CN106754684A - A kind of interstitial tissue[of testis] stem cell is separately cultured differentiation method - Google Patents
A kind of interstitial tissue[of testis] stem cell is separately cultured differentiation method Download PDFInfo
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- 230000004069 differentiation Effects 0.000 title claims abstract description 48
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- 239000012588 trypsin Substances 0.000 claims description 6
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- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
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Abstract
Differentiation method is separately cultured the invention discloses a kind of interstitial tissue[of testis] stem cell, is comprised the following steps：Step 1, the testis tissue to obtaining is separated, cultivated, expanded in vitro；Step 2, P4 is cultivated for cell, forms embryoid；Step 3, inoculation differentiation is carried out by embryoid.Research confirms that the present invention can effectively obtain interstitial tissue[of testis] stem cell, and gained stem cell has the representative configuration of interstital stem cell；Express the significant notation of interstital stem cell such as：CD29, CD166 etc.；Expression stem cell labeling is such as：OCT4, NANOG etc.；EB can be formed, can be to cell differentiations such as fat, skeletonization after induction.Confirm that the present invention can effectively obtain interstitial tissue[of testis] stem cell, and carry out induction differentiation.
Description
Technical field
The present invention relates to biological technical field, more particularly to a kind of interstitial tissue[of testis] stem cell is separately cultured differentiation method.
Background technology
Current stem cell correlation technique and achievement are continued to bring out, and a vigorous research is being experienced in stem-cell research
Upsurge.All achieved including the various stem-cell researches including embryonic stem cell, inductive pluripotent stem cells and adult stem cell
Huge progress.Adult stem cell causes it in clinical practice because of its low immunogenicity and the differentiation similarity higher with body cell
In obtain more pro-gazes.In the visual field that bone marrow interstital stem cell was put into researcher before more than 20 years, adipose stromal
This year such as stem cell also achieve great successes and considerable progress.Substantial amounts of stem cell is contained in animal testis, has removed
Outside male germ stem cells, interstitial tissue[of testis] stem cell is also particularly important available stem cell resource.However, in testis
The separation of interstital stem cell, culture, differentiation method still lack.
The content of the invention
In order to solve the above technical problems, being separately cultured differentiation method the invention provides a kind of interstitial tissue[of testis] stem cell, adopt
With by carrying out to testis tissue being separately cultured differentiation, separation, culture, the differentiation method of the interstital stem cell in testis are solved
Vacancy.
To reach above-mentioned purpose, technical scheme is as follows：A kind of interstitial tissue[of testis] stem cell is separately cultured differentiation side
Method, comprises the following steps：
Step 1, the testis tissue to obtaining is separated, cultivated, expanded in vitro；
Step 2, P4 is cultivated for cell, forms embryoid；
Step 3, inoculation differentiation is carried out by embryoid.
As a further improvement on the present invention, the method that the testis tissue that the step 1 pair is obtained in vitro separate is：
The testis tissue of acquirement is digested with trypsase and DNase, then is 10%FBS with isometric DMEM/F12 and concentration
Solution terminates enzymic digestion, and 300g*5min centrifugations, abandon supernatant at room temperature；It is 10%FBS solution with 5mL DMEM/F12 and concentration
Re-suspended cell, room temperature 300g*5min centrifugations, abandons supernatant.
As a further improvement on the present invention, the method that the testis tissue that the step 1 pair is obtained in vitro separate is：
Testis tissue 2mg/mL clostridiopetidase As, 20 μ g/mL DNase and 2mg/mL decomposition the enzymic digestions 20min, Zhi Houyong that will be obtained
1mg/mL trypsase and 10 μ g/mL DNase digestion 10min, then add 10%FBS solution to terminate with isometric DMEM/F12
Enzymic digestion, 300g*5min centrifugations, abandon supernatant at room temperature；Add 10%FBS solution re-suspended cells, room temperature with 5mL DMEM/F12
300g*5min is centrifuged, and abandons supernatant.
As a further improvement on the present invention, the method testis tissue for obtaining cultivated, expanded in vitro in step 1
For：Add 10%FBS re-suspended cells with 5mL DMEM/F12, adjust cell density, according to 100cells/60mm ware inoculating cells,
Cultivated with DMEM/F12 plus 10%FBS, interstital stem cell is taken after 6d, be re-seeded on 24 new orifice plates, carry out culture amplification.
As a further improvement on the present invention, step 2, by the good P4 of cultivation conditions in step 1 for cell through 0.25%
After Trypsin Induced, 300g*5min centrifugations, resuspended with PBS, 300g*5min is centrifuged；With DMEM/F12 plus
10%FBS re-suspended cells, blood cell counting plate is counted, and adjusts cell density, is made the droplet of 800~1000cells/25uL,
Culture 3 days is inverted, embryoid is formed.
As a further improvement on the present invention, step 3, the embryoid that will be prepared in step 2 is inoculated in 48 orifice plates, often
1 embryoid is inoculated with hole, embryoid Spontaneous Differentiation 7d is made with DMEM/F12 plus 20%FBS, triploblastica differentiation effect is detected afterwards
Really.
As a further improvement on the present invention, immunofluorescence dyeing is carried out to the cell cultivated in step 1：With 4%
PFA room temperatures fix 15 minutes, and then PBS is washed three times；Penetrating 10 minutes with 0.2%Triton X-100, PBS is washed three times；With
1%BSA room temperatures are closed 30 minutes, and primary antibody 4 spends night, and PBS is washed three times；Secondary antibody room temperature 1 hour, PBS is washed three times；Use 5ug/mL
Hoechst33342 room temperatures dye core 5min, PBS are washed three times, film recording under direct fluorescence.
As a further improvement on the present invention, Osteoinductive differentiation is carried out to the embryoid formed in step 2 or fat is lured
Lead.
By above-mentioned technical proposal, the beneficial effect of technical solution of the present invention is：Research confirms that the present invention can be obtained effectively
Interstitial tissue[of testis] stem cell is obtained, gained stem cell has the representative configuration of interstital stem cell；Express the significant notation of interstital stem cell
Such as：CD29, CD166 etc.；Expression stem cell labeling is such as：OCT4, NANOG etc.；EB can be formed, can be to fat, skeletonization etc. after induction
Cell differentiation.Confirm that the present invention can effectively obtain interstitial tissue[of testis] stem cell, and carry out induction differentiation.The inventive method simply has
Effect, by being cultivated in DMEM/F12+10%FBS with extremely low density after testis tissue is digested into single cell suspension, effectively
Interstitial tissue[of testis] stem cell is obtained, and the cells such as fat, skeletonization can be induced differentiation into, controllability is strong.
Brief description of the drawings
In order to illustrate more clearly about the embodiment of the present invention or technical scheme of the prior art, below will be to embodiment or existing
The accompanying drawing to be used needed for having technology description is briefly described, it should be apparent that, drawings in the following description are only this
Some embodiments of invention, for those of ordinary skill in the art, on the premise of not paying creative work, can be with
Other accompanying drawings are obtained according to these accompanying drawings.
Fig. 1 is the morphology photo of the interstitial tissue[of testis] stem cell of culture in the step 1 of embodiment 5.
Fig. 2 is the detection picture of the interstitial tissue[of testis] stem cell flow cytometer of culture in the step 1 of embodiment 5.
Fig. 3 is the immunofluorescence dyeing photo of the interstitial tissue[of testis] stem cell of culture in the step 1 of embodiment 5.
Fig. 4 is the induction differentiation photo of embodiment 5.
Specific embodiment
Below in conjunction with the accompanying drawing in the embodiment of the present invention, the technical scheme in the embodiment of the present invention is carried out clear, complete
Site preparation is described, it is clear that described embodiment is only a part of embodiment of the invention, rather than whole embodiments.It is based on
Embodiment in the present invention, it is every other that those of ordinary skill in the art are obtained under the premise of creative work is not made
Embodiment, belongs to the scope of protection of the invention.
Embodiment
A kind of interstitial tissue[of testis] stem cell is separately cultured differentiation method, comprises the following steps：
Step 1, the testis tissue of acquirement is digested with trypsase and DNase, then with isometric DMEM/F12
It is that 10%FBS solution terminates enzymic digestion with concentration, 300g*5min centrifugations, abandon supernatant at room temperature；With 5mL DMEM/F12 and dense
It is 10%FBS solution re-suspended cells to spend, room temperature 300g*5min centrifugations, abandons supernatant, is then cultivated, expanded；
Step 2, P4 is cultivated for cell, forms embryoid；
Step 3, inoculation differentiation is carried out by embryoid.
Embodiment 2
A kind of interstitial tissue[of testis] stem cell is separately cultured differentiation method, comprises the following steps：
Step 1, be by the method that the testis tissue of acquirement in vitro separate：The testis tissue 2mg/mL glue that will be obtained
Protoenzyme, 20 μ g/mL DNase and 2mg/mL decompose enzymic digestion 20min, afterwards with 1mg/mL trypsase and 10 μ g/mL DNase
Digestion 10min, then add 10%FBS solution to terminate enzymic digestion with isometric DMEM/F12,300g*5min centrifugations at room temperature,
Abandon supernatant；Add 10%FBS solution re-suspended cells with 5mL DMEM/F12, room temperature 300g*5min centrifugations are abandoned supernatant, then carried out
Culture, amplification；
Step 2, by the good P4 of cultivation conditions in step 1 for cell through 0.25% Trypsin Induced, 300g*5min from
Resuspended with PBS after the heart, 300g*5min is centrifuged；With DMEM/F12 plus 10%FBS re-suspended cells, blood count
Plate is counted, and adjusts cell density, is made the droplet of 800~1000cells/25uL, is inverted culture 3 days, forms embryoid；
Step 3, inoculation differentiation is carried out by embryoid.
Embodiment 3
A kind of interstitial tissue[of testis] stem cell is separately cultured differentiation method, comprises the following steps：
Step 1, be by the method that the testis tissue of acquirement in vitro separate：The testis tissue 2mg/mL glue that will be obtained
Protoenzyme, 20 μ g/mL DNase and 2mg/mL decompose enzymic digestion 20min, afterwards with 1mg/mL trypsase and 10 μ g/mL DNase
Digestion 10min, then add 10%FBS solution to terminate enzymic digestion with isometric DMEM/F12,300g*5min centrifugations at room temperature,
Abandon supernatant；Add 10%FBS solution re-suspended cells with 5mL DMEM/F12, room temperature 300g*5min centrifugations are abandoned supernatant, then carried out
Culture, amplification；
Step 2, by the good P4 of cultivation conditions in step 1 for cell through 0.25% Trypsin Induced, 300g*5min from
Resuspended with PBS after the heart, 300g*5min is centrifuged；With DMEM/F12 plus 10%FBS re-suspended cells, blood count
Plate is counted, and adjusts cell density, is made the droplet of 800~1000cells/25uL, is inverted culture 3 days, forms embryoid；
Step 3, step 3, the embryoid that will be prepared in step 2 is inoculated in 48 orifice plates, and 1 class embryo is inoculated with every hole
Body, embryoid Spontaneous Differentiation 7d is made with DMEM/F12 plus 20%FBS, and triploblastica differentiation effect is detected afterwards.
Embodiment 4
A kind of interstitial tissue[of testis] stem cell is separately cultured differentiation method, comprises the following steps：
Step 1, be by the method that the testis tissue of acquirement in vitro separate：The testis tissue 2mg/mL glue that will be obtained
Protoenzyme, 20 μ g/mL DNase and 2mg/mL decompose enzymic digestion 20min, afterwards with 1mg/mL trypsase and 10 μ g/mL DNase
Digestion 10min, then add 10%FBS solution to terminate enzymic digestion with isometric DMEM/F12,300g*5min centrifugations at room temperature,
Abandon supernatant；Add 10%FBS solution re-suspended cells with 5mL DMEM/F12, room temperature 300g*5min centrifugations are abandoned supernatant, use 5mL
DMEM/F12 adds 10%FBS re-suspended cells, adjusts cell density, according to 100cells/60mm ware inoculating cells, uses DMEM/F12
Plus 10%FBS cultures, interstital stem cell is taken after 6d, it is re-seeded on 24 new orifice plates, carry out culture amplification；
Step 2, by the good P4 of cultivation conditions in step 1 for cell through 0.25% Trypsin Induced, 300g*5min from
Resuspended with PBS after the heart, 300g*5min is centrifuged；With DMEM/F12 plus 10%FBS re-suspended cells, blood count
Plate is counted, and adjusts cell density, is made the droplet of 800~1000cells/25uL, is inverted culture 3 days, forms embryoid；
Step 3, step 3, the embryoid that will be prepared in step 2 is inoculated in 48 orifice plates, and 1 class embryo is inoculated with every hole
Body, embryoid Spontaneous Differentiation 7d is made with DMEM/F12 plus 20%FBS, and triploblastica differentiation effect is detected afterwards.
Embodiment 5
With reference to Fig. 1 to Fig. 4, a kind of interstitial tissue[of testis] stem cell is separately cultured differentiation method, comprises the following steps：
Step 1, be by the method that the testis tissue of acquirement in vitro separate：The testis tissue 2mg/mL glue that will be obtained
Protoenzyme, 20 μ g/mL DNase and 2mg/mL decompose enzymic digestion 20min, afterwards with 1mg/mL trypsase and 10 μ g/mL DNase
Digestion 10min, then add 10%FBS solution to terminate enzymic digestion with isometric DMEM/F12,300g*5min centrifugations at room temperature,
Abandon supernatant；Add 10%FBS solution re-suspended cells with 5mL DMEM/F12, room temperature 300g*5min centrifugations are abandoned supernatant, use 5mL
DMEM/F12 adds 10%FBS re-suspended cells, adjusts cell density, according to 100cells/60mm ware inoculating cells, uses DMEM/F12
Plus 10%FBS cultures, interstital stem cell is taken after 6d, it is re-seeded on 24 new orifice plates, carry out culture amplification.
Interstitial tissue[of testis] stem cell to step 1 culture carries out the Indexs measures such as morphology, surface markers, differentiation potential, sees
Fig. 1 is examined, is the morphology photo of interstitial tissue[of testis] stem cell.The good P4 of cultivation conditions is disappeared for cell through 0.25% trypsase
Change, after 300g*5min centrifugations, with PBS is resuspended, 300g*5min is centrifuged, 100uL PBS are resuspended, added according to reagent requirement
CD34, CD29, CD45, CD71, CD166, CD44 antibody, after incubation at room temperature 45min, upper machine testing draws Fig. 2 photos.To step
The cell cultivated in rapid 1 carries out immunofluorescence dyeing：15 minutes are fixed with 4%PFA room temperatures, then PBS is washed three times；With
Penetrating 10 minutes of 0.2%Triton X-100, PBS is washed three times；Closed 30 minutes with 1%BSA room temperatures, primary antibody 4 spends night, PBS
Wash three times；Secondary antibody room temperature 1 hour, PBS is washed three times；Core 5min is contaminated with the room temperatures of 5ug/mL Hoechst 33342, PBS is washed three times,
Film recording under direct fluorescence, obtains Fig. 3.With extremely low density after testis tissue is digested into single cell suspension
(50cells/60mm wares) is cultivated in DMEM/F12+10%FBS, typical interstital stem cell clone is selected after 7 days, further
Amplification Culture simultaneously detects whether it has the typical marks and function of interstital stem cell, with reference to Fig. 1 to Fig. 3, it can be seen that pass through
The above method is efficiently separated and has obtained interstitial tissue[of testis] stem cell.
Step 2, by the good P4 of cultivation conditions in step 1 for cell through 0.25% Trypsin Induced, 300g*5min from
Resuspended with PBS after the heart, 300g*5min is centrifuged；With DMEM/F12 plus 10%FBS re-suspended cells, blood count
Plate is counted, and adjusts cell density, is made the droplet of 800~1000cells/25uL, is inverted culture 3 days, forms embryoid；
Step 3, step 3, the embryoid that will be prepared in step 2 is inoculated in 48 orifice plates, and 1 class embryo is inoculated with every hole
Body, embryoid Spontaneous Differentiation 7d is made with DMEM/F12 plus 20%FBS, and triploblastica differentiation effect is detected afterwards.
Induction differentiation is detected：With reference to Fig. 4, osteogenic induction：AP is dyeed：Cell to be dyed is consolidated with 4%PFA room temperatures
Determine 15min, firm red being dissolved in the 0.1M Tris-HCl of 5mL PH8.2-8.6 of 1mg α naphthol phosphoric acid salt+5mg is made dye
Color liquid, dyeing liquor adds room temperature dyeing 15-30min, log in cell.Fat induction：Oil red O stain：Induction is thin
Born of the same parents fix 15 minutes with 4%PFA room temperatures；PBS is washed 2 times；0.3% oil red O stain 20min of Fresh；PBS is washed 2 times；It is micro-
Film recording result under mirror.The embryoid for being formed as seen in Figure 4, can be to cell differentiations such as fat, skeletonization after induction.
By above-mentioned specific embodiment, the beneficial effects of the invention are as follows：Research confirms that the present invention can effectively obtain testis
Interstital stem cell, gained stem cell has the representative configuration of interstital stem cell；Express the significant notation of interstital stem cell such as：
CD29, CD166 etc.；Expression stem cell labeling is such as：OCT4, NANOG etc.；EB can be formed, can be to cells such as fat, skeletonization after induction
Differentiation.Confirm that the present invention can effectively obtain interstitial tissue[of testis] stem cell, and broken up.The inventive method is easy and effective, passes through
Cultivated in DMEM/F12+10%FBS with extremely low density after testis tissue is digested into single cell suspension, effectively obtain testis
Interstital stem cell, and the cells such as fat, skeletonization can be induced differentiation into, controllability is strong.
The foregoing description of the disclosed embodiments, enables professional and technical personnel in the field to realize or uses the present invention.
Various modifications to these embodiments will be apparent for those skilled in the art, as defined herein
General Principle can be realized in other embodiments without departing from the spirit or scope of the present invention.Therefore, the present invention
The embodiments shown herein is not intended to be limited to, and is to fit to and principles disclosed herein and features of novelty phase one
The scope most wide for causing.
Claims (8)
1. a kind of interstitial tissue[of testis] stem cell is separately cultured differentiation method, it is characterised in that comprise the following steps：
Step 1, the testis tissue to obtaining is separated, cultivated, expanded in vitro；
Step 2, P4 is cultivated for cell, forms embryoid；
Step 3, inoculation differentiation is carried out by embryoid.
2. interstitial tissue[of testis] stem cell according to claim 1 is separately cultured differentiation method, it is characterised in that the step 1
It is to the method that the testis tissue for obtaining in vitro separate：The testis tissue of acquirement is disappeared with trypsase and DNase
Change, then be that 10%FBS solution terminates enzymic digestion with isometric DMEM/F12 and concentration, 300g*5min centrifugations, abandon at room temperature
Supernatant；It is 10%FBS solution re-suspended cells with 5mL DMEM/F12 and concentration, supernatant is abandoned in room temperature 300g*5min centrifugations.
3. interstitial tissue[of testis] stem cell according to claim 1 is separately cultured differentiation method, it is characterised in that the step 1
It is to the method that the testis tissue for obtaining in vitro separate：Testis tissue 2mg/mL clostridiopetidase As, the 20 μ g/mL that will be obtained
DNase and 2mg/mL decomposes enzymic digestion 20min, digests 10min with 1mg/mL trypsase and 10 μ g/mL DNase afterwards, then
10%FBS solution is added to terminate enzymic digestion with isometric DMEM/F12,300g*5min centrifugations, abandon supernatant at room temperature；Use 5mL
DMEM/F12 adds 10%FBS solution re-suspended cells, and supernatant is abandoned in room temperature 300g*5min centrifugations.
4. the interstitial tissue[of testis] stem cell according to Claims 2 or 3 is separately cultured differentiation method, it is characterised in that in step 1
Testis tissue to obtaining is cultivated in vitro, the method that expands is：Add 10%FBS re-suspended cells with 5mL DMEM/F12, adjust
Whole cell density, according to 100cells/60mm ware inoculating cells, is cultivated with DMEM/F12 plus 10%FBS, interstitial is taken after 6d dry thin
Born of the same parents, are re-seeded on 24 new orifice plates, carry out culture amplification.
5. interstitial tissue[of testis] stem cell according to claim 4 is separately cultured differentiation method, it is characterised in that step 2, will walk
After the good P4 of cultivation conditions is centrifuged for cell through 0.25% Trypsin Induced, 300g*5min in rapid 1, PBS weight is used
Outstanding, 300g*5min is centrifuged；With DMEM/F12 plus 10%FBS re-suspended cells, blood cell counting plate is counted, and adjustment cell is close
Degree, is made the droplet of 800~1000cells/25uL, is inverted culture 3 days, forms embryoid.
6. interstitial tissue[of testis] stem cell according to claim 5 is separately cultured differentiation method, it is characterised in that step 3, will walk
The embryoid prepared in rapid 2 is inoculated in 48 orifice plates, and 1 embryoid is inoculated with every hole, and class is made with DMEM/F12 plus 20%FBS
Idiosome Spontaneous Differentiation 7d, detects triploblastica differentiation effect afterwards.
7. interstitial tissue[of testis] stem cell according to claim 5 is separately cultured differentiation method, it is characterised in that in step 1
The cell cultivated carries out immunofluorescence dyeing：15 minutes are fixed with 4%PFA room temperatures, then PBS is washed three times；With 0.2%
Penetrating 10 minutes of Triton X-100, PBS is washed three times；Closed 30 minutes with 1%BSA room temperatures, primary antibody 4 spends night, and PBS washes three
Time；Secondary antibody room temperature 1 hour, PBS is washed three times；Core 5min is contaminated with the room temperatures of 5ug/mL Hoechst 33342, PBS washes three times, directly
Film recording under fluorescence.
8. interstitial tissue[of testis] stem cell according to claim 5 is separately cultured differentiation method, it is characterised in that in step 2
The embryoid of formation carries out Osteoinductive differentiation or fat induction.
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