CN109182269B - Culture system and method for efficiently differentiating spermatogonial stem cells to nerve cells - Google Patents
Culture system and method for efficiently differentiating spermatogonial stem cells to nerve cells Download PDFInfo
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Abstract
The invention belongs to the field of cell biology, and relates to an in vitro high-efficiency culture method for differentiating Spermatogonial Stem Cells (SSCs) into nerve cells. According to the invention, the culture vessel is treated by polylysine, the adherence capacity of the SSCs is increased, the nerve factors B27 and N2 are added, and the result shows that the SSCs can be efficiently differentiated to nerve cells under the condition of removing the growth factors. The method established by the invention is suitable for flow operation, has high repeatability, high success rate and simple operation, can achieve the effect without special conditions, can greatly save time and test materials, and has incomplete induction due to the fact that the pluripotent stem cells are applied to nerve cell induction in the past. The invention shows high efficiency and thoroughness of SSCs to neural cell differentiation by applying SSCs compared with Embryonic Stem Cells (ESCs), and the research result shows that the SSCs induction effect has obvious advantages. Can become a more optimized stem cell source applied to repairing nerve tissue damage, and becomes a new technical scheme.
Description
Technical Field
The invention belongs to the technical field of cell biology research, relates to a method for differentiating spermatogonial stem cells into nerve cells, and can be applied to the research of nerve cell differentiation and nerve tissue damage repair in the aspect of clinical medical application.
Background
Only rodent Spermatogonial Stem Cells (SSCs) are currently cultured in vitro for a long period of time, and stem cells from humans and other species are not cultured for a long period of time, so mouse SSCs are used as a universal cell model.
Spermatogonial stem cells are closely related to Neural Stem Cells (NSCs), and have many similar stem cell characteristics. First, glial cell line-derived neurotrophic factor (GDNF) is the most critical signaling factor for the maintenance of self-renewal of SSCs, while the important factor for the maintenance of in vitro proliferation of NSCs is brain-derived neurotrophic factor (BDNF); secondly, the research also finds that the stromal derived cytokine (CXCL12) plays an important role in the self-renewal control of SSCs and NSCs; thirdly, Epidermal Growth Factor (EGF) and basic fibroblast growth factor (bFGF) can also promote the proliferation of two types of stem cells; in addition, nerve cell additives (B27) and N2 play an important role in the regulation of proliferation and differentiation of NSCs, and two types of additives are usually added in the in vitro culture of SSCs, so that the additives also play an important role in promoting the in vitro self-renewal of the SSCs. Both types of stem cells exhibit many similar biological characteristics, indicating that SSCs are likely to be a better cell source for neural cell differentiation, but pluripotent stem cells and mesenchymal stem cells have been used previously to induce neural cell differentiation as a cell source for neural repair.
NSCs have the ability to differentiate into neuronal cells, astrocytes and oligodendrocytes, and are potential cells for the treatment of neurological diseases. However, the mechanism of the NSCs differentiation regulation is not well studied.
Previous research shows that the induction of the differentiation of pluripotent stem cells to nerve cells requires the addition of two reagents, nerve factor B27 and N2, but has the problems of low differentiation induction efficiency, incomplete induction and the like; in a culture system for promoting the proliferation of the mouse SSCs in vitro, two reagents, namely B27 and N2 are added, so that the culture system has an important function of promoting the proliferation of the mouse SSCs; therefore, the SSCs in vitro culture system is very close to the system induced by nerve cells, and the SSCs can possibly become ideal adult stem cells for replacing NSCs. However, previous studies have not established a method for inducing efficient neural differentiation of spermatogonial cells.
Disclosure of Invention
The present invention aims to provide a culture system that can efficiently differentiate Spermatogonial Stem Cells (SSCs) into neural cells.
The invention also provides a method for efficiently differentiating Spermatogonial Stem Cells (SSCs) into nerve cells.
The purpose of the invention is realized by the following technical means:
in one aspect, the present invention provides a culture system for differentiating SSCs into neural cells, the culture system being feeder-free, growth factor-free, and comprising L-glutamine, the neural factors B27, and N2.
The invention discovers for the first time that SSCs can be differentiated into nerve cells in a specific culture system, the technology can be applied to clinic to solve the problems of certain neurological diseases, and the SSCs can possibly become new seed cells for treating the neurological diseases.
In the prior art, the culture of SSCs is generally based on the presence of a feeder layer to promote proliferation thereof in vitro. However, the research of the invention finds that the SSCs can be better promoted to be differentiated towards the nerve under the condition without a feeder layer, and the SSCs are not found to be differentiated towards the nerve cells under the condition with the feeder layer.
Furthermore, while existing SSCs culture systems typically contain growth factors to promote SSCs proliferation, the presence of growth factors affects SSCs differentiation into neural cells, and thus, the absence of growth factors is also essential for the present invention.
Further, in the culture system, the volume fractions of the L-glutamine, the B27 and the N2 in the system are respectively 0.8-1.2%, 1-3% and 0.8-1.2%. As a preferred embodiment, the volume fractions of L-glutamine, B27 and N2 in the system are 1%, 2% and 1%, respectively.
In a preferred embodiment, the culture system of the present invention comprises the following components:
in a more preferred embodiment, the culture system comprises the following components:
in the present invention, B27 is a nutrient mixture for nerve cells, contains more than 20 substances, and has an effect of promoting proliferation of NSCs and SSCs.
The N2 is a mixture of putrescine, progesterone, transferrin, insulin and selenite.
The Stro-34 culture medium is a special culture medium for hematopoietic stem cells and can support the in vitro proliferation of SSCs.
The above substances are all commercially available and their compositions are determined.
In another aspect, the present invention provides a method for differentiating spermatogonial stem cells into neural cells, the method comprising the steps of: comprises the following steps:
(1) treating a culture vessel by adopting polylysine;
(2) seeding with Spermatogonial Stem Cells (SSCs);
(3) and (5) culturing.
Specifically, in step (1), the culture dish is treated with polylysine: and (3) adding 0.01-0.05% polylysine into a culture vessel, placing the culture vessel into an incubator when the culture vessel is required to be submerged, discarding polylysine liquid after more than 12 hours, slightly washing the culture vessel for 2-3 times by PBS phosphoric acid buffer liquid, placing the culture vessel into the incubator, and waiting for cell inoculation.
In the step (2), inoculating spermatogonial stem cells: the cultured spermatogonial stem cell single cell suspension has the cell density of 0.8-1.2 multiplied by 105And (4) inoculating the cells into a treated culture vessel.
In the step (3), the prepared neural induction culture medium is used for cell culture, and the culture condition is 5% CO2And (4) changing the liquid by half after 22-26 h at the temperature of 37 +/-1 ℃, and continuously culturing for 5-7 days to obtain the neural cells differentiated by the germ stem cells.
In a preferred embodiment, in step (1), the culture vessel is treated with 0.01% polylysine.
As a preferred embodiment, the culture system and method of the present invention are applicable to mouse spermatogonial stem cells, and of course, can also be applicable to spermatogonial stem cells of other species (such as human and pig spermatogonial stem cells), which are a kind of conserved stem cells.
The invention has the beneficial effects that:
1. the inventor of the invention finds that under the condition of a feeder layer, SSCs are not differentiated towards nerve cells, a nerve cell differentiation system is induced, and a culture solution for inducing mouse SSCs to be efficiently differentiated towards the nerve cells is finally established by combining the biological properties of the SSCs and NSCs and through experimental exploration and continuous improvement schemes, wherein the culture solution only removes growth factors on the basis of the SSCs culture system and is added with an N2 reagent (the reagent mainly plays a role in promoting differentiation towards the nerve cells); under the culture solution, the mouse SSCs are efficiently, rapidly and thoroughly differentiated to nerve cells, which indicates that the SSCs can be better repairing substitute cells for the nerve cells of NSCs;
2. the method for inducing the mouse SSCs to differentiate into the nerve cells, which is disclosed by the invention, needs short time, can generate typical neuron cells and glial cells within about one week, is suitable for flow operation, high in repeatability, high in success rate, simple to operate, capable of achieving the effect without special conditions, capable of greatly saving time and test materials, and has obvious advantages compared with the conventional method for establishing stem cells to induce the nerve cells;
3. the method has potential application values in the aspects of nerve cell differentiation and acquisition, nervous system disease repair and the like, can be applied to clinic to solve the difficult problems of certain neurological diseases, and the SSCs can possibly become new seed cells for treating the neurological diseases.
Drawings
FIG. 1 is a technical scheme showing the results of mouse SSCs differentiating into neural cells.
FIG. 2 shows the process of mouse SSCs differentiating into neural cells; wherein, A: mouse SSCs cultured without feeder; b, C and D: mouse SSCs induce 3 rd, 4 th and 5d to neural cells; e: induction of neuronal cells at 6d, appearance of neuronal synapses; f and G: induction of 6d and 7d, appearance of a large number of typical nerve cells and neurons; h: no obvious differentiation appears in the induction of the mouse SSCs under the culture of the feeder layer, and no nerve cells are generated.
FIG. 3 shows the process of inducing neural differentiation of mouse ESCs (mouse embryonic stem cells); wherein A: newly inoculating ESCs; B. c, D, E and F: induction of neural differentiation on days 2, 3, 4, 5 and 6, respectively; g: NESTIN immunofluorescent staining; h: no induced ESCs. During the culture process, the ESCs are subjected to neural differentiation, but the ESCs remain at each stage, are incompletely differentiated and have lower overall efficiency than SSCs.
FIG. 4 is a graph of the detection of mouse SSCs protein and the analysis of differentiation process; wherein, A, B and C: VASA staining is carried out on 7 d-old testicular sections at birth, and results are respectively nucleus staining, protein staining and Merge; d, E and F: immunofluorescent staining of mouse SSCs VASA protein; results for staining nuclei, protein staining and Merge, respectively; g, H and I: the ratio of the 4 th, 5 th and 6 th d expression membrane protein VASA cells of the mouse SSCs induced nerve cells is analyzed.
FIG. 5 is a photograph of immunofluorescence staining of differentiated neural cells; wherein, A and B: mouse SSCs induce neural cell NESTIN, TUJ1 immunofluorescence assay; c: and (4) a control group.
FIG. 6 results of RT-PCR detection of mouse SSCs induced neural cell gene expression.
Detailed Description
The invention is further described below with reference to the drawings and specific examples, which are not described for prior art. Specific examples of the present invention are given below, but the examples are only for the purpose of further elaborating the present invention and do not limit the claims of the present invention. The reagents and starting materials used in the following examples were all commercially available unless otherwise specified.
The raw material sources are as follows:
example 1A culture System for inducing differentiation of SSCs into neural cells
The culture solution for inducing spermatogonial stem cells to differentiate into neural cells in the embodiment comprises the following components by volume percentage:
example 2A culture System for inducing differentiation of SSCs into neural cells
The culture solution for inducing spermatogonial stem cells to differentiate into neural cells in the embodiment comprises the following components by volume percentage:
example 3A culture System for inducing differentiation of SSCs into neural cells
The culture solution for inducing spermatogonial stem cells to differentiate into neural cells in the embodiment comprises the following components by volume percent:
example 4 differentiation of SSCs into neural cells
Induction of SSCs differentiation into neural cells
In the step (1), the culture dish is treated by polylysine: and (3) adding 0.01% polylysine into a culture vessel, placing the culture vessel into an incubator when the culture vessel is required to be submerged, discarding polylysine liquid after more than 12 hours, slightly washing the culture vessel for 2 times by PBS phosphoric acid buffer solution, placing the culture vessel into the incubator, and waiting for cell inoculation.
In the step (2), the mouse spermatogonial stem cells are inoculated: the cultured mouse spermatogonial stem cell single cell suspension has the cell density of 0.8-1.2 multiplied by 105And (4) inoculating the cells into a treated culture vessel.
In step (3), the neural induction medium prepared in example 1 was used for cell culture under 5% CO2And (4) changing the liquid by half after 22-26 h at the temperature of 37 +/-1 ℃, and continuously culturing for 5-7 days to obtain the neural cells differentiated by the spermatogonial stem cells.
In MEF-feeder-free conditions, mouse SSCs can sustain long-term proliferation in vitro (see fig. 2A); however, after removing growth factors (such as GDNF, EGF, bFGF and LIF, etc.) in the culture solution, SSCs can be rapidly and efficiently differentiated towards nerve cells, and when cultured to the 3 rd, the refractive index of the cells is reduced and the cells begin to deform (as shown in FIG. 2B); culturing at 4d, the mouse SSCs extend out of the pseudopodia, do not present the growth of stem cell clone colonies, and have enhanced adherence performance (as shown in FIG. 2C); the morphology of the SSCs in the 5D round shape is basically disappeared, and the SSCs are all fibrous cells (as shown in FIG. 2D); many morphological cells resembling neurons and glial cells have been seen in culture 6d (see FIG. 2E); typical neurons and a large number of synapses appeared clearly visible by incubation to 7d (see FIGS. 2F and 2G); the culture system of the invention can promote the differentiation of the mouse SSCs to nerve cells. The culture system of the invention is shown to be capable of promoting the differentiation of the mouse SSCs to the nerve cells.
The control group was cultured using a feeder layer, and the culture system was the same as that of the test group. The control cells were cultured on the feeder layer and no significant neuronal cell production was observed, but the mouse SSCs stopped growing and decreased in number (FIG. 2H).
Comparative example 1 Process of inducing ESCs (embryonic Stem cells) into neural cells
The experimental method comprises the following steps: please supplement the detailed experimental procedures, as some procedures are the same as in example 4, it can be written that "the culture system and culture method are the same as in example 1 except that SSCs are replaced with ESCs.
The experimental results are as follows: as shown in fig. 3, where fig. 3A is a freshly inoculated ESCs; FIGS. 3B, C, D, E and F are days 2, 3, 4, 5 and 6, respectively, of inducing neural differentiation; FIG. 3G is NESTIN immunofluorescent staining; fig. 3H is no induced ESCs. As can be seen from FIG. 3, during this culture process, neural differentiation of ESCs occurred, but ESCs remained at each stage, and differentiation was incomplete, and the overall efficiency was lower than that of SSCs. Although ESCs are pluripotent stem cells, even when the culture system and method of the present invention are used, differentiation efficiency is inferior to that of SSCs, differentiation is not complete, and a large number of pluripotent stem cells remain. The culture system and method of the invention are shown to have better applicability to SSCs.
Example 5 detection of differentiation of SSCs into neural cells
In order to detect the differentiation process of the mouse SSCs to the nerve cells, the most important membrane surface specific marker VASA of germ cells is selected for analysis. By immunofluorescent staining of testis tissues of 7-day-old mice, VASA is shown to be specifically expressed on the membrane surface of SSCs of the mice at the inner side of seminiferous tubules (as shown in figures 4A, 4B and 4C) and is a germ cell specific marker protein; immunofluorescent staining was performed on mouse SSCs cultured in vitro in feeder-free conditions, and the results showed that VASA was specifically expressed on the membrane surface of mouse SSCs (see FIGS. 4D, 4E and 4F), confirming the fidelity of the VASA protein.
The experimental method is as follows:
1. preparation of Paraffin section
1) Material taking: cutting a 7 d-old mouse testis tissue which is newly taken out along a transverse axis after external fat and epididymis are removed, so that the tissue block is about 0.5mm thick; immersing into 4% paraformaldehyde for fixation for 24 h;
2) block repairing: repairing the fixed tissue to a required size, and trying to be small and thin;
3) flushing: placing the repaired blocky tissue into running water for washing for 24 hours, and washing the fixing liquid;
4) and (3) dehydrating: 50% of ethanol, 20% of n-butanol and 30% of water for 6 hours; 50% of ethanol, 35% of n-butanol and 15% of water for 4 hours; 45% of ethanol, 45% of n-butanol and 10% of water for 3 hours; 40% of ethanol, 55% of n-butanol and 5% of water for 3 hours; 25% of ethanol and 75% of n-butanol for 2 hours; 5% ethanol and 95% n-butanol for 2 h; 100% n-butanol (i), 5 h; 100% n-butanol (ii), 3 h; xylene, 10 mim; soft wax (i): 15 min; soft wax (ii): 20 min; 75% ethanol for 12h, 80% ethanol for 2h, 90% ethanol for 1h, 95% ethanol for two times, 40min each time, 100% ethanol for two times, 30min each time, two times, 30min each time. Soft wax for 30min and hard wax for 30 min.
5) Embedding: embedding the completely dehydrated tissue sample into a paraffin block;
6) cutting into blocks: cutting an ultrathin slice with the thickness of 3-5 mu m, spreading the cut ultrathin slice on a glass slide, and baking the cut ultrathin slice for later use;
2 tissue immunofluorescence
1) Dewaxing the paraffin tissue section by a conventional method, and adding water by gradient alcohol;
2) performing microwave heating on the antigen for 15min by using citric acid buffer solution with the pH of 6.0; washing with PBS for 5min three times;
3)3％H2O2sealing peroxidase in the tissue for 15 min; washing with PBS for 5min three times;
4) adding 1% BSA (10% goat serum) to block for 30min (blocking is completed without washing);
5) the corresponding 1% BSA diluted primary antibody (murine VASA Abcam-AB 13840; rabbit derived NESTIN; millipore; murine TUJ1, Sigma), left overnight at 4 ℃, washed 3 times with PBS for 5min each;
6) the corresponding 1% BSA diluted secondary antibody (green Anti-mouse secondary antibody, Goat Anti-mouse IgG Alexa 488, Invitrogen-A11001; red Anti-murine secondary antibody Donkey Anti-mouse IgG Alexa 568, Invitrogen-A10037; red Anti-rabbit secondary antibodies, coat Anti-rabbitIgG Alexa 568 and Invitrogen-A11036), reacting at 37 ℃ for 1h, washing with PBS for 3 times, and washing for 5min each time;
7) adding Hochestt 33342(Molecular Probes company) with the final concentration of 10 mug/mL to stain cell nuclei for 5-10 min, and washing with PBS for 3 times, wherein each time lasts for 3 min;
8) the anti-quencher was mounted and photographed.
3 cellular immunofluorescence assay
1) Mice SSCs were inoculated into 24-well plates for feeder-free culture (culture system and method see example 1, for cellular immunofluorescence assay;
2) fixing the cells with 4% paraformaldehyde for 10-30 min, and washing with PBS for 3 times, 3min each time;
3) adding 0.5% Triton, perforating, breaking membrane, treating for 10min, washing with PBS for 3 times, each for 3 min;
4) adding 1% BSA (10% goat serum) to block for 30min (blocking is completed without washing);
5) the corresponding 1% BSA diluted primary antibody (murine VASA Abcam-AB 13840; rabbit derived NESTIN; millipore; murine TUJ1, Sigma), left overnight at 4 ℃, washed 3 times with PBS for 5min each;
6) the corresponding 1% BSA diluted secondary antibody (green Anti-mouse secondary antibody, Goat Anti-mouse IgG Alexa 488, Invitrogen-A11001; red Anti-murine secondary antibody Donkey Anti-mouse IgG Alexa 568, Invitrogen-A10037; red Anti-rabbit secondary antibodies, coat Anti-rabbitIgG Alexa 568 and Invitrogen-A11036), reacting at 37 ℃ for 1h, washing with PBS for 3 times, and washing for 5min each time;
7) adding Hochest33342(Molecular Probes company) with the final concentration of 10 mug/mL to stain the cell nucleus for 5-10 min, and washing with PBS for 3 times, wherein each time lasts for 3 min;
8) the anti-quencher was mounted and photographed.
4 cell purity assay
1) Collecting induced 4 th, 5 th and 6 th nerve cells into a 15mL centrifuge tube, centrifuging for 3min at 178g, removing supernatant, and setting a control group in the same way;
2) adding 4% paraformaldehyde for fixation for 10-30 min, centrifuging for 3min at 178g, and washing for 3 times with PBS;
3) adding 0.5% Triton-100, perforating, breaking membrane, treating for 10min, centrifuging for 3min at 178g, and washing with PBS for 3 times;
4) adding 1% BSA (10% goat serum) and blocking for 30 min;
5) adding mouse-derived primary anti-VASA (Abcam-AB13840), and incubating at 4 deg.C for 30-60 min (adding mouse IgG to control group); washing with PBS for 5min for 3 times;
6) the test group and the control group are added with a green secondary antibody (Goat Anti-mouse IgG Alexa 488, Invitrogen-A11001) diluted by 1% BSA at the same time, placed at 37 ℃ for reaction for 1h, washed 3 times by PBS, and each time for 5 min;
7) and (4) performing detection on a flow cytometer.
The experimental results are as follows: by immunofluorescent staining of testis tissues of 7-day-old mice, VASA is shown to be specifically expressed on the membrane surface of SSCs of the mice at the inner side of seminiferous tubules (as shown in figures 4A, 4B and 4C) and is a germ cell specific marker protein; immunofluorescent staining was performed on the mouse SSCs cultured in vitro under the feeder-free condition, and the results showed that VASA was specifically expressed on the membrane surface of the mouse SSCs (see FIGS. 4D, 4E and 4F), confirming the fidelity of the VASA protein; then, cell staining is carried out on 4 th, 5 th and 6 th cells of mouse SSCs induced nerve cells, the proportion of VASA expressing cells in the process is analyzed through a flow cytometry technology, the existence proportion of spermatogonia is proved, and the results show that the proportion of the VASA expressing cells of 4 th, 5 th and 6 th cells after induction is 77.2% in sequence; 37.3% and 0.4% (as shown in FIGS. 4G, 4H and 4I), there were a large number of SSCs beginning to differentiate at the beginning of the induction of the 4 th day, and there were substantially no undifferentiated SSCs after the induction of the 6 th day, which proves that the method established by the present invention has high and thorough induction effect.
Example 6 immunofluorescence assay of induced nerve cells
The experimental method comprises the following steps: immunofluorescence was performed on cultured mouse SSCs-induced nerve cells, and the immunofluorescence detection method was the same as described in example 5.
The experimental results are as follows: the cultured nerve cells induced by the mouse SSCs are subjected to immunofluorescence detection, and specific neuron marker proteins NESTIN and tubulin TUJ1 are detected, so that the result shows that positive cells expressing NESTIN (shown in figure 5A) and TUJ1 positive cells (shown in figure 5B) exist in the nerve cells induced by the mouse SSCs, and the negative control group is negative (shown in figure 5C), which indicates that the mouse SSCs have been successfully differentiated into the nerve cells.
Example 7 induced neural cell Gene expression analysis
To determine whether the cells induced by SSCs are neural cells, neuronal marker genes (Nestin, Map-2 and Neurotensin) were tested, which also express the oligodendrocyte and astrocyte marker genes Galc and Gfap.
1 extraction of Total RNA from cells (Qiagen micro extraction kit)
1) To a micro sample, 80. mu.L of fresh lysate (lysate: 1mL of Buffer RLT contains 10 uL of mercaptoethanol, and is prepared before use);
2) adding 80 μ L70% ethanol, mixing with gun head, and centrifuging;
3) transferring the sample into Spin column provided in the kit, assembling a 2mL collecting tube, slightly covering a cover, centrifuging at 8000g for 15s, discarding the penetration liquid, and returning to the collecting tube;
4) adding 350 μ L buffer RW1, slightly covering the cover, centrifuging at 8000g for 15s, discarding the penetrating liquid, and returning to the collecting tube;
5) directly dripping 80 mu L of DNase I onto a siliceous film, standing for 15min at room temperature, and directly dripping all DNase I, otherwise, incompletely digesting DNA;
6) adding 350 μ L buffer RW1, centrifuging at 8000g for 15s, and discarding the liquid and collecting tube;
7) replacing a new collection pipe with 2mL, adding 500 μ L buffer RPE (absolute ethyl alcohol is added according to requirements), slightly covering a cover, centrifuging at 8000g for 15s, and discarding the penetrating liquid;
8) adding 500 μ L buffer RPE, slightly covering the cover, centrifuging at 8000g for 2min to completely remove ethanol, discarding the penetrating liquid and collecting tube, and removing Spin column to avoid contacting penetrating liquid, which affects recovery;
9) spin column was transferred to a new 2mL collection tube and centrifuged at high speed for 2min with the lid uncovered, and the permeate and collection tube were discarded. And replacing a new collecting pipe with 1.5mL, directly dropwise adding 20-40 mu L of RNase-free water into the center of the membrane, and centrifuging at the highest rotating speed for 2 min. The recovery amount can be increased by repeating the elution once, and the cDNA can be stored at-80 ℃ or immediately synthesized.
2 Total RNA concentration determination
Detecting whether the RNA is degraded or not by running an RNA sample through 2% agarose gel electrophoresis, detecting the concentration and the purity of the RNA by using a NANODROP 2000, controlling the purity to be 1.8-2.0 at OD260/OD280, and calculating the volume of the sample RNA adjusted to 1 mu g according to the RNA concentration.
3 Synthesis of cDNA
Reverse transcription detection 1st strand cDNA synthesis adopts a two-step method, the first step is: reaction system: sample RNA 1. mu.g, Random 6mers primer 1. mu.L, dNTP 1. mu.L, distilled water was equilibrated to 10. mu.L under the following reaction conditions: quenching on ice at 65 ℃ for 5 min; the second step is that: reaction system: mu.L of the reaction solution after the first denaturation and annealing, 4. mu.L of 5 XPrimeScript TM Buffer, 0.5. mu.L of RNase Inhibitor (40U/. mu.L), 1. mu.L of PrimeScript TM RNase (200U/. mu.L), 4.5. mu.L of distilled water, 20. mu.L of the total system, and reaction conditions: 10min at 30 ℃, 60min at 42 ℃, 15min at 70 ℃ and 1h at 4 ℃.
4 RT-PCR
20 μ L PCR System: 2 XPrimeStar Buffer/Premix 10. mu.L; upstream primer F0.2 μ L; downstream primer R0.2 μ L; template 1. mu.L and ddH2O8.6 mu L; the reaction procedure is as follows: pre-denaturation at 95 ℃ for 3 min; circulating 35 times at 95 deg.C for 30s, 60 deg.C for 30s, and 72 deg.C for 40 s; finally the total extension was 72 ℃ for 7 min. The primers used in the system are shown in Table 1 below.
TABLE 1 PCR detection primer Table
The experimental results are as follows: the RT-PCR results showed (see FIG. 6),
RT-PCR results show that nerve cells are induced to express neuron marker genes (Nestin, Map-2 and Neurotens), wherein oligodendrocyte and astrocyte marker genes Galc and Gfap are also expressed, and the fact that mouse SSCs are successfully induced to generate nerve cells is proved.
The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is specific and detailed, but not to be understood as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
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Claims (5)
1. A culture system for differentiating spermatogonial stem cells into neural cells, said culture system comprising the following components:
2. the culture system according to claim 1, comprising the following components:
3. a method of differentiating spermatogonial stem cells into neural cells, comprising the steps of:
(1) treating the culture dish with polylysine: adding polylysine with the mass fraction of 0.01-0.05% into a culture vessel, placing the culture vessel into an incubator when the culture vessel is required to be submerged, removing polylysine liquid after 10-12 hours, slightly washing the polylysine liquid with PBS (phosphate buffer solution) for 2-3 times, placing the polylysine liquid into the incubator, and waiting for cell inoculation;
(2) culturing: inoculating spermatogonial stem cells: the cultured spermatogonial stem cells exist in cell suspension singly, and the cell density is 0.8-1.2 multiplied by 105Inoculating the cells/mL into the culture vessel treated in the step (1);
(3) cell culture using a culture system according to any of claims 1-2, under conditions of 5% CO2And (4) changing the liquid by half after 22-26 h at the temperature of 37 +/-1 ℃, and continuously culturing for 5-7 days to obtain the neural cells differentiated by the spermatogonial stem cells.
4. The method of claim 3, wherein in step (1), the culture dish is treated with 0.01% polylysine.
5. The culture system according to any one of claims 1 to 2 or the method according to any one of claims 3 to 4, wherein the spermatogonial stem cells are mouse spermatogonial stem cells.
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