CN117487753A - Establishment and application of therapeutic microglial cells - Google Patents
Establishment and application of therapeutic microglial cells Download PDFInfo
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- CN117487753A CN117487753A CN202311439224.8A CN202311439224A CN117487753A CN 117487753 A CN117487753 A CN 117487753A CN 202311439224 A CN202311439224 A CN 202311439224A CN 117487753 A CN117487753 A CN 117487753A
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- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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- C12N2510/00—Genetically modified cells
Abstract
The invention discloses establishment and application of therapeutic microglial cells. According to miR-145a-5p, activation of microglial cells to M2 type can be promoted in vitro, activation of microglial cells to M1 type can be inhibited, and apoptosis of injured neurons can be reduced, and by using cultured high-purity primary microglial cells of mice to carry out M2 polarization treatment and transfecting miR-145a-5p, activation of M2 type microglial cells can be enhanced, and survival number of M2 type microglial cells in an injured spinal cord region can be increased and survival time can be prolonged.
Description
Technical Field
The invention belongs to the field of regenerative medicine, relates to biological products for treating spinal cord injury, and in particular relates to culture of primary microglia and preparation of miRNA modified M2 microglia by using the culture.
Background
Microglia can be broadly classified into resting M0 microglia, classical activated M1 microglia and alternatively activated M2 microglia. M1 microglial cells can be induced by LPS, IFN-gamma and the like, and can express marker molecules TNF-alpha, IL-6, IL-1 beta, NOS2 and the like, and have cytotoxicity and pro-inflammatory reaction effects; the M2 microglial cell can be induced by IL-4, and can express marker molecules Arg1, TGF-beta, IL-10, YM1, etc., and has neuroprotection, tissue repair promoting, and inflammatory reaction inhibiting effects. Under the action of the damaged microenvironment, microglial cells are mainly activated into M1 type and continuously exist in the damaged area of spinal cord, while M2 type microglial cells only occur briefly, which may be one of the causes of inflammatory progression and poor tissue repair in the damaged area.
Micrornas (mirnas) are non-coding RNAs of about 22 nucleotides in length that inhibit translation of a target gene protein by binding to the 3' -untranslated region (3 ' -untranslated region,3' -UTR) of the target gene mRNA, thereby regulating the biological function of the cell. MiRNA exists in various forms, the primary one is pri-miRNA, and the length is about 300-1000 bases; the pri-miRNA becomes a pre-miRNA, namely a microRNA precursor, after one-time processing, and the length is about 70-90 bases; the pre-miRNA is subjected to Dicer enzyme digestion to form mature miRNA with the length of 20-24 nt. MiRNA plays a great role in the processes of cell differentiation, biological development and disease occurrence and development, and is attracting more and more attention of researchers.
The use of cell transplantation to produce neuroprotection and regeneration is one of the most potential therapies for the treatment of spinal cord injury (spinal cord injury, SCI). Cells used for transplantation mainly include schwann cells, olfactory ensheathing cells, fibroblasts, neural stem cells, embryonic stem cells, bone marrow mesenchymal stem cells and induced pluripotent stem cells, and important roles of the cells at molecular and tissue levels include: replacement of lost neurons and glial cells, secretion of neurotrophic factors, stimulation of tissue retention and revascularization, reconstruction of neural pathways, restriction and filling of the post-injury capsule cavity, and promotion of axon regeneration and remyelination. Cells from each tissue source for transplantation are affected by the immune system in the spinal cord injury area, including affecting cell survival number and activity.
Microglial cells are important immune cells in the damaged area after spinal cord injury, and modulation of the activation state of microglial cells in the damaged area helps promote SCI repair, e.g., shuhei Kobashi et al transplanted M2 microglial cells into an animal spinal cord injury model, which was found to inhibit inflammatory responses, promote axonal regeneration and myelination. However, because of the damage microenvironment of the spinal cord injury region, the phenotype of the transplanted M2 microglial cells is weakened and the functions of the transplanted M2 microglial cells are lost, so that the search for a way capable of maintaining the M2 activation phenotype is particularly important for repairing spinal cord injury. It has been demonstrated that inhibition of M1 microglial activation and/or promotion of M2 microglial activation in the spinal cord injury zone by miRNA can reduce inflammatory response in the spinal cord injury zone, promote repair of spinal cord and recovery of hindlimb motor function in spinal cord injured animals (research on the action and mechanism of miR-145a-5p modified microglial cells in spinal cord injury treatment, DOI:10.26914/c.cnkihy.2021.070637; the fourteenth national academy of immunology, 2021-10-21), but its application in clinical treatment of spinal cord injury has yet to be studied further, and the problems that have not yet been solved include: after in situ transplantation in the spinal cord injury area, the phenotype of M2 microglial cells is not significant enough, the survival time is not long enough, and the recovery of the motor function in the later stage of spinal cord injury is affected (BMS score only reaches 4.5-5, and BMS subscore only reaches 4-4.5).
Disclosure of Invention
The invention aims to provide establishment and application of therapeutic microglial cells.
In order to achieve the above purpose, the invention adopts the following technical scheme:
a method of culturing primary microglial cells comprising the steps of:
1) Immersing the mice in pre-cooled 70% -75% alcohol, and taking brain tissues after the mice lose consciousness;
2) Cutting brain tissue into chylomorphic, then using digestive juice to digest in cell incubator at 35-37 deg.C for 15-20 min, shaking every 5-10 min; the digestive juice contains 0.125 to 0.2 percent of pancreatin and 0.02 to 0.04 percent of EDTA;
3) Repeatedly blowing with a suction tube for at least 100 times after digestion is stopped to obtain a cell suspension, filtering tissue fragments in the cell suspension with a 200-300 mesh filter screen, and centrifuging the filtered cell suspension;
4) After the step 3, the upper liquid is absorbed, and the cells are resuspended by using a culture medium containing 10 to 15 percent of serum and 1 to 1.5 percent of glutamine; mixing the obtained cell suspension with macrophage colony stimulating factor (macrophage colony stimulating factor, M-CSF) with the final concentration of 20-30 ng/mL in a culture flask, then placing the culture flask in a cell incubator with the temperature of 35-37 ℃ for culturing for 12-14 days, changing a culture medium containing 10-15% of serum and 1-1.5% of glutamine every 2-3 days during the culturing period, and adding the macrophage colony stimulating factor with the final concentration of 20-30 ng/mL;
5) After the step 4, placing the culture flask on a shaking table at 35-37 ℃ and shaking for 2-4 hours at 200-250 rpm, and collecting primary microglial cells (specifically M0 microglial cells) in culture supernatant by centrifugation after shaking.
Preferably, in step 1, the mice are selected from neonatal P1-P3C 57 mice.
Preferably, in step 1, the method for obtaining brain tissue specifically comprises the following steps: after the mice are sacrificed, the brains are taken out, the brain hemispheres at the two sides are selected to strip the pia mater, and the callus bodies connected with the brain hemispheres at the two sides in the middle are discarded.
Preferably, in step 2, the amount of digestive juice is as follows: 5mL of digestive juice is used for chylomicron brain tissue of 3-5 mice.
Preferably, in step 3 and step 5, the centrifugation conditions are: centrifuging for 4-5 min at the temperature of 4-6 ℃ and the pressure of 300-350 g.
The application of the culture method of the primary microglial cells in preparing a miRNA preparation for enhancing the phenotype of M2 microglial cells in a spinal cord injury area, wherein the miRNA preparation comprises M2 microglial cells modified by miR-145a-5p (GUCCAGUUUUCCCAGGAAUCCCU), and the M2 microglial cells are obtained by carrying out M2 activation and miRNA transfection on the primary microglial cells obtained by the culture method.
Preferably, the miRNA formulation further comprises a carrier for loading miR-145a-5p modified M2 microglial cells into the spinal cord injury zone.
Preferably, the carrier comprises matrigel.
Preferably, the miRNA preparation is in the form of injection, and each 2.5-3.5 mu L of miRNA preparation contains 2.0-3.0X10 5 And M2 type microglia modified by miR-145a-5 p.
Preferably, the preparation of the miR-145a-5p modified M2 microglial cell specifically comprises the following steps: pretreating primary microglial cells (particularly M0 microglial cells) by using macrophage colony stimulating factors, activating the pretreated M0 microglial cells by using M2 polarization stimulating factors to obtain M2 microglial cells, transfecting the M2 microglial cells by using miR-145a-5p, and then carrying out induction culture by using the macrophage colony stimulating factors to obtain miR-145a-5p modified M2 microglial cells.
Preferably, the pretreatment specifically includes the following steps: inoculating the primary microglial cells (particularly M0 microglial cells) into a cell culture plate, then culturing the cells in a cell incubator at the temperature of 35-37 ℃ for 18-24 h, adopting a culture medium containing 10-15% fetal bovine serum in the culture, and adding macrophage colony stimulating factor with the final concentration of 20-30 ng/mL.
Preferably, the transfection conditions are: the use of serum-free cultures supplemented with transfection reagents and 100-150 mM miR-145a-5p (e.g., miR-145a-5p mimics) is based on culture at 35-37℃for 6-8 h.
Preferably, the transfection reagent is lipofectamine TM 2000，lipofectamine TM The transfection ratio (volume ratio) of 2000 to miR-145a-5p is 1:2-3, so that miR-145a-5p is effectively loaded in cells, and activation of M2 microglial cells is promoted.
Preferably, the conditions of the induction culture are: after the transfection is finished, the culture which is added with macrophage colony stimulating factor with the final concentration of 20-30 ng/mL and contains 10-15% of fetal bovine serum is used for culturing for 18-20 h at the temperature of 35-37 ℃.
The beneficial effects of the invention are as follows:
when mixed glial cells are separated from sheared brain tissue, low-concentration pancreatin is adopted, and a small amount of EDTA is added into a digestive system, so that the activity of the cells is not damaged, the cells can be digested into a single-cell state better, the phenomenon of cell agglomeration is reduced, the cells are treated by combining macrophage colony stimulating factors, and finally primary microglial cells with obviously improved purity can be obtained through oscillation and centrifugation (the primary microglial cells are proved by detection of an optical microscope and a flow cytometry, and most of the primary microglial cells are M0 type microglial cells).
The preparation containing the M2 type microglial cell modified by miR-145a-5p is prepared by using the high-purity primary microglial cell obtained in the invention, the phenotype of the M2 type microglial cell can be enhanced (after miR-145a-5p is transfected, the M2 type microglial cell has a stronger anti-inflammatory and pro-repair phenotype, and the pro-inflammatory and pro-injury capacity of the M1 microglial cell is also obviously reduced), and the microglial cell can be maintained to survive for a longer time in a spinal cord injury area after the miRNA preparation is implanted, so that the recovery of the later-stage motor function of spinal cord injury is promoted.
Furthermore, the optimized transfection proportion is adopted in the invention, so that the transfection efficiency is high, and cells after transfection have stronger anti-inflammatory effect, and the activation of microglial cells in a spinal cord injury area and the regulation and control of immunoinflammatory response can be promoted, thereby improving the restoration of spinal cord injury.
Furthermore, the M2 type microglial cells modified by miR-145a-5p are wrapped in matrigel, and after the miRNA preparation is transplanted to a spinal cord injury area, the cells in the matrigel are in islands (not dispersed into single cells), so that the functions of the M2 type activated microglial cells can be effectively exerted, and an effective drug treatment path reference is provided for clinically repairing spinal cord injury.
Drawings
FIG. 1 shows the results of miR-145a-5p expression in microglia of different activation types (M0, M1 and M2 types); in the figure: * There was a statistically significant difference between the finger groups.
FIG. 2 is a graph showing the results of the effect of miR-145a-5p mimetic (mic) on microglial activation; in the figure: * There was a statistically significant difference between the finger groups.
FIG. 3 is a graph showing the effect of miR-145a-5p inhibitor (ASO) on microglial activation; in the figure: * There was a statistically significant difference between the finger groups.
FIG. 4 is an effect of miR-145a-5p modified microglial cells of different activation types on impaired neuronal (Neuron) apoptosis; in the figure: * There was a statistically significant difference between the finger groups.
FIG. 5 is a flow chart of treatment of spinal cord injury by transplantation of miR-145a-5p modified M2 microglial cells after primary microglial cell culture, miR-145a-5p mimetic modification (A) and spinal cord injury modeling (B).
Fig. 6 is the results of evaluating the influence of hindlimb motor functions in spinal cord injured mice (n=6 to 8) with reference to BMS score (Basso Mouse Scale, BMS) after transplanting miR-145a-5p modified M2 microglial cells; in the figure: # means that SCI is statistically significantly different from sci+m2; * Meaning that SCI is statistically significantly different from SCI+M2+miR-145a-5 p; what is meant is that sci+m2 has a statistically significant difference from sci+m2+mir-145a-5 p.
FIG. 7 is a graph showing the effect of improving the M2 activation of the M2 type microglial cells of miR-145a-5p modified cells transplanted before and after the improvement (7 days after the transplantation).
FIG. 8 is an illustration of the effect of transplanting miR-145a-5p modified M2 microglia cells before and after modification on glial scar and inhibitory matrix in the spinal cord injury zone of mice.
FIG. 9 shows the morphological differences of primary microglial cells cultured before and after modification.
FIG. 10 is an identification of miR-145a-5p transfection efficiency by real-time quantitative PCR before and after improvement.
FIG. 11 is an identification of miR-145a-5p transfection efficiency by immunofluorescence before and after improvement.
FIG. 12 is a graph showing the effect of the modified M2 microglial cells of miR-145a-5p on the M2 activation of microglial cells in the spinal cord injury zone of mice before and after modification (28 days after implantation).
Detailed Description
The invention is described in further detail below with reference to the drawings and examples. The examples are given solely for the purpose of illustration and are not intended to limit the scope of the invention.
1. Main reagent and instrument
DMEM medium, macrophage colony stimulating factor (macrophage colony stimulating factor, M-CSF), fetal bovine serum, glutamine, polylysine, lipofectamine TM 2000. Super clean bench, constant temperature incubator, bench type high-speed low temperature centrifuge, high pressure steam sterilizing pot, constant temperature table, real-time quantitative PCR instrument, chemiluminescent instrument, dumont system forceps, stereoscopic microscope, constant cooling frozen slicer, -20 ℃ low temperature refrigerator, -80 ℃ low temperature refrigerator and laser confocal microscope.
2. Method and results
2.1 culture of Primary microglial cells
2.1.1 soaking P1C 57BL/6J neonatal mice in pre-cooled (4deg.C) 75% (v/v) alcohol for sterilization for 5min, and taking materials after the mice lose consciousness; using an ophthalmic scissors to break the ends of the mice, and using forceps to strip the skin of the heads; peeling the skull from the back to the front along the herringbone in a precooled DMEM medium, and placing the brain in another prepared precooled DMEM medium;
2.1.2 stripping the pia with a fiber ophthalmic forceps, shearing the brain tissue of 5 exposed mice (only selecting two lateral hemispheres, discarding the callus of the middle joint two lateral hemispheres) into chylomorphic, adding 5mL of digestive juice (0.125% pancreatin+0.02% EDTA), placing in a 37 ℃ cell constant temperature incubator for digestion for 20min, and shaking every 5 min; the preparation method of the digestive juice comprises the following steps: weighing 0.125g of pancreatin powder and 0.02g of EDTA powder, adding into 100mL of sterile PBS (pH 7.0), and fully and uniformly mixing;
2.1.3 after digestion, the digestion was terminated by adding DMEM medium containing 10% (v/v) fetal bovine serum and 1.5% (g/mL) glutamine, repeatedly blowing 100 times using a glass pipette, filtering the tissue fragments in the resulting cell suspension with a 300 mesh screen, and then placing in a centrifuge at 4℃and centrifuging at 350g for 4min;
2.1.4 after the supernatant liquid was removed, 10mL of a 10% (v/v) fetal bovine was addedSerum and 1% (g/mL) glutamine in DMEM medium, and the resulting cell suspension was transferred to 75cm after repeated 100-times pipetting using a glass pipette 2 In a flask, M-CSF was added at a final concentration of 25ng/mL, placed in a 37℃incubator and mixed glial cells (mixed gli cells) were cultured according to the following procedure (FIG. 5): the DMEM medium containing 10% fetal calf serum and 1% glutamine and 25ng/mL M-CSF was replaced every 3d and co-cultured for 2 weeks;
2.1.5 After 2 weeks the flask was fixed to a thermostatic shaker at 37℃with 200rpm shaking (mechanical rocking) for 4h (FIG. 5); transferring the culture supernatant into a centrifuge tube, placing the centrifuge tube into the centrifuge tube for re-centrifugation, discarding the supernatant, and obtaining the primary microglia (microglia) which is mature in culture at the bottom of the centrifuge tube, and carrying out flow cell identification and counting. The results show that after the mixed glial cells are cultured for 2 weeks, the oscillated cells (microglial cells are dissociated and enter culture supernatant) are collected through centrifugation after shaking continuously by a shaking table, and 98% -99% of flow cell identification are M0 microglial cells, namely, the purity of primary microglial cells is 98% -99%.
The primary microglial cell culture method (before improvement for short) adopted in the early test ("effect and mechanism study of miR-145a-5p modified microglial cells in spinal cord injury treatment") is different from the culture method of primary microglial cells (after improvement for short) in the above 2.1, and mainly comprises the following steps: in the early test, the pia mater and the callus are not removed in the primary microglial cell culture operation (the callus is a fiber bundle connecting the left and right hemispheres of the brain, and the pia mater contains thicker pia mater, and blood vessels in the pia mater are rich, so that the pia mater is not thoroughly stripped, which easily causes vascular endothelial cells to be mixed in single cell suspension), and pancreatin (for example, 0.25 percent pancreatin) with higher concentration is used, but the purity of the primary microglial cells obtained after culture only reaches 90 percent.
Taking a 12-well plate as an example for cell counting, when the cell inoculation density in a cell culture plate is 70% -80%, the primary microglial obtained after improvement is less prone to aging than the primary microglial obtained before improvement, and is more transparent and more obvious in axon stretching (figure 9), the activity and the morphology of the primary microglial obtained after improvement are better, the primary microglial is beneficial to obtaining higher transfection efficiency at a later stage and survival for a longer time in a transplanted damaged spinal cord region, and thus the functions of the cells can be better exerted.
2.2 transfection and activation Induction of miR-145a-5p in microglia
Experiment one
Mature microglial cells (primary microglial cells obtained in step 2.1.5) cultured for 14 days were cultured at a ratio of 1.5X10 6 The wells were plated in 12-well plates uniformly so that the cell density was controlled at 70% -80% (mature primary microglia were not proliferated in general, so cells were seeded at 70% -80% cell density per well), three groups were set, each group was respectively cultured in 1mL DMEM medium supplemented with 25ng/mL M-CSF and containing 10% fetal bovine serum at a final concentration in a 37 ℃ incubator for 8 hours, and then M0 (PBS, ph 7.0), M1 polarization treatment, or M2 polarization treatment was performed, wherein M1 polarization treatment was performed with the addition of 100ng/mL Lipopolysaccharide (LPS) and 20ng/mL interferon- γ (IFN- γ), and M2 polarization treatment was performed with the addition of 20ng/mL interleukin-4 (IL-4). Polarization treatment was completed after further culturing for 24 hours, and the miR-145a-5p expression level of each group of microglial cells was detected, and the results are shown in FIG. 1. The results show that the actual expression conditions of miR-145a-5p in the microglia cells with different polarization types of M0, M1 and M2 are obviously different, and miR-145a-5p is low-expressed in the microglia cells with the M1 type and high-expressed in the microglia cells with the M2 type.
Experiment two
Mature microglial cells (primary microglial cells obtained in step 2.1.5) cultured for 14 days were cultured at a ratio of 1.5X10 6 Uniformly inoculating the cells into a 12-hole plate to control the cell density to be 70% -80%, and setting six groups of cells, wherein the groups are as follows: ctrl-M0, ctrl-M1, ctrl-M2, miR-145a-5p-M0, miR-145a-5p-M1 and miR-145a-5p-M2; each group was incubated with 1mL of DMEM medium containing 10% fetal bovine serum and added to a final concentration of 25ng/mL of M-CSF for 18h at 37℃in a constant temperature incubator followed by M0 (PBS, pH 7.0), M1[ lipopolysaccharide (LPS 100 ng/mL) +interferon-gamma (IFN-. Gamma.20 ng/mL)]Polarization treatment or M2[ interleukin-4 (IL-4 20 ng/mL)]Polarization treatment, sucking and discarding the culture medium after 24 hours polarization treatment, using noBacterial PBS (pH 7.0) was washed 1 time, and 1mL of serum-free DMEM medium and lipofectamine with a final concentration of 2.5. Mu.L/mL were added to each of miR-145a-5p-M0, miR-145a-5p-M1 and miR-145a-5p-M2 groups TM 2000 and 100mM miR-145a-5p mic (synthesized by Guangzhou Ruibo Biotechnology Co., ltd.) forms a transfection solution (lipofectamine) TM 2000 to miR-145a-5p mic at a 1:2 ratio by volume) for transfection (transfection) of miR-145a-5p mic into microglial cells. Similarly, control oligonucleotides (NC, 5' -UUUGUACUACACAAAAGUACUG) were added to the Ctrl-M0, ctrl-M1, ctrl-M2 groups, respectively, according to the transfection procedure described above (FIG. 5). After 6h incubation in a 37℃incubator, the transfection solution was aspirated, replaced with DMEM medium containing 10% fetal bovine serum, and M-CSF was supplemented to a final concentration of 25ng/mL, and after 18h incubation, the mRNA levels of TNF- α, IL-6, IL-1 β, NOS2 and Arg1, mrc1, YM1 and IL-10 were measured for each group of microglia. The results indicate (FIG. 2), the miR-145a-5p mimic can inhibit activation of microglia to M1 and promote activation thereof to M2.
And using the primary microglial cells obtained after the modification compared to the primary microglial cells obtained before the modification: the transfection efficiency is improved; transfection of the miR-145a-5p mimic inhibited the inflammatory response more significantly in M1 microglia and the anti-inflammatory response more significantly in M2 microglia.
Experiment three
Experimental procedure referring to experiment two, a miR-145a-5p inhibitor (ASO) was transfected into primary microglia, and the results indicated (fig. 3) that the miR-145a-5p inhibitor could promote microglia activation to M1 and inhibit activation to M2.
And using the primary microglial cells obtained after the modification compared to the primary microglial cells obtained before the modification: the transfection efficiency is improved; transfection of the miR-145a-5p inhibitor showed more pronounced inflammatory response in M1 microglia and decreased anti-inflammatory response in M2 microglia.
Experiment four
The result of detecting the transfection of miR-145a-5p mimic in microglial cells (primary microglial cells obtained in step 2.1.5) by real-time quantitative PCR is shown in FIG. 10, and the efficiency of the transfection of miR-145a-5p by the primary microglial cells obtained after the improvement is improved to about 1000 times compared with the primary microglial cells obtained before the improvement; meanwhile, the immunofluorescence detection result (figure 11) shows that the fluorescence intensity of the improved double-positive co-located cells is higher. These results indicate that the transfection efficiency of the primary microglial cells obtained after the improvement is improved.
2.3 inhibition of apoptosis of damaged neurons by miR-145a-5p modified microglia
Culturing the culture supernatant of microglial cells of Ctrl-M0, ctrl-M1, ctrl-M2 and miR-145a-5p-M0, miR-145a-5p-M1 and miR-145a-5p-M2 groups with H 2 O 2 As a result of co-culture of neurons damaged by oxidative stress after treatment, it was found (FIG. 4) that not only the miR-145a-5p-M2 group (compared with the Ctrl-M2 group) was able to inhibit apoptosis of damaged neurons, but also the miR-145a-5p-M1 group had the ability to inhibit apoptosis of damaged neurons compared with the Ctrl-M1 group.
Compared with the primary microglial cells obtained before improvement, the experimental effect of the primary microglial cells obtained after improvement is more remarkable: the ability to resist neuronal apoptosis after co-culture with oxidative stress damaged neurons is greater, allowing the damaged primary cultured neurons to survive more.
2.4 spinal cord injury animal model
Male C57BL/6J mice of 6-8 weeks were injected with 0.6% sodium pentobarbital solution per 10g body weight per 0.1mL abdominal cavity, shaved back hair after sufficient anesthesia, sterilized skin, and were in prone position, and the limbs and tail were fixed using tape. The T2 thoracic spinous process of the mouse is more prominent and can be reached by hands, and an incision with the length of about 1 cm to 1.5cm is made along the spinal spinous process line at the position of 0.5cm below the T2 thoracic vertebra. Care was taken to blunt isolate the subcutaneous fat after hemostasis, taking care not to damage the venous sinus of the neck. The muscle layers at two sides are cut along the spinous process until reaching the vertebral lamina of the vertebral body of the spine, the suture thread is used for penetrating the muscle, and the cut muscle is pulled towards two sides to expose the vertebral body.
When observed under a body microscope (operating microscope, OLYMPUS, TOKYO, JAPAN), veins exist above T6, the spinous processes of T7, T8 and T9 are sharp, the vertebral lamina is smooth, the spinous process gap between T9 and T10 is narrow, and the spinous process mutation is flat after T11, so that the thoracic vertebra of T8 is positioned. The instrument is used for biting the spinous process and the vertebral lamina of T8 under the condition of not damaging the dura mater, so that the spinal cord is fully exposed, and a longitudinal pulsating blood vessel on the back side of the spinal cord can be seen. With Dumont tie forceps, after penetrating completely into both sides of the spinal cord, perpendicular to the spinal cord jaws for 30 seconds. After sufficient hemostasis, the surgical field was flushed with saline, i.e., a spinal cord jaw injury model was established (spinal cord congestion edema was visible after jaw injury), and cell transplantation was prepared (fig. 5).
2.5 pretreatment of transplanted cells (modification of M2 microglia with miR-145a-5p before transplantation)
Pretreatment herein refers to the use of lipofectamine TM 2000, miR-145a-5p mimic was transfected into M2 polarized microglial cells after primary culture to obtain miR-145a-5p modified M2 microglial cells (see, in particular, experiment two miR-145a-5p-M2 groups).
2.6 repair experiments for in situ transplantation of miR-145a-5p modified M2 microglial cells at spinal cord injury
2.6.1 preparation of preparation containing miR-145a-5p modified M2 microglial cells
(1) Digestion of miR-145a-5p modified M2 microglia
Washing with 1 XPBS for two times after sucking and discarding the culture medium, adding digestive juice (2.5% pancreatin+0.02% EDTA), placing in a 37 DEG constant temperature incubator for digestion for 10 minutes, adding DMEM culture medium containing 10% fetal calf serum to terminate digestion, and gently beating to prepare single cell suspension;
(2) Placing in a centrifuge at 4deg.C, centrifuging at 350g for 4min, and removing supernatant;
(3) Adding 1 XPBS, shaking, mixing, centrifuging at 4deg.C for 4min, and discarding supernatant;
(4) Repeating the step 3 for three times, and then performing cell counting;
(5) Adding matrigel to make every 3 μl matrigel contain 2.5X10 5 Individual cells.
2.6.2 matrigel containing cultured mature miR-145a-5p modified M2 microglial cells is transplanted into damaged spinal cord (injection volume is 3 mu L) by a microinjector by a stereotactic instrument, and Fang Jirou groups and skin are sutured layer by layer after hemostasis and disinfection, and then the spinal column is disinfected.
2.7 MiR-145a-5p modified M2 microglial cells after transplantation promote recovery of hindlimb motor function of spinal cord injured mice
As shown in FIG. 6, the transplanted miR-145a-5p modified M2 microglial cell group (SCI+M2+miR-145 a-5 p) significantly promoted recovery of motor function in later stages in spinal cord injured mice (BMS score of 5.5-6, BMS subscore of 5-5.5) compared to sham, spinal cord injury alone (SCI) and transplanted M2 microglial cell group (SCI+M2).
2.8 enhancement of expression of M2-type marker molecule Arg1 in spinal cord injury region after transplanting miR-145a-5p modified M2-type microglial cells, reduction of GFAP-marked gliosis and reduction of CS 56-marked inhibitory matrix deposition
First, glial scar is an important obstacle affecting nerve fiber regeneration. Whereas CS 56-labeled CSPGs are an important component in gliosis. It has been found that during nerve injury, a large amount of CSPGs proteins accumulate at the injured site, limiting the occurrence and elongation of nerve synapses on the one hand, and recruiting other proteins and factors to prevent nerve regeneration after injury on the other hand. By carrying out pretreatment of transplanted cells on primary microglia obtained before and after the improvement, respectively transplanting the treated miR-145a-5p modified M2 microglia into a spinal cord injury area in an injection mode, and as shown in a result, compared with the miR-145a-5p modified M2 microglia obtained after the improvement pretreatment, the miR-145a-5p modified M2 microglia obtained after the improvement can reduce the nerve scar hyperplasia marked by GFAP (namely, the colloid scar hyperplasia marked by GFAP in the spinal cord injury area is obviously inhibited), and the deposition of an inhibitory matrix CSPGs is obviously reduced, so that the connection of nerve fibers and the restoration of nerve functions are facilitated.
Secondly, as shown in fig. 7 (CX 3CR 1-labeled microglial cells after 7 days of transplantation), compared with miR-145a-5 p-modified M2 microglial cells obtained by the modified pretreatment, miR-145a-5 p-modified M2 microglial cells obtained by the modified pretreatment can significantly enhance the expression of M2-labeled molecule Arg1 in the damaged spinal cord region, thereby promoting the repair of damaged spinal cord and the restoration of motor function by expressing stronger repair-promoting M2-labeled Arg 1.
Finally, FIG. 12 (CX 3CR 1-labeled microglial cells 28 days after transplantation) also objectively shows that the survival number of M2 microglial cells in the damaged spinal cord area after transplantation can be increased and the survival time can be prolonged by improving experimental operation.
3. Application exploration
According to the invention, based on the influence of miR-145a-5p on activation of microglia, namely, in-vitro experiments show that miR-145a-5p can promote activation of microglia to M2 type and inhibit activation of microglia to M1 type, the efficiency of transfection of miR-145a-5p into M2 type microglia is improved by optimizing culture of primary microglia, and treatment experiments are carried out on spinal cord injury of mice by transferring miR-145a-5p modified M2 type microglia through matrigel, so that the effects of remarkably inhibiting inflammatory reaction in an injured area, reducing apoptosis of neurons, reducing inhibitory matrix deposition, reducing fibrous scar hyperplasia in situ of the injured spinal cord are obtained, and the recovery of limb movement function is remarkably improved (higher BMS score is obtained). Thus, the invention provides an important reference for the induction of microglial cells for in situ repair of spinal cord injury by human autologous stem cells.
In a word, the invention uses the established standardized mouse clamp spinal cord injury model to transfer miR-145a-5p modified M2 microglial cells for in-situ transplantation of a spinal cord injury region, so that the activation proportion and number of the M2 microglial cells in the spinal cord injury region are maintained at a higher level, the activation proportion and number of the M1 microglial cells are relatively smaller, the inflammatory reaction of the spinal cord injury region is reduced, and the number of surviving neurons is increased, thereby effectively promoting the recovery of limb movement function after spinal cord injury.
Claims (10)
1. A method for culturing primary microglial cells, which is characterized in that: the method comprises the following steps:
1) Immersing the mice in pre-cooled 70% -75% alcohol, and taking brain tissues after the mice lose consciousness;
2) Cutting brain tissue into chylomorphic, then digesting for 15-20 min at 35-37 ℃ by using digestive juice, and shaking every 5-10 min in the digestion; the digestive juice contains 0.125 to 0.2 percent of pancreatin and 0.02 to 0.04 percent of EDTA;
3) Repeatedly blowing with a suction tube after digestion is stopped to obtain a cell suspension, filtering tissue fragments in the cell suspension with a filter screen, and centrifuging the filtered cell suspension;
4) After the step 3, the upper liquid is absorbed, and the cells are resuspended by using a culture medium containing 10 to 15 percent of serum and 1 to 1.5 percent of glutamine; mixing the obtained cell suspension with macrophage colony stimulating factor with the final concentration of 20-30 ng/mL, culturing at 35-37 ℃ for 12-14 days, changing a culture medium containing 10-15% serum and 1-1.5% glutamine every 2-3 days during the culturing period, and adding the macrophage colony stimulating factor with the final concentration of 20-30 ng/mL;
5) After the step 4, oscillating for 2-4 hours at the temperature of 35-37 ℃ and the rpm of 200-250 rpm, and collecting primary microglial cells in culture supernatant by centrifugation after oscillating.
2. The method for culturing primary microglial cells according to claim 1, wherein: in step 1, the mice were selected from neonatal P1-P3C 57 mice.
3. The method for culturing primary microglial cells according to claim 1, wherein: in the step 1, the material brain tissue comprises the following steps: after the mice are sacrificed, the brains are taken out, the brain hemispheres at the two sides are selected to strip the pia mater, and the callus bodies connected with the brain hemispheres at the two sides in the middle are discarded.
4. The method for culturing primary microglial cells according to claim 1, wherein: in the step 2, the consumption of the digestive juice is as follows: 5mL of digestive juice is used for chylomicron brain tissue of 3-5 mice.
5. The method for culturing primary microglial cells according to claim 1, wherein: in the step 3 and the step 5, the centrifugation conditions are as follows: centrifuging for 4-5 min at the temperature of 4-6 ℃ and the pressure of 300-350 g.
6. Use of the culture method of primary microglial cells according to any of claims 1 to 5 for the preparation of a miRNA preparation for enhancing the M2 microglial phenotype in the spinal cord injury zone, characterized in that: the miRNA preparation comprises miR-145a-5p modified M2 type microglial cells, wherein the M2 type microglial cells are obtained by carrying out M2 type activation and miRNA transfection on primary microglial cells obtained by the culture method.
7. The use according to claim 6, characterized in that: the miRNA formulation further comprises a carrier for loading miR-145a-5p modified M2 microglia cells in a spinal cord injury zone, wherein the carrier comprises matrigel.
8. The use according to claim 6, characterized in that: the miRNA preparation is in the form of injection, and each 2.5-3.5 mu L of miRNA preparation contains 2.0-3.0X10 5 And M2 type microglia modified by miR-145a-5 p.
9. The use according to claim 6, characterized in that: the preparation method of the miR-145a-5p modified M2 microglial cell specifically comprises the following steps: pretreating the primary microglial cells by using macrophage colony stimulating factors, activating the pretreated primary microglial cells by using M2 polarization stimulating factors to obtain M2 microglial cells, transfecting the M2 microglial cells by using miR-145a-5p, and then carrying out induction culture by using the macrophage colony stimulating factors to obtain miR-145a-5p modified M2 microglial cells; wherein the transfection conditions are: the serum-free culture with the transfection reagent and 100-150 mM miR-145a-5p is used for culturing for 6-8 hours at the temperature of 35-37 ℃, and the capacity ratio of the transfection reagent to miR-145a-5p is 1:2-3.
10. The use according to claim 9, characterized in that: the pretreatment specifically comprises the following steps: inoculating the primary microglial cells, culturing for 18-24 hours at 35-37 ℃, adopting a culture medium containing 10-15% of serum in the culture, and adding macrophage colony stimulating factor with the final concentration of 20-30 ng/mL; the conditions of the induction culture are as follows: after the transfection is finished, the macrophage colony stimulating factor with the final concentration of 20-30 ng/mL is added, and the culture containing 10-15% of serum is cultured for 18-20 h at the temperature of 35-37 ℃.
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