CN115927173A - Hypoxia mesenchymal stem cell exosome and application thereof - Google Patents
Hypoxia mesenchymal stem cell exosome and application thereof Download PDFInfo
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Abstract
The invention provides an anoxic mesenchymal stem cell exosome and application thereof, and belongs to the technical field of biomedicine. The hypoxia mesenchymal stem cell exosome is obtained by secreting human umbilical cord mesenchymal stem cells which are pretreated for 40-50 hours by 0.5-3% of oxygen concentration. The exosome can promote spinal cord astrocytes to be polarized to A2, enhance the expression of spinal cord tissue neurofilament protein and tubulin, further promote the repair of spinal cord tissue structures and achieve the effect of recovering the spinal cord injury movement function.
Description
Technical Field
The invention belongs to the technical field of biomedicine, and particularly relates to an anoxic mesenchymal stem cell exosome and application thereof.
Background
Spinal Cord Injury (SCI) is a devastating disease that often results in permanent paraplegia with significant physical distress and economic loss to the patient. Spinal decompression surgery and high doses of glucocorticoids (such as methylprednisolone) are currently the major clinical treatments. However, these treatments have a low probability of complete recovery of the patient and can cause a number of side effects. The pathophysiological process of spinal cord injury is closely related to astrocyte polarization. Normally, spinal astrocytes have a wide range of functions including neurotransmitter circulation, regulation of synaptogenesis, formation and maintenance of the blood-brain barrier, environmental homeostasis, and immune signaling, and once a central nervous system is injured or diseased, astrocytes are induced to activate and develop both A1 and A2 polarization states. A1 astrocytes are found to release neurotoxic factors such as complement components, inflammatory cytokines, and the like, and mediate neuronal and oligodendrocyte cell death, thereby promoting neuropathological progression. In contrast, A2 astrocytes exhibit neuroprotective functions through the production of anti-inflammatory cytokines and neurotrophic factors, thereby promoting recovery and repair of the central nervous system. Thus, the reactivity of astrocytes to different polarizations after spinal cord injury can affect the recovery of spinal cord function.
Mesenchymal stem cell-derived exosomes contain many different components including DNA, RNA, lipids, proteins and metabolites. Exosomes can reach recipient cells and deliver therapeutic molecules through respective pathways, and can play important roles in tissue repair, immune regulation, and neuroprotection. Since mesenchymal stem cells are typically in the hypoxic environment of the stem cell niche in vivo, the hypoxic environment in vitro is more conducive to maintaining their proliferation, differentiation, and self-renewal. However, there is no report on the treatment of spinal cord injury by hypoxia-pretreated mesenchymal stem cell-derived exosomes.
Disclosure of Invention
In view of the above, the present invention provides an anaerobic mesenchymal stem cell exosome and an application thereof, wherein the anaerobic mesenchymal stem cell exosome can regulate the polarization of astrocytes to A2 type, promote the recovery of spinal cord injury, and improve the motor function of rats with spinal cord injury.
In order to achieve the above purpose, the invention provides the following technical scheme:
an exosome derived from hypoxic mesenchymal stem cells is obtained by secreting human umbilical mesenchymal stem cells which are pretreated for 40-50 h by 0.5-3% of oxygen concentration.
The invention also provides a preparation method of the exosome, which comprises the step of enabling human umbilical cord mesenchymal stem cells to be subjected to 0.5-3 percent 2 Culturing for 40-50 h, collecting cell supernatant, and performing 4-6 continuous centrifugal treatments to obtain the exosome.
The invention also provides application of the hypoxia mesenchymal stem cell exosome in preparing a medicine for treating spinal cord injury.
Preferably, the hypoxic mesenchymal stem cell exosome can promote recovery of motor function of spinal cord injury.
Preferably, the hypoxia mesenchymal stem cell exosome can promote spinal cord tissue structure repair.
Preferably, the hypoxic mesenchymal stem cell exosome can promote expression of neural filament protein in spinal cord tissue.
Preferably, the hypoxic mesenchymal stem cell exosome can enhance expression of spinal tissue neural tubulin.
Preferably, the hypoxic mesenchymal stem cell exosomes promote spinal astrocytes to A2 polarization.
Preferably, the injection dosage of the hypoxia mesenchymal stem cell exosome is 2.0-2.5 mg.
Preferably, the medicament comprises an active ingredient of the hypoxia mesenchymal stem cell exosome and a pharmaceutically acceptable carrier.
Compared with the prior art, the invention has the following beneficial effects:
the invention provides an anoxic mesenchymal stem cell exosome and application thereof, wherein the anoxic mesenchymal stem cell exosome is obtained by secreting human umbilical cord mesenchymal stem cells which are pretreated for 40-50 h by 0.5-3% of oxygen concentration. The exosome can promote spinal cord astrocytes to be polarized to A2, enhance the expression of spinal cord tissue neurofilament protein and tubulin, further promote the repair of a spinal cord tissue structure and achieve the effect of recovering the spinal cord injury movement function.
Drawings
Fig. 1 is an identification of mesenchymal stem cell-derived exosomes (NEx) and hypoxia-pretreated mesenchymal stem cell-derived exosomes (HEx); a is representative images of NEx and HEx under a transmission electron microscope; b, tracking and analyzing the size distribution of exosomes by adopting nanoparticles; c Westernblot verifies that the exosome markers comprise CD63, CD9, alix and TSG101 and an exosome negative biomarker calnexin;
FIG. 2 shows the results of NEx and HEx promoting the recovery of hindlimb motor function in spinal cord injured SD rats; a is motor function BBB score of SD rat at different time after spinal cord injury; b is a representative image of the result of the ink test of the fourth circumferential movement of the SD rat after the operation; c is the step size analysis result of the SD rat in the fourth week after operation; d is the step width analysis result of the SD rat in the fourth week after the operation;
FIG. 3 shows the results of NEx and HEx promoting spinal cord tissue repair in SD rats; a is a representative image of HE staining of spinal cord tissues; b is the result of immunohistochemical staining of spinal cord tissue neurofilament protein (NF); c is the tissue immunofluorescence result of spinal cord tissue neural tubulin (MAP 2); d is a representative image of a Westernblot for detecting NF and MAP2 of spinal cord tissues; e, carrying out gray scanning statistical analysis on the D picture Westernblot result;
FIG. 4 shows the results of NEx and HEx promoting the polarization of rat spinal astrocytes towards A2; a is a representative diagram of a western blot for detecting astrocyte polarization indexes C3 and S100A10 of spinal cord tissues; b is the expression of quantitative PCR detection astrocyte polarization indexes C3, H2-T23, sgrn, S100A10, emp1 and SPHK 1; c is a representative image of a double positive cell of a tissue immunofluorescence detection spinal cord tissue C3 and GFAP; d is a representative image of tissue immunofluorescence detecting spinal cord tissue S100a10 and GFAP dicaryon cells.
Detailed Description
The invention provides an exosome derived from an anoxic mesenchymal stem cell, which is obtained by secreting human umbilical cord mesenchymal stem cells which are pretreated for 40-50 hours by 0.5-3% of oxygen concentration.
The invention also provides a preparation method of the exosome, which preferably comprises the following steps: human umbilical cord mesenchymal stem cells at 0.5% -3% 2 Culturing for 40-50 h, collecting cell supernatant, and performing 4-6 continuous centrifugal treatments to obtain the exosome. The oxygen concentration is more preferably 1%, and the culture time is more preferably 48h; the centrifugation temperature is preferably 0 to 10 ℃, more preferably 4 ℃, the centrifugation frequency is preferably 5, and more preferably, the centrifugation treatment comprises: centrifuging the cell supernatant 500g for 10min, centrifuging the cell supernatant 2000g for 10min, centrifuging the cell supernatant 10000g for 30min, then ultrafiltering the cell supernatant 2000g for 30min, concentrating, finally centrifuging the concentrated solution 100000g for 70min, and finally resuspending the cell supernatant with PBS to obtain the exosome.
The invention also provides application of the hypoxia mesenchymal stem cell exosome in preparing a medicine for treating spinal cord injury.
In the present invention, the hypoxic mesenchymal stem cell exosome can promote recovery of motor function in spinal cord injury. In animal experiments, after successful construction of the Sham, PBS, NEx and HEx rat models, BBB run function scores were performed at week 1, week 2, week 3 and week 4, and the average BBB scores of the Sham, PBS, NEx and HEx rats were 21,7, 12 and 15, respectively. The scoring result shows that the exosome can promote the motor function recovery of a spinal cord injury rat, and the exosome has a better recovery effect compared with NEx.
In the invention, the hypoxia mesenchymal stem cell exosome can promote spinal cord tissue structure repair. In animal experiments, gait analysis is carried out on rats of a Sham group, a PBS group, a NEx group and a HEx group by adopting a rat ink experiment, the dragging area of hind limbs of the rats is reduced, the step length is increased and the step width is reduced after exosome treatment. Experimental results show that the treatment of the exosome can improve the hindlimb motor function of a rat with spinal cord injury, and the exosome has a better spinal cord tissue structure repairing effect compared with NEx.
In the present invention, the hypoxic mesenchymal stem cell exosome can promote the expression of neural filament protein and neural tubulin of spinal cord tissue. In animal experiments, spinal cord was collected from Sham, PBS, NEx and HEx groups and HE staining, neurofilament paraffin sections immunohistochemical staining and neurotubulin (MAP 2) immunofluorescence staining were performed, respectively. Experimental results show that the exosome treatment can promote the expression of neural thread protein and neural tubulin in of spinal cord tissues, and the exosome has a better enhancing effect relative to NEx.
In the present invention, the hypoxic mesenchymal stem cell exosomes can promote spinal astrocytes to A2 polarization. In animal experiments, tissue protein detection, tissue RNA detection and astrocyte immunofluorescence staining are carried out on the Sham group, the PBS group, the NEx group and the HEx group, the astrocyte A2 marker of spinal cord tissues of rats in the NEx group and the HEx group is increased compared with that of the PBS group, and the spinal cord tissues of rats in the HEx group are increased more obviously. Experimental results show that the exosome can further promote spinal astrocytes to be polarized to A2.
In the present invention, the injection dosage of the hypoxic mesenchymal stem cell exosome is preferably 2.0-2.5 mg, and more preferably 2.4mg.
In the present invention, the medicament preferably comprises an active ingredient of the hypoxic mesenchymal stem cell exosome and a pharmaceutically acceptable carrier.
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Example 1
Extraction and identification of mesenchymal stem cell-derived exosomes (NEx) and hypoxia-pretreated mesenchymal stem cell-derived exosomes (HEx)
1. Preparation of exosomes
Digesting human umbilical cord mesenchymal stem cells with 0.25% pancreatin for 1min, stopping digestion with a-MEM nutrient solution containing 10% fetal calf serum, centrifuging at 800rpm, suspending with a-MEM nutrient solution containing 10% fetal calf serum, inoculating into cell culture dish, and culturing at 5% CO 2 Culturing in an incubator for 24h, and using when the cell growth fusion degree reaches 40-60 percentWashing with PBS 3 times, replacing serum-free mesenchymal stem cell nutrient solution, and placing normal group cells into 21% 2 ,5%CO 2 An incubator is used for 48h; the cells of the anoxic pretreatment group were placed in 1% 2 ,5%CO 2 Incubate for 48h, then collect cell supernatant.
Firstly, centrifuging two groups of collected cell supernatants at 4 ℃ and 500g for 10min to remove dead cells; centrifuging the collected cell supernatant at 4 deg.C and 2000g for 10min to remove cell debris; then collecting the supernatant, centrifuging at 4 deg.C and 10000g for 30min to remove organelles; transferring the supernatant to a 100kDaMWCO ultrafiltration centrifugal tube, centrifuging at 4 ℃ for 30min and concentrating at 2000 g; repeatedly concentrating and centrifuging to appropriate volume to obtain corresponding concentrated solution. Then centrifuging the concentrated solution at 100000g at 4 deg.C for 70min; discarding the supernatant, washing with PBS, centrifuging at 4 deg.C for 70min at 100000 g; finally adding PBS filtered by a 0.22 mu m filter, blowing, beating and resuspending to respectively obtain HEx and NEx; and finally, filtering and sterilizing the mixture by using a sterile filter membrane with the diameter of 0.22 mu m, subpackaging the mixture by using a sterile 1.5mL EP tube with high pressure, and storing the subpackaged mixture at the temperature of minus 80 ℃.
2. Transmission electron microscope for observing basic form of exosome
Respectively taking 20 mu L of NEx and HEx, fully and uniformly mixing, dropwise adding the mixture on a sample-carrying copper net with the diameter of 2mm, standing at room temperature for 5min, slightly sucking residual liquid at the edge of the copper net by using filter paper, then reversely covering the copper net on 30g/L of phosphotungstic acid (pH6.8) liquid drops, carrying out negative dyeing at room temperature for 5min, finally drying the copper net under an incandescent lamp, and placing the copper net under a transmission electron microscope for observation and photographing. The specific results are shown in FIG. 1A.
From the results in FIG. 1A, it can be seen that there is no significant difference in the shape of the two groups of exosomes.
3. NTA measurement of particle size distribution
Diluting 1 mu of LNEx and HEx with purified water 500-1000 times (the specific dilution ratio is related to the exosome concentration) respectively to measure Nanosight, and measuring the particle size to be generally 150-200nm. The specific results are shown in FIG. 1B.
From the results in FIG. 1B, it is clear that there is no significant difference in size between the two groups of exosomes.
4. Exosome protein extraction and concentration determination
(1) Putting 45 mu L of exosomes of LNEx and HEx in an EP tube respectively, and putting on ice;
(2) Adding an equal volume of RIPA-PMSF-PIC cell lysate;
(3) After shaking with a vortex mixer for 1min, the mixture was immediately placed on ice and left to stand for 10min, and this step was repeated 5 times. Protein concentration was determined by BCA kit instructions after lysis. Then adding 4xSDS loading buffer with the volume of 1/3, uniformly mixing, boiling in boiling water for 10min, subpackaging and storing in a refrigerator at the temperature of-20/70 ℃.
The concentration of NEx protein is 30 mug/mu L by using a BCA method; the concentration of the HEx protein is 45 mug/mu L, and for the convenience of subsequent use, the HEx is diluted to 30 mug/mu L by PBS for standby.
5. Protein identification of exosomes: western blotting (western blot)
(1) Preparing SDS-PAGE separation gel in advance according to the concentration of the NEx and HEx exosome proteins; clamping the gel plate, pulling out the gel comb, washing the hole by using a freshly prepared 1 xSDS-PAGE electrophoresis solution, completely throwing, and filling the inner tank with the freshly prepared 1 xSDS-PAGE electrophoresis solution;
(2) The samples were slowly loaded vertically at a protein loading of 30. Mu.g per well. And adding a proper amount of electrophoretic fluid into the outer tank to ensure that liquid level difference exists between the inside and the outside. The laminated gel is subjected to electrophoresis at a voltage of 60V, and the separation gel is subjected to electrophoresis at a voltage of 80V;
(3) Half-drying SDS-PAGE gel for 2 hours under the constant current condition of 350mA, and transferring the target protein to a PVDF membrane;
(4) Sealing the PVDF membrane in 5% skimmed milk for at least 1h at room temperature;
(5) PVDF membrane was cut into appropriate bands by molecular weight, diluted with 5% skim milk primary antibody, and incubated overnight at 4 ℃. The primary anti-dilution ratio is: β -actin (1;
(1) negative index Calnexin, albumin; positive indexes are as follows: alix, TSG101, CD81, CD9 and CD63. The sample loading amount is about 150-200 μ g.
(2) After the extracted exosome protein and source cell protein are subjected to WB, four-transmembrane proteins such as CD9, CD63 and CD81, multivesicular bodies such as Alix and TSG101 are detected to synthesize related proteins, and cytoplasmic proteins such as beta-actin and annexin are detected to detect a Calnexin exosome negative marker.
(3) The extracted exosome surface marker protein is detected, MSC cells are used as a control, negative markers Calnexin and positive markers Alix, CD9, CD63 and CD81 are used.
(6) The next day, the strips were removed and returned to room temperature, followed by 5min washes with 1 × TBS/T for 4 times. The secondary antibody is prepared according to the proportion of 1:2000 diluted in blocking solution and incubated for 1h at 37 ℃ in an incubator. Wash 5 times for 8min at 1 × TBS/T. Exposure analysis is carried out after exposure liquid is prepared freshly.
The protein of NEx and HEx was identified according to the above procedure, and the results are shown in fig. 1C.
From the results in FIG. 1C, it can be seen that both exosomes express a positive indicator and do not express a negative indicator.
Example 2
SD rat spinal cord injury model construction, motor function evaluation and spinal cord tissue recovery evaluation
1. Construction of SD rat spinal cord injury model
Adult female SD rats weighing 200-250 g were selected and randomly divided into sham, PBS, NEx and HEx groups.
In the process of model building, 20g/L pentobarbital sodium (40 mg/kg) is used for abdominal anesthesia of female SD rats, the female SD rats are fixed on a dissecting microscope platen in a prone position, the chest and the back are shaved and prepared into skin, the female SD rats are disinfected conventionally, the female SD rats are positioned at the T10 spinous process position, a central incision of the back is made by taking the position as the center, the length is about 3cm, skin and subcutaneous tissues are cut, paraspinal muscles on two sides are separated, the T10 spinous process and vertebral laminas on two sides are slightly bitten by a straight mosquito type toothed vascular clamp to the articular process, the dura mater is fully exposed, the length is about 5mm, the T9 spinous process and the T11 spinous process are respectively clamped by a mosquito type vascular clamp and pulled to two sides to fix the spinal cord, the corresponding area of the T10 is used as an injury area, the posterior median vessel is used as the center, and a self-made improved Allen device is used for preparing an acute spinal contusion animal model.
Vertically impacting a weight with the weight of 5g and the diameter of 2mm with spinal cord tissue, staying for 3 seconds, then flushing a wound, scattering a small amount of penicillin powder on the wound surface, suturing paraspinal muscle and skin layer by using 3-0 number wires, and exposing the spinal cord by using the sham method without damaging the spinal cord. The PBS group was injected with 80 μ LPBS at 7 different depths of 3mm position before and after the injury site, and the NEx group and HEx group were injected with 80 μ LNEx and HEx at 7 different depths of 3mm position before and after the injury site, respectively. After injury, the rats are raised in a single cage, naturally irradiated, periodically and regularly added with sufficient feed, drinking water is not limited, and the rats are manually massaged for urination twice a day in the morning and at night until the bladder recovers to urinate automatically. The padding is replaced every 2-3 days and kept dry.
2. BBB run function score
BBB functional scores were performed at weeks 1, 2, 3, and 4 post-surgery, with bladder emptying before evaluation and an observation period of 4min. The BBB score was an observation of the hip, knee, ankle joints walking, trunk movement and coordination of the animals.
BBB scoring (Basso, beattie & bresnahanalocomatoratingscale, bbbsacle), (rat spinal cord injury) scoring criteria:
0 minute: no visible hind limb movement;
1 minute: slight motion of one or both joints, typically the hip and/or knee;
and 2, dividing: one joint moves greatly or one joint moves greatly and the other joint moves slightly;
and 3, dividing: the two joints move greatly;
and 4, dividing: all three joints of hind limbs can move slightly;
and 5, dividing: two joints move slightly, and the third joint can move greatly;
6 min: two joints move greatly, and the third joint can move slightly;
7 min: all three joints of hind limbs can move greatly;
8 min: the palm surface of the claw can land on the ground under the condition of non-load bearing;
9 min: sole is only in the weight bearing position, or occasionally/frequently/continuously walking with instep load, walking without sole load; carrying out load bearing: when the sole is in the weight bearing position or only when the rear trunk is lifted, the HL extensor contracts;
10 min: the palm surface of the occasional claw bears the weight and moves; no coordination action of front and back limbs;
and (3) 11 min: the palm surface can bear weight and move more, but the coordination action of the front limbs and the rear limbs is not available;
12 min: the palm surface can be seen to move in a bearing way, and the coordination of the front limb and the rear limb is occasionally seen;
13 min: the common palm surface can bear weight and move, and the common fore-and-aft limb can coordinate actions;
14 min: continuous palm bearing movement and coordination of fore-and-aft limbs; or common palm surface movement, continuous coordination of front and rear limbs, and even dorsal movement of the paw occur;
15 min: continuous palm surface movement and continuous coordination action of forelimbs and hindlimbs, and no or European ground grasping is performed in the advancing process of the forelimbs; the position of the driving claw is parallel to the body during initial contact;
16 min: continuous tunnel face movement and continuous coordination of forelimbs and hindlimbs can be seen in gait, and the forelimbs are usually grabbed by claws in the advancing process; the position of the driving claw is parallel to the body during initial contact, and the driving claw rotates after load transfer;
17 min: continuous tunnel face movement and continuous coordination of forelimbs and hindlimbs can be seen in gait, and the forelimbs are usually grabbed by claws in the advancing process; the position of the driving claw is parallel to the body during initial contact and after load transfer;
18 min: continuous tunnel face movement and continuous coordination of forelimbs and hindlimbs can be seen in gait, and the forelimbs can be continuously grabbed by claws in the advancing process; the position of the driving claw is parallel to the body during initial contact, and the driving claw rotates after load transfer;
and 19 min: continuous tunnel face movement and continuous coordination actions of forelimbs and hindlimbs can be seen in gait, and the forelimbs can be continuously grabbed by the claws in the advancing process; the position of the driving claw is parallel to the body during initial contact and after load transfer. The tail sometimes or always droops;
and 20 min: the continuous movement of the palm surface, the continuous coordination of gait, the continuous grasping of the toes, the parallel of the position of the driving claw with the body during the initial contact and after the load transfer, the unstable trunk and the continuous tilting of the tail;
and (3) 21 minutes: the continuous movement of the palm surface, the continuous coordination of the gait, the continuous gripping of the toes, the position of the driving claw which is always parallel to the body in the moving process, the continuous stability of the trunk and the continuous tilting of the tail.
In the scoring, it needs to be able to correctly judge whether the movement of the rat hind limb is the dragging of the body or the autonomous movement of the hind limb of the body, where the dragging is the movement of the body to drive the hind limb, and the autonomous movement of the hind limb may be the movement of the hind limb itself in twitching, or the flexion and extension of the rat paw, or the movement of the hip, knee, ankle joint.
BBB scoring was performed on Sham, PBS, NEx and HEx rats using the scoring method described above, and the results are shown in fig. 2A.
From the results of fig. 2A, it can be observed that the average BBB scores of the Sham, PBS, NEx and HEx groups of rats were 21,7, 12 and 15, respectively. The scoring result shows that the exosome can promote the motor function recovery of a spinal cord injury rat, and the exosome provided by the invention has a better recovery effect compared with NEx.
3. Ink experiment in rats after spinal cord injury (gait analysis)
(1) Training phase normal rats are placed on one side of a narrow plate and a dark box is placed on the other side of the narrow plate every day, and the rats run from one side to the dark box on the other side to complete 1 training due to the darkness preference of the rats. Training is carried out 3 times a day, with the interval of 5-10min for 3 days. After the daily training is completed, the animals are returned to the rearing room.
(2) In the experimental stage, white paper with the same length and width as the narrow plate for training animals is laid on the narrow plate, and the serial numbers and the test dates of the animals are recorded on the white paper; after the animal is adapted, the red ink is smeared on the dorsum of the hind limb of the animal, and the black ink is smeared on the sole of the foot; immediately placing the animal on one side of the white paper to enable the animal to walk towards the magazine; after the animal enters the hidden box, the white paper is collected and dried; the above steps were continued until all animals were analyzed.
(3) The footprints of each group of rats are scanned into pictures, and the step length and the step width can be obtained according to the footprints.
(4) The step length is the distance between two steps on the same side in the continuous walking process of the rat. The step width is the distance between the left and right feet in two consecutive steps. The step size and the step width can reflect the weight bearing capacity of hind limbs to the body when the animal walks with the weight bearing. Generally, a smaller stride length and a larger stride width represent a reduction in the ability of the animal to bear weight on the hind limb.
Gait analysis was performed on Sham, PBS, NEx and HEx rats using the rat ink test described above, and the results are shown in fig. 2B-C.
From the results of fig. 2B-C, it can be seen that the hindlimb dragging area of the rat is reduced, the step size is increased, and the step width is reduced after the exosome treatment, which indicates that the treatment can improve the hindlimb motor function of the spinal cord injured rat, and the exosome of the present invention recovers better than NEx.
Experimental example 3
Spinal cord sampling and spinal cord tissue repair evaluation
1. Taking materials and fixing
After the fourth week of exosome intervention, chloral hydrate (1.5 mL/kg) with a volume fraction of 10% was intraperitoneally anesthetized, and after anesthesia was successful, the chest was cut open and the heart was exposed. The physiological saline bag is connected with the medical infusion apparatus and the filling needle, and the physiological saline bag is arranged at a high position. The heart is held by the left hand, the blunt tip needle is inserted from the apex of the heart to the ascending aorta by the right hand, and the vessel forceps are used for preventing the perfusion liquid from being lost. A small opening is cut at the right atrium by a pair of scissors, an infusion switch is opened, the rat mouth and paws are infused by 0.9% normal saline until the rat mouth and paws gradually become white, and when colorless liquid flows out from the right atrium, the infusion liquid is replaced by 4% paraformaldehyde solution, and the infusion is continued. Successful immobilization was indicated when the rats had extremities and muscles throughout the body, with constant twitching until the tail and the body became hardened. The original operation incision is entered and cut sequentially to separate soft tissues, a model rat is removed to expose a T7-T9 vertebral plate and a T7-T9 spinal cord, the spinal cord is rapidly taken out after the injury part is determined to be marked in advance, the spinal cord is placed in 20% sucrose solution, and the solution is placed in a refrigerator at 4 ℃ overnight until the spinal cord tissue is settled in the sucrose solution. Frozen immunofluorescence sections were stored. Each specimen was prepared into 6 frozen sections of 5 μm from the head, middle and tail of the spinal cord injury area, and the remaining spinal cord specimens were fixed with 10% neutral formalin for dehydration and paraffin embedding.
The Sham, PBS, NEx and HEx rats were treated as described above to obtain sections.
2. HE staining
(1) Slicing the Sham group, PBS group, NEx group and HEx group into distilled water, and staining in hematoxylin water solution for 2-3min.
(2) Separating color in acid water and ammonia water for 10-30 s each.
(3) Washing with running water for 1 hr, and adding distilled water for 1-3min.
(4) Dehydrating in 70% and 90% ethanol for 10min respectively.
(5) Dyeing for 2-3min in alcohol eosin dyeing solution.
The dyed slices are dehydrated by pure alcohol and then are transparent by xylene. The transparent sections were dropped with Canadian gum and mounted with a coverslip. And after the gum is slightly dried, sticking a label to obtain a corresponding section specimen.
The slice specimen pictures of Sham, PBS, NEx and HEx sets are detailed in figure 3A.
From the results in fig. 3A, it can be seen that NEx treatment can promote the repair of rat spinal cord tissue structure, and HEx has better repairing effect.
3. Immunohistochemical staining of neurofilament paraffin sections
Slices from Sham, PBS, NEx and HEx groups were deparaffinized to water: (Paraffin section staining was preceded by a 60 ℃ oven for 10 h). Xylene I and xylene II, each for 10min. Gradient alcohol hydration: 100%,2min → 95%,2min → 80%,2min → 70%,2min. Washing with distilled water: 5min,2 times (placed on a shaker). Hydrogen peroxide blocks endogenous peroxidase: 3% of H 2 O 2 Room temperature for 10min (protected from light). Washing with distilled water: 5min,2 times (placed on a shaker).
The method for antigen retrieval comprises the following steps: placing in plastic or temperature-resistant glass container with plastic slicing frame, submerging sodium citrate buffer solution into slices, and selecting medium-high or high-grade slices for 5min; taking out and supplementing the preheated sodium citrate buffer solution; then, selecting medium-high or high-grade PBS for 5min (the optimal temperature is 92-95 ℃): 5min,2 times (placed on a shaker). Normal serum blocking: taking out the section from the staining jar, wiping off water on the back of the section and water around the front tissue of the section (keeping the tissue in a moist state), adding normal goat or rabbit serum (homologous animal serum with a second antibody) dropwise, and treating at 37 deg.C for 15min. Dropping a first neurofilament protein antibody: the blocking serum was removed by blotting with filter paper, and the primary antibody was added dropwise directly without washing at 37 ℃ for 2 hours. (it can also be left in a refrigerator at 4 ℃ overnight). PBS:5min,2 times (placed on a shaker). Biotinylated secondary antibody was added dropwise at 37 ℃ for 40min. PBS:5min,2 times (placed on a shaker). Three antibodies (SAB complex) were added dropwise at 37 ℃ for 40min. PBS:5min,2 times (placed in a shaking table). DAB coloration, under-lens observation, and timely termination (stop of tap water flushing). Tap water (fine water) was thoroughly rinsed. Hematoxylin counterstain, room temperature, 30 seconds, tap water rinse. And washing with tap water to return blue for 15min. Gradient alcohol dehydration: 80%,2min → 95%,2min → 100%,2 times, 5min. And (3) xylene transparency: i, II (xylene) each 5min. Mounting: canadian gum (or neutral gum) seals. And finally, scanning the tablet in a digital scanning machine, and judging the expression of the neurofilament protein according to the brown shade.
Immunohistochemical staining of neurofilament paraffin sections was performed on Sham, PBS, NEx and HEx groups as described above, and the results are detailed in figure 3B.
From the results in fig. 3B, it can be seen that exosome therapy can promote the expression of neurofilament protein in spinal cord tissue, and the present invention has a more significant effect compared to NEx.
4. Neurotubulin (MAP 2) immunofluorescence staining
The Sham, PBS, NEx and HEx sections were baked, dewaxed to water, endogenous peroxidase inactivated, heat-induced antigen retrieval and blocking, respectively, and the tissues were immunochemically stained. Followed by overnight incubation at 4 ℃ with the primary tubulin antibody, and washing with histochemical PBS. The subsequent steps were all performed away from light, and the corresponding fluorescent antibody was incubated at room temperature for 45min, monkey anti-rabbit lgG 555 fluorescent secondary antibody/goat anti-mouse lgG FITC secondary antibody (dilution factor 1. The composed PBS was washed gently. Staining for 5min at room temperature, dapi (1. And sealing, and capturing and shooting by using a laser confocal microscope.
The neurotubulin (MAP 2) immunofluorescent staining was performed on the Sham, PBS, NEx and HEx groups as described above, and the results are detailed in FIG. 3C.
From the immunofluorescence results of the tissues in fig. 3C, it can be seen that the exosome treatment can enhance the expression of the neural tubulin in the spinal cord tissue, and the exosome of the present invention has a better enhancing effect relative to NEx.
Experimental example 4
Spinal cord tissue astrocyte polarization evaluation
The astrocyte marker is Glial Fibrillary Acidic Protein (GFAP), the A1-type astrocyte marker includes complement component 3 (C3), histocompatibility 2, T region locus 23 (H2-T23), and hemoglobinizing hormone (Srgn), and the A2-type astrocyte marker includes calbindin a10 (calceinbingprotein a10, S100a 10), epidermal membrane protein (Emp 1), and sphingosine kinase 1 (SPHK 1).
1. Protein and mRNA level tissue protein detection
Placing a small amount of spinal cord tissue blocks into a 1.5mL centrifuge tube, adding 500. Mu.L of strong RIPA lysis solution containing PMSF, shearing the tissue blocks as much as possible with clean scissors, continuously blowing and sucking with a 1mL gun head, further breaking the tissue with a homogenizer, and finally carrying out ultrasonic lysis with an ultrasonic instrument. Subsequently, the centrifuge tube was placed in a 4-degree centrifuge, centrifuged at 12000rpm for 15min, the supernatant was taken and 1/3 loadingbuffer was added thereto, heated in boiling water for 10min, and the mixture was dispensed into 1.5mL centrifuge tubes and stored at-80 ℃. Subsequent expression of astrocyte type A1 and A2 proteins in tissues was examined using a Westernblot.
2. Tissue RNA detection
Adding liquid nitrogen into a mortar, cutting spinal cord tissues into small pieces, grinding the small pieces into powder in the liquid nitrogen, taking 50-100mg of tissue powder by using a medicine spoon precooled by the liquid nitrogen, adding the tissue powder into an EP (ultra Violet) tube filled with 1mL of Trizol liquid (note that the total volume of the tissue powder cannot exceed 10% of the volume of the used Trizol), and fully and uniformly mixing. After 5min at room temperature, 200. Mu.L of chloroform were added, the EP tube was capped and shaken vigorously for 15 seconds. Centrifuge at 12000rpm for 10min, take the upper aqueous phase in a new EP tube (ten million don't mix the middle pellet and the lower liquid, otherwise re-centrifuge), add 500. Mu.L isopropanol, mix by gentle inversion. The mixture was then left at room temperature for 10min and centrifuged at 12000rpm for 10min. The supernatant was carefully discarded, 1mL of 75% ethanol was added, vortexed and mixed, and centrifuged at 12000rpm for 5min at 4 ℃. The operation was repeated once. The supernatant was discarded (residual liquid was removed as much as possible),drying at room temperature or under vacuum for 5-10min (taking care not to dry too much, otherwise the solubility of RNA will be reduced). The RNA was solubilized with 20. Mu.L of DEPC-treated water and subsequently reverse transcribed according to the reverse transcription reagent instructions. Then, a real-time fluorescent quantitative PCR experiment is carried out according to the instruction, firstly, a premixing system is prepared, and each sample is provided with 3 compound wells. 18 μ L premix system per well: 10 μ LSYBGreenMix, 7 μ L RNase-free ddH 2 O, 0.5. Mu.L of the forward primer for H2-T23 or Emp1 or SPHK1 or S100a10 or β -actin or C3 or Srgn and 0.5. Mu.L of the backward primer, the bottom of the octal tubing was added, and finally 2. Mu.L of the pre-diluted cDNA was added. Covering an eight-tube-connected tube cover, instantly separating and mixing the sample, carrying out reaction on an ABI 7500 fluorescence quantitative PCR instrument, pre-searching the annealing temperature of the primer to be 60 ℃, and carrying out PCR program setting by adopting a two-step method in the experiment: pre-denaturation at 95 ℃ for 10min, denaturation at 95 ℃ for 15s, annealing/extension at 60 ℃ for 1min, and 40 cycles of denaturation and annealing/extension; finally, melting curve analysis was carried out at 95 ℃ 15s,60 ℃ 1min,95 ℃ 15s, and 60 ℃ 15s. The primer sequences are as follows:
H2-T23:
forward primer (SEQ ID NO: 1): CTGTGGTAGTGCCTTCTGGG;
rear primer (SEQ ID NO: 2): CTGGAAAGCCTTGCTGCCTA;
Emp1:
forward primer (SEQ ID NO: 3): accattggccaacgtctggat;
rear primer (SEQ ID NO: 4): AGCATCATCATTGCCGTAGC;
SPHK1:
forward primer (SEQ ID NO: 5): GGCACGAGCAGGTCACTAAT;
rear primer (SEQ ID NO: 6): CGTGAAACGAATCCCCCCCCCCCCCA;
S100a10:
forward primer (SEQ ID NO: 7): GAGTGCTCATCGAAAGGGAGT;
rear primer (SEQ ID NO: 8): CTTTTCCATCTCGGCATGGCACTGG;
β-actin:
forward primer (SEQ ID NO: 9): CACGAAACTACCTTCAACTCC;
rear primer (SEQ ID NO: 10): TGTGGGTGGTATCCTGTGGA;
C3:
forward primer (SEQ ID NO: 11): ATCGAGGATGGTTCAGGGGA;
rear primer (SEQ ID NO: 12): GCCTCTACCATGTCGCTACC;
Srgn:
forward primer (SEQ ID NO: 13): CTTCGTCCTGGTTTTGGGGAT;
rear primer (SEQ ID NO: 14): TCGAACCGTGTCCTTCTC.
3. Spinal cord tissue astrocyte immunofluorescent staining
The Sham, PBS, NEx and HEx groups were sliced, dewaxed to water, endogenous peroxidase inactivated, heat-induced antigen retrieval and blocking, respectively, as described above, incubated at 4 ℃ for overnight, GFAP antibody and C3 antibody were co-incubated; GFAP antibody and S100A10 were incubated together. The histochemical PBS is washed, and the secondary antibody is added dropwise and incubated for 30min at room temperature. The histochemical PBS was washed gently. Nuclei were stained at room temperature for 5min, dapi (1. And sealing, and capturing and shooting by using a laser confocal microscope.
From the results of FIGS. 4A-C, it can be seen that the spinal cord tissue of rats in NEx group showed an increase in astrocyte A2 marker compared to that of PBS group, and that of rats in HEx group showed an increase in spinal cord tissue. The experimental result shows that the exosome can further promote the polarization of spinal astrocytes to A2 relative to NEx.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (10)
1. An exosome derived from hypoxic mesenchymal stem cells, which is obtained by secreting human umbilical mesenchymal stem cells which are pretreated for 40-50 h at the oxygen concentration of 0.5% -3%.
2. The method for producing exosome according to claim 1, wherein human umbilical cord mesenchymal stem cell is subjected to 0.5% -3% o 2 Culturing for 40-50 h, collecting cell supernatant, and carrying out 4-6 continuous centrifugal treatments to obtain the exosome.
3. Use of the hypoxic mesenchymal stem cell exosome according to claim 1 or the exosome obtained by the preparation method according to claim 2 in preparation of a medicament for treating spinal cord injury.
4. The use according to claim 2, wherein the injection dose of the hypoxic-mesenchymal stem cell exosome is 2.0-2.5 mg.
5. The use of claim 2, wherein the hypoxic-mesenchymal stem cell exosomes promote recovery of spinal cord injury motor function.
6. The use of claim 2, wherein the hypoxic mesenchymal stem cell exosome is capable of promoting spinal cord tissue architecture repair.
7. The use of claim 2, wherein the hypoxic mesenchymal stem cell exosome is capable of promoting expression of spinal tissue neurofilament protein.
8. The use according to claim 2, wherein the hypoxic-mesenchymal stem cell exosomes are capable of enhancing expression of spinal tissue neurotubulin.
9. The use of claim 2, wherein the hypoxic-mesenchymal stem cell exosomes promote spinal astrocytes to A2 polarization.
10. The use according to claim 2, wherein the medicament comprises an active ingredient hypoxic mesenchymal stem cell exosome and a pharmaceutically acceptable carrier.
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