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Construction and application of model for differentiating stem cells into testicular interstitial cells

A technology of Leydig cells and embryonic stem cells, which is applied in the fields of developmental biology and pharmacology, can solve problems such as the screening of unseen pro-cell differentiation agents, and achieve the effect of improving application prospects

Inactive Publication Date: 2010-11-24
ZHEJIANG UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The male reproductive system is derived from the mesoderm, and there are no reports on the directional differentiation of ES cells into Leydig cells and the mechanism of its impact on differentiation and development, let alone the use of this model to evaluate the function of regulating the secretion of testosterone by Leydig cells as a target. Differentiation agent screening

Method used

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  • Construction and application of model for differentiating stem cells into testicular interstitial cells
  • Construction and application of model for differentiating stem cells into testicular interstitial cells
  • Construction and application of model for differentiating stem cells into testicular interstitial cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] Example 1: Directed differentiation of ES cells into Leydig cells in vitro

[0018] 1. ES cell transfection

[0019] ES cells were cultured without a feeder layer, and plasmid transfection was performed according to the instructions of effectene transfection regeagent (Qiagen) (effective transfection reagent manual). 4×10 per well in a 6-well culture plate 5 ES cells, and operate according to the following reagent volume. Take 2 μg of each of the two plasmids to be transfected, SF-1 and LHR, and dissolve them in 100 μl of EC buffer, then add 16 μl of enhancer enhancer solution, vortex for 1 second to mix, and place at room temperature for 2-5 minutes; then add 25 μl of effective transfection solution effectene transfection regeagent , vortex for 10s, and place at room temperature for 5-10min; then add 0.6ml of culture medium and mix well, add it to ES cells in a 6-well plate, set the final volume to 2.2ml, and place at 37°C, 5% CO 2 Cultivate in the incubator for 24 ...

Embodiment 2

[0025] Example 2: Real-time PCR detection of gene expression of luteinizing hormone LH induced directed differentiation of ES cells into Leydig cells

[0026] First, according to step 1 of Example 1, ES cells were transfected for 24 hours and the culture medium was replaced, then the inducer was added, 100ng / mLLH and 1×10 -3mol / L 8-Br-cAMP co-induced ES cells transfected with SF-1 and LHR plasmids to differentiate into Leydig cells. Collect the cells on the second day of induced differentiation, extract RNA routinely, and then prepare a reaction solution in a 0.2ml PCR tube: 3 μg of total RNA, 0.5 μl of Oligo-dT (15 μg / μl), 1 μl of dNTP mix (10 mmol / L), double Distilled water (DEPC) 10 μl, put the reaction tube in a PCR instrument, and pre-denature at 65°C for 5 minutes. After the denaturation reaction, prepare the cDNA first-strand synthesis reaction system in the PCR tube, place the PCR tube in a PCR instrument for 50 min at 42°C, denature at 70°C for 15 min (to inactivate ...

Embodiment 3

[0035] Example 3: WB detection of protein expression of luteinizing hormone LH induced directed differentiation of ES cells into Leydig cells

[0036] Collect the cells on the 2nd day of induced differentiation as in Example 2, extract protein as usual, run 12.5% ​​SDS-PAGE electrophoresis, then take out the nitrocellulose membrane after transfer, and place it in 0.1% of 5% (w / v) skimmed milk powder. In % Tween-PBS solution, room temperature for 1h or overnight at 4°C; take out the overnight nitrocellulose membrane, wash with 0.5% Tween-PBS solution for 5min / 3 times; add primary antibody, room temperature for 1h; wash with 0.5% Tween-PBS solution Wash for 5 min / 3 times; add secondary antibody, and place at room temperature for 30 min; wash with 0.1% Tween-PBS solution for 5 min / 3 times; prepare ECL solution; add ECL solution to the membrane, avoid drying, room temperature for 1 min; Membrane wraps the membrane well; X-film exposes and analyzes the results.

[0037] The result...

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Abstract

The invention provides a method for constructing a model of oriented differentiation of embryonic stem (ES) cells into testicular interstitial cells. The method comprises the following steps of: firstly, cotransfecting a steroidogenic factor 1 and a luteinizing hormone receptor plasmid into an ES cell; then inducing the ES cell by a compound or growth factor to directionally differentiate the ES cell into the testicular interstitial cell. In the invention, the construction and the application of the model for in-vitro oriented differentiation of the stem cells into the testicular interstitialcells are demonstrated at the cellular level; a great deal of biology information is discussed initially; the constructed cell model is a substituted model for researching the influencing factor of the secretory regulation function of the testicular interstitial cell and can be used for initially selecting and evaluating the medical effect of the pharmaceutical drug using the testicular interstitial cell as the target cells; the transplanting of the testicular interstitial cell differentiated from the stem cells is used as a new method for supplying testosterone, and the application prospect of the renewable alternative treatment of the testicular interstitial cell is improved.

Description

technical field [0001] The invention belongs to the field of developmental biology and pharmacology, and relates to the construction and application of a model, in particular to the construction of a model of stem cells directed differentiation into Leydig cells in vitro, and its induction with the aim of regulating the secretory function of Leydig cells Differentiation agent screening application. Using the transplantation of Leydig cells differentiated from stem cells as a new method of supplementing testosterone will improve the application prospect of Leydig cell regeneration and transplantation. Background technique [0002] Andropause syndrome, that is, partial androgen deficiency in middle-aged and elderly men (PADAM) or hypogonadism in middle-aged and elderly men is a social problem of general concern at present. At present, chemically synthesized androgen is mainly used in clinic for replacement therapy. However, the long-term application of chemically synthesized...

Claims

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Application Information

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IPC IPC(8): C12N5/0735C12N5/10C12Q1/02
Inventor 朱丹雁葛仁山楼宜嘉张莹莹周立民崔荣张翔南
Owner ZHEJIANG UNIV
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