseases related to low testosterone levels use of the composition.

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 mesenchymal stem cells expressing nestin and its application. Background technique [0002] With the process of social aging, the health of middle-aged and elderly people has received extensive international attention. Late-onset hypogonadism (LOH) has become one of the important diseases seriously affecting the health and quality of life of middle-aged and elderly men. The core mechanism of LOH is that Leydig cells that secrete testosterone decrease in number and function with age, resulting in a lack of androgen in the body. Insufficient testosterone can eventually lead to a decline in libido and sexual function, and can also cause symptoms such as fatigue, loss of muscle volume, increase in fat volume, osteoporosis, and anemia. At the same time, it affects mood and mental decline, seriously affects quality of lif...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/10A61K35/28A61P5/26
Inventor 项鹏姜美花蔡炳臧志军汪建成
Owner SUN YAT SEN UNIV
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