CN115844927A - Application of stem cells in preparation of preparation for treating leukoencephalopathy - Google Patents
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Abstract
The invention discloses an application of stem cells in preparing a preparation for treating leukoencephalopathy, wherein neural stem cells are directionally differentiated into oligodendrocyte precursor cells in vitro, the oligodendrocyte precursor cells are loaded by taking hydrogel microspheres as a carrier to obtain a cell microcarrier, and the cell microcarrier and pharmaceutically acceptable auxiliary materials are prepared into an injectable preparation, wherein the hydrogel microspheres are prepared from gelatin derivatives and carboxymethyl chitosan. The cell microcarrier provided by the invention can provide a supporting and protecting effect for cells, ensure the integrity of the cells, improve the effective injection rate of the cells, promote the development and maturation of oligodendrocyte precursor cells, and improve the differentiation efficiency and myelination of the oligodendrocyte precursor cells; the cell microcarrier and pharmaceutically acceptable auxiliary materials are prepared into an injectable preparation, and the injectable preparation shows excellent myelin repair effect when used for treating the leukoencephalopathy.
Description
Technical Field
The invention belongs to the technical field of medicines, and particularly relates to application of stem cells in preparation of a preparation for treating leukoencephalopathy.
Background
Leukoencephalopathy is a group of demyelinating diseases of the central nervous system of which the specific etiology is not yet clear. The cell therapy is a method for repairing tissues and organs by using active cells, and the cells used for the cell therapy mainly comprise stem cells, mature functional cells, somatic cells, xenogenic cells and transdifferentiated cells, and the stem cells are directionally differentiated into oligodendrocyte precursor cells in vitro and are used for treating the leukoencephalopathy, which is the key research direction of technicians in the related field. Oligodendrocyte Precursor Cells (OPCs) are precursor cells of oligodendrocytes in the central nervous system, can proliferate and differentiate during brain development to form oligodendrocytes, which form myelin sheaths in the central nervous system, and the transplantation of in-vitro amplification cultured OPCs brings hope for remyelination.
The patent with the application number of 201310455895.3 provides an induction medium for inducing neural stem/progenitor cells to be differentiated into oligodendrocyte precursor cells, an induction method and application thereof, the patent differentiates the human neural stem/progenitor cells into the oligodendrocyte precursor cells under the action of the induction medium after pretreatment culture, wherein the key components of the induction medium are bFGF, PDGF-AA and NT-3, and the oligodendrocyte precursor cells prepared by the method can be applied to the preparation of medicines for treating nervous system injury diseases. Cell therapy generally needs to be carried out through the processes of in vivo extraction, in vitro activation culture and in vivo infusion, while cell injection is a common method for infusing cells into a patient, but the cell injection process has some defects, such as serious cell loss, low cell retention rate, insufficient effective cells and low cell survival rate, so that not only is cell source wasted, but also the effect and effect of the cells infused into the body are influenced. A cell microcarrier matrix and an application thereof are provided in the patent with the application number of 202011505673.4, the cell microcarrier matrix is prepared by taking methacrylated gelatin as an effective component, cells are wrapped in the matrix to provide a three-dimensional supporting effect for the cells, the cells are prevented from being damaged in the injection and transplantation processes, and more growth factors can be attached to the matrix to better promote the expression and release of the cells.
Therefore, in order to improve the effective injection rate of cell injection, ensure the integrity and survival rate of cells and promote the in vivo differentiation and myelination of OPCs, the search for a suitable cell microcarrier or scaffold material is of practical significance.
Disclosure of Invention
In view of the defects of the prior art, the invention aims to provide the application of the stem cells in preparing a preparation for treating the leukoencephalopathy.
The technical scheme adopted by the invention for realizing the purpose is as follows:
the application of the stem cells in preparing the preparation for treating the leukoencephalopathy is to directionally differentiate the neural stem cells into oligodendrocyte precursor cells in vitro, load the oligodendrocyte precursor cells by taking hydrogel microspheres as a carrier to obtain a cell microcarrier, and prepare the cell microcarrier and pharmaceutically acceptable auxiliary materials into an injectable preparation; the hydrogel microspheres are prepared from gelatin derivatives and carboxymethyl chitosan, wherein the structural formula of the gelatin derivatives is as follows:
The preparation method of the gelatin derivative comprises the following steps:
s1, dispersing gelatin in deionized water, stirring and dissolving at 50-65 ℃, cooling to 35-45 ℃, and adding potassium periodate (KIO) 4 ) Reacting in dark for 5-8h, adding ethylene glycol, stirring for 0.5-1h, dialyzing, and freeze dryingDrying to obtain aldehyde gelatin;
s2, dissolving miconazole in N, N-dimethylformamide to obtain a solution 1, dissolving trans-3-chloroacrylic acid in deionized water to obtain a solution 2, dropwise adding the solution 2 into the solution 1 in a nitrogen atmosphere, stirring, heating to 30-35 ℃ after dropwise adding is completed within 0.5-1h, continuously stirring for 7-9h to obtain a reaction mixture, and performing rotary evaporation, extraction, concentration and drying to obtain a miconazole derivative;
and S3, dispersing the aldehyde gelatin obtained in the step S1 into deionized water, heating to 60-75 ℃, stirring for 20-30min, sequentially adding sodium hydroxide, the miconazole derivative obtained in the step S2 and benzyltriethylammonium chloride, continuously stirring for 3-4h, adjusting the pH to 6-7 by using hydrochloric acid, and dialyzing, freezing and drying to obtain the gelatin derivative.
The gelatin is a protein obtained by partial hydrolysis of collagen, has good biodegradability, biocompatibility and histocompatibility, has active groups capable of being connected with other ligands, can be used for preparing micro-capsules or microspheres by using methods such as single aggregation and complex aggregation, has higher encapsulation efficiency and drug-loading rate, and has no obvious change after long-term storage (more than 25 years) of some drug-loaded microspheres. Gelatin has many excellent properties and performance but inevitably has some disadvantages and thus needs to be compensated for and eliminated by some means or to have new properties and performance. The gelatin is chemically modified, aldehyde groups and miconazole are introduced into the gelatin structure, and aldehyde group reaction sites are provided for the formation of dynamic imine bonds. The invention takes gelatin as a reaction raw material, takes potassium periodate as an oxidant, oxidizes hydroxyl in a gelatin structure into aldehyde group to obtain aldehyde-based gelatin, the miconazole reacts with trans-3-chloroacrylic acid to obtain a miconazole derivative, and the aldehyde-based gelatin and the miconazole derivative carry out N-alkylation reaction under the catalysis of sodium hydroxide and benzyltriethylammonium chloride to obtain the gelatin derivative.
In order to obtain the aldehyde-modified gelatin, the mass ratio of the gelatin to the deionized water to the potassium periodate to the ethylene glycol in the step S1 is 1.1-1.3; in order to introduce miconazole into a gelatin structure and ensure the consistency of products, the mass concentration of miconazole in the solution 1 in the step S2 is 0.16-0.20g/mL, the mass concentration of trans-3-chloroacrylic acid in the solution 2 is 0.1-0.15g/mL, the molar ratio of miconazole to trans-3-chloroacrylic acid is 1; in the step S3, the mass ratio of the aldehyde gelatin to the sodium hydroxide to the miconazole derivative to the benzyltriethylammonium chloride is 20-25.
The preparation method of the cell microcarrier comprises the following steps:
(1) Dispersing gelatin derivatives and carboxymethyl chitosan in deionized water, performing high-speed homogenization treatment and vacuum filtration sterilization, adding a cell suspension containing oligodendrocyte precursor cells, and uniformly blowing to obtain a preformed solution, wherein the addition amount of the gelatin derivatives in the preformed solution is 0.03-0.06g/mL, the addition amount of the carboxymethyl chitosan in the preformed solution is 0.02-0.04g/mL, and the high-speed homogenization treatment is carried out at 1000-1200r/min for 10-15s;
(2) Dripping the prepared solution obtained in the step (1) into sterile mineral oil, heating to 36-38 ℃, preserving heat for 10-12h to obtain hydrogel microspheres loaded with cells, filtering, collecting, and washing with sterile Phosphate Buffer Solution (PBS) to obtain the injectable cell microcarrier.
The carboxymethyl chitosan has excellent water solubility, antibacterial property, moisture retention and oxidation resistance, has the characteristics of hydrophilicity, gelling property, permeability promotion, biocompatibility, degradability and the like when being used as a medicine carrier, can prolong the detention time of a medicine at a specific part in a body and improve the bioavailability of the medicine, and can be used as a novel injection material after being compounded with other materials. The dynamic covalent chemistry is established on the basis of a mixture of generating interconversion compounds by dynamic covalent bonds, the reversible reaction involved in the preparation process of the mixture is very fast, the mixture has tolerance to various functional groups and has activity under mild conditions, the most common reversible reactions in the dynamic covalent chemistry comprise imine exchange reaction, ester exchange reaction, acetal exchange reaction and the like, and the hydrogel material constructed based on the dynamic covalent chemistry concept has excellent three-dimensional network structure and self-repairing property. According to the invention, the gelatin derivative and the carboxymethyl chitosan are compounded, and the hydrogel material is obtained in a dynamic imine bond combination mode, has excellent three-dimensional network structure, self-repairing function, controlled slow release effect and anti-deformation capability, can provide a three-dimensional supporting effect for cells, prevents the cells from being damaged in the injection or transplantation process, and can perform self-repairing in a short time to protect the integrity of the cells when the hydrogel material cracks under the action of external force; the miconazole structure in the gelatin derivative can also cooperate with oligodendrocyte precursor cells to promote the oligodendrocyte precursor cells to mature and improve the differentiation efficiency and myelination.
The invention has the following beneficial effects: the invention carries out chemical modification on gelatin, introduces aldehyde group and miconazole into gelatin molecules, and obtains the gelatin derivative which has good surface activity and can be well compatible with cells and active factors; the gelatin derivative and the carboxymethyl chitosan are combined in a dynamic imine bond mode to prepare the hydrogel microsphere, and the hydrogel microsphere has an excellent three-dimensional network structure, a self-repairing function, a controlled slow release effect and an anti-deformation capacity; the method comprises the following steps of (1) loading oligodendrocyte precursor cells by taking hydrogel microspheres as a carrier to obtain a cell microcarrier, wherein the cell microcarrier can provide a supporting and protecting effect for the cells, ensure the integrity of the cells, improve the effective injection rate of the cells, simultaneously promote the development and maturation of the oligodendrocyte precursor cells, and improve the differentiation efficiency and myelination of the oligodendrocyte precursor cells; the cell microcarrier and pharmaceutically acceptable auxiliary materials are prepared into an injectable preparation, and the injectable preparation shows excellent myelin repair effect when used for treating the leukoencephalopathy.
Drawings
FIG. 1 is a test chart of the self-healing performance of hydrogel materials prepared according to the process described in example 2.
FIG. 2 is an SEM image of a hydrogel material prepared according to the process described in example 2.
FIG. 3 shows in vitro experimental myelin basic protein expression of oligodendrocyte precursor cells and cell microcarriers.
FIG. 4 shows experimental myelin basic protein expression in oligodendrocyte precursor cells and cell microcarriers.
Detailed Description
The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the embodiments of the present application, and it is obvious that the described embodiments are only a part of the embodiments of the present application, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present application.
The raw materials used in the following examples are all common commercial products. Phosphate Buffered Saline (PBS), pH6.0, sterile, available from Shanghai Shangbao Biotech Co., ltd; du's phosphate buffer (DPBS, calcium and magnesium free), pH7.4, sterile, purchased from Condits chemical (Hubei) Inc.; gelatin, pharmaceutical grade, model CP2020, carboxymethyl chitosan, pharmaceutical grade, all purchased from shanxi brocade pharmaceutic adjuvant ltd; miconazole CAS number 22916-47-8; trans-3-chloroacrylic acid CAS number 2345-61-1; benzyltriethylammonium chloride CAS number 56-37-1.
Example 1
The culture medium source is as follows: CTS Stem Pro Medium: thermo Fisher, cat.no. a1033201; advanced DMEM/F12: thermo Fisher, cat. No. 12634028; neurobasal medium: thermo Fisher, cat.no. 21103049; growth factors of clinical standard: CTS N2 (Thermo Fisher, cat. No.: 1370701), CTS B27 (Thermo Fisher, cat. No.: 1486701), GMP bFGF (Peprotech, cat. No.: GMP 100-18B), and GMP PDGF-AA (Peprotech, cat. No.: GMP 100-13A); material sources are as follows: bone marrow was derived from 30 day old clean-grade mice (purchased from Shanghai laboratory animal center).
A method of preparing oligodendrocyte precursor cells comprising the steps of:
s1, preparing bone marrow mesenchymal stem cells: diluting 1mL of clean mouse bone marrow by using 9mL of CTS Stem Pro Medium, adding the diluted bone marrow into a10 cm cell culture dish suitable for cell adherent culture for culture, and defining the day of adherent culture of the bone marrow as the first day (D0) of the differentiation process of oligodendrocyte precursor cells; 48hRemoving the culture Medium in the cell culture dish, washing the nonadherent cells by using DPBS, adding 10mL of CTS Stem Pro Medium, and then replacing the CTS Stem Pro Medium every 72 hours to continue culturing; bone marrow mesenchymal stem cell (BMSC) clones were observed in the culture dish when cultured to D6-D7; when the culture is carried out to D8-D10, the culture Medium is aspirated and 3mL of DPBS is used for flushing nonadherent cells in a culture dish, the DPBS is removed, 1mL of CTS Tryple Select is added into the culture dish, the culture dish is incubated for 5min at 37 ℃, the digested cell solution is collected and centrifuged for 5min at 200g at room temperature to obtain a precipitate, and CTS Stem Pro Medium is used for re-suspending the centrifugal precipitate; the cells were cultured at 4X 10 4 Individual cell/cm 2 Is inoculated to a cell culture dish of 6cm or 10cm, and is subcultured when the BMSC in the culture dish reaches 80-90% fusion, wherein the BMSC subculture cannot exceed 10 generations; the following three groups of markers were detected using flow cytometry: a: BMSC marker: STRO-1, CD90 and CD73; b: neural stem cell markers: nestin; c: hematopoietic stem cell markers: CD45; when the cells in the dish exhibited greater than 90% positive for the group a marker, greater than 5% positive for the group B marker, and less than 1% for the group C marker, the cells in the dish were available for further manipulation;
s2, preparing neural stem cells: preparing an Advanced DMEM/F12 culture medium containing GlutaMAX with a final concentration of 1%, B27 with a final concentration of 2%, bFGF with a final concentration of 40ng/mL, EGF with a final concentration of 40ng/mL, IGF-1 with a final concentration of 20ng/mL and P/S with a final concentration of 1% as a neural stem cell culture medium; when step S1 BMSCs passed between passages 3-10 (preferably D15), BMSCs were digested with CTS Tryple Select, resuspended in neural stem cell culture media after centrifugation, and resuspended at 1X 10 4 Individual cell/cm 2 The density of (A) is inoculated in a non-adherent culture 6-well plate; replacing the neural stem cell culture medium every 48h, forming neural spheres by the neural stem cells and suspending the neural spheres in the neural stem cell culture medium along with the increase of the culture time, and centrifuging the culture medium for 5min at 200g on the non-adherent culture day 5-7 to obtain the neural stem cells in the neural sphere form; taking bone marrow mesenchymal stem cells as a control, scattering neurospheres cultured on non-adherent days 5, detecting the content of cells which positively express Nestin and GFAP in the neurospheres by using immunofluorescence technology, counting, and countingThe amount of cells in the pellet that positively express Nestin and GFAP was higher than 75%.
S3, preparing oligodendrocyte precursor cells: coating a 6-well plate with poly-L-ornithine (PLO) and human source laminin with the concentration of 10 mug/mL, adding an oligodendrocyte precursor cell differentiation culture medium into the 6-well plate, and then inoculating the neural stem cells obtained in the step S2, wherein the inoculation density is 4 multiplied by 10 4 Individual cell/cm 2 (ii) a Renewing an oligodendrocyte precursor cell differentiation culture medium every 48 hours after inoculation, digesting and collecting oligodendrocyte precursor cells by using CTS (clear to send) Tryple Select from the 8 th day of the day when the neural stem cells are inoculated to the differentiation culture medium containing the first group of oligodendrocyte precursor cells; wherein the oligodendrocyte precursor cell differentiation medium is prepared by mixing Advanced DMEM/F12 and Neurobasal medium in a volume ratio of 1; when in use, the oligodendrocyte precursor cells are dispersed in PBS to prepare cell suspension, and each mu L of PBS in the cell suspension contains 10 cells 5 And (4) respectively.
Example 2
A method for preparing a gelatin derivative, comprising the steps of:
s1, dispersing gelatin in deionized water, stirring and dissolving at 60 ℃, cooling to 35 ℃, adding potassium periodate, reacting for 6 hours in a dark place, adding ethylene glycol, continuing stirring for 0.5 hour, dialyzing for 72 hours by using a dialysis bag with a cut-off molecular weight of 8000-14000D, replacing the deionized water every 12 hours, collecting a solution in the dialysis bag, and freeze-drying to obtain aldehyde-based gelatin, wherein the mass ratio of the gelatin to the deionized water to the potassium periodate to the ethylene glycol is 1.2;
s2, dissolving miconazole in N, N-dimethylformamide to obtain a solution 1, dissolving trans-3-chloroacrylic acid in deionized water to obtain a solution 2, dropwise adding the solution 2 into the solution 1 under a nitrogen atmosphere, stirring, heating to 30 ℃ after 1h of dropwise addition is finished, continuously stirring for 8h to obtain a reaction mixture, adding deionized water into the reaction mixture to ensure that the volume ratio of the N, N-dimethylformamide to the deionized water in the reaction mixture is 1;
s3, dispersing the aldehyde gelatin obtained in the step S1 in deionized water, heating to 60 ℃, stirring for 30min, sequentially adding sodium hydroxide, the miconazole derivative obtained in the step S2 and benzyltriethylammonium chloride, continuously stirring for 4h, adjusting the pH to 6-7 by using hydrochloric acid, dialyzing for 72h by using a dialysis bag with the molecular weight cutoff of 8000-14000D, replacing the deionized water every 12h, collecting the solution in the dialysis bag, and freeze-drying to obtain the gelatin derivative, wherein the mass ratio of the aldehyde gelatin to the sodium hydroxide to the miconazole derivative to the benzyltriethylammonium chloride is 25.15 g/mL.
A method for preparing a cell microcarrier, comprising the steps of:
(1) Dispersing gelatin derivatives and carboxymethyl chitosan in deionized water, performing high-speed homogenization treatment and vacuum filtration sterilization, adding a cell suspension (obtained in example 1) containing oligodendrocyte precursor cells, and uniformly blowing to obtain a preformed solution, wherein the addition amount of the gelatin derivatives in the preformed solution is 0.035g/mL, the addition amount of the carboxymethyl chitosan in the preformed solution is 0.035g/mL, and the high-speed homogenization treatment is performed at 1000r/min for 10s;
(2) And (2) dripping the prepared solution obtained in the step (1) into sterile mineral oil through an injector, heating to 37 ℃, preserving heat for 12 hours to obtain hydrogel microspheres loaded with cells, filtering, collecting, and washing with a sterile phosphate buffer solution to obtain the injectable cell microcarrier.
The synthetic route of the hydrogel microspheres prepared in this example is as follows:
wherein
Is the main chain of the gelatin molecule.
Example 3
A process for the preparation of a gelatin derivative, prepared according to the method of example 2, except that: the cooling temperature in the step S1 is 40 ℃; a method for preparing a cell microcarrier was performed according to example 2.
Example 4
A process for the preparation of a gelatin derivative, as prepared in example 2, with the difference that: the cooling temperature in the step S1 is 45 ℃; a method for preparing a cell microcarrier was performed according to example 2.
Example 5
A process for the preparation of a gelatin derivative, prepared according to the method of example 2, except that: in the step S3, the continuous stirring time is 3 hours; a method for preparing a cell microcarrier was performed according to example 2.
Comparative example 1
A process for the preparation of a gelatin derivative, prepared according to the method of example 2, except that: omitting steps S2 and S3; a method for preparing a cell microcarrier was performed according to example 2.
Comparative example 2
A method of preparing a cell microcarrier, prepared according to the method of example 2, except that: the gelatin derivative was replaced with gelatin.
Comparative example 3
The cell suspension containing oligodendrocyte precursor cells obtained in example 1 is combined with pharmaceutically acceptable excipients (e.g., physiological saline) to prepare an injectable formulation.
The hydrogel material prepared according to the process described in example 2 (gelatin derivative and carboxymethyl chitosan are dispersed in deionized water, and subjected to high-speed homogenization treatment, and the hydrogel material is subjected to heat preservation at 37 ℃ for 12 hours, and the hydrogel material is obtained), wherein one of two hydrogel materials with the same volume and shape is dyed with reactive dye B, and the other is not dyed, and is cut into two symmetrical parts by a blade, the sections of two hydrogels with different colors are combined together, the fracture surfaces are in contact with each other, the spliced hydrogel sample is placed in a polyethylene culture dish, the self-healing test is performed under the conditions that the temperature is 36.5 ℃ and the humidity is 80%, the hydrogel sample is subjected to a simple stretching experiment at a preset time, and the healing condition of the hydrogel is recorded, and as shown in fig. 1, after 0.5 hour, the two samples can be combined into a single entity, and no external stimulation is required, the boundary between samples with different colors becomes fuzzy, and the diffusion of the reactive dye B from the dyed part to the non-dyed part can be clearly observed, and the hydrogel can be kept intact after simple stretching, and the hydrogel can be observed, and the molecular motion between adjacent samples can be illustrated.
The hydrogel material prepared according to the process described in example 2 (gelatin derivative and carboxymethyl chitosan are dispersed in deionized water, and subjected to high-speed homogenization treatment, and then the mixture is subjected to heat preservation at 37 ℃ for 12 hours, so that the hydrogel material is obtained), after the hydrogel material is subjected to freeze drying, the microscopic morphology of a sample is observed by using a Scanning Electron Microscope (SEM), the sample is broken in a liquid nitrogen environment before observation, and the cross section is subjected to gold spraying treatment, so that the hydrogel material has more regular pore size distribution and thick wall, and most of the hydrogel material is through holes, and the hydrogel material has excellent permeability and higher specific surface area due to the porous structure, so that the hydrogel material has great advantages for loaded cells.
Cyclophosphamide is added into the hydrogel microspheres to test the controlled-release effect of the hydrogel microspheres, and the preparation method comprises the following steps: dispersing gelatin derivatives, carboxymethyl chitosan and cyclophosphamide in deionized water, carrying out high-speed homogenization treatment to obtain a preformed solution, dripping the preformed solution into sterile mineral oil through an injector, carrying out heat preservation at 37 ℃ for 12 hours, filtering and collecting to obtain the gelatin chitosan gel, wherein the addition amount of the gelatin derivatives in the preformed solution is 0.035g/mL, the addition amount of the carboxymethyl chitosan in the preformed solution is 0.035g/mL, the addition amount of the cyclophosphamide in the preformed solution is 0.02g/mL, and the high-speed homogenization treatment is carried out at 1000r/min for 10s. Preparing hydrogel microspheres into an aqueous solution (the mass fraction is 2%), taking water (1L) as dialysate, dialyzing with a 8000-14000D dialysis bag, stirring, measuring the phosphorus content in the dialysate by a molybdenum blue method at selected time points (1 h, 2h, 4h, 8h, 16h and 24 h), and calculating the cumulative phosphorus release rate, wherein the test results are shown in table 1, the hydrogel microspheres exhibit a rapid release phenomenon within 0-4h, the hydrogel microspheres exhibit a slow release phenomenon within 4-24h, the sustained-release period lasts for a longer time, and the hydrogel microspheres exhibit better controlled and sustained-release effects.
In vitro experiments were performed on the oligodendrocyte precursor cells obtained in example 1 and the cell microcarriers obtained in examples 2-5 and comparative examples 1-2. A CTS Neurobasal Medium containing CTS GlutaMAX at a final concentration of 1%, CTS B27 at 2%, 2-mercaptoethanol at 10 μ M, P/S at 1%, and T3 at a final concentration of 10 μ M was prepared, and the oligodendrocyte precursor cells obtained in example 1 and the cell microcarriers obtained in examples 2-5 and comparative examples 1-2 were co-cultured with neurons in the CTS Neurobasal Medium for 15 days (the final concentration of oligodendrocyte precursor cells was 4X 10) 4 Individual cells/ul), and the expression level of Myelin Basic Protein (MBP) was measured by Western blot using a culture medium without oligodendrocyte precursor cells as a negative control group, and the results are shown in fig. 3, wherein the expression level of MBP protein (expressed as the ratio of absorbance values of a target band and an internal reference band) was significantly different between 8 groups, and the expression levels of MBP protein in examples 2 to 5 were significantly higher than those in example 1, comparative examples 1 to 2 and the negative control group, wherein the expression level of MBP protein in example 2 was the highest.
The cell microcarriers obtained in examples 2-5 and comparative examples 1-2 are mixed with pharmaceutically acceptable auxiliary materials (such as physiological saline) to prepare injectable preparations, and the preparations obtained in examples 2-5 and comparative examples 1-3(final oligodendrocyte precursor concentration 4X 10 4 Individual cells/ul) were tested in vivo. Subject: a cleaning-grade shiver newborn mouse is obtained by a pregnant mother mouse in an animal room of general navy hospital of the liberation army (provided by Beijing Wintolite, china), genotype detection is carried out on the mouse once the mouse is born, 56 mice with shi-/- (complete deletion of MBP gene) are selected as a cerebral leukosis model and are averagely divided into 8 groups, the central nerves of the mice are respectively injected with the preparations obtained in examples 2-5 and comparative examples 1-3 within 24h after birth, the preparation without oligodendrocyte precursor cells is taken as a negative control group, when the mouse is 8 weeks old, western blot detection is carried out on mouse brain tissues, positive expression of Myelin Basic Protein (MBP) is detected (expressed by the ratio of absorbance values of a target strip and an internal reference strip), the result is shown in figure 4, the expression of MBP protein in examples 2-5 is higher than 0.55, wherein the MBP protein expression of example 2 is the highest and is 0.64, the MBP protein expression of 1-3 is 0.25-0.30, the limited MBP protein expression is higher than the microsphere protein expression of MBP protein in the comparative example 2, the MBP protein expression of the contrast gene is capable of improving the myelin forming of the oligodendrocyte precursor cells, the mouse, the MBP gene, the synergistic effect of improving the demyelination of the myelin forming of the mouse is improved, and the myelin forming of the MBP is improved.
Although embodiments of the present application have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the application, the scope of which is defined in the appended claims and their equivalents.
Claims (8)
1. The application of the stem cells in preparing the preparation for treating the leukoencephalopathy is characterized in that the neural stem cells are directionally differentiated into oligodendrocyte precursor cells in vitro, the oligodendrocyte precursor cells are loaded by taking hydrogel microspheres as a carrier to obtain a cell microcarrier, and the cell microcarrier and pharmaceutically acceptable auxiliary materials are prepared into an injectable preparation; the hydrogel microspheres are prepared from gelatin derivatives and carboxymethyl chitosan, wherein the structural formula of the gelatin derivatives is as follows:
2. Use of stem cells according to claim 1 for the preparation of a preparation for the treatment of leukoencephalopathy, wherein said gelatin derivative is prepared by:
s1, dispersing gelatin in deionized water, stirring and dissolving at 50-65 ℃, cooling to 35-45 ℃, adding potassium periodate, reacting for 5-8h in a dark place, adding ethylene glycol, continuously stirring for 0.5-1h, dialyzing, and freeze-drying to obtain aldehyde gelatin;
s2, dissolving miconazole in N, N-dimethylformamide to obtain a solution 1, dissolving trans-3-chloroacrylic acid in deionized water to obtain a solution 2, dropwise adding the solution 2 into the solution 1 in a nitrogen atmosphere, stirring, heating to 30-35 ℃ after dropwise adding is completed within 0.5-1h, continuously stirring for 7-9h to obtain a reaction mixture, and performing rotary evaporation, extraction, concentration and drying to obtain a miconazole derivative;
and S3, dispersing the aldehyde gelatin obtained in the step S1 into deionized water, heating to 60-75 ℃, stirring for 20-30min, sequentially adding sodium hydroxide, the miconazole derivative obtained in the step S2 and benzyltriethylammonium chloride, continuously stirring for 3-4h, adjusting the pH to 6-7 by using hydrochloric acid, and dialyzing, freezing and drying to obtain the gelatin derivative.
3. Use of the stem cells according to claim 2 in the preparation of a preparation for treating leukoencephalopathy, wherein the mass ratio of the gelatin, the deionized water, the potassium periodate and the ethylene glycol in step S1 is 1.
4. Use of the stem cells according to claim 2 in the preparation of a preparation for treating leukoencephalopathy, wherein the mass concentration of miconazole in the solution 1 in the step S2 is 0.16 to 0.20g/mL, the mass concentration of trans-3-chloroacrylic acid in the solution 2 is 0.1 to 0.15g/mL, and the molar ratio of miconazole to trans-3-chloroacrylic acid is 1.
5. Use of stem cells according to claim 2 for preparing a preparation for treating leukoencephalopathy, wherein deionized water is added to the reaction mixture before rotary evaporation in step S2, so that the volume ratio of N, N-dimethylformamide to deionized water in the reaction mixture is 1.
6. The use of the stem cells according to claim 2 for preparing a preparation for treating leukoencephalopathy, wherein the mass ratio of the aldehyde gelatin, the sodium hydroxide, the miconazole derivative and the benzyltriethylammonium chloride in the step S3 is 20-25.
7. Use of stem cells according to claim 2 for the preparation of a preparation for the treatment of leukoencephalopathy, wherein the cell microcarriers are prepared by a method comprising:
(1) Dispersing gelatin derivatives and carboxymethyl chitosan in deionized water, performing high-speed homogenization treatment and vacuum filtration sterilization, adding a cell suspension containing oligodendrocyte precursor cells, and uniformly blowing to obtain a prefabricated solution;
(2) Dripping the prepared solution obtained in the step (1) into sterile mineral oil, heating to 36-38 ℃, preserving heat for 10-12h to obtain hydrogel microspheres loaded with cells, filtering, collecting, and cleaning with sterile phosphate buffer solution to obtain the injectable cell microcarrier.
8. Use of the stem cells according to claim 7 for preparing a preparation for treating leukoencephalopathy, wherein the gelatin derivative is added in the pre-prepared solution in the step (1) in an amount of 0.03-0.06g/mL, the carboxymethyl chitosan is added in the pre-prepared solution in an amount of 0.02-0.04g/mL, and the high-speed homogenization treatment is carried out at 1000-1200r/min for 10-15s.
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