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Method and application for inducing human umbilical cord mesenchyme stem cells to be differentiated into testicular interstitial cells

A technology for Leydig cells and Leydig stem cells is applied in the field of inducing human umbilical cord mesenchymal stem cells to differentiate into Leydig cells. Efficiency, the effect of good testosterone secretion capacity

Inactive Publication Date: 2011-09-07
JINAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

But bone marrow mesenchymal stem cells in vitro proliferation ability, multi-directional differentiation ability and cell stability will be gradually weakened or even lost to varying degrees with age or the impact of disease. This method has low differentiation efficiency and low amount of gonadal hormones secreted , it is necessary to find a method to efficiently differentiate stem cells into Leydig cells

Method used

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  • Method and application for inducing human umbilical cord mesenchyme stem cells to be differentiated into testicular interstitial cells
  • Method and application for inducing human umbilical cord mesenchyme stem cells to be differentiated into testicular interstitial cells
  • Method and application for inducing human umbilical cord mesenchyme stem cells to be differentiated into testicular interstitial cells

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Experimental program
Comparison scheme
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Embodiment 1

[0038] Example 1 Isolation and Expansion of Human Umbilical Cord Mesenchymal Stem Cells

[0039] Tissue block adherent separation method was used. Obtain the umbilical cord under sterile conditions, soak it in 0.25% iodophor for 3 minutes for disinfection, rinse with normal saline, remove the surface of the umbilical cord and blood clots in the blood vessel, cut the umbilical cord, tear off the Wharton's Jelly around the blood vessel wall with tweezers, Cut it into 1.5~2.5mm 3 The size of the tissue block was placed in a 24-well plate, and 800ml of culture medium was added (the composition of the culture medium was 10% (v / v) fetal bovine serum (FBS), 5ng / ml bFGF, 216μg / ml glutamate, 2mg / ml NaHCO 3 , 100U / ml penicillin, 100μg / ml streptomycin and 1μg / ml amphotericin B LG-DMEM culture fluid), put in the incubator, 37 ℃, 5% CO 2 culture under static conditions. After 12-15 days, microscopic examination can see a large number of long spindle cells dissociated from around the ad...

Embodiment 2

[0040] Example 2 Cell Morphology Observation

[0041] After the umbilical cord Walton jelly tissue block was inoculated into a 24-well plate and cultured for 10 days, a small amount of cells could be gradually dissociated from the tissue block, adhered to the wall, and continued to proliferate ( figure 1 a). Observed under a microscope, it can be seen that the cells are in the shape of a uniform long spindle, which is a typical shape of fibroblasts, growing in a parallel arrangement or in a spiral shape, and fused into a single layer of adherent cells with the continuous expansion of the colony growth. Conventional subculture, the cells can be seen to appear swirl after overgrowth ( figure 1 b).

Embodiment 3

[0042] Example 3 Identification of Cell Surface Antigens by Flow Cytometry

[0043] The normally cultured umbilical cord mesenchymal stem cells were taken, and the expression of cell surface antigens were detected by flow cytometry, including CD105, CD73, CD90, CD45, CD34, CD14, CD19, and HLA-DR.

[0044] (1) After the cells grow to 80-90% confluent, they are digested with 0.25% trypsin solution with mass volume ratio, collected by centrifugation, washed twice with PBS solution (1X, pH=7.2), and the concentration of the cell suspension is adjusted to 10 6 cells / ml.

[0045] (2) Add 2ml of PBS solution (1X, pH=7.2) containing 2.5% FBS by volume, mix the cells by pipetting, and centrifuge at 200g.

[0046] (3) Antibodies used include CD105, CD73, CD90, CD45, CD34, CD14, CD19 and HLA-DR antibodies. Non-specific backgrounds were incubated with mouse anti IgG1 / PE, mouse anti IgG1 / FITC as controls, all at 1:50 Ratio, after dilution with PBS solution (1X, pH=7.2), incubate on ice f...

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Abstract

The invention discloses a method and application for inducing human umbilical cord mesenchyme stem cells to be differentiated into testicular interstitial cells. The method comprises the following step of culturing human umbilical cord mesenchyme stem cells of patients suffering from adenovirus and carrying mice steroidogenic factor-1 genes in a DMEM-F12 culture solution containing 0.3-3ng / ml of luteinizing hormone, 200-800mu M of dibutyryl cyclic adenosine monophosphate, 5*10<-6>-5*10<-4>M of all-trans retinoic acid (ATRA), 10mU / ml of human chorionic gonadotropin and 2.4uM of adrenocorticotrophic hormone for a week. Induced by the method in the invention, the human umbilical cord mesenchyme stem cells can be differentiated into testicular interstitial cells in vitro and provides important sources of cells for treating testosterone shortage by the cell replacing method or the genetic method.

Description

technical field [0001] The invention relates to the field of biological technology of inducing stem cell differentiation, in particular to a method and application for inducing human umbilical cord mesenchymal stem cells to differentiate into testicular Leydig cells. Background technique [0002] With the prolongation of life expectancy and the problem of population aging, the number of middle-aged and elderly male partial androgen deficiency (PADAM) patients is increasing. At present, exogenous androgen replacement therapy (ART) is mainly used clinically, the purpose is to maintain the physiological concentration of testosterone in serum to replace the physiological function of endogenous testosterone. However, long-term use of hormones has relatively large side effects. Firstly, there are many target cells of androgen in the body. Long-term use of exogenous androgen therapy is likely to cause toxic and side effects, especially in the prostate gland; Frequent blood tests a...

Claims

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Application Information

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IPC IPC(8): C12N5/0775C12N15/861C12N7/01C12N5/077C12P33/00
Inventor 魏星白杨
Owner JINAN UNIVERSITY
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