CN109182269A - A kind of cultivating system and method making the efficient differentiating into nerve cells of stem spermatogonium - Google Patents
A kind of cultivating system and method making the efficient differentiating into nerve cells of stem spermatogonium Download PDFInfo
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Abstract
The invention belongs to cell biologies, are related to the external high-efficient culture method of stem spermatogonium (SSCs) differentiating into nerve cells.The present invention handles culture vessel by poly-D-lysine, increases the adherent ability of SSCs, and under the conditions of adding neural factor B27 and N2, and removal growth factor, SSCs can efficient differentiating into nerve cells as the result is shown.The method that the present invention establishes is suitble to procedure operation, repeatability height, success rate high, easy to operate, effect can be reached without the condition of becoming privileged, time and test material can be greatly saved, mostly carry out nerve cell induction using multipotential stem cell in the past, there are the no thoroughnesses of induction.Present invention application SSCs, comparison embryonic stem cell (ESCs) show that SSCs differentiating into nerve cells high efficiency and completeness, this result of study show that SSCs inducing effect obviously has advantage.It is likely to become a kind of more optimized source of human stem cell for being applied to and repairing neural tissue injury, becomes a new technical solution.
Description
Technical field
The invention belongs to RESEARCH ON CELL-BIOLOGY technical fields, are related to the method for stem spermatogonium differentiating into nerve cells,
Neural cellular differentiation and neural tissue injury's reparation be can be applied in the research of clinical medicine application aspect.
Background technique
Only rodent stem spermatogonium (spermatogonial stem cells, SSCs) is obtained in vitro at present
Long-term cultivation, people and other species stem cells are still unable to long-term cultivation, so mouse SSCs becomes general cell model and uses.
Stem spermatogonium and neural stem cell (neural stem cells, NSCs) have very there is close connection
Cells and characteristic of stem as multiphase.Firstly, the neurotrophic factor (GDNF) of glia cell line-derived is that SSCs maintains self-renewing most
Crucial signal factor, and it is brain derived neurotrophic factor (BDNF) that NSCs, which maintains the important factor of in-vitro multiplication,；Secondly, research
It has also been found that substrate source cell factor (CXCL12) is all played an important role in SSCs and NSCs self-renewing regulation；Again,
Epidermal growth factor (EGF) and Basic Fibroblast Growth Factor (bFGF) can also promote two class stem cells hyperplasias；In addition, nerve is thin
Born of the same parents' additive (B27) and N2 are proliferated NSCs and break up regulation and play an important role, and then cultivate in SSCs in vitro
Usually two class additives of addition, this maintains self-renewing also for SSCs in vitro has important facilitation.Two class stem cell exhibitions
Revealing many similar biological properties, display SSCs is likely to be a kind of better cell origin of neural cellular differentiation, but
Previously mostly broken up using versatile stem cell and mescenchymal stem cell inducing nerve cell, the cell origin as neural restoration.
NSCs has the ability for being divided into neuronal cell, astroglia and oligodendroglia, is nervous system
The potential cell of disease treatment.But the regulatory mechanism research of NSCs differentiation is not also thorough.
In the past researches show that differentiating into nerve cells is induced multi-potent stem cell, need to add two kinds of neural factor B27 and N2
Reagent is induced, but there is the problems such as low induction differentiation efficiency and no thoroughness；It is external that mouse SSCs is promoted to be proliferated in
In cultivating system, two kinds of reagents of B27 and N2 can be added, play the role of important promotion for mouse SSCs proliferation；It is therefore seen that
SSCs culture system in vitro and the system of nerve cell induction are very close, and it is ideal that SSCs is likely to become substitution NSCs
Adult stem cell.But there is no establish efficiently induction method of the spermatogonium to Neural Differentiation for previous research.
Summary of the invention
The purpose of the present invention is to provide a kind of cultivating system, which can make stem spermatogonium (SSCs) efficient
Differentiating into nerve cells.
The present invention also provides the methods of the efficient differentiating into nerve cells of stem spermatogonium (SSCs).
The purpose of the present invention is realized by following technological means:
On the one hand, the present invention provides a kind of cultivating system for making SSCs differentiating into nerve cells, the cultivating system is without feeding
Layer, no growth factor are supported, and contains L-Glutamine, neural factor B27 and N2.
Present invention firstly discovers that SSCs in specific cultivating system, can be divided into nerve cell, this technology may
It is applied in clinic and removes to solve the problems, such as some neurogenic diseases, SSCs is likely to become the new seed of neurogenic disease treatment
Cell.
In the prior art, the culture of SSCs has generally been built upon on the basis of feeder layer and has promoted its in-vitro multiplication.But
The research of the invention finds that without can preferably promote SSCs to Neural Differentiation under the conditions of feeder layer, under the conditions of there are feeder layer,
SSCs differentiating into nerve cells is not found.
In addition, existing SSCs cultivating system, typically contains growth factor, to promote SSCs to be proliferated, and for the present invention
For, the presence of growth factor will affect SSCs differentiating into nerve cells, therefore, no growth factor and necessary.
Further, in the cultivating system, the volume fraction in system point of L-Glutamine, B27 and N2
It Wei 0.8~1.2%, 1~3%, 0.8~1.2%.Implement embodiment as preferred, L-Glutamine, B27 and N2
Volume fraction in system is respectively 1%, 2%, 1%.
As preferred embodiment, in cultivating system of the invention, contain following components:
Embodiment more preferably in the cultivating system, contains following components:
In the present invention, the B27 is a kind of nutritional blend of neural class cell, contains 20 many kinds of substance, to NSCs
Play the role of promoting proliferation with SSCs.
The N2 is putrescine, progesterone, transferrins, the mixture of insulin and selenite.
The Stro-34 culture medium is candidate stem cell special culture media, can support SSCs in-vitro multiplication.
Above-mentioned substance can be commercially available, what ingredient determined.
On the other hand, the present invention also provides a kind of method for making stem spermatogonium differentiating into nerve cells, this method packets
It includes following steps: comprising the steps of:
(1) culture vessel is handled using poly-D-lysine；
(2) it is inoculated with stem spermatogonium (SSCs)；
(3) it cultivates.
Specifically, in step (1), handle culture dish using poly-D-lysine: the poly of configuration 0.01~0.05% relies ammonia
Acid is added to culture vessel, it is desirable that did not had culture vessel, and was put into incubator, and after 12h, discarded poly-D-lysine liquid, PBS
Phosphate buffer liquid gently washs 2-3 times, is put into incubator, cell to be accessed.
In step (2), it is inoculated with stem spermatogonium: by cultured stem spermatogonium single cell suspension, cell density 0.8
~1.2 × 105A/mL is linked into the culture vessel handled well.
In step (3), cell culture is carried out using the Neuronal induction media of above-mentioned preparation, condition of culture is 5%CO2With
37 ± 1 DEG C, liquid is changed using half amount after 22~26h, continues culture 5~7 days, it can the nerve for obtaining germline stem cell differentiation is thin
Born of the same parents.
As preferred embodiment, in step (1), culture vessel is handled using 0.01% poly-D-lysine.
As preferred embodiment, cultivating system and method, the stem spermatogonium being applicable in the present invention are mouse
Stem spermatogonium is readily applicable to the stem spermatogonium of other species certainly, and (such as people and pig stem spermatogonium) essence is former
Stem cell is a kind of conservative stem cell, according to previous the study found that stem spermatogonium gene expression and life between different plant species
Object is in nature without essential distinction, therefore this method is readily applicable to the research of other species stem spermatogonium inducing neurals differentiation
Using can especially attempt to be applied in the stem spermatogonium of the mankind, another height can be provided for neurotrosis patient in this way
Imitate stem cell recovery scenario.
Beneficial effects of the present invention:
1, the inventors found that under the conditions of there are feeder layer, SSCs differentiating into nerve cells is not found, is induced
Neural cellular differentiation system by experimental exploring and continuously improves scheme in conjunction with SSCs and NSCs biological property, final to establish
A kind of culture solution without feeder layer culture of efficient differentiating into nerve cells of inducing mouse SSCs is played, the culture solution is only in SSCs
Growth factor is eliminated on the basis of cultivating system, be added to N2 reagent (reagent main effect in the culture solution is to promote more
Forward to neural cellular differentiation)；The efficient differentiating into nerve cells quickly and thoroughly of mouse SSCs, this explanation under this culture solution
SSCs may be that the nerve cell of substitution NSCs preferably repairs substitution cell；
2, the method for inducing mouse SSCs differentiating into nerve cells that the present invention establishes, needs the time short, at about one week
Between typical neuronal cell and spongiocyte can occur, the method for the present invention is suitble to that procedure operation, repeatability be high, success rate
It is high, easy to operate, effect can be reached without the condition of becoming privileged, can greatly save time and test material, comparison is built in the past
The method of vertical stem cell inducing nerve cell has a clear superiority；
3, method of the invention has potential in neural cellular differentiation and acquisition and the nervous system disease reparation etc.
Application value, the method for the present invention, which can be applied in clinic, goes to solve the problems, such as that some neurogenic diseases, SSCs are likely to become
The new seed cell of neurogenic disease treatment.
Detailed description of the invention
Fig. 1 is the result Technology Roadmap of mouse SSCs differentiating into nerve cells.
Fig. 2 is mouse SSCs differentiating into nerve cells process；Wherein, A: the mouse SSCs without feeder layer culture；B, C and D:
Mouse SSCs induces the 3rd, 4 and 5d to nerve cell；E: inducing nerve cell 6d there is nerve synapse；F and G: induction the
There is a large amount of typical nerve cells and neuron in 6d and 7d；H: obviously breaking up does not occur in mouse SSCs induction under feeder layer culture,
Impassivity cell generates.
Fig. 3 is mouse ESCs (mouse embryo stem cell) to neural induction process；Wherein A: newly it is inoculated with ESCs；B,C,
D, E and F: being respectively that inducing neural breaks up the 2nd, 3,4,5 and 6 day；G:NESTIN immunofluorescence dyeing；H: the ESCs without induction.
In this incubation, there is Neural Differentiation in ESCs, but each period, there are still ESCs residual, differentiation is not exclusively, overall
Efficiency is lower than SSCs.
Fig. 4 is that mouse SSCs Protein Detection and atomization are analyzed；Wherein, A, B and C: birth 7d age testis is sliced VASA
Dyeing, respectively dye core, protein staining and Merge result；D, E and F: mouse SSCs VASA protein immunization fluorescent staining；Respectively
To contaminate core, protein staining and Merge result；G, H and I: the 4th, 5 and 6d of mouse SSCs inducing nerve cell expresses memebrane protein
The analysis of VASA cell proportion.
Fig. 5 is the nerve cell immunofluorescence dyeing figure of differentiation；Wherein, A and B: mouse SSCs inducing nerve cell
NESTIN, TUJ1 Immunofluorescence test；C: control group.
The nerve cell gene expression RT-PCR testing result of Fig. 6 mouse SSCs induction.
Specific embodiment
It is further discussed below the present invention in the following with reference to the drawings and specific embodiments, the present invention does not address place and is suitable for existing skill
Art.It is given below specific embodiments of the present invention, but embodiment is not intended to limit this merely to this explanation is described in further detail
The claim of invention.Reagent used in following embodiment or raw material derive from commercially available unless otherwise specified.
Raw material sources:
A kind of cultivating system for inducing SSCs differentiating into nerve cells of embodiment 1
The culture solution of the induction stem spermatogonium differentiating into nerve cells of the present embodiment, is calculated with percent by volume, including
Following components:
A kind of cultivating system for inducing SSCs differentiating into nerve cells of embodiment 2
The culture solution of the induction stem spermatogonium differentiating into nerve cells of the present embodiment, is calculated with percent by volume, including
Following components:
A kind of cultivating system for inducing SSCs differentiating into nerve cells of embodiment 3
The culture solution of the induction stem spermatogonium differentiating into nerve cells of the present embodiment, is calculated with percent by volume, including
Following components:
The process of embodiment 4SSCs differentiating into nerve cells
Induce SSCs differentiating into nerve cells
In step (1), handle culture dish using poly-D-lysine: the poly-D-lysine for configuring 0.01% is added to incubator
Ware, it is desirable that do not had culture vessel, and be put into incubator, and after 12h, discarded poly-D-lysine liquid, PBS phosphate buffer liquid is light
Fine laundering is washed 2 times, and incubator, cell to be accessed are put into.
In step (2), Mice Inoculated stem spermatogonium: by cultured spermatogonial stem cells into mouse single cell suspension, cell
Density is 0.8~1.2 × 105A/mL is linked into the culture vessel handled well.
In step (3), the Neuronal induction media that Application Example 1 is prepared carries out cell culture, and condition of culture is 5%
CO2With 37 ± 1 DEG C, liquid is changed using half amount after 22~26h, continues culture 5~7 days, it can obtains stem spermatogonium differentiation
Nerve cell.
In the condition of no MEF feeder layer, mouse SSCs can maintain in-vitro multiplication (such as Fig. 2A) for a long time；But in its training
Growth factor is removed in nutrient solution such as after (such as GDNF, EGF, bFGF and LIF etc.), SSCs can rapidly and efficiently to nerve cell
Differentiation, when culture is to 3d, the decline of cell refractivity starts to deform (such as Fig. 2 B)；4d is cultivated, mouse SSCs stretches out pseudopodium,
Stem cell clone colony growth be not presented, adherent performance also enhances (such as Fig. 2 C)；It is basic to cultivate the circular SSCs form of 5d
It disappears, is all into the cell (such as Fig. 2 D) of threadiness；Cultivate 6d and can see many similar neurons and spongiocyte
Morphological cellular (such as Fig. 2 E)；Cultivate to 7d be clear that typical neuron and a large amount of cynapses occur (such as Fig. 2 F and
2G)；Show that cultivating system of the invention can promote mouse SSCs differentiating into nerve cells.Show that cultivating system of the invention can
To promote mouse SSCs differentiating into nerve cells.
The control group feeder layer culture, cultivating system are identical with test group.Cellular control unit culture on feeder layer, and
Do not find that apparent neural class cell generates, but mouse SSCs stops growing, quantity reduces (such as Fig. 2 H).
The process that comparative example 1ESCs (embryonic stem cell) is induced to nerve cell
Experimental method: please supplement specific experimental method, and such as certain steps are same as Example 4, can write " cultivating system
With cultural method with embodiment 1, except SSCs replaces with ESCs.
Experimental result: as shown in figure 3, wherein Fig. 3 A is new inoculation ESCs；Fig. 3 B, C, D, E and F are respectively inducing neural point
Change the 2nd, 3,4,5 and 6 day；Fig. 3 G is NESTIN immunofluorescence dyeing；Fig. 3 H is the ESCs without induction.From the figure 3, it may be seen that at this
In incubation, there is Neural Differentiation in ESCs, but each period, there are still ESCs residual, not exclusively, overall efficiency is low for differentiation
In SSCs.Although ESCs is multipotential stem cell, even using the cultivating system and method in the present invention, differentiation effect
Rate is but not so good as SSCs, while breaking up there are no thoroughness, and multipotential stem cell largely remains.Show cultivating system and side of the invention
Method has better applicability to SSCs.
The detection of embodiment 5SSCs differentiating into nerve cells process
In order to detect mouse SSCs differentiating into nerve cells process, the most important film surface specificity of reproduction cell has been selected
Label VASA is analyzed.By 7 day-old Mice testis tissue immunofluorescence dyeings, show the expression of VASA specificity in production of sperm
Mouse SSCs film surface (such as Fig. 4 A, 4B and 4C) on the inside of tubule, is the marker protein of reproduction cell specificity；To in vitro without feeding
It supports the mouse SSCs cultivated under the conditions of layer and carries out immunofluorescence dyeing, VASA special expression is in mouse SSCs film table as the result is shown
Face (such as Fig. 4 D, 4E and 4F), it was confirmed that the fidelity of VASA albumen.
Experimental method is as follows:
1. prepared by paraffin section
1) draw materials: the 7d age mouse testis tissue that will newly take after removing external adipose and epididymis, cuts along horizontal axis, makes group
Knit block thickness about 0.5mm；It is immersed in 4% paraformaldehyde and fixes for 24 hours；
2) it repairs block: fixing organization being repaired to required size, is made every effort to small and thin；
3) it washes by water: the block-like tissue fixed being put into flowing water and is rinsed for 24 hours, fixer wash clean；
4) it is dehydrated :+30% water of+20% n-butanol of 50% ethyl alcohol, 6h；+ 15% water of+35% n-butanol of 50% ethyl alcohol, 4h；
+ 10% water of+45% n-butanol of 45% ethyl alcohol, 3h；+ 5% water of+55% n-butanol of 40% ethyl alcohol, 3h；The positive fourth of 25% ethyl alcohol+75%
Alcohol, 2h；+ 95% n-butanol of 5% ethyl alcohol, 2h；100% n-butanol (i), 5h；100% n-butanol (ii), 3h；Dimethylbenzene,
10mim；Soft wax (i): 15min；Soft wax (ii): 20min；75% ethyl alcohol 12h, 80% ethyl alcohol 2h, 90% ethyl alcohol 1h, 95% ethyl alcohol
Twice, each 40min, 100% ethyl alcohol twice, each 30min, twice, each 30min.Soft wax 30min, hard wax 30min.
5) it embeds: being embedded into paraffin mass complete tissue sample has been dehydrated；
6) stripping and slicing: the ultra-thin section that 3~5 μm of thickness is taped against on slide after cutting and dries for use；
2 histogenic immunity fluorescence
1) paraffin tissue sections routinely dewax, and enter water through graded ethanol；
2) 6.0 citrate buffer microwave heating antigen retrieval 15min of PH；PBS is washed three times, each 5min；
3) 3%H2O2Close the peroxidase 15min in tissue；PBS is washed three times, each 5min；
4) 1%BSA (10% lowlenthal serum) closing 30min (closed and do not had to wash) is added；
5) the corresponding diluted primary antibody of 1%BSA (source of mouse VASA Abcam-AB13840 is added；Rabbit source NESTIN；
Millipore；Source of mouse TUJ1, Sigma), it is placed in 4 DEG C overnight, PBS is washed 3 times, each 5min；
6) the corresponding diluted secondary antibody of 1%BSA (the anti-mouse secondary antibody Goat Anti-mouse IgG Alexa of green light is added
488, Invitrogen-A11001；The anti-mouse secondary antibody Donkey Anti-mouse IgG Alexa 568, Invitrogen- of feux rouges
A10037；Feux rouges anti-rabbit secondary antibody Goat Anti-rabbit IgG Alexa 568, Invitrogen-A11036), it is placed in 37 DEG C
1h is reacted, PBS is washed 3 times, each 5min；
7) Hochest33342 (Molecular Probes company) of final concentration of 10 μ g/mL is added to nuclear targeting
5~10min, PBS are washed 3 times, each 3min；
8) anti-quencher mounting, takes pictures.
The detection of 3 cellular immunofluorescences
1) without feeder layer culture, (cultivating system and method are shown in embodiment 1, carry out for Mice Inoculated SSCs progress in 24 orifice plates
Cellular immunofluorescence detection；
2) after fixing 10~30min of cell with 4% paraformaldehyde, PBS is washed 3 times, each 3min；
3) 0.5%Triton perforation rupture of membranes is added and handles 10min, PBS is washed 3 times, each 3min；
4) 1%BSA (10% lowlenthal serum) closing 30min (closed and do not had to wash) is added；
5) the corresponding diluted primary antibody of 1%BSA (source of mouse VASA Abcam-AB13840 is added；Rabbit source NESTIN；
Millipore；Source of mouse TUJ1, Sigma), it is placed in 4 DEG C overnight, PBS is washed 3 times, each 5min；
6) the corresponding diluted secondary antibody of 1%BSA (the anti-mouse secondary antibody Goat Anti-mouse IgG Alexa of green light is added
488, Invitrogen-A11001；The anti-mouse secondary antibody Donkey Anti-mouse IgG Alexa 568, Invitrogen- of feux rouges
A10037；Feux rouges anti-rabbit secondary antibody Goat Anti-rabbit IgG Alexa 568, Invitrogen-A11036), it is placed in 37 DEG C
1h is reacted, PBS is washed 3 times, each 5min；
7) Hochest33342 (Molecular Probes company) of final concentration of 10 μ g/mL is added to nuclear targeting
5~10min, PBS are washed 3 times, each 3min；
8) anti-quencher mounting, takes pictures.
The detection of 4 cell purities
1) induction the 4th, 5 and 6d nerve cell are collected into 15mL centrifuge tube, 178g is centrifuged 3min, removes supernatant, and same
Control group is set；
2) after 4% paraformaldehyde of addition fixes 10~30min, 178g is centrifuged 3min, and PBS is washed 3 times；
3) 0.5%Triton-100 perforation rupture of membranes is added and handles 10min, 178g is centrifuged 3min, and PBS is washed 3 times；
4) 1%BSA (10% lowlenthal serum) is added and closes 30min；
5) source of mouse primary antibody VASA (Abcam-AB13840) is added, is placed in 4 DEG C of 30~60min and is incubated for (control group addition mouse
IgG)；It is washed 3 times with PBS afterwards, each 5min；
6) the diluted green light secondary antibody of 1%BSA (Goat Anti-mouse IgG is added in test group and control group simultaneously
Alexa 488, Invitrogen-A11001), 37 DEG C of reaction 1h are placed in, PBS is washed 3 times, each 5min；
7) machine testing on flow cytometer.
Experimental result: by 7 day-old Mice testis tissue immunofluorescence dyeings, show the expression of VASA specificity in production of sperm
Mouse SSCs film surface (such as Fig. 4 A, 4B and 4C) on the inside of tubule, is the marker protein of reproduction cell specificity；To in vitro without feeding
It supports the mouse SSCs cultivated under the conditions of layer and carries out immunofluorescence dyeing, VASA special expression is in mouse SSCs film table as the result is shown
Face (such as Fig. 4 D, 4E and 4F), it was confirmed that the fidelity of VASA albumen；Next to mouse SSCs induction nerve cell the 4th,
5 and 6d carries out cell dyeing, and the ratio of expression VASA cell during this is analyzed by Flow Cytometry, it was demonstrated that essence is former thin
There are ratios by born of the same parents, and as the result is shown after induction, 4,5 and 6d expression VASA cell proportion is followed successively by 77.2%；37.3% He
0.4% (such as Fig. 4 G, 4H and 4I) starts in induction 4d, has a large amount of SSCs to start to break up, after inducing 6d substantially
There is no undifferentiated SSCs, it was demonstrated that the method inducing effect that the invention patent is established is high, thoroughly.
The Immunofluorescence test of 6 inducing nerve cell of embodiment
Experimental method: Immunofluorescence test, Immunofluorescence test are carried out to the nerve cell of the mouse SSCs induction of culture
Method in embodiment 5 the same as recording.
Experimental result: the nerve cell of the mouse SSCs induction of culture carries out Immunofluorescence test, specific neuron
Marker protein nestin NESTIN and tubulin TUJ1 are detected, and are deposited in the nerve cell of mouse SSCs induction as the result is shown
In the positive cell (such as Fig. 5 A) and TUJ1 positive cell (such as Fig. 5 B) of expression NESTIN, negative control group is negative (as schemed
5C), this illustrates that mouse SSCs is successfully divided into nerve cell.
7 inducing nerve cell gene expression analysis of embodiment
To determine that the cell of SSCs induction is nerve cell, neuronal marker gene (Nestin, Map- are had detected herein
2 and Neurotensin), wherein also expressing oligodendroglia and star spongiocyte marker gene Galc and Gfap.
The extraction (the micro extraction agent box of Qiagen) of 1 cell total rna
1) 80 μ L of the lysate (lysate: containing 10 μ L sulfydryls in the Buffer RLT of 1mL newly prepared is added in micro-example
Ethyl alcohol uses preceding preparation)；
2) ethyl alcohol of 80 μ L 70% is added, is mixed, is not centrifuged with pipette tips；
3) sample is transferred in the Spin column provided in kit, assembles 2mL collecting pipe, gently closes the lid,
8000g is centrifuged 15s, discards and penetrates liquid, puts back to collecting pipe；
4) 350 μ L buffer RW1 are added, gently close the lid, 8000g is centrifuged 15s, and abandoning penetrates liquid, puts back to collecting pipe；
5) 80 μ L DNase I are directly dripped on siliceous film, is stored at room temperature 15min, directly must all dripped, otherwise
DNA can digest not exclusively；
6) 350 μ L buffer RW1,8000g are added and are centrifuged 15s, abandoning penetrates liquid and collecting pipe；
7) 500 μ L buffer RPE (joined dehydrated alcohol as required) are added, gently in the 2mL collecting pipe more renewed
It closes the lid, 8000g is centrifuged 15s, and abandoning penetrates liquid；
8) 500 μ L buffer RPE are added, gently close the lid, 8000g is centrifuged 2min, completely removes ethyl alcohol, abandoning penetrates
Liquid and collecting pipe avoid contact to when removing Spin column and penetrate liquid, and ethyl alcohol will affect recycling；
9) Spin column is moved to new 2mL collecting pipe, and uncaps and is high speed centrifugation 2min, abandoning penetrates liquid and collection
Pipe.The 1.5mL collecting pipe more renewed, directly 20~40 μ L RNase-free water of dropwise addition are in center membrane, maximum speed centrifugation
2min.Yield can once be increased by repeating elution, and -80 DEG C save or synthesize immediately cDNA.
The measurement of 2 total rna concentrations
2% agarose gel electrophoresis runs whether RNA sample detection RNA degrades, and NANODROP 2000 detects the concentration of RNA
And purity, Pureness control are to calculate sample rna between 1.8~2.0 according to RNA densimeter and be adjusted to 1 μ g's in OD260/OD280
Volume.
3 synthesis cDNA
Reverse transcription detection 1st strand cDNA synthesis uses two step method, step 1: reaction system: sample rna
1 μ g, Random 6mers primer, 11 μ L of μ L, dNTP, distilled water trim to 10 μ L, reaction condition are as follows: 65 DEG C of 5min, ice
Upper chilling；Step 2: reaction system: by 10 μ L, 5 × PrimeScript TM Buffer of reaction solution after first step denaturation, annealing
4 μ L, Rnase Inhibitor (40U/ μ L) 0.5 μ L, PrimeScript TM Rnase (200U/ul) 1 μ L, 4.5 μ of distilled water
L, 20 μ L of total system, reaction condition: 30 DEG C of 10min, 42 DEG C of 60min, 70 DEG C of 15min, 4 DEG C of 1h.
4 RT-PCR
20 μ L PCR systems: 2 × PrimeStar Buffer/Premix, 10 μ L；0.2 μ L of upstream primer F；Downstream primer
R 0.2μL；Template 1 μ L and ddH2O 8.6μL；Response procedures are as follows: 95 DEG C of 3min of initial denaturation；95℃ 30s,60℃ 30s,72
DEG C 40s recycles 35；72 DEG C of 7min of last overall elongation.The primer the following table 1 in system.
1 PCR detection primer table of table
Experimental result: RT-PCR as the result is shown (see Fig. 6),
RT-PCR the results show that inducing nerve cell expression neuronal marker gene (Nestin, Map-2 and
Neurotensin), wherein also expressing oligodendroglia and star spongiocyte marker gene Galc and Gfap, it was demonstrated that small
Mouse SSCs is successfully induced and is produced nerve cell.
Each technical characteristic of embodiment described above can be combined arbitrarily, for simplicity of description, not to above-mentioned reality
It applies all possible combination of each technical characteristic in example to be all described, as long as however, the combination of these technical characteristics is not deposited
In contradiction, all should be considered as described in this specification.
The embodiments described above only express several embodiments of the present invention, and the description thereof is more specific and detailed, but simultaneously
It cannot therefore be construed as limiting the scope of the patent.It should be pointed out that coming for those of ordinary skill in the art
It says, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to protection of the invention
Range.Therefore, the scope of protection of the patent of the invention shall be subject to the appended claims.
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Claims (10)
1. a kind of cultivating system for making stem spermatogonium differentiating into nerve cells, which is characterized in that the cultivating system is without feeding
Layer, no growth factor are supported, and contains L-Glutamine, B27 and N2.
2. cultivating system according to claim 1, percent by volume of the N2 in the system is 0.8~1.2%.
3. cultivating system according to claim 2, percent by volume of the N2 in the system is 1%.
4. cultivating system according to claim 1, percent by volume of the B27 in the system is 1~3%.
5. cultivating system according to claim 4, percent by volume of the B27 in the system is 2%.
6. cultivating system according to claim 1, which is characterized in that contain component below:
7. according to cultivating system as claimed in claim 6, which is characterized in that contain component below:
8. a kind of method for making stem spermatogonium differentiating into nerve cells, which is characterized in that comprise the steps of:
(1) handle culture dish using poly-D-lysine: the poly-D-lysine of configuration quality score 0.01~0.05% is added to training
Support vessel, it is desirable that do not had culture vessel, and be put into incubator, and after 10~12h, discarded poly-D-lysine liquid, PBS phosphoric acid is slow
Fliud flushing is gently washed 2~3 times, and incubator, cell to be accessed are put into；
(2) it cultivates: cultured stem spermatogonium individually inoculation stem spermatogonium: being had into cell suspension, cell density 0.8
~1.2 × 105A/mL is linked into the culture vessel that step (1) is handled well；
(3) application cultivating system as claimed in claim 1 to 7 carries out cell culture, and condition of culture is 5%CO2With 37 ± 1
DEG C, liquid is changed using half amount after 22~26h, continues culture 5~7 days, it can obtains the nerve cell of stem spermatogonium differentiation.
9. method according to claim 8, which is characterized in that
In step (1), culture dish is handled using 0.01% poly-D-lysine.
10.-the 9 any cultivating system or method according to claim 1, which is characterized in that the stem spermatogonium is
Spermatogonial stem cells into mouse.
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