CN111454895A - Experimental method for repairing spinal cord by inducing microglia polarization through exosome - Google Patents

Abstract

The invention relates to the technical field of medical experiments, in particular to an experimental method for repairing spinal cord by inducing microglia polarization through exosome, which comprises the following experimental steps: step one, cell culture and hypoxia model establishment; step two, separating exosomes; step three, exosome identification; step four, establishing a mouse spinal cord injury model; step five, RNA sequencing; step six, scoring the movement function of the mouse; seventhly, carrying out real-time quantitative PCR analysis; step eight, performing immunofluorescence evaluation; ninth, western blot analysis; step ten, plasmid construction and transfection; step eleven, taking up exosomes by BV2 microglia; step twelve, enzyme linked immunosorbent assay; thirteen, detecting dual luciferase reporter genes; step fourteen, statistical analysis, the experimental method provided by the invention is very intuitive, and the fact that the exon processed by the ADSCs containing lncGm37494 can more effectively repair the spinal cord injury through the transfer of microglia M1/M2 polarization is proved.

Description

Experimental method for repairing spinal cord by inducing microglia polarization through exosome

Technical Field

The invention relates to the technical field of medical experiments, in particular to an experimental method for repairing spinal cord by inducing microglia polarization through exosome.

Background

Mesenchymal stem cells are pluripotent stem cells that have all of the commonalities of stem cells, namely self-renewal and multipotential differentiation capacity. Clinically, mesenchymal stem cells have been used for rapid recovery of blood cells after trauma or chemotherapy in patients. After a patient receives a large dose of chemotherapy, the loss of blood cells is serious, and a doctor inputs mesenchymal stem cells and hematopoietic stem cells into the patient body together, so that the recovery time of the blood cells of the patient can be obviously accelerated, and the patient is safe and has no adverse reaction. There is increasing evidence that Mesenchymal Stem Cell (MSC) transplantation may be an effective therapy for treating SCI. In particular, studies have shown that this therapy can promote sensory and functional recovery and reduce proinflammatory factor levels by shifting glial M1 to M2 polarization, but limitations and challenges are not negligible when transplanting MSCs directly into target tissues. Studies report that the survival rate of transplanted stem cells is very low. Hypoxic pretreatment or transfection with anti-stress molecules can enhance the therapeutic effect of stem cells. Intensive research has shown that the role of MSCs in tissue regeneration suggests that paracrine mechanisms may be involved in the underlying MSC mechanism of action in the treatment of various diseases, and that exosomes may play an important role in this process. Exosomes (Exos) are membranous lipid vesicles (40-100 nm in diameter) containing functional proteins, mRNA, long non-coding rna (lncrna), microRNA and substances, and are involved in the process of information transfer between cells. Previous studies found that exosomes from bone mesenchymal stem cells protected in spinal cord injury by inhibiting synthesis and release of complement mRNA and by inhibiting SCI-activated NF- κ B by binding to microglia. It was found that MSC exosomes could inhibit the development of inflammation by altering the microglia/macrophage phenotype in neurogenic diseases. However, the specific molecular mechanism still needs to be further determined, and research shows that the hypoxic pretreated ADSCs increase the lnc gm37494 content in Exos. The lncGm37494 modified ADSCs treated exon can repair spinal cord injury more effectively by transferring microglia M1/M2 polarization, but no effective experimental method is provided for the strong demonstration of the above conclusion. In view of this, we propose an experimental method for repairing spinal cord by inducing microglia polarization by exosome.

Disclosure of Invention

In order to make up for the defects, the invention provides an experimental method for repairing spinal cord by inducing microglia polarization by exosome.

The technical scheme of the invention is as follows:

an experimental method for repairing spinal cord by inducing microglia polarization by exosome comprises the following experimental steps:

step one, cell culture and hypoxia model establishment;

step two, separating exosomes;

step three, exosome identification;

step four, establishing a mouse spinal cord injury model;

step five, RNA sequencing;

step six, scoring the movement function of the mouse;

seventhly, carrying out real-time quantitative PCR analysis;

step eight, performing immunofluorescence evaluation;

ninth, western blot analysis;

step ten, plasmid construction and transfection;

step eleven, taking up exosomes by BV2 microglia;

step twelve, enzyme linked immunosorbent assay;

thirteen, detecting dual luciferase reporter genes;

and step fourteen, carrying out statistical analysis.

As a preferred embodiment of the present invention, there are included experimental materials of wild type C57B L/6 mice and knockout C57B L/6 mice.

The invention preferably comprises the following reagent preparations of PBS solution, 10% SDS, 1.0M Tris-HCl, 30% stock sol, 10% AP, 10 × electrophoresis buffer solution, blocking solution, 10 × membrane transferring buffer solution, 1 × TBST buffer solution and 4% paraformaldehyde.

Preferably, the cell culture and hypoxia model establishment specifically comprises the following steps:

s1, extracting and culturing the adipose-derived stem cells, which comprises the following specific operations:

(1) preparing before experiment, including experiment article disinfection, ultraviolet disinfection, hand cleaning and the like;

(2) taking 3 wild type C57B L/6 mice, after anesthesia succeeds, fixing the mice in a supine position on an operating table, cutting the skin of the inguinal region of the mice by about 2cm, taking subcutaneous adipose tissues, suturing wounds on two sides, and putting the adipose tissues into a PBS solution for cleaning;

(3) putting the cleaned adipose tissues into a glass beaker, adding 0.075% type II collagenase with the dosage of 200U/ml, placing the glass beaker in a 37 ℃ thermostat for 30 minutes, oscillating the glass beaker once every 5 to 10 minutes, and stopping digestion of the collagenase after 30 minutes by using normal saline;

(4) placing the mixed solution into a centrifugal tube, centrifuging for 10 minutes at 1200 g/1200 r/min, removing supernatant and undigested fat, using 10% FBS DMEM to resuspend cell precipitates, using 0.16 mol/L ammonia chloride to dissolve residual red blood cells, filtering the collected cell suspension through a copper net, centrifuging for 10 minutes at 1200r/min, adding culture solution, blowing, uniformly mixing, sampling and counting, and adjusting the cell concentration to be 104 cells/ml according to the counting result;

(5) inoculating and culturing: inoculating 1ml of 4ml of culture solution with cell suspension amount in each 10cm2 culture bottle, and culturing in a 5% CO2 incubator at 37 deg.C;

(6) ADSC of 3 rd to 5 th generation is selected for the next experiment;

s2, establishing a cell hypoxia and glucose-starvation model, and specifically operating as follows:

(1) taking the neuron cells which grow and mature for one week, and inoculating the neuron cells into a multi-well plate according to the density of 1 × 106 cells/m L;

(2) sucking out the original culture solution, washing with PBS for three times, and adding a sugar-free culture medium containing drugs with corresponding concentrations;

(3) placing the culture dish in an anoxic device, introducing mixed gas of 95% nitrogen and 5% carbon dioxide, filling the device with the mixed gas, sealing the device with a sealing film, and placing the device in a 37 ℃ incubator for incubation for 3-6 hours;

(4) taking out the culture dish, and carrying out the next experiment;

s3, BV2 microglia cell lines were cultured in DMEM/high glucose medium containing 10% FBS and 1% pen/strep L PS was co-cultured with BV2 microglia cells for 24h, and then exosomes were added to the different groups of media.

As a preferred aspect of the present invention, the isolation of exosomes specifically comprises the steps of:

(1) when the ADSCs reach 80% confluence, the culture medium is replaced by FBS which is poor in exosomes, the culture is continued for 48 hours, and the culture is carried out under the normal oxygen or low oxygen condition;

(2) the medium was collected and centrifuged at 300 × g for 10 minutes, then at 2000 × g for 10 minutes at 4 ℃, after which the cell supernatant was filtered from whole cells and cell debris using a 0.22 μm sterile filter;

(3) adding the filtered supernatant to the upper compartment of the centrifugal filter device and centrifuging at 4000 × g until the volume of the upper layer is reduced to 200 μ L;

(4) the ultrafiltered supernatant was washed twice with PBS and, for purification of exosomes, the liquid was loaded on top of a 30% sucrose/D2O buffer in a sterile tube and centrifuged at 100,000 × g for 60 minutes at 4 ℃ in an ultracentrifuge;

(5) the fraction containing ADSC-Exos was recovered using an 18-G needle, then diluted in PBS and centrifuged at 4000Xg in a centrifugal filter unit at 4 ℃ until the final volume reached 200. mu. L and the exosomes were stored at-80 ℃ or immediately used in subsequent experiments.

As a preference of the present invention, the immunofluorescence evaluation specifically comprises the following steps:

(1) spinal cord tissue samples were taken on day 3 post-surgery;

(2) all samples were immersed in 1% bovine serum albumin and 0.3% triton x-100 for 1 hour to prevent non-specific reactions, followed by overnight incubation with an antibody at 4 ℃ including: anti-iNOS, anti-Ibal, anti-Arg 1, anti-NeuN;

(3) the next day, all samples were washed with PBS and incubated with the corresponding secondary antibody for 2 h: a secondary antibody in which Dylight (Dy)488 and Dy594 are mixed;

(4) nuclei were stained with DAPI and imaging was performed using an inverted microscope or confocal microscope.

As a preference of the present invention, the Western blot analysis specifically comprises the following steps:

(1) according to the molecular weight of the target protein, preparing 8% or 10% of separation gel according to a reagent specification formula, and filling 5% of concentrated gel after the separation gel is solidified;

(2) calculating the volume according to the concentration of the sample, and loading the sample by using a 10ul pipette;

(3) electrophoresis: firstly, carrying out electrophoresis for 30 minutes by using a voltage of 80V, and then adjusting the voltage to 120V until the electrophoresis is finished;

(4) film transfer: cutting a PVDF membrane with a proper size, soaking the PVDF membrane in formaldehyde, prying a glass plate by using a plastic warping plate after activation, cutting off concentrated glue, taking out the separated glue, placing the separated glue on filter paper of a rotating membrane clamp cathode, covering the PVDF membrane, placing the PVDF membrane into a rotating membrane tank, burying the rotating membrane tank in an ice box, and rotating the membrane by using a 300mA constant current, wherein the membrane rotating time is the same as the molecular weight of protein;

(5) and (3) sealing: blocking with 5% BSA for 1 hour at room temperature;

(6) primary antibody incubation: diluting the antibody according to the antibody specification, washing the PVDF membrane for 3 times by using TBST, cutting the PVDF membrane according to the molecular weight of target protein by using a Marker, putting the PVDF membrane into an incubation box, adding a primary antibody, and standing overnight in a shaking table at 4 ℃;

(7) incubating a second antibody; recovering the primary antibody in the incubation box, shaking the TBST for 3 times, diluting the secondary antibody by using 5% skimmed milk powder at a ratio of 1:100, and incubating for 1 hour at room temperature;

(8) and (3) exposure, namely discarding the secondary antibody, placing the incubation box on a shaking table, shaking and washing for 3 times, exposing by using a gel presentation system, mixing EC L luminescent solution A and B at a ratio of 1:1, and dripping 200ul of luminescent solution on the PVDF membrane for exposure.

As a preferred aspect of the present invention, the uptake of exosomes by BV2 microglia specifically comprises the steps of:

(1) adding a Dil solution of 4mg/m L to PBS containing exosomes and incubating;

(2) ultracentrifugation at 100,000 × g for 1h at 4 ℃ to remove excess fluorescent label from the labeled exosomes;

(3) the pellet was then resuspended in PBS, and finally washed and resuspended in PBS to wash the exosome pellet 3 times;

(4) these Dil-labeled exosomes were co-cultured with BV2 microglia for 24 hours, then the cells were washed with PBS and fixed in 4% paraformaldehyde;

(5) finally, the extent of uptake of Dil-labeled Exos and HExos by BV2 microglia was observed by confocal laser microscopy and the fluorescence intensity of Dil was measured with ZENlite software at different time points in both groups.

Compared with the prior art, the invention has the beneficial effects that:

the experimental method provided by the invention is very intuitive to prove that the exon processed by the ADSCs containing lncGm37494 can more effectively repair the spinal cord injury by transferring microglia M1/M2 polarization.

Drawings

FIG. 1 is a comparison graph I of various indexes and data after the experiment of the present invention;

FIG. 2 is a second comparison graph of various indexes and data after the experiment of the present invention;

FIG. 3 is a third comparison graph of various indexes and data after the experiment of the present invention;

FIG. 4 is a comparison graph of various indexes and data after the experiment of the present invention;

FIG. 5 is a graph showing comparison of various indexes and data after the experiment of the present invention;

FIG. 6 is a graph showing comparison between various indexes and data after the experiment of the present invention.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, 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 invention.

Referring to fig. 1-6, the present invention provides a technical solution:

an experimental method for repairing spinal cord by inducing microglia polarization by exosome comprises the following experimental steps:

step one, cell culture and hypoxia model establishment;

step two, separating exosomes;

step three, exosome identification;

step four, establishing a mouse spinal cord injury model;

step five, RNA sequencing;

step six, scoring the movement function of the mouse;

seventhly, carrying out real-time quantitative PCR analysis;

step eight, performing immunofluorescence evaluation;

ninth, western blot analysis;

step ten, plasmid construction and transfection;

step eleven, taking up exosomes by BV2 microglia;

step twelve, enzyme linked immunosorbent assay;

thirteen, detecting dual luciferase reporter genes;

and step fourteen, carrying out statistical analysis.

As a preferred embodiment of the present invention, there are included experimental materials of wild type C57B L/6 mice and knockout C57B L/6 mice.

The invention preferably comprises the following reagent preparations of PBS solution, 10% SDS, 1.0M Tris-HCl, 30% stock sol, 10% AP, 10 × electrophoresis buffer solution, blocking solution, 10 × membrane transferring buffer solution, 1 × TBST buffer solution and 4% paraformaldehyde.

It should be added that the preparation process of the above reagent is as follows:

preparation of PBS solution 1600ml of double distilled water measured by a measuring cylinder is poured into a glass beaker, then 16g of NaCl, 0.4KCl, 2.88g of Na2HPO4 and 0.48KH2PO4 are sequentially weighed by an electronic balance, the above solutions are sequentially added into the glass beaker and stirred by a stirrer until the solutions are completely dissolved, the pH of the solution is adjusted to 7.4 by hydrochloric acid, the double distilled water is added to 2L, and the solution is stored at room temperature after high temperature and high pressure sterilization.

Preparation of 10% SDS: 270ml of double distilled water measured by a measuring cylinder is poured into a glass beaker, 30g of SDS powder is weighed by an electronic balance, poured into the beaker and stirred by a stirrer until the SDS powder is completely dissolved, hydrochloric acid is added to adjust the pH value of the solution to 7.2, and the double distilled water is added to make up the solution to 300ml and stored at room temperature.

1.0 preparation of MTris-HCl, 800ml of double distilled water is measured with a measuring cylinder and poured into a glass beaker, 121.1g of Tris powder is weighed with an electronic balance, the Tris powder is poured into the glass beaker and stirred with a stirrer until completely dissolved, hydrochloric acid is added to adjust the pH of the solution to 6.8, and finally the solution is made up to 1L and stored at room temperature.

Preparation of 30% stock sol: and (3) taking 270ml of double distilled water out of the measuring cylinder, pouring the double distilled water into a glass beaker, weighing 87.0g of acrylamide and 3.0g of methylene-bis-propylene by using an electronic balance, simultaneously adding the reagents into the glass beaker, stirring the reagents by using a stirrer until the reagents are completely dissolved, adding the double distilled water solution, keeping the constant volume to 200ml, and storing the solution in a brown bottle at room temperature.

Preparation of 10% AP: dripping 2ml of double distilled water in western region of a pipette into an EP tube, weighing 0.2gAP powder by an electronic balance, adding into the EP tube, mixing uniformly by using an oscillator, and storing at-20 ℃.

10 × preparation of electrophoresis buffer solution, weighing 800ml double distilled water by using a measuring cylinder, pouring into a glass beaker, weighing 30.2g Tris, 188g glycine and 10g SDS10g by using an electronic balance, adding into ddH2O for dissolving, heating and stirring the mixed solution in a stirrer until the mixed solution is completely dissolved, adding the double distilled water to make up the solution to 1L, and storing at room temperature.

Preparation of blocking solution (BSA or 5% skim milk powder): 100ml of TBST solution is measured out of a measuring cylinder and poured into a glass beaker, 5g of BSA or skimmed milk powder is weighed by an electronic balance, stirred until completely dissolved and stored in a refrigerator at 4 ℃.

10 × preparation of the transmembrane buffer, weighing 800ml of double distilled water by using a measuring cylinder, pouring the double distilled water into a glass beaker, weighing Tris58g, glycine 29g and SDS3.7g of powder by using an electronic balance, putting the powder into the glass beaker, heating and stirring the powder by using a stirrer until the powder is completely dissolved, complementing the solution to 1L by using the double distilled water, and storing the solution at room temperature.

Preparation of 1 × TBST 800ml of double distilled water was measured using a measuring cylinder and poured into a glass beaker, Tris2.42g and NaCl8.0g were weighed using an electronic balance, the mixture was poured into the beaker, heated and stirred using a stirrer until completely dissolved, the pH of the solution was adjusted to 7.6 with hydrochloric acid, and finally 2ml of Tween-20 was added.

Preparation of 4% paraformaldehyde: 800ml of double distilled water was measured by using a measuring cylinder and poured into a beaker, 4g of paraformaldehyde was weighed by an electronic balance, and the powder was added into a glass beaker and stirred with a stirrer until completely dissolved. Hydrochloric acid was added to adjust the pH of the solution to 7.3. Finally, the solution was made up to 100ml and stored at room temperature.

Preferably, the cell culture and hypoxia model establishment specifically comprises the following steps:

s1, extracting and culturing the adipose-derived stem cells, which comprises the following specific operations:

(1) preparing before experiment, including experiment article disinfection, ultraviolet disinfection, hand cleaning and the like;

(2) taking 3 wild type C57B L/6 mice, after anesthesia succeeds, fixing the mice in a supine position on an operating table, cutting the skin of the inguinal region of the mice by about 2cm, taking subcutaneous adipose tissues, suturing wounds on two sides, and putting the adipose tissues into a PBS solution for cleaning;

(3) putting the cleaned adipose tissues into a glass beaker, adding 0.075% type II collagenase with the dosage of 200U/ml, placing the glass beaker in a 37 ℃ thermostat for 30 minutes, oscillating the glass beaker once every 5 to 10 minutes, and stopping digestion of the collagenase after 30 minutes by using normal saline;

(4) placing the mixed solution into a centrifugal tube, centrifuging for 10 minutes at 1200 g/1200 r/min, removing supernatant and undigested fat, using 10% FBS DMEM to resuspend cell precipitates, using 0.16 mol/L ammonia chloride to dissolve residual red blood cells, filtering the collected cell suspension through a copper net, centrifuging for 10 minutes at 1200r/min, adding culture solution, blowing, uniformly mixing, sampling and counting, and adjusting the cell concentration to be 104 cells/ml according to the counting result;

(5) inoculating and culturing: inoculating 1ml of 4ml of culture solution with cell suspension amount in each 10cm2 culture bottle, and culturing in a 5% CO2 incubator at 37 deg.C;

(6) ADSC of 3 rd to 5 th generation is selected for the next experiment;

s2, establishing a cell hypoxia and glucose-starvation model, and specifically operating as follows:

(1) taking the neuron cells which grow and mature for one week, and inoculating the neuron cells into a multi-well plate according to the density of 1 × 106 cells/m L;

(2) sucking out the original culture solution, washing with PBS for three times, and adding a sugar-free culture medium containing drugs with corresponding concentrations;

(3) placing the culture dish in an anoxic device, introducing mixed gas of 95% nitrogen and 5% carbon dioxide, filling the device with the mixed gas, sealing the device with a sealing film, and placing the device in a 37 ℃ incubator for incubation for 3-6 hours;

(4) taking out the culture dish, and carrying out the next experiment;

s3, BV2 microglia cell lines were cultured in DMEM/high glucose medium containing 10% FBS and 1% pen/strep L PS (1. mu.g/ml) was co-cultured with BV2 microglia cells for 24h, and then exosomes (200. mu.g/ml) were added to the different groups of media.

As a preferred aspect of the present invention, the isolation of exosomes specifically comprises the steps of:

(1) when the ADSCs reach 80% confluence, the culture medium is replaced by FBS which is poor in exosomes, the culture is continued for 48 hours, and the culture is carried out under the normal oxygen or low oxygen condition;

(2) the medium was collected and centrifuged at 300 × g for 10 minutes, then at 2000 × g for 10 minutes at 4 ℃, after which the cell supernatant was filtered from whole cells and cell debris using a 0.22 μm sterile filter;

(3) adding the filtered supernatant to the upper compartment of the centrifugal filter device and centrifuging at 4000 × g until the volume of the upper layer is reduced to 200 μ L;

(4) the ultrafiltered supernatant was washed twice with PBS and, for purification of exosomes, the liquid was loaded on top of a 30% sucrose/D2O buffer in sterile Ultra-clean tubes and centrifuged at 100,000 × g for 60 min at 4 ℃ in an optima L-100 XP ultracentrifuge;

(5) the fraction containing ADSC-Exos was recovered using an 18-G needle (under normoxic conditions), then diluted in PBS and centrifuged at 4000Xg in a centrifugal filter unit at 4 ℃ until the final volume reached 200. mu. L and the exosomes were stored at-80 ℃ or immediately used in subsequent experiments.

As a preference of the present invention, the immunofluorescence evaluation specifically comprises the following steps:

(1) spinal cord tissue samples were taken on day 3 post-surgery;

(2) all samples were immersed in 1% bovine serum albumin and 0.3% triton x-100 for 1 hour to prevent non-specific reactions, followed by overnight incubation with an antibody at 4 ℃ including: anti-iNOS, anti-Ibal, anti-Arg 1, anti-NeuN;

(3) the next day, all samples were washed with PBS and incubated with the corresponding secondary antibody for 2 h: a secondary antibody in which Dylight (Dy)488 and Dy594 are mixed;

(4) nuclei were stained with DAPI and imaging was performed using either a nikon EC L IPSETi inverted microscope or an OlympusFV1000 confocal microscope.

As a preference of the present invention, the Western blot analysis specifically comprises the following steps:

(1) according to the molecular weight of the target protein, preparing 8% or 10% of separation gel according to a reagent specification formula, and filling 5% of concentrated gel after the separation gel is solidified;

(2) calculating the volume according to the concentration of the sample, and loading the sample by using a 10ul pipette;

(3) electrophoresis: firstly, carrying out electrophoresis for 30 minutes by using a voltage of 80V, and then adjusting the voltage to 120V until the electrophoresis is finished;

(4) film transfer: cutting a PVDF membrane with a proper size, soaking the PVDF membrane in formaldehyde, prying a glass plate by using a plastic warping plate after activation, cutting off concentrated glue, taking out the separated glue, placing the separated glue on filter paper of a rotating membrane clamp cathode, covering the PVDF membrane, placing the PVDF membrane into a rotating membrane tank, burying the rotating membrane tank in an ice box, and rotating the membrane by using a 300mA constant current, wherein the membrane rotating time is the same as the molecular weight of protein;

(5) and (3) sealing: blocking with 5% BSA for 1 hour at room temperature;

(6) primary antibody incubation: diluting the antibody according to the antibody specification, washing the PVDF membrane for 3 times by using TBST, cutting the PVDF membrane according to the molecular weight of target protein by using a Marker, putting the PVDF membrane into an incubation box, adding a primary antibody, and standing overnight in a shaking table at 4 ℃;

(7) incubating a second antibody; recovering the primary antibody in the incubation box, shaking the TBST for 3 times, diluting the secondary antibody by using 5% skimmed milk powder at a ratio of 1:100, and incubating for 1 hour at room temperature;

(8) and (3) exposure, namely discarding the secondary antibody, placing the incubation box on a shaking table, shaking and washing for 3 times, exposing by using a gel presentation system, mixing EC L luminescent solution A and B at a ratio of 1:1, and dripping 200ul of luminescent solution on the PVDF membrane for exposure.

As a preferred aspect of the present invention, the uptake of exosomes by BV2 microglia specifically comprises the steps of:

(1) adding a Dil solution of 4mg/m L to PBS containing exosomes and incubating;

(2) ultracentrifugation at 100,000 × g for 1h at 4 ℃ to remove excess fluorescent label from the labeled exosomes;

(3) the pellet was then resuspended in PBS, and finally washed and resuspended in PBS to wash the exosome pellet 3 times;

(4) these Dil-labeled exosomes were co-cultured with BV2 microglia for 24 hours, then the cells were washed with PBS and fixed in 4% paraformaldehyde;

(5) finally, the extent of uptake of Dil-labeled Exos and HExos by BV2 microglia was observed by confocal laser microscopy and the fluorescence intensity of Dil was measured with ZENlite software at different time points in both groups.

It is supplementary to the operation of exosome identification as follows:

vesicle diameter distributions from Exos and HExos were analyzed using the Nanosight L M10 system, morphology of exosomes obtained under normoxic and hypoxic conditions was observed using transmission electron microscopy, and specific exosome surface markers, such as TSG101, CD63 and CD8, were determined using Western blotting.

In addition, the operation process for establishing the mouse spinal cord injury model is as follows:

the mice were modeled after 12-16 weeks female wild-type C57B L/6 mice and knockout C57B L/6 mice were intraperitoneally injected with 0.35% sodium pentobarbital 35mg/kg before surgery, and whether anesthesia was successful was judged by pupillary and corticotranscription.after anesthesia was successful, the mice were fixed on an operating table, dorsal hair was removed using depilatory cream, sterilized with 75% alcohol, a surgical incision was made approximately 3cm long along the midline centering around T10 (the apex of the back), the skin was incised, the perispinous glass soft tissue was layered, T10 vertebral lamina was exposed, interspinous ligament and vertebral lamina were cut off using scissors, the vertebral lamina was opened to expose dura mater.the mice were placed on a striker table with their backs facing up, a striker needle was used with a 10g striker, a striker T10 segment spinal cord was freely dropped from a height of 5cm, e.g., forced lower limb convulsions occurred immediately with the tail swinging like one side of the body, which was considered as successful modeling, mice were housed in single cages with 2U daily intramuscular injections of penicillin injections, and the mice were treated with 200U injections of 200 u.g of penicillin immediately after urinary bladder excretion (200 u.200 g) injections).

It should be added that the procedure for RNA sequencing is as follows:

for lncRNA-seq experiments, Illumina-based RNA-seq was performed on RNA using TruSeq chain mRNA L T sample preparation kit Illumina HiSeq2500(2 × 50bp) was used to sequence the RNA-seq library, each sample reading 70-100 × 106, then KEGG was used for the comprehensive analysis of RNA-seq data.

It should be added that the operation process of the mouse motor function scoring is as follows:

the content of the score of Bresnahan by adopting Basso Beattie mainly comprises the following steps: the lower limb weight bearing condition, the coordination of the front and rear limb movements, the movable joint number, the joint movable range, the front and rear grab and the tail movement condition are Germany; the mechanism comprises 22 grades, wherein the grade 0 is invisible hind limb movement, the grade 21 is continuous volar movement, continuous coordinated gait is realized, toes continuously grab the ground, the position of a driving claw is always parallel to the body in the movement process, the trunk is continuously stable, and the tail is continuously tilted.

It should be added that the real-time quantitative pcr (qpcr) analysis is performed as follows:

configuring a PCR system according to the RT-PCR kit instruction:

(1)2×SYBR Green 5ul；

(2) upstream primer + downstream primer 0.2ul +0.2 ul;

(3) DEPC water 4.4 ul;

(4)cDNA0.2ul；

(5) placing the system into an RT-PCR instrument, and denaturing at 95 ℃ for 30 seconds;

(6) denaturation at 95 ℃ for 5 seconds; annealing at 60 ℃ for 30 seconds; extension at 72 ℃ for 10 min; the steps are circulated for 30 times;

(7) relative quantitative analysis: GAPDH was used as an internal control in this study, and 2- (. DELTA.CT) was used to calculate the relative expression of the gene of interest.

It should be added that the procedures for plasmid construction and transfection are as follows:

overexpression of lncGm37494 was achieved by plasmid transfection, target cells were transfected with L ipofectamine 2000, plasmid pcDNA-lncGm37494 (full length lncGm37494 was cloned into pcDNA vector in full length), and cells transfected with empty plasmid pcDNA served as controls.

It should be added that the enzyme-linked immunosorbent assay (E L ISA) is performed as follows:

the expression levels of I L-6, I L-1 β, tumor necrosis factor- α (TNF- α) were evaluated using the commercial E L ISA kit, following the manufacturer's instructions.

It is added that the operation process of the dual luciferase reporter gene detection is as follows:

the reporter plasmid was produced by inserting lncGm37494 or PPAR γ 3' -UTR sequences into the pmirG L O vector L ipofectamine 2000 was used to co-transfect the reporter plasmid and miR-130b-3p mimic into the cells after 48 hours of culture using the dual luciferase reporter assay system as a standard.

It should be added that the operation procedure of the statistical analysis is as follows:

all projects of this study were subjected to 3 independent replicates, and differences between different groups were compared using unpaired t-test or one-way ANOVA, while homogeneity of variance analysis was performed. With GraphPad Prism 7, there were significant differences at P < 0.05.

Results of the experiment

1. In vivo experiments, exosomes (Exos) injected with hypoxia-pretreated adscs (hexos) after SCI were shown to inhibit inflammatory factor expression and promote the conversion of microglia from M1 to M2 polarization, showing the effect of promoting functional recovery.

1.1, to demonstrate the protective effect of ADSCs-Exos on SCI. We isolated ADSCs from mouse adipose tissue. Exos of the ADSCs or the anoxic pretreated ADSCs are then isolated. TEM shows typical round nanoparticles, ranging from 50 to 150nm in diameter, with no significant difference in size, shape or electron density between the two groups of Exos (fig. 1A). WB showed that isolated exosomes could express the exosome surface markers TSG101, CD63 and CD81 (fig. 1B). NTA also showed similar size distribution at 100nm in the normoxic and hypoxic groups (fig. 1C). To examine whether BV2 microglia absorbed exosomes from normal oxygen or hypoxic conditions, exosomes were labeled with Dil dye and then co-cultured with the target BV2 for 24 hours. Fluorescence microscopy was used for monitoring and exosomes in both groups were found to be taken up by BV2 (fig. 1D).

FIG. 1 Exos form of ADSC. (A) Morphology of Exos observed by TEM. Scale bar: 500 nm. (B) Exos markers CD63, CD81, TSC101 were expressed by WB. Data are presented as mean ± SD. P <0.001 compared to Exos group. (C) Particle size distribution as measured by NTA. (D) Uptake of Exos labeled with the red fluorescent dye Dil into BV2 microglia. Data are presented as mean ± SD. P <0.001 compared to Exos group.

1.2, to determine the protective effect of Exos and HExos on SCI mice, we examined BBB (FIG. 2A) and BMS (FIG. 2B) scores of SCI mice after injection of Exos, which were found to be significantly lower after SCI.Exos treatment was scored higher, indicating partial recovery of nerve function, but HExos treatment had better functional improvement compared to PBS-treated and Exos-treated mice.immunofluorescence was performed using NeuN staining, it was seen that neurons surrounding myelopathy were injured, the number and morphology of three groups of neurons were observed.results show that NeuN-positive neurons increased after Exos treatment compared to PBS and Exos groups, HExos treatment had a greater effect on increasing neuronal numbers (FIG. 2D), E L ISA method detects inflammatory factors in spinal cord tissue, and found that inflammatory factors TNF- α, I L-6 and I8937 were more strongly expressed after injury to TNF- α, I-L-6 and I L-1 when treated with HExos, and I-3538 showed a greater effect on increasing glial cell activation in the glial cell activation by TNF-7, suggesting that TNF-7 cells after injury, TNF-6, TNF-3626, and TNF-11 cells were more strongly expressed in a map, and a more strongly expressed by TNF-7.

Figure 2 exosomes (Exos) of hypoxia-pretreated adscs (HExos) were bbb (a) and bms (B) scored by inhibiting inflammatory factor expression and promoting microglia transition from M1 polarization to M2 polarization with greater function promoting functional recovery after SCI than Exos under normal conditions (a and B) to assess neurological function 28 days after PBS, Exos and HExos injury, data are presented as mean ± SD. compared to PBS group, P < 0.01. (C and D) representative immunostaining image of NeuN-positive cells and number of NeuN-positive cells in different groups after SCI, P < 0.001. P <0.001vs Exos group (E-G) E ISA test showed TNF- α (E), I3-6 (F), I < 42-461. G (iv) data were presented as mean ± SD. compared to PBS group, P <0.001v s Exos group (E-G) E2 ISA test showed TNF- α (E), I3-6 (F), I < 42- β (I) data were presented as mean v β -x 2 test showed that P < M < v β -x 2P < 7 x β -x 2, P < x β -x 2, P < I < x 2, P < I < x 2, P < I.

2, L ncGm37494 are upregulated in HExos and can be delivered to BV2 cells via exosomes

2.1, it was found that lncRNA plays an important regulatory role after SCI [23, 24 ]. To determine if lncRNA plays a role in exosome-mediated neuroprotection after SCI. High-throughput sequencing found a large number of abnormally expressed lncRNA. Among these, 66 lncRNA up-regulated and 58 lncRNA down-regulated differed by > 1.5 fold between Exos and ADSC-derived HExos (FIG. 3A). qRT-PCR detection showed that the expression of lncGm37494 was significantly higher than Exos in HExos (FIG. 3B). To determine the effect of inc gm37494 on SCI. We constructed lnc gm37494 overexpression vector and transfected ADSCs. qRT-PCR detection revealed that the expression of lncGm37494 was significantly increased in Exos overexpressing ADSC compared to wild-type ADSC (FIG. 3C).

FIG. 3L ncGm37494 is upregulated in HExos and can be delivered via exosomes to BV2 (A)66 upregulated lncRNA and 58 downregulated lncRNA heatmaps with differences of > 1.5 fold between Exos and ADSC derived HExos data expressed as mean. + -. SD. compared to the Exos group, P < 0.001. (B) qRT-PCR assay showed lncRNA expression in both Exos and HExos (C) qRT-PCR assay showed expression of lncGm37494 in either ADOS form of Exos or lncGm37494 overexpressing ADSC.

L PS treatment promoted the expression of inflammatory factors TNF- α (FIG. 4A), I L-6 (FIG. 4B), I L-1 β (FIG. 4C) in the supernatant of BV2 cells high levels of Exos (lncExos) had a greater significance of the inhibition of inflammatory factor expression by lncGm37494 than by Exos Werstern blot assay showed that lncExos treatment significantly inhibited iNOS but promoted Arg1 expression suggesting that Hexos polarized microglia from M1 to M2 by delivering lncGm 37494.

Figure 4L ncGm37494-Exos therapy more effectively inhibited inflammatory responses by shifting microglia/macrophage polarization from M1 to M2, (a-C) E L ISA assays showed expression of TNF- α (a), I L-6 (B), I L-1 (C) in BV2 cell supernatants under L PS assay conditions data expressed as mean ± sd.p <0.05, P <0.01, P <0.001vs Ctrl group P <0.05, P <0.001vs L PS group (D-F) western blot assays showed protein levels of M1 and M2 related proteins iNOS and Arg1, respectively data expressed as mean ± sd.p < 0.p <0.05, P <0.001vs Ctrl group P <0.05, P <0.001vs group P <0.01, P <0. L.

3. ADSCs Exos containing L ncGm37494 promote microglia/macrophage polarization from M1 to M2 by inhibiting miR-130b-3p and enhancing PPAR γ expression.

Bioinformatics analysis shows that miR-130b-3p is a downstream target of lncGm 37494. Thus, we constructed luciferase reporter vectors. The results of the luciferase reporter assay showed that miR-130B-3p is a downstream lncGm37494 binding target (fig. 5A), and lncGm37494 inhibited luciferase activity in wild-type but unmutated cell lines (fig. 5B). Bioinformatic analysis also showed that PPAR γ was the target of miR-130b-3p, indicating that miR-130b-3p interacts directly with the PPAR γ 3' -UTR to inhibit its mRNA expression (figure 5C). In the luciferase reporter assay, PPAR γ inhibited luciferase activity in the wild type but not in the mutant cell line (fig. 5D). qRT-PCR assays showed that overexpression of lncGm37494 with BV2 cells promoted the expression of lncGm37494, but that up-regulation of miR-130b-3p or down-regulation of PPAR γ did not reverse the levels of lncGm37494 (FIG. 5E). Suggesting that miR-130b-3p and PPAR γ are both located downstream of lncGm 37494. qRT-PCR assays also found that lncGm37494 overexpression suppressed miR-130b-3p (FIG. 5F), but promoted PPAR γ expression (FIG. 5G). Western blot detection showed that lncGm37494 overexpression inhibited the expression of iNOS and promoted the expression of Arg 1. It is suggested that lncGm37494 overexpression promotes the transfer of microglial polarization from M1 to M2. However, over-expressed miR-130b-3p or downregulated PPAR γ reversed the stimulatory effect of lncGm37494 on M2 microglia (FIGS. 5H-J). The combined results show that lncGm37494 overexpression promotes microglial polarization to be converted from M1 to M2 by targeting miR-130b-3p/PPAR γ axis.

After SCI, ADSC (lncExos) from the ADSCs containing lncGm37494 has a remarkable effect of promoting functional recovery after injury compared with ADSCs Exos

4.1, FIG. 5ADSCs delivered L ncGm37494 Exos transfer microglia/macrophage polarization from M1 to M2 by inhibition of miR-130B-3P and enhanced PPAR γ expression (A) prediction of miR-130B-3P binding site in lncGm 37494A mutant version of lncGm37494 is presented (B) prediction of relative luciferase activity measured 48 hours after transfection of HEK293T cells with miR-130B-3P mimetics/NC or lncGm37494 wild type/Mut. data are presented as mean + -SD.P <0.001 (C) prediction of miR-130B-3P binding site within 3'UTR of PPAR γ. A mutant form of 3' -UTR-PPAR γ (D) is presented as 3'-UTR-PPAR γ mutant form of protein (D) comparison of miR-130B-3P mimetics/UTR or 3' wild-HEγ/3P for miR 3P mimetics/UTR < 0.001. C. the binding site of wild-PPAR γ/3P. A relative luciferase protein expression (D) is presented as mean + -7B-3P mimetics/UTR < 9. the mean 3P > 2. the mean 3P < 9. the mean 3P 15. the map is presented and.

4.2 ADSC (lncExos) containing lncGm37494 was more able to promote recovery of nerve function than ADSCs Exos after SCI, to determine the protective effect of lncGm37494 on SCI mice, we examined SCI mice after exosome injection for BBB (FIG. 6A) and BMS (FIG. 6B) scores and found a significant reduction in post-SCI scores Exos treatment fractions but, lncExos treatment had a better functional improvement compared to PBS and Exos group mice.

Figure 6. exons from lncGm 37494-modified adsc (lncexos) had greater effect on promoting functional recovery after in vivo SCI than ADSCs Exos (a and B) bbb (a) score and bms (B) score to assess neurological function 21 days after PBS, Exos and HExos injury, data expressed as mean ± SD. versus PBS group, P < 0.01. (C and D) representative immunostaining images of NeuN-positive cells and numbers of NeuN-positive cells in different groups after SCI, data expressed as mean ± SD. versus PBS group, P <0.05, P < 0.001. P <0.001vs Exos group, (E-G) E2 ISA test showed TNF- α (E), I L-6 (F), I395-1 (G) β (G), expression of I39L-1 (G), data expressed as mean ± 0.001 vs. sd group, sd < 0.01. E-G) E3875 (E-G) after injury, data expressed as mean ± 0.75G < PBS group, i.001. P < 0.001. isn < PBS group, P < 0.8678, P < 0.75. isn < PBS group, data expressed as mean ± SD., P < 0.001H < PBS group, I < 0.001.

The foregoing shows and describes the general principles, essential features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, and the preferred embodiments of the present invention are described in the above embodiments and the description, and are not intended to limit the present invention. The scope of the invention is defined by the appended claims and equivalents thereof.

Claims (8)

1. An experimental method for repairing spinal cord by inducing microglia polarization by exosome is characterized in that: the method comprises the following experimental steps:

step one, cell culture and hypoxia model establishment;

step two, separating exosomes;

step three, exosome identification;

step four, establishing a mouse spinal cord injury model;

step five, RNA sequencing;

step six, scoring the movement function of the mouse;

seventhly, carrying out real-time quantitative PCR analysis;

step eight, performing immunofluorescence evaluation;

ninth, western blot analysis;

step ten, plasmid construction and transfection;

step eleven, taking up exosomes by BV2 microglia;

step twelve, enzyme linked immunosorbent assay;

thirteen, detecting dual luciferase reporter genes;

and step fourteen, carrying out statistical analysis.

2. The experimental method for the secretion body to induce microglia polarization to repair spinal cord as claimed in claim 1, which comprises the following experimental materials, wild type C57B L/6 mouse and knockout C57B L/6 mouse.

3. The experimental method for the secretion-induced polarization of microglia to repair spinal cord according to claim 1, which comprises the following reagent preparation, including PBS solution, 10% SDS, 1.0M Tris-HCl, 30% stock sol, 10% AP, 10 × electrophoresis buffer, blocking solution, 10 × trans-membrane buffer, 1 × TBST buffer and 4% paraformaldehyde.

4. The experimental method for the secretion-induced polarization of microglia to repair spinal cord according to claim 1, wherein: the cell culture and hypoxia model establishment specifically comprises the following steps:

s1, extracting and culturing the adipose-derived stem cells, which comprises the following specific operations:

(1) preparing before experiment, including experiment article disinfection, ultraviolet disinfection, hand cleaning and the like;

(2) taking 3 wild type C57B L/6 mice, after anesthesia succeeds, fixing the mice in a supine position on an operating table, cutting the skin of the inguinal region of the mice by about 2cm, taking subcutaneous adipose tissues, suturing wounds on two sides, and putting the adipose tissues into a PBS solution for cleaning;

(3) putting the cleaned adipose tissues into a glass beaker, adding 0.075% type II collagenase with the dosage of 200U/ml, placing the glass beaker in a 37 ℃ thermostat for 30 minutes, oscillating the glass beaker once every 5 to 10 minutes, and stopping digestion of the collagenase after 30 minutes by using normal saline;

(4) placing the mixed solution into a centrifugal tube, centrifuging for 10 minutes at 1200 g/1200 r/min, removing supernatant and undigested fat, using 10% FBS DMEM to resuspend cell precipitates, using 0.16 mol/L ammonia chloride to dissolve residual red blood cells, filtering the collected cell suspension through a copper net, centrifuging for 10 minutes at 1200r/min, adding culture solution, blowing, uniformly mixing, sampling and counting, and adjusting the cell concentration to be 104 cells/ml according to the counting result;

(5) inoculating and culturing: inoculating 1ml of 4ml of culture solution with cell suspension amount in each 10cm2 culture bottle, and culturing in a 5% CO2 incubator at 37 deg.C;

(6) ADSC of 3 rd to 5 th generation is selected for the next experiment;

s2, establishing a cell hypoxia and glucose-starvation model, and specifically operating as follows:

(1) taking the neuron cells which grow and mature for one week, and inoculating the neuron cells into a multi-well plate according to the density of 1 × 106 cells/m L;

(2) sucking out the original culture solution, washing with PBS for three times, and adding a sugar-free culture medium containing drugs with corresponding concentrations;

(3) placing the culture dish in an anoxic device, introducing mixed gas of 95% nitrogen and 5% carbon dioxide, filling the device with the mixed gas, sealing the device with a sealing film, and placing the device in a 37 ℃ incubator for incubation for 3-6 hours;

(4) taking out the culture dish, and carrying out the next experiment;

s3, BV2 microglia cell lines were cultured in DMEM/high glucose medium containing 10% FBS and 1% pen/strep L PS was co-cultured with BV2 microglia cells for 24h, and then exosomes were added to the different groups of media.

5. The experimental method for the secretion-induced polarization of microglia to repair spinal cord according to claim 1, wherein: the exosome separation specifically comprises the following steps:

(1) when the ADSCs reach 80% confluence, the culture medium is replaced by FBS which is poor in exosomes, the culture is continued for 48 hours, and the culture is carried out under the normal oxygen or low oxygen condition;

(2) the medium was collected and centrifuged at 300 × g for 10 minutes, then at 2000 × g for 10 minutes at 4 ℃, after which the cell supernatant was filtered from whole cells and cell debris using a 0.22 μm sterile filter;

(3) adding the filtered supernatant to the upper compartment of the centrifugal filter device and centrifuging at 4000 × g until the volume of the upper layer is reduced to 200 μ L;

(4) the ultrafiltered supernatant was washed twice with PBS and, for purification of exosomes, the liquid was loaded on top of a 30% sucrose/D2O buffer in a sterile tube and centrifuged at 100,000 × g for 60 minutes at 4 ℃ in an ultracentrifuge;

(5) the fraction containing ADSC-Exos was recovered using an 18-G needle, then diluted in PBS and centrifuged at 4000Xg in a centrifugal filter unit at 4 ℃ until the final volume reached 200. mu. L and the exosomes were stored at-80 ℃ or immediately used in subsequent experiments.

6. The experimental method for the secretion-induced polarization of microglia to repair spinal cord according to claim 1, wherein: the immunofluorescence evaluation specifically comprises the following steps:

(1) spinal cord tissue samples were taken on day 3 post-surgery;

(2) all samples were immersed in 1% bovine serum albumin and 0.3% triton x-100 for 1 hour to prevent non-specific reactions, followed by overnight incubation with an antibody at 4 ℃ including: anti-iNOS, anti-Ibal, anti-Arg 1, anti-NeuN;

(3) the next day, all samples were washed with PBS and incubated with the corresponding secondary antibody for 2 h: a secondary antibody in which Dylight (Dy)488 and Dy594 are mixed;

(4) nuclei were stained with DAPI and imaging was performed using an inverted microscope or confocal microscope.

7. The experimental method for the secretion-induced polarization of microglia to repair spinal cord according to claim 1, wherein: the western blot analysis specifically comprises the following steps:

(1) according to the molecular weight of the target protein, preparing 8% or 10% of separation gel according to a reagent specification formula, and filling 5% of concentrated gel after the separation gel is solidified;

(2) calculating the volume according to the concentration of the sample, and loading the sample by using a 10ul pipette;

(3) electrophoresis: firstly, carrying out electrophoresis for 30 minutes by using a voltage of 80V, and then adjusting the voltage to 120V until the electrophoresis is finished;

(4) film transfer: cutting a PVDF membrane with a proper size, soaking the PVDF membrane in formaldehyde, prying a glass plate by using a plastic warping plate after activation, cutting off concentrated glue, taking out the separated glue, placing the separated glue on filter paper of a rotating membrane clamp cathode, covering the PVDF membrane, placing the PVDF membrane into a rotating membrane tank, burying the rotating membrane tank in an ice box, and rotating the membrane by using a 300mA constant current, wherein the membrane rotating time is the same as the molecular weight of protein;

(5) and (3) sealing: blocking with 5% BSA for 1 hour at room temperature;

(6) primary antibody incubation: diluting the antibody according to the antibody specification, washing the PVDF membrane for 3 times by using TBST, cutting the PVDF membrane according to the molecular weight of target protein by using a Marker, putting the PVDF membrane into an incubation box, adding a primary antibody, and standing overnight in a shaking table at 4 ℃;

(7) incubating a second antibody; recovering the primary antibody in the incubation box, shaking the TBST for 3 times, diluting the secondary antibody by using 5% skimmed milk powder at a ratio of 1:100, and incubating for 1 hour at room temperature;

(8) and (3) exposure, namely discarding the secondary antibody, placing the incubation box on a shaking table, shaking and washing for 3 times, exposing by using a gel presentation system, mixing EC L luminescent solution A and B at a ratio of 1:1, and dripping 200ul of luminescent solution on the PVDF membrane for exposure.

8. The experimental method for the secretion-induced polarization of microglia to repair spinal cord according to claim 1, wherein: the BV2 microglia cell exosome uptake method specifically comprises the following steps:

(1) adding a Dil solution of 4mg/m L to PBS containing exosomes and incubating;

(2) ultracentrifugation at 100,000 × g for 1h at 4 ℃ to remove excess fluorescent label from the labeled exosomes;

(3) the pellet was then resuspended in PBS, and finally washed and resuspended in PBS to wash the exosome pellet 3 times;

(4) these Dil-labeled exosomes were co-cultured with BV2 microglia for 24 hours, then the cells were washed with PBS and fixed in 4% paraformaldehyde;

(5) finally, the extent of uptake of Dil-labeled Exos and HExos by BV2 microglia was observed by confocal laser microscopy and the fluorescence intensity of Dil was measured with ZENlite software at different time points in both groups.

Images