CN105331579A - Separation and culture method and application of human testis mesenchymal stem cells - Google Patents
Separation and culture method and application of human testis mesenchymal stem cells Download PDFInfo
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- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
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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
The present invention relates to stem cell and tissue engineering technique field, be specifically related to the separation of Human Testis interstital stem cell, cultural method and uses thereof.
Background technology
Delayed onset hypogonadism (LOH) refers to that middle-aging male with age, and interstitial glands quantity reduces and deterioration and cause testosterone levels in body to reduce gradually, and it has a strong impact on quality of life.At present, the primary treatments of LOH is androgen replacement, but this therapy exists supplementary dosage to be difficult to grasp, cannot to simulate the secretion characteristic of physiological testosterone, to need the shortcomings such as long-term prescription.Along with social population's aging process is accelerated, LOH patient increases day by day, therefore, explores and makes the androgen levels of LOH patient return to normal methods for the treatment of, become important scientific problems in the urgent need to address.
Interstitial tissue[of testis] stem cell (stemLeydigcells, SLCs) has self-renewal capacity, is the direct cell derived being divided into interstitial glands (Leydigcells, LCs), is the seed cell of desirable LOH stem-cell therapy.Very few to the research of SLCs at present, be confined to isolate SLCs in the animal testis such as mouse, rat.Major part obtains the method employing density gradient centrifugation (GeRS of interstitial glands traditionally, DongQ, SottasCM, PapadopoulosV, ZirkinBR, HardyMP.InsearchofratstemLeydigcells:identification, isolation, andlineage-specificdevelopment.ProcNatlAcadSciUSA2006; 103:2719-2724; StanleyE, LinCY, JinS, LiuJ, SottasCM, GeR, etal.Identification, proliferation, anddifferentiationofadultLeydigstemcells.Endocrinology20 12; 153:5002-5010.), the centrifugal abstraction and purification being mainly used in the different sample component of density of isopycnic banding, this method requires higher to cell, not only there is requirement to whizzer and centrifugal rotor, and the medium being separated viable cell is had higher requirements, such as: 1) can density gradient be produced, and during density height, viscosity is not high; 2) pH is neutral or be easily adjusted to neutrality; 3) when concentration is large, osmotic pressure is little.Its main drawback is isopycnic zone centrifugation is a kind of equilibrium centrifugation, reaches starting time very long, usually wants tens even tens hours, and cell loss is too much, so limit its universal use.Some sample is exposed in the gradient liquid of high density in addition, may cause damage.Studies have reported that recently, utilize nestin-GFP transgenic mice, the SLCs efficiency of separation that fluidic cell sorting obtains is high, there is stronger self and multi-lineage potential, and ripe LCs can be divided in transplanting animal body, promote secretion and the spermatogenesis of testosterone, for testosterone replacement therapy provides important scientific basis (JiangMH, CaiB, TuoY, etal.CharacterizationofNestin-positivestemLeydigcellsasa potentialsourceforthetreatmentoftesticularLeydigcelldysf unction.CellRes.2014.24 (12): 1466-85), but because nestin is intracellular protein, because which limit its clinical application.In addition, the separation and ientification of Human Testis interstital stem cell (humanstemLeydigcells, hSLCs) is studied at present have no relevant report.
CD51 is also called integrin protein alpha v, is a member in integrin superfamily protein, and its essence is that heterodimer AQP-CHIP is made up of α chain and β chain two portions.α v β 1, α v β 3, α v β 5, α v β 6 and α v β 8 totally 5 hypotypes can be subdivided into further.In regulation and control, embryonic organ is formed, and vasculogenesis, vascular permeability, cancer evolution, plays a significant role and will act in the inflammation of epithelium and fibrosis.Studies have reported that, CD51 is distributed widely in periodontal ligament, the surface of MSC like cell in human cord blood and magnificent Tong Shi glue.Simultaneously, also find that CD51 is present in the surface of most people's marrow MSC cell, think the mark (PinhoS of MSC, LacombeJ, HanounMetal.PDGFRalphaandCD51markhumannestin+sphere-form ingmesenchymalstemcellscapableofhematopoieticprogenitorc ellexpansion.TheJournalofexperimentalmedicine2013; 210:1351-1367).This laboratory is demonstrated by the method such as flow cytometer showed and immunostaining and expresses CD51 at mouse testis nestin+SLC, and may be the mark of mouse testis SLC.But, not yet there is the relevant report that CD51 expresses in Human Testis at present.
Summary of the invention
The object of this invention is to provide a kind of Human Testis interstital stem cell (humanleydigstemcells, hSLCs) separation and cultural method, for hypogonadism patient especially patient LOH provide new more close to the scheme of the supplementary testosterone of physiological conditions.
The present invention is separated Human Testis interstital stem cell using CD51 as cell sign thing, obtains the Human Testis interstital stem cell of CD51+.Concrete steps are: digested by the Obstructive azoospermia patient testis sample collagenase IV that biopsy is separated, and filter postdigestive cell suspension and organize agglomerate to remove, obtain Human Testis cell; By CD51 streaming antibody labeling Human Testis cell, utilize flow cytometer to carry out sorting, collect the cell of expressing CD51, thus isolate the Human Testis interstital stem cell of the CD51 positive.
The concrete steps of the method for the Human Testis interstital stem cell of the separation of C D51 positive are as follows:
(1), after taking off Human Testis biopsy sample, the ice DMEM/F12 substratum containing 15 μ g/ml gentamicins is placed in immediately.Process tissue in 2 hours: little scissors shreds tissue part, puts into the IV Collagenase Type solution of 1mg/ml subsequently, in 37 DEG C, 5%CO
2hatch 30min under condition, the PBS adding 3 times of volumes dilutes the activity of collagenase, the centrifugal 5min of 1500rpm; Abandoning supernatant adds 15mlPBS, and is the nylon net filter cell suspension of 40 μm with aperture, removes indigested convoluted seminiferous tubule, collects unicellular, with the centrifugal 5min of 1500rpm, obtain Human Testis cell;
(2) CD51 antibody labeling is carried out.Get above-mentioned Human Testis cell suspension to mix with fluorescently-labeled CD51 antibody, both volume ratios are CD51 antibody: Human Testis cell suspension is 1:200,4 DEG C of micro-revolving hatch 30 minutes, abandon clear liquid, PBS buffer solution 2 times, simultaneously separately get above-mentioned Human Testis cell suspension and IgG carries out hatching as negative control, collect through airflow classification the cell that fluorescence intensity is 10 times of the fluorescence intensity of negative control cell or more, thus isolate CD51 positive human interstitial tissue[of testis] stem cell (CD51+hSLCs).
The present invention also improves on the basis of DMEM/F12 basic medium, optimizes passage cell substratum, the Human Testis interstital stem cell obtained with culture of isolated, produces the cell of self and propagation.The CD51 positive cell collected by airflow classification is placed 2 hours in primary culture medium (be specially in DMEM-F12 substratum and add 5%FBS), is changed to serum-free passage cell substratum afterwards.Described serum-free passage cell substratum is specially and adds 1nM dexamethasone, 2ng/mlLIF, 50ng/ml Regular Insulin, 50ng/ml Transferrins,iron complexes, 50pg/ml Sodium Selenite, 5% (V/V) chick embryo extract, 1% (V/V) non-essential amino acid, 2% (V/V) B27 serum-free additive, 20ng/mlbFGF, 20ng/mlEGF, 20ng/mlPDGFBB in DMEM-F12 substratum.Experiment shows, this substratum is used for the cultivation of Human Testis interstital stem cell of the present invention, and cell proliferation effect is very good.
The present invention carries out the detection of interstitial tissue[of testis] stem cell line (CD51+hSLCs) and stem cell correlative protein expression to being separated the CD51 positive cell obtained, the molecular biology characteristics of CD51+hSLCs is analyzed from molecular level, the self of further this cell of analysis and Multidirectional Differentiation ability, thus this kind of stem cell is identified.Result shows, CD51+hSLCs is the growth of clone ball sample.CD51+hSLCs PDGF-B expression R-α, P75, SSEA-4, LIFR of cultivating, but do not express 3 β-HSD, LHR etc.CD51+hSLCs cell has self-renewal capacity.Analytical Chemical Experiment result shows, under specific differentiation-inducing condition, CD51+hSLCs can be divided into adipocyte, scleroblast; Also interstitial glands can be divided into, and Testosterone Secretion.
The CD51+hSLCs of separation is transplanted to the rat animal model (Apoptosis Model of the interstitial glands that the specificity antiapoptotic inductor ethane dimethane sulfonate (EDS) applying Leydig Cells is set up) removing interstitial glands by the present invention, evaluates the effect of this stem cell in testis microenvironment.Found that, the concentration of serum testosterone after this stem cell transplantation, can be improved.Therefore, the CD51+hSLCs that method of the present invention obtains may be used for the medicine preparing the low relative disease for the treatment of people's testosterone levels.
The present invention isolates the Human Testis interstital stem cell (CD51+hSLCs) of the CD51 positive first and carries out cultivation to it and make it breed.CD51+hSLCs of the present invention in vitro in specific inducing culture, can obtain the markers of interstitial tissue[of testis] stem cell and the ability of testosterone secretion gradually.Being transplanted to by CD51+hSLCs of the present invention eliminates in the rat model of interstitial glands with EDS, and CD51+hSLCs can be divided into interstitial glands, and can improve the level of serum testosterone of animal pattern.
Accompanying drawing explanation
Fig. 1 is the cultivation figure of CD51+hSLCs vitro culture.
Fig. 2 is CD51+hSLCs interstitial tissue[of testis] stem cell Research of predicting markers expression figure.
Fig. 3 is the CD51+ Human Testis interstital stem cell cultivated rises to clone ball white-light visualization figure from individual cells.
Fig. 4 is that the CD51+hSLCs that cultivates is in vitro to osteoblast differentiation (A), figure to Adipocyte Differentiation (B).
Fig. 5 is that CD51+hSLCs is to external interstitial glands differentiation figure.
Fig. 6 is the amount of Testosterone Secretion under CD51+hSLCs condition of in vitro culture.
Fig. 7 is that CD51+hSLCs is transplanted to EDS rat model after 10 days, level of serum testosterone schematic diagram.Wherein EDS (+)/saline (+) is model group, and EDS (+)/Cell (+) is groups of cells.
Embodiment
Be understandable that, particular implementation described here represents by way of example, and it is not as limitation of the present invention.When not deviating from the scope of the invention, principal character of the present invention may be used for various embodiment.One skilled in the art will appreciate that and maybe can confirm, only use normal experiment, many equivalents can be applied in particular step described herein.These equivalent places of being considered within the scope of the present invention, and cover by claim.
An aspect of of the present present invention provides a kind of separation method of Human Testis interstital stem cell, it is characterized in that:
(1) by the IV Collagenase Type digestion of Human Testis Tissue biopsy samples, filter postdigestive cell suspension, obtain Human Testis cell suspension;
(2) with the Human Testis cell that fluorescently-labeled CD51 antibody labeling step (1) obtains, utilize flow cytometer to carry out sorting, collect the cell of expressing CD51, thus isolate the Human Testis interstital stem cell of the CD51 positive.
In a specific embodiment, above-mentioned steps (1) is specially in the ice DMEM/F12 substratum be positioned over by Human Testis Tissue biopsy samples containing 15 μ g/ml gentamicins; Process tissue in 2 hours: shred tissue, put into 1mg/mlIV Collagenase Type solution subsequently, in 37 DEG C, 5%CO
2hatch 30min under condition, the PBS adding 3 times of volumes dilutes collagenase, the centrifugal 5min of 1500rpm; Abandoning supernatant, enters 15mlPBS, and is the nylon net filter cell suspension of 40 μm with aperture, and the filtrate of collection, with the centrifugal 5min of 1500rpm, obtains Human Testis cell
In a specific embodiment, containing a small amount of DNase enzyme (450U) in described IV Collagenase Type solution.
In a specific embodiment, " collecting the cell of expressing CD51 " described in above-mentioned steps (2) is collect the cell that fluorescence intensity is 10 times of the fluorescence intensity of Human Testis cell negative control or more.In further embodiment, Human Testis cell negative control is the Human Testis cell suspension that the step (1) of hatching with IgG obtains.
In a specific embodiment, above-mentioned steps (2) is specially gets above-mentioned Human Testis cell suspension and mixes with fluorescently-labeled CD51 antibody, both volume ratios are CD51 antibody: Human Testis cell suspension is 1:200,4 DEG C of micro-revolving hatch 30 minutes, abandon clear liquid, PBS buffer solution 2 times, simultaneously separately get above-mentioned Human Testis cell suspension and IgG carries out hatching as negative control, collect through selected by flow cytometry apoptosis the cell that fluorescence intensity is 10 times of the fluorescence intensity of negative control cell or more, thus isolate CD51 positive human interstitial tissue[of testis] stem cell.
Another aspect of the present invention provides and is separated by above-mentioned separation method the Human Testis interstital stem cell obtained, and it is characterized in that, described Human Testis interstital stem cell is the CD51 positive.
In a specific embodiment, Human Testis interstital stem cell PDGF-B expression R-α, P75, LIFR, SSEA-4 of the described CD51 positive, but do not express 3 β-HSD, LHR.
Another aspect of the present invention provides a kind of method of cultivating CD51 positive human interstitial tissue[of testis] stem cell of the present invention, it is characterized in that: the composition of the substratum used for add 1nM dexamethasone, 2ng/mlLIF, 50ng/ml Regular Insulin, 50ng/ml Transferrins,iron complexes, 50pg/ml Sodium Selenite, 5% (V/V) chick embryo extract, 1% (V/V) non-essential amino acid, 2% (V/V) B27 serum-free additive, 20ng/mlbFGF, 20ng/mlEGF, 20ng/mlPDGFBB in DMEM-F12 substratum.
In a preferred embodiment, the substratum cultivating CD51 positive human interstitial tissue[of testis] stem cell of the present invention is serum free medium.
Another aspect of the present invention provides a kind of external method by Human Testis interstital stem cell directed differentiation behaviour interstitial glands, it is characterized in that: CD51 positive human interstitial tissue[of testis] stem cell of the present invention is broken up in interstitial glands differentiation culture liquid, and described interstitial glands differentiation culture liquid for add 2% (V/V) FCS, 1nMT3,1ng/mlLH, 70ng/mlIGF-I, 10ngPDGF-BB and 50ng/ml Regular Insulin, 50ng/ml Transferrins,iron complexes, 50pg/ml Sodium Selenite in DMEM-F12 substratum.
Another aspect of the present invention provides CD51 positive human interstitial tissue[of testis] stem cell of the present invention to improve the application in the medicine of testosterone levels in preparation, and treats the application in the medicine of the relative disease that people's testosterone levels lowly causes in preparation.
In a specific embodiment, CD51 positive human interstitial tissue[of testis] stem cell of the present invention can improve level of serum testosterone.
Below in conjunction with embodiment, embodiment of the present invention are described in detail, but it will be understood to those of skill in the art that the following example only for illustration of the present invention, and should not be considered as limitation of the scope of the invention.
Embodiment 1 is separation of C D51 positive cell from the testis sample of Obstructive azoospermia tissue of patient biopsy
Obstructive azoospermia patient, part sample is positioned in the ice DMEM/F12 substratum containing 15 μ g/ml gentamicins, 0-4 DEG C of preservation after taking out by Testicular biopsy.In 2 hours, process tissue sample: shred tissue to the length of every root convoluted seminiferous tubule with microscissors and be about 1mm, put into the IV Collagenase Type solution that 1mg/ml contains DNase enzyme (450U) subsequently, in 37 DEG C, 5%CO
2hatch 30min under condition, the PBS adding 3 times of volumes dilutes collagenase, the centrifugal 5min of 1500rpm; Abandoning supernatant, enters 15mlPBS, and is the nylon net filter cell suspension of 40 μm with aperture, and the filtrate of collection, with the centrifugal 5min of 1500rpm, obtains Human Testis cell
Carry out CD51 antibody labeling.Fluorescently-labeled CD51 antibody (1:200 volume ratio) is mixed with above-mentioned Human Testis cell suspension, 4 DEG C of micro-revolving hatch 30 minutes, abandon clear liquid, PBS buffer solution 2 times, simultaneously separately get above-mentioned Human Testis cell suspension and IgG carries out hatching as negative control.Use flow cytometer (BDinfluxcellsorter) first to carry out streaming loading to IgG negative control group cell suspension, select negative fluorescent signal region as negative control.Carry out streaming loading to CD51 positive cell suspension subsequently, sorting is purified into the cell that fluorescence intensity is 10 times of negative control or more, collects the cell sub-elected with substratum.
The qualification of embodiment 2CD51 positive human interstitial tissue[of testis] stem cell self and multiplication capacity
The qualification of a.CD51 positive human interstitial tissue[of testis] stem cell self-renewal capacity:
1) each single hole that positive for the CD51 being separated acquisition in embodiment 1 hSLCs is placed in 6 orifice plates is respectively cultivated.Nutrient solution composition for add 1nM dexamethasone, 2ng/mlLIF, 50ng/ml Regular Insulin, 50ng/ml Transferrins,iron complexes, 50pg/ml Sodium Selenite, 5% (V/V) chick embryo extract, 1% (V/V) non-essential amino acid, 2% (V/V) B27 serum-free additive, 20ng/mlbFGF, 20ng/mlEGF, 20ng/mlPDGFBB in DMEM-F12 substratum.The formational situation of observation of cell clone, when cell clone size reach more than 50 μm, density reaches 70%, with the 0.05% trypsinase-EDTA of 37 DEG C to cell dissociation 30 seconds, go down to posterity.It is adherent in the medium that Fig. 1 shows the primary cell that sorting obtains, and grows in shuttle-type, forms clone ball growth after going down to posterity.
2) apply the method for immunofluorescence dyeing and carry out comprising PDGFR-α, LIFR, CD51, embryonic stem cell marker as the dyeing of SSEA-4, interstitial glands mark LHR and 3 β-HSD, the cell section solid 20min of 4% paraformaldehyde, 5min × 3 time are embathed in PBS damping fluid, 0.2%Triton-X-100 penetrates 30 minutes, and Normal Goat Serum incubated at room 20 minutes, adds primary antibodie, in humidifying box, 4 DEG C are spent the night, add two to resist, incubated at room 1 hour, fluorescence microscope result.
The immunofluorescence dyeing result of the mark that CD51 positive human interstitial tissue[of testis] stem cell ball is relevant with above-mentioned stem cells hyperplasia reprogramming shows, as shown in Figure 2, CD51 positive human interstitial tissue[of testis] stem cell ball expresses P75, PDGFR-α, LIFR, SSEA4, but does not express LHR, 3 β-HSD.
The qualification of b.CD51 positive human interstitial tissue[of testis] stem cells hyperplasia ability:
In order to demonstrate the self-renewal capacity of CD51 positive human interstitial tissue[of testis] stem cell, obtaining unicellular from P4 for the digestion of CD51 positive human interstitial tissue[of testis] stem cell, carrying out unicellular spheroid and forming test.Fig. 3 shows that single cell culture can form clone after 10 days.Result shows, CD51 positive human interstitial tissue[of testis] stem cell has stronger self-renewal capacity.
The qualification of c.CD51 positive human interstitial tissue[of testis] differentiation of stem cells ability:
In order to demonstrate the differentiation capability of CD51 positive human interstitial tissue[of testis] stem cell, making CD51 positive human interstitial tissue[of testis] stem cell at conventional skeletonization nutrient solution, becoming in fat nutrient solution to break up.As shown in Figure 4, after 21 days, can to scleroblast (Fig. 4 A), adipocyte (Fig. 4 B) differentiation.
Differentiation 12 days in specific interstitial glands differentiation culture liquid (adding 2% (V/V) FCS, 1nMT3,1ng/mlLH, 70ng/mlIGF-I, 10ngPDGF-BB and 50ng/ml Regular Insulin, 50ng/ml Transferrins,iron complexes, 50pg/ml Sodium Selenite in DMEM-F12 substratum).Carry out immunostaining results analysis, as shown in Figure 5 and Figure 6, cell expressing GATA-4, P450C17, StAR, 3 β-HSD, SF-1, LHR after CD51 positive human interstitial tissue[of testis] differentiation of stem cells; Testosterone ELISA detected result finds, a large amount of testosterone of CD51+hSLCs secreted in vitro after differentiation, shows that CD51+hSLCs can be divided into ripe interstitial glands under specific differentiation condition.
Embodiment 3 is observed CD51 positive human interstitial tissue[of testis] stem cell and is acted in tissue repair in vivo
Stem cell is an important attribute to the regenerative power of damaged tissue in body.Previous research shows that processing 4 days with thecytotoxinethanedimethanesulfonate (EDS) may exhaust interstitial glands.Therefore, set up the rat model (EDS model) of interstitial glands disappearance, whether investigation CD51 positive human interstitial tissue[of testis] stem cell can promote that interstitial glands recovers in EDS model.Select two groups of adult rats, be respectively model group, groups of cells.Model group (EDS (+)/saline (+)) rats by intraperitoneal injection EDS, and intratesticular injection 20 μ l physiological saline (the one-sided testis of 10 μ l/) after 4 days.In the rat abdominal cavity of groups of cells (EDS (+)/Cell (+)), injection EDS (75mg/kg body weight) is after 4 days, CD51 positive human interstitial tissue[of testis] stem cell (1.5x10 embodiment 1 obtained
6be resuspended in the one-sided testis of 20 μ lPBS/) be transplanted in rat testicle.Serum testosterone concentrations is measured when the 10th day after transplanting.Result as shown in Figure 7, transplants the level that CD51+hSLCs can improve serum testosterone.
Claims (10)
1. a separation method for Human Testis interstital stem cell, is characterized in that comprising the following steps:
(1) by the IV Collagenase Type digestion of Human Testis Tissue biopsy samples, filter postdigestive cell suspension, obtain Human Testis cell suspension;
(2) with the Human Testis cell that fluorescently-labeled CD51 antibody labeling step (1) obtains, utilize flow cytometer to carry out sorting, collect the cell of expressing CD51, thus isolate the Human Testis interstital stem cell of the CD51 positive.
2. separation method according to claim 1, wherein " collecting the cell of expressing CD51 " described in step (2) is collect the cell that fluorescence intensity is 10 times of the fluorescence intensity of Human Testis cell negative control or more.
3. be separated according to the separation method of claim 1 or 2 the Human Testis interstital stem cell obtained, it is characterized in that, described Human Testis interstital stem cell is the CD51 positive.
4. Human Testis interstital stem cell according to claim 3, is characterized in that: described Human Testis interstital stem cell PDGF-B expression R-α, P75, LIFR, SSEA-4, but does not express 3 β-HSD, LHR.
5. cultivate a method for the Human Testis interstital stem cell of claim 3 or 4, it is characterized in that: the composition of the substratum used for add 1nM dexamethasone, 2ng/mlLIF, 50ng/ml Regular Insulin, 50ng/ml Transferrins,iron complexes, 50pg/ml Sodium Selenite, 5% (V/V) chick embryo extract, 1% (V/V) non-essential amino acid, 2% (V/V) B27 serum-free additive, 20ng/mlbFGF, 20ng/mlEGF, 20ng/mlPDGFBB in DMEM-F12 substratum.
6. method according to claim 5, wherein said substratum is serum free medium.
7. an external method by Human Testis interstital stem cell directed differentiation behaviour interstitial glands, it is characterized in that: the Human Testis interstital stem cell of claim 3 or 4 is broken up in interstitial glands differentiation culture liquid, described interstitial glands differentiation culture liquid for add 2% (V/V) FCS, 1nMT3,1ng/mlLH, 70ng/mlIGF-I, 10ngPDGF-BB and 50ng/ml Regular Insulin, 50ng/ml Transferrins,iron complexes, 50pg/ml Sodium Selenite in DMEM-F12 substratum.
8. the Human Testis interstital stem cell of claim 3 or 4, the method for claim 5 or 6 cultivate the application of Human Testis interstital stem cell in the medicine of preparation raising testosterone levels obtained.
9. the Human Testis interstital stem cell of claim 3 or 4, the method for claim 5 or 6 cultivate the application of Human Testis interstital stem cell in the medicine preparing the relative disease that treatment testosterone levels lowly causes obtained.
10. the application of claim 8 or 9, wherein said testosterone levels is level of serum testosterone.
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| 张柳平等: "成年小鼠睾丸间质细胞的分离、鉴定及功能检测", 《动物医学进展》 * |
| 脱颖等: "Nestin-GFP转基因小鼠睾丸中P75NTR表达的研究", 《中华男科学杂质》 * |
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| WO2017088829A1 (en) * | 2015-11-27 | 2017-06-01 | 中山大学 | Methods for separating and culturing human testicular mesenchymal stem cells, and use of human testicular mesenchymal stem cells |
| CN106754684A (en) * | 2017-01-05 | 2017-05-31 | 厚朴生物科技（苏州）有限公司 | A kind of interstitial tissue[of testis] stem cell is separately cultured differentiation method |
| CN108588004A (en) * | 2018-04-09 | 2018-09-28 | 深圳市莱利赛生物科技有限公司 | The method of induction differentiation interstitial glands and its application in sexual function recovery |
| CN111394302A (en) * | 2020-02-24 | 2020-07-10 | 中山大学 | Method for separating and culturing human testicular interstitial stem cells |
| WO2021169041A1 (en) * | 2020-02-24 | 2021-09-02 | 中山大学 | Separation and culture method of human testicular mesenchymal stem cells |
| CN112094884A (en) * | 2020-08-07 | 2020-12-18 | 温州医科大学附属第二医院、温州医科大学附属育英儿童医院 | Method for detecting inhibition of differentiation of mesenchymal stem cells to Leydig cells by phthalate |
| CN114181885A (en) * | 2021-10-11 | 2022-03-15 | 温州医科大学附属第二医院（温州医科大学附属育英儿童医院） | Method for testing influence of midazolam on in-vitro cultured testicular interstitial cell development |
| CN114181885B (en) * | 2021-10-11 | 2022-09-20 | 温州医科大学附属第二医院（温州医科大学附属育英儿童医院） | Method for testing influence of midazolam on in-vitro cultured testicular interstitial cell development |
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