CN101638633B - Method for in-vitro separation and culture of goat male germ stem cells - Google Patents
Method for in-vitro separation and culture of goat male germ stem cells Download PDFInfo
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Abstract
The invention discloses a method for in-vitro separation and culture of goat male germ stem cells. Spermatogonia are obtained from the testicular convolutedtubules of a male goat. A method for separating and purifying the goat male germ stem cells adopts 0.2% of gelatin and a Matrigel differential adherence combined cloning method to differentiate and separate un-adhered goat male germ stem cells from other cells for adherence culture; and the culture of the separated goat male germ stem cells is MEF feeder-layer culture of feeder-layer-free culture. The separated goat male germ stem cells have ES cell characteristics and the potential of differentiation into nerve cells, cardiac myocytes and sperm sample cells.
Description
Technical field
The invention belongs to biological technical field, relate to in-vitro multiplication, differentiation, the separation of stem cell animal, particularly a kind of in-vitro separation of goat male germ stem cells and cultural method.
Background technology
The research of the generation of sexual cell, propagation, differentiation is one of important topic of life science.In the animal reproduction breeding, high yield, the fine genetic resources sexual cell that places one's entire reliance upon is accomplished heredity; The mankind, because the infertility that the generation of spermatid, dysmaturity etc. cause perplexs numerous patients' a realistic problem especially; In fundamental research, sperm is one of the most important research object of developmental biology research especially; Therefore the generation of sexual cell, the research of differentiation have great importance in livestock industry production and biomedical research.Yet the generation of higher mammal sexual cell, transfer, differentiation are accomplished fully in vivo; Therefore be difficult to dynamic real-time ground and detect and study molecular events (Hua etc. wherein; Recentadvances in the derivation of germ cells from the embryonic stem cells.StemCells and Development; 2008,17:399-411).
Stem cell is one type and has self, the highly propagation and the cell colony of multidirectional differentiation potential.Embryonic stem cell (ES cell or ESCs) is to be separated and next multipotential stem cell by body early embryo inner cell mass (ICM) or archeocyte (PGCs).Because unique biological characteristics of embryonic stem cell; Make stem cell at cell replacement treatment, histoorgan reparation and reconstruction, gene therapy and developmental biology model, new drug development and toxicological experiment etc., nearly all life science and biomedicine field all have great importance.
In recent years, some famous researchs (Sesndel etc., Generation of functional multipotentadult stem cells from GPR125
+Germline progenitors.Nature; 2007,449:346-350; Bukovsky etc.; Generation of pluripotent stem cells from adult humantestis.Nature; 456,344-349) find from embryonic stage the animal sex-ridge archeocyte or male testis can obtain being similar to the male germ stem cells (mGSCs) of the versatility differentiation potential of ES cell.It is generally acknowledged; These male germ stem cells (mGSCs) derive from archeocyte (primordial germ cells, PGCs), in the testis after the being present in property differentiation throughout one's life; In the spermatogeny process, has the cell general name of self duplication and differentiation potential after the differentiation of comprising property; Be different from stem spermatogonium (SSCs), mGSCs can be divided into all cell types of body, and stem spermatogonium refers to undifferentiated sperma-togonium A, it is generally acknowledged that it has only the potential (Ko etc., 2009) to the sperm differentiation.But also the someone think present germline stem cell origin and unclear (Yu etc., Pluripotent stem celllines.Genes Dev, 2008,22:1987-1997).
Because the spermatogonium of adult animals testis is under external appropriate condition; Possibly be converted into the male germ stem cells (Matsui etc. of versatility; Derivation of pluripotential embryonic stemcells from murine primordial germ cells in culture.Cell; 1992,70:841-847; Dong Wuzi etc., cultivation and the detection of new born bovine male sex-cell source class ES, journal of animal science and veterinary medicine, 2007,38 (2): 144-148; Guan etc., Pluripotency of spermatogonial stem cellsfrom adult mouse testis.Nature, 2006,440:1199-1203; Sesndel etc., Generationof functional multipotent adult stem cells from GPR125
+Germline progenitors.Nature; 2007,449:346-350; Kanatsu-Shinohara etc., Pluripotency of a singlespermatogonial stem cell in mice.Biol Reprod., 2008,78 (4): 681-687; Kanatsu etc., Generation of pluripotent stem cells from neonatal mouse testis.Cell, 2004,119:1001-1012; Bukovsky etc., Generation of pluripotent stem cells from adulthuman testis.Nature, 456,344-349; Kanatsu-Shinohara etc., Pluripotency of asingle spermatogonial stem cell in mice.Biol Reprod., 2008,78 (4): 681-687).So mGSCs becomes the ideal carrier cell of transgenic animal and cloned animal, and compared with normal ES cell, mGSCs cell source quantity is many, cultivates relatively easy.But study for sexual cell; What is more important mGSCs can break up effectively and produces sperm (Ko etc.; Induction ofpluripotency in adult unipotent germline stem cells.Cell Stem Cell, 2009,5:87-96).
MGSCs has obtained some progress on nematode, fruit bat and mouse isotype animal; As set up the culture system of stem spermatogonium; Mechanism (the Ko etc. that the various animal male germ stem cells of basic understanding are kept; Induction of pluripotency in adult unipotent germline stem cells.Cell StemCell, 2009,5:87-96).But germline stem cell to big domestic animal such as ox, sheep, pig etc.; It is still not enough that people understand; Rarely seen several pieces of research reports relate to the separation and Culture of mGSCs cell, but do not see the stdn of germline stem cell isolation cultivation method is arranged (Dong Wuzi etc., goat fetus testicular cell in-vitro separation is cultivated and the growth behavior of arrenotoky lineage stem cells. Journal of Agricultural Biotechnology; 2004,12 (6): 648-652.; Dong Wuzi etc., the separation and Culture and the versatility research of tire ox male germ stem cells source class ES cell. biotechnology journal, 2007,23 (4): 751-755; Dong Wuzi etc., cultivation and the detection of new born bovine male sex-cell source class ES. journal of animal science and veterinary medicine, 2007,38 (2): 144-148).
Goat is a kind of important economic animal, produces economic products such as meat, suede, milk, is particularly utilizing goat mammary gland aspect bio-reactor production biological products, to have huge market potential.How to obtain, keep fine blastogenesis resource, existing population is improved, become the pressing issues of goat large-scale farming, the research of goat sexual cell is become the important means of solution blastogenesis resource.
Summary of the invention
The problem of solution of the present invention is to provide a kind of in-vitro separation and cultural method of goat male germ stem cells; Testis in-vitro separation clone male germ stem cells from male goat; And the goat male germ stem cells isolated culture to being separated to, for the sexual cell of studying goat lays the foundation.
The present invention realizes through following technical scheme:
A kind of extracorporeal separation method of goat male germ stem cells may further comprise the steps:
1) isolated cell
To from the goat testis of aseptic collection, shred to fragment by isolating convoluted seminiferous tubule,, obtain lower floor's convoluted seminiferous tubule cell mass after leaving standstill with resuspended 2～3 the convoluted seminiferous tubule fragments of DMEM nutrient solution that comprise volume(tric)fraction 10%FBS; Then with the mixed solution digestion process lower floor convoluted seminiferous tubule cell mass 2～3 times of the Collagenase, 10 μ g/ml DNase and massfraction 0.1% Unidasa that comprise massfraction 0.1%; Each 20～30 minutes; DMEM nutrient solution with comprising volume(tric)fraction 10%FBS is resuspended, separates to obtain spermatogonium;
2) male germ stem cells former is commissioned to train foster
With the spermatogonium that obtains with 5 * 10
5The density of individual/mL is seeded in the 37 ℃ of plastic culture dish that encapsulate 30min of gelatin through massfraction 0.2%, and nutrient solution is a goat male germ stem cells separation and Culture liquid;
Said goat male germ stem cells separation and Culture liquid is basic medium with the DMEM high glucose medium, also comprises following component: the Basic Fibroblast Growth Factor of the foetal calf serum of volume(tric)fraction 20%, 2mmol/L L-glutaminate, 1mmol/L Sodium.alpha.-ketopropionate, 0.1mmol/L beta-mercaptoethanol, 0.1mmol/L non-essential amino acid, 5～10ng/ml, the LIF of 1000U/ml, 100IU/ml penicillium mould and 100mg/mL Streptomycin sulphate;
Forward not adherent cell to encapsulated 1 hour through 4 ℃ of Matrigel plastic culture dish behind the cultivation 12h and cultivate, nutrient solution is that goat male germ stem cells separation and Culture liquid or human embryo stem cell serum-free do not have the feeder layer nutrient solution;
After not adherent cell shifts for the first time and cultivates 2h; The plastic culture dish that encapsulated 1 hour through 4 ℃ of Matrigel is cultivated in the parietal cell transfer of not pasting once more after the transfer cultivation cultivated, nutrient solution is that goat male germ stem cells separation and Culture liquid or human embryo stem cell serum-free do not have the feeder layer nutrient solution;
After attached cell does not shift cultivation for the second time, nutrient solution of replacing in 2 days; The cell growth began to occur fine and close embryonic stem cell appearance colony after 3～7 days;
3) cultivation of going down to posterity of male germ stem cells
When treating that fine and close embryonic stem cell appearance colony grows to periphery and begins noble cells to occur, with the sucking-off of fine and close embryonic stem cell appearance colony, washing is 2 times in PBS; Move on to again in the trypsinase of TrypLE cell dissociation buffer or massfraction 0.05% and digest 2～3min, and pressure-vaccum 5～20 times; The discrete unicellular or small cell cluster of opening is transferred to the petridish cultivation that encapsulates 1 hour plastics through 4 ℃ of Matrigel, and nutrient solution is a goat male germ stem cells separation and Culture liquid; 37 ℃, 5%CO
2Cultivate under the saturated humidity condition;
Cultivate after 3～7 days, begin to occur fine and close embryonic stem cell appearance colony, be goat male germ stem cells.
Further, the invention also discloses the extracorporeal culturing method that separates the goat male germ stem cells that obtains:
Goat male germ stem cells is inoculated on the MEF feeder layer; Nutrient solution is a basic medium with the DMEM high glucose medium, also comprises following component: the ITS of 1% non-essential amino acid of the blood serum substituting article of the foetal calf serum of volume(tric)fraction 10%, volume(tric)fraction 10%, 2mM Stimulina, 0.1mM beta-mercaptoethanol, 100IU/ml penicillium mould, 100mg/mL Streptomycin sulphate, massfraction, 1000IU/ml LIF, 5～10ng/ml Basic Fibroblast Growth Factor, massfraction 1%; Changed a nutrient solution in 2 days;
Perhaps goat male germ stem cells being inoculated into plastic culture dish that 4 ℃ of Matrigel encapsulated 1 hour does not have feeder layer and cultivates, and nutrient solution does not have the feeder layer nutrient solution for the human embryo stem cell serum-free, changes a nutrient solution in 2 days.
Compared with prior art, the present invention has following beneficial technical effects:
1, the goat male germ stem cells of separating clone forms typical fine and close embryonic stem cell appearance colony; Has the ES cell characteristics; Its SEAP (AP) is positive, and OCT4, SSEA1, SSEA 3, SSEA 4, β 1-INTEGRIN, NANOG, CD49f, CD133, C-MYC, SOX2, KLF4 also are positive;
2, the goat male germ stem cells of separating clone has tridermic differentiation potential, can be differentiated to form neurocyte, myocardial cell and sperm like cell; Especially the differentiation potential that has a sperm like cell has great importance for acquisition, maintenance, the improvement of goat high-quality germ plasm resource;
3, the goat male germ stem cells of separation and Culture is bigger, and cell is bright, opacifying property is strong, nuclear is big, cultivates the back and forms typical fine and close cell clone, similar ES cell clone in 3～7 days.
Description of drawings
Fig. 1 is the biological characteristics and dyeing detected result of goat male germ stem cells; Wherein, figure A is single goat male germ stem cells, and bigger, cell is bright, opacifying property is strong, nuclear is big; Figure B, C are the goat male germ stem cells colony, densification; Figure D, E are that alkaline phosphatase staining shows that goat male germ stem cells takes on a red color, and are the AP positive; Figure F is the Gimesa dyeing of goat male germ stem cells colony, and nuclear is big; Bar=100 μ m.
Fig. 2 is the immunofluorescence chemical detection result of goat male germ stem cells surface markers; Wherein, figure A is expressed as the OCT4 positive; Figure B is expressed as the SSEA1 positive; It is weak positive that figure C is expressed as SSEA 3; Figure D is expressed as SSEA 4 positives; It is positive that figure E is expressed as β 1-INTEGRIN; Figure F is expressed as the NANOG positive; Figure G is expressed as the CD49f positive; Figure H is expressed as the CD133 positive; It is weak positive that figure I is expressed as VASA; Figure J representes that C-MYC is positive, figure K representes that SOX2 is positive, figure L representes that KLF4 is positive; Bar=20 μ m.
Fig. 3 is that the differentiation potential of goat male germ stem cells detects, wherein, and the embryoid (EB) that figure A forms for the goat male germ stem cells suspension culture; Figure B is that EB adherent culture rear perimeter edge differentiates neural like cell; Figure C is that goat male germ stem cells differentiates neural like cell; Figure D is the myotube spline structure that the cardiac-like muscle cell that differentiates after the EB adherent culture merges; Figure E is that the cardiac-like muscle cell that differentiates after the EB adherent culture is myocardium α-actin positive; Figure F is that the cardiac-like muscle cell that differentiates after the EB adherent culture is the myocardium specificity TnT positive; Figure G is that the fat-like cell that differentiates after the EB adherent culture is the oil red O positive; Figure H is the sperm like cell that differentiates after the EB adherent culture; Figure I is that the sperm like cell that differentiates after the EB adherent culture is the Analine Blue positive; Figure J is that the cell that goat male germ stem cells differentiates is the VASA positive; Figure K is that the cell that goat male germ stem cells differentiates is the FE-J1 positive; Figure L is that the cell that goat male germ stem cells differentiates is the EMA1 positive.
Fig. 4 does not present more typical fine and close embryonic stem cell appearance colony for there being the goat male germ stem cells of raising layer vitro culture.
Embodiment
The present invention provides a kind of culture system of high efficiency separation clone goat male germ stem cells, comprises the separation of goat male germ stem cells, the detection and the vitro culture of goat male germ stem cells.
With 2 monthly ages above tire sheep, newborn sheep or adult Testis Caprae seu Ovis originate as starting materials; The separation purification method of goat male germ stem cells adopts 0.2% gelatin and the adherent combination PCR cloning PCR of Matrigel differential, with other cell differentiation of not adherent goat male germ stem cells and adherent culture, separate.
The extracorporeal separation method of a, goat male germ stem cells specifically may further comprise the steps:
1) separates goat testicle cells
With the aseptic saline water that contains two anti-(500IU/ml penicillium mould, 500mg/mL Streptomycin sulphates) with the goat testis washing of aseptic collection 3 times; Again with containing two anti-PBS (phosphate buffered saline buffer) washings 3 times; In sterile petri dish, peel off epiorchium; Separate convoluted seminiferous tubule and testis beam, remove girder, obtain convoluted seminiferous tubule;
Convoluted seminiferous tubule is shredded the fine grained chippings to 1mm3; DMEM (Dulbecco ' s Modified Eagle Media) nutrient solution with comprising volume(tric)fraction 10%FBS resuspended (centrifugal below 500 commentaries on classics) convoluted seminiferous tubule fragment; Repeat 2～3 times; After leaving standstill 2～5 minutes, obtain lower floor's convoluted seminiferous tubule cell mass;
Then with the mixed solution digestion process 2～3 times of the Collagenase, 10 μ g/ml DNase and massfraction 0.1% Unidasa that comprise massfraction 0.1%; Each 20～30 minutes; Use the resuspended nutrient solution of DMEM+10%FBS (centrifugal below 500 commentaries on classics) again, separate obtaining spermatogonium;
If cell mass is still bigger, then using massfraction is 0.25% trypsin treatment 3～5 minutes, resuspended with the DMEM nutrient solution that comprises volume(tric)fraction 10%FBS, cell counting;
2) male germ stem cells former is commissioned to train foster
With the spermatogonium that obtains with 5 * 10
5It is 37 ℃ of plastic culture dish that encapsulate 30min of gelatin of 0.2% that the density of individual/mL is seeded in through massfraction, adds goat male germ stem cells separation and Culture liquid;
Said goat male germ stem cells separation and Culture liquid is basic medium with the DMEM high glucose medium; Also comprise following component: the foetal calf serum of volume(tric)fraction 20%, 2mmol/L L-glutaminate, 1mmol/L Sodium.alpha.-ketopropionate, 0.1mmol/L beta-mercaptoethanol, 0.1mmol/L non-essential amino acid (NEAA, American I nvitrogen Company products), the Basic Fibroblast Growth Factor (bFGF) of 5～10ng/ml, the LIF (mLIF) of 1000U/ml, 100IU/ml penicillium mould, 100mg/mL Streptomycin sulphate;
Forwarding not adherent cell to encapsulated 1 hour through 4 ℃ of Matrigel matrix membranes (BD company) plastic culture dish behind the cultivation 12h cultivates; Nutrient solution is that goat male germ stem cells separation and Culture liquid or human embryo stem cell serum-free do not have feeder layer nutrient solution (mTeSR nutrient solution; Commercial hESC's nutrient solution, Stemcell technology company produces);
After not adherent cell shifts for the first time and cultivates 2h; Not adherent once more cell transfer after the transfer cultivation is cultivated the petridish that encapsulated 1 hour through 4 ℃ of Matrigel matrix membranes cultivate, nutrient solution is that goat male germ stem cells separation and Culture liquid or human embryo stem cell serum-free do not have the feeder layer nutrient solution;
After attached cell does not shift cultivation for the second time, nutrient solution of replacing in 2 days; The cell growth began to occur more typical fine and close embryonic stem cell appearance colony after 5～7 days;
3) cultivation of going down to posterity of male germ stem cells
Treat that fine and close embryonic stem cell appearance colony grows to periphery and begins to occur noble cells, it is big that promptly peripheral cell becomes, and when boundary was obvious, with the sucking-off of fine and close embryonic stem cell appearance colony, washing was 2 times in 100 μ l PBS; Move on to 100 μ l TrypLE cell dissociation buffer (commercial cell dissociation buffer, the TrypLE of Invitrogen company again
TMExpress) or in the trypsinase of massfraction 0.05% digest 2～3min, and with 5～20 discrete cells of 10 μ l liquid-transfering gun pressure-vaccums; The discrete unicellular or small cell cluster of opening is moved on to the plastic culture dish cultivation that encapsulates 1 hour through Matrigel4 ℃, and nutrient solution is a goat male germ stem cells separation and Culture liquid; 37 ℃, 5%CO
2Cultivate under the saturated humidity condition;
The cell growth began more typical fine and close embryonic stem cell appearance colony to occur after 3～7 days, was and separated the goat male germ stem cells that obtains.
The cell detection of b, the goat male germ stem cells that is separated to
1) SEAP detects
The goat male germ stem cells of the fine and close embryonic stem cell appearance colony that the culture of isolated that goes down to posterity is arrived with the fixing 3～5min of 4% Paraformaldehyde 96 after; PBS with no calcium magnesium washes twice; With SEAP dye liquor (in the firm red Tris-C1 solution that is dissolved in 10ml PH 8.2～8.4 of 2mg naphthyl alcohol phosphoric acid salt and 10mg) dyeing 8min; Sucking-off staining fluid, PBS are washed 2 times, photographic recording;
The goat male germ stem cells of separating clone has the biological characteristics of ES cell characteristics and germline stem cell, forms typical colony, shown in Figure 1B～F; The alkaline phosphatase staining of goat male germ stem cells takes on a red color, and shown in figure D～E, is SEAP (AP) positive, promptly has characteristic (Hua etc., 2009 of multipotent stem cells; Ko etc., 2009).
2) immunohistochemical methods or immunofluorescence detect
The goat male germ stem cells of cultivating with the fixing 15min of 4% Paraformaldehyde 96, is washed twice with PBS, 3%H
2O
2Incubated at room 10min, PBS wash twice, 10% foetal calf serum (PBS dilution) sealing; Incubated at room 30min, the serum deprivation that inclines drips OCT4 (1: 500), SSEA1 (1: 200), SSEA3 (1: 200), SSEA4 (1: 200), β 1-INTEGRIN (1: 200), NANOG (1: 200), CD49f (1: 200), CD133 (1: 500), VASA (1: 1000), C-MYC (1: 200), SOX2 (1: 200), KLF4 (1: a 200) anti-working fluid respectively; 4 ℃ are spent the night, and PBS washes twice, drip two anti-(goat-anti rabbit FITC or sheep anti mouse FITC; 1: 500) working fluid, incubated at room 1h, PBS wash twice; Each 5min, observation photographic recording result under the fluorescent microscope;
One anti-sign of above-mentioned immunofluorescence is the mark of ES cell and germline stem cell; Detected result is as shown in Figure 2 respectively; OCT4 (A), SSEA1 (B), SSEA4 (D), β 1-INTEGRIN (E), NANOG (F), CD49f (G), CD133 (H), C-MYC (J), SOX2 (K), KLF4 (L) tests positive; SSEA3 (C), VASA (I) detect weak positive, show that goat male germ stem cells has germline stem cell and ES cell characteristics (Hua etc., 2009; Ko etc., 2009).
3) cells in vitro differentiation potential
Cultivate making embryoid (EB, Hua etc., 2009) with the droplet hanging drop of 600 cells/25 μ l, stereoscopic microscope checks that down embryoid forms situation after 3 days, and the embryoid (EB) that the goat male germ stem cells suspension culture forms is shown in Fig. 3 A; Collect EB; (substratum is for removing the goat male germ stem cells parting liquid of bFGF and LIF on the 48 empty culture plates that 0.1% gelatin attaches in cultivation; Be basic medium promptly with the DMEM high glucose medium; Add foetal calf serum, 2mmol/LL-Stimulina, 1mmol/L Sodium.alpha.-ketopropionate, 0.1mmol/L beta-mercaptoethanol, 0.1mmol/L non-essential amino acid, 100IU/ml penicillium mould and the 100mg/mL Streptomycin sulphate of volume(tric)fraction 20%), the cell type that 1～3 weekly check EB comes off and;
The goat male germ stem cells of separating clone has tridermic differentiation potential; Can be differentiated to form EB (shown in Fig. 3 A), neurocyte (shown in Fig. 3 B, the C), myocardial cell (shown in Fig. 3 D, E, the F), fat-like cell (shown in Fig. 3 G, oil red O is positive) and sperm like cell (shown in Fig. 3 H, the I); Fig. 3 J, K, L are respectively noble cells and are sperm and sexual cell specific marker VASA (Fig. 3 J), FE-J1 (Fig. 3 K) and EMA1 (Fig. 3 L), and the goat male germ stem cells that shows separating clone has and is divided into spermaioid potential (Toyooka etc., 2003; Hua etc., 2009), this screening study for goat high-quality germ plasm resource is even more important.
C, for the goat male germ stem cells that separation obtains, its external isolated culture method is:
1) there is feeder layer to cultivate
It (is the MEC feeder layer, with the white MEC warp of conceived 12-16 days elder brother 10 μ g/mL MTCs processing 2-3 hour, with 50000/cm that goat male germ stem cells is inoculated on the MEF feeder layer
2Density shop be attached on the plastic culture plate that 0.1% gelatin is handled); Nutrient solution is a basic medium with the DMEM high glucose medium; Also comprise following component: the blood serum substituting article (KSR of the foetal calf serum of volume(tric)fraction 10% (FBS), volume(tric)fraction 10%; Invitrogen), 2mM Stimulina, 0.1mM beta-mercaptoethanol, 100IU/ml penicillium mould, 100mg/mL Streptomycin sulphate, 1% non-essential amino acid (NEAA), 1000IU/ml LIF (LIF), 5～10ng/ml Basic Fibroblast Growth Factor (bFGF), 1%ITS (Regular Insulin+Transferrins,iron complexes+Sodium Selenite, Sigma company);
Cultivate and changed a nutrient solution in 2 days, the cell growth is after 3～7 days, and the more typical fine and close germline stem cell colony that begins to occur is shown in Figure 1B.
2) no feeder layer is cultivated
Perhaps goat male germ stem cells being inoculated into 4 ℃ of plastic culture dish that encapsulated 1 hour of Matrigel (BD company) does not have the feeder layer cultivation, and nutrient solution does not have feeder layer nutrient solution (mTeSR nutrient solution, Stem cells company) for the human embryo stem cell serum-free;
Cultivate and changed a nutrient solution in 2 days; The cell growth is after 3～7 days; The more typical fine and close germline stem cell colony that begins to occur; The goat male germ stem cells colony immunostaining that the goat male germ stem cells colony that no feeder layer shown in Fig. 4 A is cultivated, Fig. 4 B do not have the feeder layer cultivation is the AP positive.
Claims (1)
1. the extracorporeal separation method of a goat male germ stem cells is characterized in that, may further comprise the steps:
1) isolated cell
To from the goat testis of aseptic collection, shred to fragment by isolating convoluted seminiferous tubule,, obtain lower floor's convoluted seminiferous tubule cell mass after leaving standstill with resuspended 2～3 the convoluted seminiferous tubule fragments of DMEM nutrient solution that comprise volume(tric)fraction 10%FBS; Then with the mixed solution digestion process lower floor convoluted seminiferous tubule cell mass 2～3 times of the Collagenase, 10 μ g/ml DNase and massfraction 0.1% Unidasa that comprise massfraction 0.1%; Each 20～30 minutes; DMEM nutrient solution with comprising volume(tric)fraction 10%FBS is resuspended, separates to obtain spermatogonium;
2) male germ stem cells former is commissioned to train foster
With the spermatogonium that obtains with 5 * 10
5The density of individual/mL is seeded in the 37 ℃ of plastic culture dish that encapsulate 30min of gelatin through massfraction 0.2%, and nutrient solution is a goat male germ stem cells separation and Culture liquid;
Said goat male germ stem cells separation and Culture liquid is basic medium with the DMEM high glucose medium, also comprises following component: the Basic Fibroblast Growth Factor of the foetal calf serum of volume(tric)fraction 20%, 2mmol/L L-glutaminate, 1mmol/L Sodium.alpha.-ketopropionate, 0.1mmol/L beta-mercaptoethanol, 0.1mmol/L non-essential amino acid, 5～10ng/ml, the LIF of 1000U/ml, 100IU/ml penicillium mould and 100mg/mL Streptomycin sulphate;
Forward not adherent cell to encapsulated 1 hour through 4 ℃ of Matrigel matrix membranes plastic culture dish behind the cultivation 12h and cultivate, nutrient solution is that goat male germ stem cells separation and Culture liquid or human embryo stem cell serum-free do not have the feeder layer nutrient solution;
After not adherent cell shifts for the first time and cultivates 2h; Not adherent once more cell transfer after the transfer cultivation is cultivated the plastic culture dish that encapsulated 1 hour through 4 ℃ of Matrigel matrix membranes cultivate, nutrient solution is that goat male germ stem cells separation and Culture liquid or human embryo stem cell serum-free do not have the feeder layer nutrient solution;
After attached cell does not shift cultivation for the second time, nutrient solution of replacing in 2 days; The cell growth began to occur fine and close embryonic stem cell appearance colony after 3～7 days;
3) cultivation of going down to posterity of male germ stem cells
When treating that fine and close embryonic stem cell appearance colony grows to periphery and begins noble cells to occur, with the sucking-off of fine and close embryonic stem cell appearance colony, washing is 2 times in PBS; Move on to again in the trypsinase of TrypLE cell dissociation buffer or massfraction 0.05% and digest 2～3min, and pressure-vaccum 5～20 times; The discrete unicellular or small cell cluster of opening is transferred to the plastic culture dish that encapsulated 1 hour through 4 ℃ of Matrigel matrix membranes cultivate, nutrient solution is a goat male germ stem cells separation and Culture liquid; 37 ℃, 5%CO
2Cultivate under the saturated humidity condition;
Cultivate after 3～7 days, begin to occur fine and close embryonic stem cell appearance colony, be goat male germ stem cells.
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| CN106754724A (en) * | 2016-12-09 | 2017-05-31 | 西北农林科技大学 | A kind of ox stem spermatogonium system of immortalization and its construction method |
| CN107201343B (en) * | 2017-07-14 | 2021-05-11 | 阜阳师范学院 | Goat cell clone embryo culture solution and culture method |
| CN108795850B (en) * | 2018-06-26 | 2021-08-31 | 西北农林科技大学 | Long-term culture method for spermatogonial stem cells without feed layer in vitro |
| CN111304155B (en) * | 2019-11-25 | 2021-08-20 | 内蒙古自治区农牧业科学院 | Separation culture method and culture solution for pluripotent stem cells in free state in sheep tissue |
| CN111024660A (en) * | 2019-11-29 | 2020-04-17 | 南阳师范学院 | Method for rapidly identifying nucleated sperms and spermatogonial cells in spermary of silkworms |
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