CN113846060B - Application of nanomaterial in promoting differentiation of embryonic stem cells to neural precursor cells - Google Patents
Application of nanomaterial in promoting differentiation of embryonic stem cells to neural precursor cells Download PDFInfo
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- YIXJRHPUWRPCBB-UHFFFAOYSA-N magnesium nitrate Chemical compound [Mg+2].[O-][N+]([O-])=O.[O-][N+]([O-])=O YIXJRHPUWRPCBB-UHFFFAOYSA-N 0.000 claims description 6
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Abstract
The invention relates to an application of a nano material in promoting differentiation of embryonic stem cells to neural precursor cells. According to the invention, firstly, the nano layered double hydroxide composed of different metal ions is synthesized by adopting a coprecipitation method at normal temperature, the toxicity of the nano layered double hydroxide to embryonic stem cells is proved to be low by a cell viability detection experiment, and the differentiation can be obviously promoted by adding the nano layered double hydroxide into a system for inducing the embryonic stem cells to differentiate into the neural precursor cells by detecting the expression of the neural precursor cell marker gene or protein, and the addition time and the concentration of the nano layered double hydroxide are further optimized, so that the differentiation induction effect is obviously improved. The invention provides a new strategy for promoting the differentiation of embryonic stem cells to neural precursor cells and creates conditions for the treatment of related diseases of the nervous system.
Description
Technical Field
The invention relates to induction differentiation of stem cells, in particular to application of a nanomaterial in promoting differentiation of embryonic stem cells to neural precursor cells.
Background
Stem cell transplantation brings new hope for treating central nervous system diseases, and regulation and mechanism analysis of stem cell fate are the leading edge and hot spot of research in the field. Embryonic Stem Cells (ESCs) with differentiated totipotency can differentiate into specific neural cell types including neural precursor cells (neural progenitor cells, NPCs), dopaminergic neurons, motor neurons, oligodendrocyte precursor cells, etc. under appropriate external signal induction conditions, providing a series of solutions for treatment after nerve injury. Wherein NPCs not only can self-renew and differentiate into all types of nerve cells, but also can migrate and integrate into the damaged part of the central nervous system, thus becoming an ideal treatment means for the related diseases of the nervous system. Therefore, it is of great scientific significance to find new methods for regulating and controlling the differentiation of ESCs to NPCs and to reveal the mechanism thereof.
The prior art discloses a method for differentiating NPCs from ESCs, for example, patent document CN107254442A discloses a method for differentiating neural precursor cells from embryonic stem cells, and in-vitro culture of the embryonic stem cells is carried out in a colony form, and the cells in the middle of the colony are compact, so that the interaction among the cells is stronger, and peripheral cells are easier to differentiate; journal paper (Li Bin, chen Hongwei, hu Zhixing, et al. Directed differentiation of human embryonic stem cells into neural precursor cells [ J ]. Animal studies 2007,28 (3)) discloses that a monolayer adherent differentiation method is used to induce directed differentiation of human embryonic stem cells cultured in a homogenous feeder layer under serum-free conditions, resulting in a high proportion of neural precursor cells; journal paper (Hu Zhixing, geng Jumin, liang Daoming. Hepatocyte growth factor promotes differentiation of human embryonic stem cells to neural precursor cells [ J ]. Journal of pathophysiology, 2010,26 (4)) discloses that serum-free neural differentiation systems containing Hepatocyte Growth Factor (HGF) and G5 can effectively induce neural differentiation of hESCs.
Since the nano material has similar size to biological molecules, the nano material is easy to interact with DNA or protein in organs, tissues or cells, and research for regulating and controlling the fate of ESCs through the physicochemical properties of the nano material is an important research direction in the field. In recent years, there has been an increasing search for the use of biochemically stable nanomaterials to regulate the neural differentiation process of ESCs. The nanomaterial can induce differentiation of ESCs into motor neurons, dopaminergic neurons, and glial cells. At present, no report on the differentiation of ESCs to NPCs regulated by nano materials is available.
Disclosure of Invention
The invention aims at overcoming the defects in the prior art and provides a new application of nano layered double hydroxide.
It is a further object of the present invention to provide a method for promoting differentiation of embryonic stem cells into neural precursor cells.
In order to achieve the first object, the invention adopts the following technical scheme:
Use of a nanolayered double hydroxide for promoting differentiation of embryonic stem cells into neural precursor cells.
As a preferred example of the present invention, the divalent metal cation of the nano-layered double hydroxide is selected from Mg 2+、Zn2+ and Ca 2+.
As another preferred example of the present invention, the trivalent metal cation of the nano layered double hydroxide is selected from Al 3+ and Fe 3+.
As another preferred example of the present invention, the interlayer anion of the nano layered double hydroxide is selected from Cl -、NO3 - and SO 4 2-.
As another preferable example of the present invention, the molar ratio of divalent metal ion to trivalent metal ion of the nano-layered double hydroxide is (0.1 to 5): 1.
As another preferred example of the present invention, the nano layered double hydroxide has the general formula of [ M 2+ (1-x)M3+ x(OH)2]x+[An-]x/n·zH2 O, wherein M 2+ is a divalent metal cation, M 3+ is a trivalent metal cation, A n- is an anion with an interlayer valence of n, x is the molar ratio of the trivalent cation to all cations, and z is the number of crystal water between each nano layered double hydroxide molecule.
In order to achieve the second purpose, the invention adopts the following technical scheme:
a method for promoting differentiation of embryonic stem cells into neural precursor cells, comprising the step of adding nano layered double hydroxide to a system of embryonic stem cells into neural precursor cells.
As a preferred example of the present invention, the nano-layered double hydroxide is added on days 3-5 of the induced differentiation culture.
As another preferable example of the present invention, the concentration of the nano layered double hydroxide in the differentiation system is 1 to 40. Mu.g/mL.
More preferably, the nanolayered double hydroxide is present in the differentiation system at a concentration of 10 μg/mL.
The invention has the advantages that:
1. According to the invention, the specific induction of the ESCs to NPCs is carried out by adopting the nano layered double hydroxide for the first time, so that the novel function of the nano layered double hydroxide on ESCs fate regulation is defined, and a strategy is provided for optimizing the ESCs to NPCs differentiation system.
2. According to the invention, the nano layered double hydroxide and ESCs are co-cultured, materials are added into a differentiation system of ESCs according to concentration gradients and different time windows, and the expression level of corresponding NPCs marker genes and proteins is detected, so that the proper concentration and key time window for promoting NPCs differentiation of the nano layered double hydroxide are found, and the differentiation induction effect is remarkably improved.
3. The invention adopts immunoblotting means to determine the effect of the nano lamellar material in promoting the differentiation of ESCs to NPCs, and provides a new strategy for optimizing the neural differentiation system of ESCs.
Drawings
Fig. 1: characterization of nanomaterial LDH and effect on the proliferation capacity of mESCs cells. (a) TEM detects the structure of both LDHs; (B) Zeta potential detection of the material surface; (C) CCK8 measures cell viability after 24h and 48h LDH treatment of mESCs at different concentrations.
Fig. 2: qPCR was performed to examine the effect on the expression levels of NPCs marker genes Sox1, pax6, N-cadherein and Map2 after addition of MgFe-LDH and MgAl-LDH materials to NPCs differentiation of ESCs.
Fig. 3: screening MgFe-LDH material promotes mESCs to NPCs differentiation optimal time window. (A) qPCR (quantitative polymerase chain reaction) detection of expression levels of NPCs marker genes Sox1, pax6, N-cadherein and Map2 after MgFe-LDH is added to days 1-5 in NPCs differentiation of ESCs; (B) Immunofluorescence detects the fluorescent signals of the marker proteins Sox1, pax6 and N-cadherein.
Fig. 4: the MgFe-LDH material promotes optimal concentration screening of ESCs towards NPC differentiation. (A) qPCR (quantitative polymerase chain reaction) detection of the expression level of NPCs marker gene after materials with different concentrations are added on day 5 of NPCs differentiation process; (B) Western blot detection NPCs of expression of marker proteins; (C) quantitative statistics of Western blot.
Detailed Description
The following detailed description of the invention provides specific embodiments with reference to the accompanying drawings.
Mouse Embryonic Stem Cells (ESCs) are derived from blastula cell clusters, are multipotent stem cells with self-renewal and multipotent differentiation potential, are induced into Neural Precursor Cells (NPCs) with higher survival rate and plasticity in neural transplantation, and can reduce the risk of teratoma formation. Therefore, a complete NPCs differentiation induction culture system can be established to provide a basis for future clinical cell transplantation treatment.
In the following examples, DMEM, fetal bovine serum, GMEM, serum analogs (KOSR), nonessential amino acids, glutamine, and diabodies were used as essential components of cell culture medium for maintaining normal growth of cells, and were commercially available from Gibco, usa, and other non-self-made reagents and raw materials were commercially available.
Example 1
1. Successfully preparing LDH materials with two different metal elements, and characterizing and detecting biocompatibility
(1) Preparation of magnesium aluminum and magnesium iron LDH nano material
Step one, a 60mL salt solution was prepared with 6mmol magnesium nitrate and 2mmol aluminum nitrate or 2mmol ferric nitrate, wherein the molar ratio between Mg 2+ and Al 3+ or Fe 3+ was 3:1.
Step two, 40mL of a 0.016M NaOH solution was prepared using ddH 2 O (double distilled water) with CO 2 removed as a solvent.
Step three, 60mL of the mixed salt solution prepared in the step one was added to 40mL of the 0.016M NaOH solution prepared in the step two with vigorous stirring (rotation speed of 400 rpm) while continuously administering N 2, to obtain a first suspension.
And step four, transferring the first suspension to a hydrothermal synthesis kettle, and heating for 16 hours at 100 ℃ to obtain a second suspension.
Step five, 20000g is centrifugated for 15min to collect the product, and the product is washed twice with double distilled water without CO 2, and then the gelatinous material is placed in a refrigerator at 4 ℃ for storage.
And step six, drying the second suspension in a vacuum drying oven to obtain magnesium aluminum LDH (MgAl-LDH) or magnesium iron LDH (MgFe-LDH).
The structure of the material was examined by Transmission Electron Microscopy (TEM), two LDHs were formulated as a 0.1mg/mL suspension, and the samples were sonicated for 30min to make a homogeneous solution, FIG. 1A is a transmission electron microscopy topography of the two LDHs. From FIG. 1A, it is found that MgAl-LDH and MgFe-LDH both have hexagonal layered structures, the distribution is relatively uniform, and the particle size distribution is about 80-120nm (FIG. 1A).
(2) Surface potential measurement of nano LDH
Preparing two LDH into 1mg/mL suspension, performing ultrasonic dispersion for 30min to make the sample into uniform solution, and performing particle size detection and surface Zeta potential detection by using a particle size analyzer, wherein the detection result is shown in figure 1B. FIG. 1B shows the surface potentials of two LDHs at a concentration of 0.5mg/mL, showing that the MgAl-LDH surface potential is about 24.4mV and the MgFe-LDH surface potential is about 13.4mV, indicating that the Zeta potentials of both LDH materials in this example are positive potentials, are stable, and facilitate contact with negatively charged cell membranes and entry into cells.
(3) Determination of cell viability of ESCs after nanoLDH treatment
Step one, culturing ESCs of the embryo stem cells of the mice. ESCs were seeded on 96-well plates, plated at 8000 pieces/well, and incubated with medium at 37 ℃ and 5% co 2 for 24h for subsequent drug or material treatment. The ESCs culture medium comprises the following raw materials: DMEM; FBS; glutamine; and (3) double antibodies. In this experiment, the incubation time was 24h.
Step two, five solutions were prepared at concentrations of 2.5. Mu.g/mL, 5. Mu.g/mL, 10. Mu.g/mL, 20. Mu.g/mL and 40. Mu.g/mL in this order.
And thirdly, discarding the supernatant of the cultured ESCs in the first step, adding LDH solutions with different concentrations prepared in the second step into each hole respectively, culturing for 24 hours, adding 10 mu L of CCK-8 solution with the concentration of 5mg/mL into each hole, placing for 2 hours in a dark place, shaking for 10 seconds in a dark place, and measuring an OD (optical density) value at 455nm wavelength by using an enzyme-labeled instrument, wherein the detection result is shown in figure 1C. The two LDH materials were treated with mESCs in concentration gradients of 2.5. Mu.g/mL, 5. Mu.g/mL, 10. Mu.g/mL, 20. Mu.g/mL and 40. Mu.g/mL for 7 days, respectively, and the results showed that the cell proliferation ability was not affected.
2. MgFe-LDH can better promote differentiation of mESCs to NPCs than MgAl-LDH material
Step one, culturing ESCs of the embryo stem cells of the mice. ESCs were inoculated on 6cm suspension dishes, plated at a density of 2 x 10 5 per well, and differentiated cultured with Neural Precursor Cells (NPCs) medium at 37 ℃ under 5% co 2 for 1-5 days for subsequent drug or material treatment. The NPCs culture medium comprises the following raw materials: GMEM;8% KOSR;1% glutamine; 1% of diabody. The medium components were all commercially available from Gibco corporation.
Step two, mgFe-LDH and MgAl-LDH solutions are respectively prepared, the concentrations are 2.5 mug/mL, 5 mug/mL and 10 mug/mL respectively, after ESCs are differentiated into NPCs, two LDH materials with different concentrations are added for 48 hours, cells at each time point are collected, total RNA is extracted after Trizol is cracked, cDNA is obtained through reverse transcription by using a cDNA reverse transcription kit, and expression of NOC differentiation related genes (Sox 1, pax6, N-cadherein and Map 2) is detected through real-time quantitative PCR.
Thirdly, the qPCR detection finds NPCs marker gene expression, and the result of FIG. 2 shows that NPCs treated by MgFe-LDH under various concentration conditions can better promote the expression of NPCs marker genes Sox1, pax6, N-cadherein and Map2 compared with a control group and a MgAl-LDH group, so that the material is adopted in the subsequent experiment.
3. Optimal time point for MgFe-LDH nano material to promote ESCs to NPCs to differentiate
Step one, ESCs were plated on 6cm suspension dishes at a plating density of 2×10 5/well, and considering that NPCs differentiation reached a higher maturity at substantially day 5, we added 10 μg/mL MgFe-LDH material to NPCs differentiation days 1-5, and replaced the new culture broth every 3 days and harvested cells to examine the differentiation efficiency of NPC day 7 by qPCR, and fig. 3A is the effect on NPCs differentiation marker genes Sox1, pax6, N-cadherein and Map2 after MgFe-LDH material addition on days 1,2,3, 4 and 5, respectively, and the results indicate that the differentiation thereof was able to promote the expression of Sox1, pax6, N-cadherein and Map2 after the early (days 1-2) and late (days 3-5) addition of NPCs differentiation.
Step two, mgFe-LDH material was added on days 1, 3 and 5 of NPCs differentiation, each group of cells was harvested on day 7 of differentiation, and differentiation markers of NPCs after each group of treatment, including Sox1, pax6 and N-cadherein, were detected by immunofluorescent staining. Cells were fixed with 4% paraformaldehyde for 10min, washed three times with PBS and then permeabilized with 0.25% Triton X-100 (polyethylene glycol octylphenyl ether) for 10min, then blocked with 5% goat serum for 1h to inhibit non-specific antibody binding, and then incubated overnight at 4℃with Sox1, pax6 and N-cadherein primary antibodies (1:500 dilution), washed three times with PBS and incubated with fluorescent secondary antibodies (1:200 dilution) for 1h at normal temperature, washed three times with PBS and incubated with DAPI (37℃for 15 min) for nuclear staining, and finally observed with a fluorescent confocal microscope. The antibodies used are commercially available from Abcam. According to the results of FIG. 3B, it was shown that the fluorescence intensity of Sox1, pax6 and N-cadherein in NPCs after addition of the material was lower than that of the control group (without addition of the material treatment), the fluorescence intensity of Pax6 and N-cadherein was higher than that of the control group on day 3, and the protein fluorescence intensity of Pax6 and N-cadherein was highest on day 5. From this, it was shown that the optimal time point for MgFe-LDH nanomaterial to promote differentiation of ESCs to NPCs was day 5 of differentiation.
4. Optimal concentration screening for promoting differentiation of ESCs to NPC by nano LDH material
Step one, adding MgFe-LDH materials of 2.5, 5, 10, 20 and 40 mug/mL on day 5 in the NPCs differentiation process, detecting NPCs differentiation efficiency after MgFe-LDH treatment under different concentration conditions by using qPCR (FIG. 4A) and WB (FIG. 4B) on day 7, wherein the qPCR results in FIG. 4A show that the expression level of marker genes Sox1, pax6, N-cadherein and Map2 corresponding to NPCs is higher than that of other concentration conditions under the concentration condition of 10 mug/mL;
Step two, mgFe-LDH materials of 2.5, 5, 10, 20 and 40 mug/mL are respectively added on the 5 th day in the NPCs th differentiation process, the influence on NPCs marker proteins after the MgFe-LDH materials are treated under different concentration conditions is detected by using a protein immunoprecipitation (WB) on the 7 th day, polyacrylamide gel electrophoresis is carried out according to the same protein amount after quantitative detection is carried out on total protein extracted from each group of cells, the gel is subjected to membrane transfer, closure and incubation corresponding to primary and secondary antibodies after the electrophoresis is finished, and corresponding protein strips are exposed by using ECL luminous solution, and the expression of NPCs marker proteins Sox1, pax6 and N-cadherein after the treatment is compared. The WB level detection results in FIG. 4B also demonstrate that the protein expression levels of the corresponding N-cadhererin and Pax6 are more than those of the other groups under the condition of consistent internal reference protein expression at the concentration of 10 mug/mL, and the quantitative statistics in FIG. 4C also indicate that the optimal concentration condition of MgFe-LDH for promoting the differentiation of mESCs to NPCs is 10 mug/mL.
The foregoing is merely a preferred embodiment of the present invention, and it should be noted that modifications and additions may be made to those skilled in the art without departing from the method of the present invention, which modifications and additions are also to be considered as within the scope of the present invention.
Claims (4)
1. The application of the nano layered double hydroxide in promoting the differentiation of embryonic stem cells to neural precursor cells in vitro is characterized in that the nano layered double hydroxide is prepared by the following method:
Step one, preparing 60mL of salt solution by using 6mmol of magnesium nitrate and 2mmol of ferric nitrate, wherein the mol ratio between Mg 2+ and Fe 3+ is 3:1;
Step two, preparing 40mL of 0.016M NaOH solution by using double distilled water with CO 2 removed as a solvent;
Step three, adding 60mL of the mixed salt solution prepared in the step one into 40mL of 0.016M NaOH solution prepared in the step two while continuously giving N 2 to obtain a first suspension;
Transferring the first suspension to a hydrothermal synthesis kettle, and heating at 100 ℃ for 16 hours to obtain a second suspension;
Step five, centrifuging 20000g for 15min, collecting a product, washing twice with double distilled water without CO 2, and then placing the gelatinous material in a refrigerator at 4 ℃ for storage;
step six, drying in a vacuum drying oven to obtain the magnesium-iron nano layered double hydroxide;
The nano layered double hydroxide is added on the 3 rd to 5 th days of embryonic stem cell induced differentiation culture; the concentration of the nano layered double hydroxide in the differentiation system is 5-20 mug/mL.
2. The use according to claim 1, wherein the nanolayered double hydroxide has the general formula [M2+ (1-x)M3 + x(OH)2]x+[An-]x/n·zH2O, wherein M 2+ is a divalent metal cation, M 3+ is a trivalent metal cation, a n- is an anion having an interlayer valence of n, x is the molar ratio of trivalent cations to all cations, and z is the number of water of crystallization per nanolayered double hydroxide molecule.
3. A method for promoting the differentiation of embryonic stem cells to neural precursor cells in vitro is characterized by comprising the step of adding nano layered double hydroxide into a system for differentiating embryonic stem cells to neural precursor cells,
Step one, preparing 60mL of salt solution by using 6mmol of magnesium nitrate and 2mmol of ferric nitrate, wherein the mol ratio between Mg 2+ and Fe 3+ is 3:1;
Step two, preparing 40mL of 0.016M NaOH solution by using double distilled water with CO 2 removed as a solvent;
Step three, adding 60mL of the mixed salt solution prepared in the step one into 40mL of 0.016M NaOH solution prepared in the step two while continuously giving N 2 to obtain a first suspension;
Transferring the first suspension to a hydrothermal synthesis kettle, and heating at 100 ℃ for 16 hours to obtain a second suspension;
Step five, centrifuging 20000g for 15min, collecting a product, washing twice with double distilled water without CO 2, and then placing the gelatinous material in a refrigerator at 4 ℃ for storage;
step six, drying in a vacuum drying oven to obtain the magnesium-iron nano layered double hydroxide;
the nano layered double hydroxide is added on the 3 rd to 5 th days of embryonic stem cell induced differentiation culture, and the concentration of the nano layered double hydroxide in a differentiation system is 5-20 mug/mL.
4. A method according to claim 3, wherein the nanolayered double hydroxide is present in the differentiation system at a concentration of 10 μg/mL.
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