CN106492190B - Method for inducing and maintaining selective polarization of microglia by Hpx protein and application thereof - Google Patents

Abstract

The invention belongs to the fields of genetic engineering and medicine, and particularly relates to a method for specifically inducing selective polarization of microglia by using a Hemopexin protein and application of the Hemopexin protein in treating spinal cord injury repair. The main technical scheme of the invention is as follows: transplanting microglia into spinal cord of Hemopexin knockout mice to block the microglia from selectively polarizing. And (II) incubating the Hemopexin protein and the microglia can induce and maintain the selective polarization of the microglia. Hemopexin induces microglia to present an M2 phenotype, and can effectively promote the repair of spinal nerve injury.

Description

Method for inducing and maintaining selective polarization of microglia by Hpx protein and application thereof

Technical Field

The invention belongs to the fields of bioengineering and medicine, and particularly relates to microglia under the action of plasma protein Hpx (hepexin) and treatment application thereof in spinal cord injury repair.

Background

The mortality and disability rate of central nerve injuries, especially spinal cord injuries, is high, and an ideal treatment scheme is lacking at present. The reasons for poor prognosis of spinal cord injury include at least focal neurodegeneration, demyelination, and remyelination disorders caused by impairment of oligodendrocyte precursor cell differentiation.

Microglia and macrophages play an important role in central injury repair, with their different polarization states involved in the pathological progression of secondary spinal cord injury. It has now been found that microglial polarization plays an important role in the regulation of neuronal and oligodendrocyte precursor cell function. Microglia in different polarization states secrete different cytokines, which serve different functions in the process of disease and repair. The number of M1 type microglia is positively correlated with the damage degree of the axon, while M2 type promotes differentiation of oligodendrocyte precursor cells and protects neurons from damage. Studies have shown that, following spinal cord injury, central microglia begin to polarize to M1 type and persist; whereas M2-type microglia appear only transiently within one week after injury. The rapid decrease of M2 microglia after a transient increase is probably an important reason for the poor prognosis of spinal cord injury. In view of this, modulation of the polarization state of microglia is a potential strategy for treating spinal cord injury. However, there is currently no effective means for polarizing microglia to a specific state.

Hemopexin (Hpx) is an acute phase protein. The sequence of Hpx (hepexin, GeneID: GI:184497, GenBank: AAA58678) is shown in SEQ ID NO: 1. Hpx binds Heme (Heme) non-covalently and with high affinity. It is generally believed that the role of Hpx is to bind to Heme and transport it to the liver for metabolism. Research also shows that Hpx has direct action on neurons and can directly weaken the cytotoxicity of the Heme; hpx reduces the Heme-induced Th17 differentiation and macrophage inflammatory response.

At present, the polarization effect of the Hpx protein on microglia is not reported.

Disclosure of Invention

The invention aims to provide a new medical application of hepxin (Hpx) protein, in particular to an application of Hpx protein in preparing a medicine for treating spinal cord injury; and in promoting the microglia to be converted into the M2 polarization state.

It is another object of the present invention to provide a method for promoting the transformation of microglia into M2 polarized state.

The invention also aims to provide application of the method for promoting the microglia to be converted into the M2 polarization state in preparing a medicament for treating spinal cord injury

In a first aspect of the invention, the application of the Hpx protein in preparing a medicament for treating spinal cord injury is provided.

The sequence of the Hpx protein is shown as SEQ ID NO: 1.

Applicants found that Hpx protein (hepxin, GeneID: 184497, GenBank: AAA58678) was differentially expressed in focal tissues of spinal cord injury model mice at different disease stages and was synchronized with the expression level of a marker protein of M2 type microglia (hereinafter, microglia refer to mouse microglia unless otherwise specified). Both microglia from Hpx knockout mice and their blood-derived precursor cell, monocyte, after spinal cord injury are more prone to M1 polarization. Down-regulating MSX3 levels in microglia accelerated mouse demyelination and neurodegeneration. Whereas transplantation of microglia pre-activated in vitro to M1 type into the spinal cord of Hpx-deficient mice down-regulated the rate of microglia polarization to M2. Further, the function recovery of the Hpx knockout mouse is obviously poor after spinal cord injury, and the damaged myelopathy tissue is obviously enlarged. More interestingly, the Hpx protein converts the M1 type microglia activated classically in the TLR-4 pathway into M2 type microglia, promotes the differentiation of the oligodendrocytes, and relieves the apoptosis of the oligodendrocytes and neurons. The study reveals a blood borne factor dependent mechanism driving polarization of microglia M2, and Hpx is expected to become a new target for promoting remyelination therapy.

In a second aspect of the invention, there is provided the use of Hpx to promote the switching of microglia to the M2 polarized state.

The application also provides a method for promoting the microglia to be converted into the M2 polarization state.

The application is the application of Hpx in preparing the reagent for promoting the microglia to be converted into the M2 polarization state.

The application is as follows: 0.5-50ng/ml of Hemopexin protein is co-hatched with microglia to obtain the microglia in the M2 polarization state.

The main technical scheme of the invention is as follows:

(one) incubating the Hpx protein with microglia.

And (II) co-culturing the conditioned supernatant obtained by using the microglia and neuron or oligodendrocyte precursor cells.

And (III) transplanting the microglia into spinal cords of the mice with spinal cord injuries.

The "conditioned supernatant" of the invention: and (3) after the Hpx protein and the microglia are incubated for 12 hours, replacing fresh culture solution for the microglia, and collecting culture solution after 72 hours, namely conditioned supernatant.

The method for promoting the microglia to be converted into the M2 polarization state comprises the following specific steps:

the day before the experiment, 1X 10 inoculation is carried out⁵Putting the microglia into a 24-hole culture plate, wherein the volume of the added culture medium is 500 mu L, and the fusion rate of the cells is about 30-50%;

adding three proteins with different gradients into corresponding holes of each group, wherein the concentrations of the added proteins are 0.5ng/mL, 5ng/mL and 50ng/mL respectively;

the culture plate is gently shaken in the horizontal direction to fully mix the culture medium and the protein, then the cell plate is put back into the incubator for incubation at 37 ℃ for 12 hours, and then the fresh culture solution is changed, and after 72 hours, the microglia (functional microglia) in the M2 polarization state is obtained.

The invention transplants the functional microglia into the spinal cord of a spinal cord injury model, which comprises the following steps:

centrifuging 400g of the microglia in the M2 polarization state for 5 minutes to prepare 2 mu L of cell suspension;

injecting 2 μ L of cell suspension in the range of 1mm from the head and tail sides of the core plane of the injured spinal cord tissue (such as in a spinal cord hemisection model, in a spinal cord cutting plane), injecting at an injection speed of 0.5 μ L/min for 4 minutes, standing for 5 minutes, withdrawing the needle for 2 minutes, and suturing the meninges, the muscle, the subcutaneous tissue and the skin layer by layer.

In a third aspect of the present invention, there is provided a method of promoting the transition of microglia to M2 polarization state, the method including but not limited to:

after 0.5-50ng/mL of Hpx protein was incubated with microglia for 12 hours, fresh culture medium was changed for the microglia, and the microglia were collected after 72 hours.

The sequence of Hpx (hepexin, GeneID: GI:184497, GenBank: AAA58678) is shown in SEQ ID NO: 1.

Further, the invention also provides application of the method for promoting the microglia to be converted into the M2 polarization state in preparation of treating spinal cord injury.

The conditioned supernatant obtained from the microglia acted by the Hpx protein is co-cultured with neurons or oligodendrocyte precursor cells, and the viability, differentiation and degeneration of the neurons or oligodendrocyte precursor cells are detected. The conditioned supernatant of microglia acted by the Hpx protein has a protective effect on neurons and promotes the differentiation and maturation of oligodendrocyte precursor cells, and the Hpx knockout mouse microglia inhibits the differentiation and maturation of the oligodendrocyte precursor cells and delays the repair of spinal cord injury.

And detecting the Hpx related cell factors in the microglia by adopting Q-PCR and an immunoblotting experiment. The result shows that the microglia acted by the Hpx can promote the polarization of the microglia M2, inhibit the polarization of M1, promote the survival, the differentiation and the growth of neurite of the oligodendrocyte and promote the repair of spinal cord injury by regulating LRP-1 related signal pathways.

In a fourth aspect of the present invention, there is provided a functional microglia obtained by the method for promoting the transformation of microglia into M2 polarization state.

In a fifth aspect of the present invention, there is provided the use of the above functional microglia cell for the treatment of spinal cord injury. And (III) transplanting the microglia into spinal cords of the mice with spinal cord injuries.

As previously mentioned, microglia in different polarization states secrete different cytokines that serve different functions in the process of pathology and repair. The number of M1 type microglia is positively correlated with the damage degree of the axon, while M2 type promotes differentiation of oligodendrocyte precursor cells and protects neurons from damage. The polarization mechanism of microglia is unclear at present. Our study found that Hpx deficient mice had a tendency to polarize M1 type in microglia (fig. 2) and demonstrated inhibition of demyelinating lesion remyelination and neurite outgrowth from in vitro and in vivo experiments (fig. 4, 6, 7). More interestingly, exogenous administration of Hpx protein to microglia caused microglia to polarize through LRP-1 signaling pathway to M2 type, significantly alleviating spinal cord injury. In conclusion, exogenous Hpx therapy is expected to become a new means for promoting spinal cord injury repair therapy. Namely, the invention also provides the application of Hpx as an agonist of LRP-1 signaling pathway.

In addition, the inventor has also made relevant studies on the inhibition of M1 polarization by Hpx.

The main technical scheme of the invention is as follows: first, a spinal cord injury model was prepared from Hpx knockout mice already available in the laboratory. And (II) detecting the proportion of polarized cells after spinal cord injury of the knockout mouse by using histochemistry and flow cytometry. And thirdly, behaviorally evaluating the influence of mouse knockout Hpx on the recovery of the motor function of the mouse. (IV) evaluating scar formation and neuron damage of the Hpx knockout mouse by using a histochemical method.

Experiments show that mouse microglia knocked out by Hpx is more prone to be converted into an M1 polarization state, and the mouse microglia knockouts tumor necrosis factors, accelerates neuronal damage, and delays recovery of spinal motor function.

In combination with our experimental results, it was found that the hepexin protein in blood was synchronized with the expression of the marker protein of M2 type microglia in the course of spinal cord injury, and increased early but gradually decreased later (fig. 1). The absence of the hepexin protein caused microglial inclination to M1 type polarization (fig. 2), which impaired neurons by secreting inflammatory factors including TNF-a (fig. 3) (fig. 5), hampered the recovery of spinal cord motor function (fig. 4). In vitro experiments, Hpx regulated microglia M1-M2 polarization by modulating LRP-1 signaling pathway (FIGS. 7, 9), inhibited neuronal and oligodendrocyte apoptosis (FIGS. 7, 8), and promoted their differentiation and maturation (FIG. 8). The study reveals a blood borne factor regulation mechanism driving microglia M2 polarization, and discusses the role in spinal cord injury repair.

Drawings

FIG. 1, the level of hepexin protein in the course of spinal cord injury was synchronized with the level of polarization of M2 microglia; (A, D) Q-PCR to observe the level of Hpx and microglial polarization during the course of spinal cord injury. (B, C, E, F) levels of Hpx and microglial polarization marker proteins during spinal cord injury.

Figure 2, flow cytometry analysis of the effect of Hpx knockdown on mouse microglial cell polarization after spinal cord injury. (A, B) schematic diagram of microglial cell sorting. (C, D, G) M1 polarizes the proportion of microglia in spinal cord lesions. (E, F, H) M2 polarizes the proportion of microglia in spinal cord lesions.

FIG. 3, Hpx deficiency results in upregulation of mouse microglial inflammatory factor TNF-a expression after spinal cord injury. (A, B) TNF-alpha at lesions 4 days after spinal cord injury⁺Microglial cell number, (C, D) TNF-alpha at lesion 7 days after spinal cord injury⁺Microglial cell number, (E) TNF-alpha⁺Microglial cell occupancy of Iba-1⁺The proportion of microglia.

Figure 4, Hpx deficiency resulted in delayed recovery of mouse motor function following spinal cord injury. (A, B) BMS scoring. (C, D) swimming function score.

Figure 5, Hpx deficiency resulted in a reduction in mouse functional neurons after spinal cord injury. (A, B, C, D, EF) NeuN in the proximal (A, C) and peripheral (B, D) gray matter of spinal cord injury⁺Total number of neurons and number of ventral anterior horn neuronsAmount of the compound (A).

FIG. 6, exogenous supplementation with Hpx induced the conversion of LPS-activated microglia type M1 to type M2. (A-D) Q-PCR detection of microglia polarization marker level, and (E-H) immunoblotting detection of microglia polarization marker level.

FIG. 7, Hpx inhibits neuronal apoptosis by modulating microglial M1-M2 polarization. (A) Experimental flow charts for collecting conditioned supernatant of microglia and incubating the supernatant with neurons, (B-E) Hpx antagonizes pro-inflammatory effects of LPS on microglia, the cell supernatant reduces apoptotic numbers of co-incubated neurons, and (F) apoptotic neurons (TUNEL)⁺/NeuN⁺) Accounts for total neurons (NeuN)⁺) Percentage of (c).

FIG. 8, Hpx inhibits oligodendrocyte apoptosis and promotes differentiation and maturation by modulating microglia M1-M2 polarization. (A) Experimental flow sheet, (B-F) cytochemical experiment quantitative NG2⁺TUNEL in OPC⁺Percentage of apoptotic cells, (G-L) cytochemical experiments quantitate MBP⁺Proportion of differentiated OPCs in different treatment groups.

FIG. 9, Hpx regulates microglial M1-M2 polarization by modulating the LRP-1 signaling pathway. (A-D) Q-PCR (polymerase chain reaction) detection of a microglia polarization marker level, (E-H) immunoblotting detection of a microglia polarization marker level, and (A-H) RAP blocking LRP-1 pathway and antagonizing polarization regulation of the microglia by Hpx.

Detailed Description

The invention will now be further described with reference to examples and figures, but the practice of the invention is not limited thereto.

Example 1: spinal cord injury modeling, detecting the correlation between Hpx and microglia polarization markers.

1. Spinal cord injury model

Mice were anesthetized by intraperitoneal injection with 4% chloral hydrate and then prepped on a foam board. Passively bending the spine of the mouse, calculating 1-2 stages from the most obvious postero-convex part of the thoracolumbar region to the cephalic side, and taking a longitudinal operation incision. The skin is incised, the bilateral musculature is separated, the spinous process and the bilateral vertebral plates are exposed, and the vertebral plate stage is located. At the 9-10 chest, the 9 thoracic lamina was removed by rongeur biting to fully expose the spinal cord tissue. The spinal cord is organized into bright white, and the median posterior aspect is seen as the longitudinal artery. If the operation is the contrast operation of laminectomy, the operation incision can be sutured at the moment; in case of a spinal cord crush injury operation, a forceps with a minimum closing gap of 0.4mm is transversely erected at the spinal cord for 15 seconds, the forceps are loosened to show obvious hematoma traces on two sides of the spinal cord, and then an operation incision is sutured. And observing the bleeding condition of the operation wound after the operation. Bladder compression micturition was performed daily after surgery until mice could urinate by themselves.

2. Preparation of spinal cord tissue samples

4% chloral hydrate is injected into abdominal cavity of a mouse which is molded for a certain number of days for anesthesia, the mouse is fixed on a foam board after being completely anesthetized and lying backwards, and the upper limb and the lower limb are pulled longitudinally as much as possible to ensure that the spinal structure is fully extended. A U-shaped incision is made in the chest, the skin is cut open, the ribs are cut open, the heart is exposed, and the right auricle is cut open. Then puncture the left ventricle with the syringe needle, use 1 x PBS solution to inject continuously first, use about 20 ml; perfusion was continued with 4% PFA, approximately 20 ml. After fixation was complete, the spine of the mice (along with bone and spinal cord tissue) was surgically removed for WB and RNA extraction.

3. Extracting total RNA from tissue, reverse transcription

(1) mRNA extraction and reverse transcription into cDNA by Trizol: adding 1ml Trizol into each well of a 24-well plate, fully cracking cells, and standing in an EP tube for 5 min; centrifuging at 12,000g for 5min, and discarding the precipitate; adding 200ul chloroform/1 ml Trizol, mixing, standing at room temperature for 15 min; centrifuging at 4 deg.C for 15min at 12,000g, and sucking the upper water phase into another centrifuge tube without sucking the middle layer; adding isopropanol with equal volume, mixing, and standing at room temperature for 5-10 min; centrifuging at 4 deg.C for 10min at 12,000g, removing supernatant, and precipitating RNA at the bottom of the tube; adding 1ml of 75% ethanol, and suspending RNA precipitate; centrifuging at 4 deg.C and 8000g for 5min, and removing supernatant; drying at room temperature or vacuum drying for 5-10 min; adding 20ul DEPC water, and dissolving in a water bath at 60 ℃; reverse transcription into cDNA was performed according to the Reveraid TM First Strand cDNAsynthesis Kit.

(2) RT-PCR: reaction system: SYBR Green Mix 10 ul; 2ul of each upstream and downstream primer (2 uM); 1ul of cDNA; ddH2O 5ul (Total System 20ul)

Step 1:95 ℃ for 5min

Step 2: at 95 ℃ for 15s, at 60 ℃ for 15s, at 72 ℃ for 45s, for 40 cycles;

and step 3: dissolution Curve analysis

The amplification of the target gene was normalized by the internal control GAPDH, and the relative expression level of the target gene was calculated by the 2-ddct method.

4. Detection of Hpx and Arginase-1 nucleic acid and protein levels in spinal cord injury tissues.

A. A PCR method is adopted to obtain a linearized template from the mouse microglia total RNA by fishing a target gene, and the method comprises the following steps: reaction system: p10.4. mu.L, P20.4. mu.L, 10 Xbuffer 2.0. mu.L, MgCl20.5. mu.L, dNTPs (2.5mmol/L) 0.8. mu.L, Taq polymerase 0.2. mu.L, template 1. mu.L (negative control group not added), configured to 20. mu.L by ddH 2O. And (3) PCR reaction conditions: hot start at 94 ℃ for 30s → denaturation at 94 ℃ for 30s → renaturation at 60 ℃ for 30s → extension at 72 ℃ for 30s, 30 cycles, and finally polymerization at 72 ℃ for 6 min.

The Q-PCR primers used were:

B. spinal cord tissue was digested with RIPA supplemented with protease inhibitor (Roche) to extract proteins. The protein levels were measured with anti-Hpx and anti-Arginase-1 antibodies, respectively. (see Yu Z, Sun D, Feng J, Tan W, Fang X, ZHaoM, ZHao X, Pu Y, Huang A, Xiaoing Z, Cao L, He C. MSX3 Switches Microglia polarizationj protection from information-Induced Demyelation. J Neurosis. 2015 Apr 22; 35(16):6350-65.)

The results are shown in fig. 1, the level of the hemepexin protein in the course of spinal cord injury is synchronized with the level of polarization of M2 microglia; (A, D) Q-PCR detection finds that the levels of Hpx and a microglia polarization marker Arginase-1 are synchronously up-regulated and down-regulated in the course of spinal cord injury. (B, C, E, F) immunoblotting experiments find that the Hpx and microglia polarization marker protein Arginase-1 level are synchronously up-regulated and down-regulated in the course of spinal cord injury.

Example 2: flow cytometry analysis

And (3) sorting microglia in the injured spinal cord of the Hpx knockout mouse by using a flow cytometer, and detecting the polarization ratio.

A. Collection of monocytes

4% chloral hydrate is injected into abdominal cavity of a mouse which is molded for a certain number of days for anesthesia, the mouse is fixed on a foam board after being completely anesthetized and lying backwards, and the upper limb and the lower limb are pulled longitudinally as much as possible to ensure that the spinal structure is fully extended. The anterior "U" incision was made, the skin was cut open, the ribs were cut open, the heart was exposed, the right auricle was cut open, the left ventricle was then punctured with a syringe needle and the injection was continued with 1 x PBS solution, approximately 40 ml. Then, under a microscope, spinal cord tissues (including tissues 1mm each above and below the spinal cord injury) at the spinal cord injury site were surgically removed and placed in a 4 ℃ dissecting solution. The removed spinal cord tissue was cut to pieces, each piece being less than 1mm 3. 0.125% pancreatin for 15 minutes, and then the pancreatin digestion was stopped with DF-12 containing 10% FBS. Repeatedly blowing and sucking with glass suction tube to disperse the tissue blocks to single cell state. The cell suspension was filtered through a 100um sieve to remove tissue debris. 900g, 10min centrifugation, discard supernatant and sort cells using Percoll. White myelin on the liquid surface was removed, 8ml of PBS was added, mixed well, centrifuged for 10min, and the supernatant was discarded. Resuspended in 1.5ml PBS + 0.5% FBS and placed in a 1.5ml EP tube. All monocytes were thus obtained.

B. Staining of monocytes

The above monocytes were centrifuged (900g, 30min), the supernatant discarded and resuspended in 0.05ml of PBS + 0.5% FBS. To this, 1ul each of cell surface marker antibodies (anti-CD 11b, CD45, CD16/32) was added, and 1ml of PBS + 0.5% FBS was added after 15 minutes at room temperature, and centrifuged to remove the supernatant. 100ul of fixative was added and resuspended at room temperature for 20 minutes. Then 1ml of the punch solution was added, centrifuged, and the supernatant was discarded. 100ul of punch solution was resuspended. This was divided into two portions, one portion was added with 1ul of intracellular antibody (Mouse anti-Arginase-1), the other portion was added with 1ul of isotype control antibody (purified Mouse control), and the mixture was centrifuged at room temperature for 15 minutes, 1ml of the punch solution was added, and the supernatant was discarded. Each was resuspended in 50ul of the punch and the corresponding secondary antibody was added for 15 minutes at room temperature. Then 1ml of the punch was added, centrifuged (900g, 10min), the supernatant discarded and 300ul of punch resuspended. And (4) flow analysis.

The results are shown in FIG. 2, where the effect of Hpx knockdown on mouse microglial polarization after spinal cord injury prompted an increase in microglial M1/M2. (A, B) schematic diagram of microglial cell sorting. (C, D, G) proportion of M1 polarized microglia in spinal cord lesions: the Hpx knockout mice were significantly higher than the control mice. (E, F, H) proportion of M2 polarized microglia in spinal cord lesions: the Hpx knockout mice were significantly lower than the control mice. It was demonstrated that Hpx caused polarization of mouse microglia M2, and that lack of Hpx caused polarization of mouse microglia M1.

Example 3: histochemical test

The expression level of M1 polarization marker molecule TNF-a of spinal cord injury Hpx knockout mouse microglia is analyzed by an immunohistochemical method, and the regulation and control of the polarization of the microglia by Hpx are further verified.

A. Step of grouping and dyeing

Placing the frozen slices in an oven at 37 ℃ for 1h, and then sequentially carrying out the following steps: washing the slices with PBS for 5min twice; PBS diluted anti-TNF-alpha primary antibody was incubated overnight at 4 ℃; standing the slices at normal temperature for 40 minutes; washing the slices with normal-temperature PBS for 5 minutes each time for three times; adding a secondary antibody, and incubating for 70 minutes at normal temperature; washing the slices with normal-temperature PBS for 5 minutes each time for three times; then 1: 1000 Hoechst for 7 minutes; washing the slices with normal-temperature PBS for 5 minutes each time for three times; sealing the film and storing in dark.

B. Counting of TNF-a by immunofluorescence assay⁺/Iba-1⁺Cell occupancy of Iba-1⁺The proportion of microglia.

The results are shown in FIG. 3, where Hpx deficiency results in upregulation of mouse microglial inflammatory factor TNF-a expression after spinal cord injury.

Example 4 Overall behavioral testing of mice

To demonstrate the effect of this difference on secondary spinal cord injury, we analyzed behavioral indicators of mice after injury, and compared the spinal cord tissue injury after injury. The BMS score, the BMS sub-score and the swimming function score are adopted for behavioral observation, and the NeuN staining is adopted for observing the neuron survival quantity under the condition of tissue injury.

BMS scoring, BMS sub-scoring and swimming function scoring, and NeuN staining is adopted to observe the neuron survival quantity in the tissue damage condition. (for behavioral scores see Basso, D.M., Fisher, L.C., Anderson, A.J., Jakeman, L.B., McTigue, D.M., Popovich, P.G., 2006.Baso Mouse scales for coordination detection and correction in recovery after coil in real heart common motion estimation and journal of neural 23,635-659. mjen, D.D., Klusann, S.eber, S.S., Zuliani, C.Stiel, B.M., Metzger, C.Hirt, U.A., Walck, H.email, Falk, W.S., Essest, M, Edirer, L.M., catalog, N.7. C., and P.7. environmental test and 7. environmental test, C.3611. environmental test and 7. environmental test No. 11. Ca, Na, K.R. Falsen, K, W.S.S.S., C.D.S. Klusi., C., C.D.S. Pub., Klauser, C.S. C. Pub. K. No. C. J. C. No. 7, C. 7, C. E. J. C. No. 4, C. 7, C. E. C. 7, C. E. No. E. C. E. C. E. No. C. E. C. E. No. E. C. E.

The results show that the Hpx knockout mice had a significant decrease in BMS score, BMS sub-score (as in fig. 4A-B), and swimming function score (as in fig. 4C-D) after spinal cord injury compared to control mice. Further, both the number of neurons in the entire spinal cord section (fig. 5) and the number of motor neurons at the anterior angle of the spinal cord (fig. 5) were significantly reduced.

Example 5, in vitro microglia polarization assay.

1. Microglial cell purification preparation

And (3) preparing mouse microglia by primary culture.

2. Preparation of microglial Total RNA

The total RNA of the human epidermal cells is extracted by a general guanidinium isothiocyanate method by using a total RNA extraction kit of Shanghai biological engineering Co. The method comprises the following steps:

taking the microglia growing in a monolayer on a culture dish with the diameter of 3.5cm, directly removing the culture medium, adding 1ml of TRIzol to dissolve the cells, and removing the cell lysate by using a pipette after the cells are fully dissolved. And incubating the cell lysis sample for 5 minutes at 15-30 ℃ to completely decompose the nucleoprotein body. 0.2ml of chloroform was added to 1ml of TRIzol, the sample tube cap was closed, the tube was shaken by hand for 15 seconds and incubated at 30 ℃ for 2 to 3 minutes. Freezing and centrifuging for 15 minutes at the centrifugal force of not more than 12,000 Xg under the condition of 2-8 ℃. The mixture after centrifugation can be divided into three layers: the lower layer is a red phenol-chloroform layer, the middle layer and the upper colorless water sample layer. RNA was present in the upper aqueous layer at a capacity of approximately 60% of the volume of TRIzol added. The aqueous layer was transferred to a DEPC-treated, RNase-free tube and RNA was precipitated by mixing with isopropanol. 0.5ml of isopropanol was added per 1ml of TRIzol. Incubating the mixed sample at 15-30 ℃ for 10 minutes and then carrying out high-speed refrigerated centrifugation at 2-8 ℃ for 10 minutes under a centrifugal force of not more than 12,000 Xg. The RNA pellet may form a gel-like pellet attached to the wall and bottom of the tube after centrifugation. The RNA pellet was washed once with 75% ethanol (prepared with DEPC treated ultra pure water) and at least 1ml of 75% ethanol was added per 1ml of TRIzol. Mixing the sample by vortex oscillation, and carrying out high-speed refrigerated centrifugation for 5 minutes at the centrifugal force of not more than 7,500 Xg under the condition of 2-8 ℃. And (5) air-drying for 5-10 minutes to precipitate RNA. Failure to completely dry the RNA pellet reduces its solubility. RNA samples were dissolved with DEPC treated ultrapure water.

4. Co-incubating Hpx with LPS pre-stimulated microglia;

good growth of the cells was ensured before the experiment, and 1X 105 cells were seeded one day before the experiment in a 24-well culture plate in a medium volume of 500. mu.L. (the fusion rate of the cells is about 30 to 50%).

Adding three proteins with different gradients into corresponding holes of each group, wherein the concentrations of the added proteins are 0.5ng/mL, 5ng/mL and 50ng/mL respectively; and slightly shaking the culture plate in the horizontal direction to fully and uniformly mix the culture medium and the protein, then putting the cell plate back into the incubator for incubation at 37 ℃ for 12 hours, replacing fresh culture solution, and collecting after 72 hours.

Determination of the levels of M1 and M2 types in microglia cells before and after Hpx incubation

The measurement method was the same as in example 1.

The results are shown in fig. 6, where Hpx regulates mouse glial polarization state. Hpx down-regulates microglia IL-1 beta, iNOS and TNF-alpha, inhibits microglia M1 polarization, and simultaneously up-regulates Arginase-1 level and promotes M2 polarization.

Example 6: cell experiments (in vitro experiments)

Cell viability and myelination are analyzed from multiple aspects such as cell morphology, myelin associated protein expression (MBP) and the like by using biological experimental methods such as immunocytochemistry and the like.

The specific method comprises the following steps:

1) immunocytochemistry method for detecting OPC differentiation and neuron growth

(1) As shown in FIG. 7A, the Hpx and LPS pre-stimulated microglia cells were cultured, and the supernatant of the collection conditions was co-cultured with OPC (Oligodendrocyte precursor cells) (10000 cells/well inoculated in 24-well plate) or cortical neurons (10000 cells/well inoculated in 24-well plate). Each group of cells was plated in 3 replicates per time point and the experiment was repeated 3 times. The cells are fixed.

(2) Microglia were divided into 4 groups: blank control, LPS-stimulated, Hpx-incubated, Hpx + LPS group.

(3) 4% BSA blocked non-specific binding sites.

(4) Neurons were programmed with a 1: primary anti-NeuN, OPC was added at a concentration of 200 in a 1: primary anti-MBP or TUNEL was added at a concentration of 200.

(5) Incubated at 37 ℃ in the dark for half an hour.

(6) PBS was washed 3 times.

(7)1: secondary antibody was added at a concentration of 500.

(8) PBS was washed 3 times.

(9) And (6) sealing and microscopic examination.

The experimental results (as shown in fig. 7 and 8) show that Hpx can inhibit neuronal apoptosis and oligodendrocyte apoptosis by regulating polarization of microglia M1-M2, and promote differentiation and maturation of the microglia.

Example 7: correlation upstream Signal Path Studies

Microglia were divided into 6 groups: placebo, LPS-stimulated, RAP (LRP-1 pathway stimulator) + LPS-stimulated, Hpx + LPS, RAP + Hpx + LPS, RAP.

The microglia polarization marker was detected by Q-PCR and immunoblotting as described in example 1 above.

Experimental results As shown in FIG. 9, Hpx regulates microglial M1-M2 polarization by modulating the LRP-1 signaling pathway.

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, which are given by way of illustration of the principles of the present invention, and that various changes and modifications may be made without departing from the spirit and scope of the invention as defined by the appended claims. The scope of the invention is defined by the appended claims and equivalents thereof.

Claims (6)

1. Application of Hemopexin protein in promoting conversion of microglia into M2 polarization state in vitro.
2. 2. The use of the Hemopexin protein according to claim 1 in promoting the conversion of microglia into M2 polarization state in vitro, wherein the Hemopexin protein is co-incubated with the microglia at a concentration of 0.5-50ng/ml to obtain the microglia in M2 polarization state.
3. 3. The use of a Hemopexin protein according to claim 1 to promote microglial transformation into M2 polarization state in vitro, wherein the use comprises the following steps:

   A. incubating Hemopexin protein 0.5-50ng/ml with microglia for 12 hr;

   B. replacing fresh culture solution for microglia, and culturing for 72 hours to obtain the microglia in M2 polarization state.
4. 4. The use of a Hemopexin protein according to claim 3 in promoting microglial transformation into M2 polarization state in vitro, wherein the step A is as follows:

   inoculation of 1X 10⁵Putting the microglia into a 24-hole culture plate, wherein the volume of the added culture medium is 500 mu L, and the fusion rate of the cells is about 30-50%;

   adding 0.5-50ng/mL of Hemopexin protein into corresponding holes of each group;

   the plate was gently shaken in the horizontal direction to mix the medium and protein well, and the plate was returned to the incubator and incubated at 37 ℃ for 12 hours.
5. 5. The use of a Hemopexin protein according to claim 3 in promoting microglial transformation into M2 polarization state in vitro, wherein the step B comprises the following steps:

   culturing microglia in a 24-hole culture plate at 37 ℃ for 12h, observing the cell state, removing cell supernatant, and replacing with fresh culture medium;

   after continuously culturing for 3-4 days, observing the growth condition of the cells; for cells with slow growth state, shortening observation interval, changing liquid in the middle, and maintaining activity of the cells; microglia in the polarization state of M2 were obtained.
6. 6. A method of promoting the switching of microglia to M2 polarization state, the method comprising the steps of:

   A. incubating Hemopexin protein 0.5-50ng/ml with microglia for 12 hr;

   B. replacing fresh culture solution for microglia, and culturing for 72 hours to obtain the microglia in M2 polarization state.

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