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Separation and culture method and application of human testis mesenchymal stem cells

A testicular interstitial, separation method technology, applied to animal cells, vertebrate cells, bone/connective tissue cells, etc., can solve the problems of no CD51, no related reports on separation and culture, long equilibration time, etc. Effects of Serum Testosterone Levels

Inactive Publication Date: 2016-02-17
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Its main disadvantage is that isopycnic zonal centrifugation is a balanced centrifugation method, and it takes a long time to achieve equilibrium, usually more than ten or even dozens of hours, and the loss of too many cells limits its popularization and use.
In addition, some samples are exposed to high concentration gradient solution, which may cause damage
Recent studies have reported that using nestin-GFP transgenic mice, SLCs obtained by flow cytometry sorting have high sorting efficiency, strong self-renewal and multi-lineage differentiation potential, and can be differentiated into mature LCs in transplanted animals. The promotion of testosterone secretion and spermatogenesis provides an important scientific basis for testosterone replacement therapy (JiangMH, CaiB, TuoY, et al. Characterization of Nestin-positive stem Lydig cells as a potential source for the treatment of testicular Leydig cell dysfunction. , thus limiting its clinical application
In addition, there are no relevant reports on the isolation and culture of human testicular Leydig cells (hSLCs)
However, there is no report on the expression of CD51 in human testis

Method used

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  • Separation and culture method and application of human testis mesenchymal stem cells
  • Separation and culture method and application of human testis mesenchymal stem cells
  • Separation and culture method and application of human testis mesenchymal stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1 Isolation of CD51 positive cells from testicular specimens of obstructive azoospermia patient tissue biopsy

[0038] For patients with obstructive azoospermia, after the testicular tissue biopsy was taken out, some specimens were placed in iced DMEM / F12 medium containing 15 μg / ml gentamicin, and stored at 0-4°C. Within 2 hours, process tissue samples: use micro scissors to cut the tissue until the length of each seminiferous tubule is about 1 mm, then put in 1 mg / ml type IV collagenase solution containing DNase enzyme (450 U), at 37 °C , 5%CO 2 Incubate under conditions for 30 min, add 3 times the volume of PBS to dilute collagenase, and centrifuge at 1500 rpm for 5 min; discard the supernatant, add 15 ml of PBS, and filter the cell suspension with a nylon mesh with a pore size of 40 μm, and centrifuge the collected filtrate at 1500 rpm for 5 min to obtain human testicular cells

[0039] CD51 antibody labeling was performed. Mix the fluorescently labeled CD...

Embodiment 2

[0040] Example 2 Identification of CD51-positive human testicular mesenchymal stem cells self-renewal and proliferation ability

[0041] a. Identification of self-renewal ability of CD51-positive human testicular mesenchymal stem cells:

[0042] 1) The CD51-positive hSLCs isolated in Example 1 were placed in each single well of a 6-well plate for culture. The composition of the culture solution is to add 1nM dexamethasone, 2ng / ml LIF, 50ng / ml insulin, 50ng / ml transferrin, 50pg / ml sodium selenite, 5% (V / V) chicken embryo in DMEM-F12 medium Extract, 1% (V / V) non-essential amino acids, 2% (V / V) B27 serum-free supplement, 20 ng / ml bFGF, 20 ng / ml EGF, 20 ng / ml PDGFBB. Observe the formation of cell clones. When the size of cell clones reaches more than 50 μm and the density reaches 70%, the cells are digested with 0.05% trypsin-EDTA at 37° C. for 30 seconds, and then passaged. figure 1 It shows that the sorted primary cells adhere to the wall in the culture medium, grow in a spind...

Embodiment 3

[0050] Example 3 Observation of the role of CD51-positive human testicular mesenchymal stem cells in tissue repair in vivo

[0051]The ability of stem cells to regenerate damaged tissues in the body is an important attribute. Previous studies have shown that treatment with thecytotoxinethanedimethylsulfonate (EDS) for 4 days may deplete Leydig cells. Therefore, a rat model of Leydig cell loss (EDS model) was established to investigate whether CD51-positive human Leydig stem cells could promote the recovery of Leydig cells in the EDS model. Two groups of adult rats were selected, namely the model group and the cell group. Rats in the model group (EDS(+) / saline(+)) were intraperitoneally injected with EDS, and 4 days later, 20 μl of normal saline was injected into the testis (10 μl / unilateral testis). After 4 days of intraperitoneal injection of EDS (75 mg / kg body weight) in the rats of the cell group (EDS(+) / Cell(+)), the CD51-positive human testicular mesenchymal stem cells ...

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Abstract

The invention discloses a separation and culture method and application of human testis mesenchymal stem cells. The separation method comprises the steps that an obstructive azoospermia patient testis specimen which is processed through tissue biopsy separation is digested with type IV collagenase, digested cell suspension is filtered to remove tissue clumps, and human testis cells are obtained; the human testis cells are marked with a CD51 antibody, separation is performed with a flow cytometer, the cells expressing the CD51 are collected, and then the human testis mesenchymal stem cells with the positive CD51 are separated. The invention further provides the human testis mesenchymal stem cells which are obtained through the separation method and have the positive CD51, the culture method of the stem cells and application of the stem cells in preparation of medicine for treating related diseases caused by low testosterone level.

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 human testicular mesenchymal stem cells and its application. Background technique [0002] Late-onset hypogonadism (LOH) refers to the gradual decrease in testosterone levels in the body due to the decrease in the number and function of Leydig cells in middle-aged and elderly men as they grow older, which seriously affects the quality of life. At present, the main treatment method for LOH is androgen replacement therapy, but this therapy has disadvantages such as difficulty in mastering supplementary doses, inability to simulate the secretion characteristics of physiological testosterone, and the need for long-term medication. With the acceleration of the aging process of the social population, the number of patients with LOH is increasing day by day. Therefore, exploring a treatment method to restore the androgen le...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0775A61K35/52A61P5/26
CPCA61K35/52
Inventor 项鹏姜美花张敏臧志军
Owner SUN YAT SEN UNIV
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