CN116139259A - Application of TIMP2 in preparation of medicine for preventing or treating traumatic brain injury - Google Patents

Abstract

The invention relates to the field of medicines, in particular to application of matrix metalloproteinase endogenous inhibitor-2 (Tissue inhibitor metalloproteinases-2, TIMP 2) in preparing medicines for preventing or treating traumatic brain injury. The TIMP2 protein can improve the dropping latency of a rod-rotating experiment of a brain trauma mouse; improving the movement balance capacity of the brain trauma mice on balance beams; improving the nerve function injury of mice with brain trauma; reducing Evan's blue permeability of brain tissue. In brain microvascular endothelial cells HBMEC, TIMP2 protein can be involved in maintaining the integrity of the endothelial barrier, reversing the loss of expression and changes in localization of the junction complex caused by the ex vivo brain trauma model, and reducing luciferase leakage. The invention also provides application of the TIMP2 protein serving as an Intergrin alpha 3 beta 1 ligand in preparing a drug for treating central nervous system diseases caused by blood brain barrier disorder, wherein the acting of the TIMP2 protein is required to regulate and control VE-Cadherin phosphorylation by combining with a cell membrane receptor Intergrin alpha 3 beta 1. The TIMP2 protein has good application prospect in treating traumatic brain injury blood brain barrier dysfunction.

Description

Application of TIMP2 in preparation of medicine for preventing or treating traumatic brain injury

Technical Field

The invention relates to the field of medicines, in particular to application of matrix metalloproteinase endogenous inhibitor-2 (Tissue inhibitor metalloproteinases-2, TIMP 2) in preparing medicines for preventing or treating traumatic brain injury.

Background

Traumatic brain injury (traumatic brain injury, TBI), a disease that causes severe damage to brain tissue due to various traumas, is one of the common emergency in the neurosurgery field, and has become the first cause of disability and death in young and young people worldwide. TBI causes a series of clinical symptoms, such as dyskinesia, cognitive dysfunction, epilepsy, etc., which are caused by impaired nerve function, and furthermore, places a heavy economic and mental burden on the home and society. However, the clinical therapeutic strategies for TBI are limited at present, and therapeutic drugs for TBI have not been approved by the FDA.

The blood brain barrier (blood brain barrier, BBB) is a special barrier that exists between brain blood circulation and nervous tissue, whose main function is to maintain brain tissue homeostasis, regulate the balance of mass exchange within the brain, and protect brain tissue from damage. The pathological process of brain trauma is divided into primary injury and secondary injury, wherein the primary injury causes serious injury of brain tissues and dysfunction of blood brain barrier, and then a large amount of inflammatory factors and immune cells in peripheral blood enter the brain tissues to start immune response, so that secondary injury of central nervous system is caused. Thus, targeting the blood brain barrier, maintaining its integrity is one of the important strategies for TBI treatment.

The blood brain barrier is a cell complex composed of brain microvascular endothelial cells, pericytes and astrocyte podophyllum, wherein the brain microvascular endothelial cells are the main components of the blood brain barrier, and the cell-cell is connected with the protein related to adhesion by tight junction protein to form a connecting complex, so that the high transmembrane resistance and low penetration of the paracellular pathway are maintained, and the integrity of the endothelial barrier is maintained. The ligation complex is composed of cytoplasmic adhesion proteins (zonula occludens protein, ZO), transmembrane proteins Occludin, claudin, and adhesion connexins. It has now been found that in various TBI models, the expression and localization of the relevant components of the ligation complex are altered, thereby disrupting the integrity of the blood brain barrier. A significant down-regulation of Claudin-5, ZO-1 expression was observed both in the hydraulic impact and in the controlled cortical impact injury models. And after TBI occurs, various secondary pathological factors can further influence the expression and positioning of critical components of the blood brain barrier: (1) Under the condition of hypoxia, the expression of the mouse brain microvascular endothelial cell bEND.3Claudin-5 is down-regulated, the transmembrane cell resistance is reduced, and the paracellular permeability is increased. (2) TBI is usually accompanied by an inflammatory response in which inflammatory factor IL-1β down regulates the expression of Occludin and ZO-1 at the cell junction, and not only promotes neutrophil infiltration, but also causes changes in cell localization and decreased expression of the tight junction molecules Occludin, ZO-1, claudin-5, VE-cadherein.

Endogenous inhibitors of matrix metalloproteinases-2 (Tissue inhibitor metalloproteinases-2, TIMP 2) are members of the TIMP family, are secreted proteins that inhibit Matrix Metalloproteinases (MMPs), and have the amino acid sequence shown in FIG. 1.TIMP 2 has been found to be capable of physiological function with non-MMP inhibitory activity in addition to inhibiting MMP2, MMP14 activity and thus reducing extracellular matrix degradation. TIMP2 activates AKT pathway after binding to MT1-MMP, inhibiting tumor cell apoptosis; TIMP2 can resist EGF-induced a549 proliferation and cell adhesion reduction by up-regulating E-cadherein; TIMP2 activates cAMP/Rap1/ERK pathway to promote neuronal differentiation; ala+TIMP2, without MMP inhibitory activity, can inhibit VEGF-A induced activation of the VEGFR2 downstream pathway; TIMP2 also binds to intelgrinα3β1 and inhibits VEGF-induced increases in vascular permeability via the Shp-1-cAMP/PKA pathway. However, whether TIMP2 has a protective effect in the field of vascular injury-related neurological diseases, particularly traumatic brain injury, and whether TIMP2 has a regulation and control effect on the blood brain barrier are not reported.

The invention mainly aims to take a traumatic brain injury model as an example, explore whether TIMP2 can be used as a secretory protein to protect blood brain barrier dysfunction caused by central nervous system diseases by regulating and controlling vascular integrity, and provide effective drug targets and treatment strategies for treating and intervening in brain injury and blood brain barrier injury related neuropsychiatric system diseases.

The invention comprises the following steps:

the invention solves the technical problem of providing an application of matrix metalloproteinase endogenous inhibitor-2 in preparing medicines for preventing or treating traumatic brain injury.

In order to solve the technical problems of the invention, the invention provides the following technical scheme:

in a first aspect, the invention provides an application of an endogenous inhibitor-2 of matrix metalloproteinase in preparing a medicament for preventing or treating traumatic brain injury.

Wherein the amino acid sequence of the matrix metalloproteinase endogenous inhibitor-2 is as follows: the amino acid sequence shown in SEQ ID NO.1-SEQ ID NO. 12.

In a second aspect, the present invention provides an application of a nucleic acid molecule encoding an endogenous inhibitor-2 of matrix metalloproteinase in preparing a medicament for preventing or treating traumatic brain injury.

The sequence of the nucleic acid molecule is as follows: the nucleotide sequence shown in SEQ ID No.13-SEQ ID No.30 or the nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID No.13-SEQ ID No.30 in the sequence table.

In a third aspect, the present invention provides an application of an expression vector containing the nucleic acid molecule of the second aspect in preparing a medicament for preventing or treating traumatic brain injury. Such expression vectors include, but are not limited to, adeno-associated viral vectors, adenoviral vectors, retroviral vectors, exosomes, liposome complexes, cationic polymers, chitosan polymers, inorganic nanoparticles.

According to a fourth aspect of the present invention there is provided the use of a host cell comprising a nucleic acid molecule according to the second aspect or an expression vector according to the third aspect in the manufacture of a medicament for the prevention or treatment of traumatic brain injury.

The host cell is selected from the group consisting of bacteria, yeast, aspergillus, plant cells, insect cells, or mammalian cells.

The traumatic brain injury according to the first to fourth aspects includes blood brain barrier dysfunction due to traumatic brain injury, and central nervous system diseases associated with blood brain barrier dysfunction.

The beneficial technical effects are as follows:

animal experiments prove that the TIMP2 protein can improve the rod rotating and falling latency period of a brain trauma mouse; improving the movement balance capacity of the brain trauma mice on balance beams; improving the nerve function injury of mice with brain trauma; reducing Evan's blue permeability of brain tissue.

Cell experiments prove that the TIMP2 protein can be combined with a cell membrane Integlin alpha 3 beta 1 receptor in a brain trauma in-vitro model, and the condition of damaged intercellular connection in the brain trauma in-vitro model can be relieved. Particularly, up-regulating the expression of the tight junction complex and reducing the leakage of luciferase.

The Ala+TIMP2 protein without MMP inhibiting activity can maintain the integrity of vascular endothelial barrier in the whole brain trauma and in an in vitro model.

The invention provides application of the TIMP2 protein serving as an Intergrin alpha 3 beta 1 ligand in preparing a medicine for treating central nervous system diseases caused by blood brain barrier disorder, including but not limited to ischemic stroke, hemorrhagic stroke, alzheimer disease and the like. The invention prompts that the TIMP2 protein has application prospect in treating the acute blood brain barrier injury of brain trauma.

Drawings

FIG. 1 effect of TIMP2 protein on neuro-behaviours of brain-trauma mice (A) drop latency of mice of each group; (B) mice balance beam score for each group; (C) mice of each group were scored for mNSS.

FIG. 2 effect of TIMP2 protein on blood brain barrier permeability in brain-trauma mice.

FIG. 3 protection of barrier integrity in HBMEC ex vivo brain trauma model by TIMP2 protein (A) exogenous addition of TIMP2 protein can reverse loss of cell-connexin complex expression and changes in localization; (B) exogenous addition of TIMP2 protein reduces luciferase leakage.

FIG. 4 TIMP2 protein regulates VE-Cadherin phosphorylation in an HBMEC ex vivo brain trauma model.

FIG. 5 effect of TIMP2 protein and Ala+TIMP2 protein on neuro-behaviours of brain-trauma mice (A) drop latency of mice of each group; (B) mice balance beam score for each group; (C) mice of each group were scored for mNSS.

FIG. 6 effect of TIMP2 protein and Ala+TIMP2 protein on blood brain barrier permeability in brain-trauma mice.

FIG. 7 sets up a primary three-dimensional blood brain barrier model, and luciferase leakage experiments examined the protective effect of TIMP2 protein and Ala+TIMP2 protein on barrier integrity in an ex vivo brain trauma model.

FIG. 8 shows TIMP2 and Integlin alpha 3 beta 1 interaction experiments to verify that (A) TIMP2 antibodies were immunoprecipitated with HBMEC cell lysates and Western blots detection results were performed with TIMP2, integlin alpha 3 and Integlin beta 1 antibodies; (B) Immunoprecipitation of the Integrin alpha 3 antibody and HBMEC cell lysate, and Western blotting detection results with TIMP2, integrin alpha 3 and Integrin beta 1 antibodies; (C) Immunoprecipitation of the Integlin beta 1 antibody with HBMEC cell lysate was performed, and Western blots detection results were performed using TIMP2, integlin alpha 3, and Integlin beta 1 antibodies.

FIG. 9 TIMP2 protein regulates cell-tight junction protein complex expression through membrane complex Integrin α3β1 and paracellular permeability (A) after Integrin α3 or Integrin β1 knockdown, TIMP2 protein fails to regulate cell-tight junction protein complex expression; (B) After knocking down Integrinα3 or Integrinβ1, the TIMP2 protein cannot reverse the increased luciferase leakage phenotype.

Detailed Description

EXAMPLE 1 protective Effect of TIMP-2 protein on mice with brain trauma

1. Experimental animal

SPF grade male C57BL/6J mice 22-25g, male, purchased from Peking Vitre Liwa laboratory animal technologies Co.

2. Brain trauma model making

After isoflurane anesthesia of the mice, the scalp was cut at a position of 0.8mm behind the right coronary suture and 1.3mm beside the midline, and a bone hole with a diameter of 3mm was drilled. The scalp was sutured by a modified Feeney free-fall injury device, impacting the striker bar with a hammer of 20g weight, free-fall from 25cm, striking depth 3 mm. Sham mice were sutured to the scalp after cutting the scalp holes without hammer blows.

3. Grouping and administration of animals:

mice were divided into sham surgery, model, 100. Mu.g/kg TIMP-2 protein, TIMP2 knockout mice. Immediately after the operation, PBS or TIMP2 protein is injected into the tail vein, and the administration is continued for three days.

4. Study of behavioural

Rotating rod experiment: the mice are placed on a bar rotating instrument with the diameter of 3.5cm, the initial speed of the rotating wheel is adjusted to 4rpm/min, the mice are evenly accelerated to 35rpm/min within 180s after being placed on the rotating wheel, the latency period of 3 times of falling is recorded, 180s is a demarcation value, the recording is carried out according to 180s for more than 180s, and the steps are repeated for 3 times.

Balance beam experiment: the mice were placed on wooden balance beams 6mm wide and 1m long, and the balance beams were suspended at a height of 30cm. (1) Grading and evaluating the balance capacity of the mice on the balance beam by 7-division balance beam, wherein the 7-division balance beam is characterized in that the left hind limb of the mice slides down for less than two times in the process of passing through the balance beam; 6, the sliding times of the left hind limb are less than 50 percent; 5 is divided into the left hind limb sliding times of more than 50 percent but less than 100 percent; 4, the left hind limb slides off completely; 3, the left hind limb is not on the balance beam; 2, the mice can be balanced on the balance beam, but cannot pass through the balance beam; the mice divided 1 cannot balance on the balance beam.

mNSS score: the mNSS neurological deficit score was used at three time points 24h, 48h, 72h after molding, and was 0 to 18 points (0 points were normal and 18 points were maximum neurological deficit). It contains comprehensive assessment of rat motor, sensory, reflex and balance functions. The higher the score, the more severe the damage. The detection method and the scoring standard are shown in the table below.

Mouse neurological score (mNSS)

5. Blood brain barrier permeability assay

The mice were injected with 3ml/kg of 4% Evan's blue solution intravenously. After 2h of anesthesia, the heart was perfused with normal saline, the brain was removed by head breaking, the hemispheres of the brain were rapidly separated on ice, weighed and placed in 1ml of formamide solution, incubated in a constant temperature incubator at 45℃for 48h, and absorbance values were determined using an enzyme-labeled instrument (wavelength 632 nm). The concentration of evans blue in the solution was calculated according to the linear regression equation.

6. Statistical analysis of data

Data are all expressed as mean ± standard error (mean ± SEM), statistical analysis is by single factor analysis of variance, "#" is compared to sham-operated group, where ^## P<0.01; ", represents the comparison with the model group, wherein ^** P<0.01， ^* P<0.05。

7. Results

The inventor detects the exercise ability of the brain trauma mice through a rotating rod experiment. The movement time of mice in the brain trauma model group on the rotating stick is obviously reduced. The continuous three days of 100 mug/kg TIMP2 protein treatment can obviously improve the movement ability of the mice on the rotating rod in the second day and the third day, which shows that the TIMP2 protein treatment can reduce the damage of the movement ability of the rotating rod of the mice with brain trauma. The drop latency of TIMP2 knockout mice was significantly lower on the second and third days than in the model group, indicating that TIMP2 knockout further aggravated the motor ability of brain trauma mice on the rotating stick (fig. 1A).

The inventor detects the balance ability of the mice with brain trauma through a balance beam experiment, and the specific detection index is the balance beam score. The balance score of mice in the TIMP2 treated group was significantly higher than that of mice in the model group for three days following three days of 100 μg/kg TIMP2 protein treatment (fig. 1B). The results show that TIMP2 protein treatment can significantly improve the balance ability of mice with brain trauma.

The inventors assessed the overall neurological function of brain-injured mice by mNSS neurological deficit score. The mNSS score of mice in the TIMP2 treatment group is obviously lower than that of mice in the model group three days after 100 mug/kg of TIMP2 protein treatment, which indicates that the TIMP2 protein treatment can reduce the nerve function damage of the mice with brain trauma. TIMP2 knockout mice had significantly higher mNSS scores on the third day than the model group, indicating that TIMP2 knockout further aggravated neurological impairment in brain trauma mice (fig. 1C).

The inventors tested the blood brain barrier permeability of mice by means of an evans assay. The Evansan content of mice in the brain trauma model group is obviously increased. The continuous three days of 100 mug/kg TIMP2 protein treatment significantly reduced the Evanserin content in the injured side brain tissue of the mice, while the TIMP2 knockout mice had significantly increased the Evanserin content in the injured side brain tissue compared to the model group. The above results demonstrate that TIMP2 protein significantly reduced blood brain barrier damage in brain-injured mice (fig. 2).

EXAMPLE 2 protective Effect of TIMP2 on blood brain Barrier injury in ex vivo brain trauma model

1. Ex vivo brain trauma model

An in vitro model of brain trauma is established by using an anoxic cell: IL-1 beta is added into HBMEC cell culture medium to a final concentration of 20ng/ml, each group of cell culture dishes are placed into a stemcell anoxic small chamber, a large culture dish filled with 10ml of sterile water is placed at the lowest layer of the small chamber, and 95% nitrogen and 5% CO are introduced into the anoxic small chamber for 10 minutes continuously ₂ The gas was mixed to ensure complete placement of the cells in an anoxic environment, after which the cells were placed in a 37℃incubator for 24 hours.

2. Cell permeability test

HBMEC was inoculated into a rat tail collagen I coated 0.4 μm Transwell chamber, the chamber was placed in a 24-well plate, an ex vivo brain trauma model was established according to the grouping and TIMP2 protein treatment was given, and FITC-Dextran with a molecular weight of 70Kd was added to the upper chamber at a final concentration of 1mg/ml. After 2h, the medium in the wells below the cells was collected and the amount of FITC-Dextran leakage was measured by a fluoroenzyme-labeled instrument.

3. Extraction of cell membrane protein

In vitro cell experiments adopt a biotin labeling and Neutravidin bead enrichment extraction method to extract envelope proteins.

Cells were washed three times with DPBS, 0.2mg/ml of Sulfo-NHS-SS-Biotin was added, protected from light at 4℃for 30min, and neutralization solution (7.5 g glycine/1000 ml TBS) was added to neutralize residual bio-protein not bound to the cell membrane, protected from light at 4℃for 10min. Cells were collected and 1ml of NP-40 lysate was added and the mixture was kept on ice for 30min. The supernatant was centrifuged at 13000rpm and 20. Mu.l of Neutravidin beads were added overnight at 4 ℃. The next day, centrifugation was performed at 5000rpm for 1min at 4℃and the supernatant was discarded. 1ml of lysate was added and centrifuged at 5000rpm at 4℃for 1min, the supernatant was discarded and repeated five times. Mu.l of a 2×loading buffer was added thereto at 99℃for 10min. Centrifuging at 5000rpm for 1min, and collecting supernatant to obtain membrane protein.

VE-Cadherin phosphorylation assay

After scraping with a cell scraper and washing with pre-chilled PBS, lysates (50 mM Tris-HCl,150mM were addedNaCl,1% NP-40, pH 7.5), was lysed on ice for 30min, centrifuged at 13,000rpm for 20min at 4℃and the supernatant was taken to determine the protein concentration, the total amount of protein used was controlled between 0.8-1mg per experimental group. Mu.l of magnetic beads (Dynabeads) ^TM Protein G) was added with 5. Mu.l of VE-Cadherin antibody and incubated in 600. Mu.l of PBS for 15min at room temperature with rotation. The antibody-conjugated magnetic beads were incubated overnight at 4℃with each histone lysate. The next day, the column was washed with 1ml of cell lysate, 5 times each, unbound protein was washed away, 1 Xloading buffer was added, heating was performed at 70℃for 10min, the pole was left to stand to collect the supernatant, heating was performed at 99℃for 10min to denature, and Western blots were assayed for VE-Cadherin phosphorylation with p-Tyr antibody.

5. Statistical analysis of data

Data are expressed as mean ± standard error (mean ± SEM), statistical analysis is by single factor analysis of variance, "#" is compared to control group, wherein ^## P<0.01; ", represents the comparison with the model group, wherein ^** P<0.01， ^* P<0.05。

6. Results

Under the condition of IL-1β+ hypoxia, HBMEC increased fluorescein leakage, and exogenous addition of TIMP2 protein reversed the damage in a concentration-dependent manner, indicating that TIMP2 can alleviate the damage of endothelial barrier function in an ex vivo brain trauma model (FIG. 3A).

Under the condition of IL-1β+hypoxia, the expression of the tight junction proteins ZO-1 and Occludin, claudin-5 is down-regulated by HBMEC, the expression of VE-Cadherin is not obviously changed, but the expression of VE-Cadherin on a cell membrane is down-regulated. 3 μg/ml TIMP2 protein reversed the loss of expression of the tight junction protein components while inhibiting VE-Cadherin transfer from the membrane to the cytoplasm (FIG. 3B).

Under the condition of IL-1β+ hypoxia, the phosphorylation level of VE-Cadherin is obviously increased by HBMEC. Exogenous addition of TIMP2 protein significantly inhibited VE-cadherein phosphorylation (fig. 4). It is suggested that TIMP2 may regulate the posttranscriptional modification of VE-Cadherin as a mechanism for regulating the localization of VE-Cadherin cells.

The results indicate that TIMP2 protein can maintain vascular endothelial barrier integrity by regulating expression and localization of the ligation complex in an ex vivo brain trauma model.

EXAMPLE 3 protective Effect of TIMP2 protein and Ala+TIMP2 protein on brain trauma mice

1. Experimental animal

Same as in example 1

2. Brain trauma model making

Same as in example 1

3. Grouping and administration of animals:

mice were divided into sham surgery groups, model groups, 100. Mu.g/kg TIMP2 protein group, 100. Mu.g/kg Ala+TIMP2 protein group. Immediately after surgery, PBS, TIMP2 protein or Ala+TIMP2 protein group was injected into the tail vein and administered for three consecutive days.

4. Study of behavioural

Rod rotation experiment, balance beam experiment, mNSS scoring blood brain barrier permeability determination

Same as in example 1

5. Blood brain barrier permeability assay

Same as in example 1

6. Statistical analysis of data

Data are all expressed as mean ± standard error (mean ± SEM), statistical analysis is by single factor analysis of variance, "#" is compared to sham-operated group, where ^## P<0.01; ", represents the comparison with the model group, wherein ^** P<0.01， ^* P<0.05。

7. Results

The inventor detects the exercise ability of the brain trauma mice through a rotating rod experiment. The movement time of mice in the brain trauma model group on the rotating stick is obviously reduced. Administration of 100 μg/kg TIMP2 protein or 100 μg/kg ala+timp2 protein treatment for three consecutive days significantly improved the locomotor ability of mice on the rotor bar both the second and third days, indicating that ala+timp2 protein treatment reduced the rotor bar locomotor ability impairment in brain trauma mice (fig. 5A).

The inventor detects the balance ability of the mice with brain trauma through a balance beam experiment, and the specific detection index is the balance beam score. Treatment with 100 μg/kg TIMP2 protein or 100 μg/kg ala+timp2 protein was administered three consecutive days, and the balance score of mice in the model group was significantly increased for each three days (fig. 5B). The results show that Ala+TIMP2 protein treatment can significantly improve the balance ability of mice with brain trauma.

The inventors assessed the overall neurological function of brain-injured mice by mNSS neurological deficit score. The mNSS score of both mice groups was significantly lower than that of the mice in the model group for three consecutive days with 100 μg/kg TIMP2 protein or 100 μg/kg Ala+TIMP2 protein treatment, indicating that Ala+TIMP2 protein treatment reduced the neurological impairment in the brain-trauma mice (FIG. 5C).

The inventors tested the blood brain barrier permeability of mice by means of an evans assay. The Evansan content of mice in the brain trauma model group is obviously increased. The treatment of 100 mug/kg of TIMP2 protein or 100 mug/kg of Ala+TIMP2 protein can obviously reduce the Evansan content of brain tissue on the injured side of the mice after three consecutive days. The above results demonstrate that the ala+timp2 protein significantly reduces blood brain barrier damage in brain-injured mice (fig. 6).

The results show that Ala+TIMP2 without MMP inhibiting activity can relieve the damage of blood brain barrier of mice with brain injury and improve the neurological function defect of mice with brain injury. The non-MMP inhibitory function of TIMP2 was suggested to be involved in protecting the blood brain barrier integrity of brain-trauma mice and improving neurological impairment in mice.

EXAMPLE 4 protection of TIMP2 protein and Ala+TIMP2 protein against blood brain Barrier injury in ex vivo brain trauma model 1. Culture of Primary mouse microvascular endothelial cells

Separating and shearing cerebral cortex into pieces of 1mm ³ The tissue mass was digested with collagenase type II and DNase at 37℃for 2 hours, centrifuged at 1000g for 20 minutes with 20% BSA-DMEM to remove phospholipids, and the pellet was digested with collagenase-dispase and DNase at 37℃for 1 hour with shaking, and centrifuged at 1000g for 20 to obtain brain microvascular endothelial cells. Primary brain microvascular endothelial cells were obtained by culturing in DMEM/F12 (supplemented with 10% FBS, 1.5ng/ml bFGF, 100. Mu.g/ml heparin, 5. Mu.g/ml insulin, 5. Mu.g/ml transferrin, 5ng/ml sodium selenite, 4. Mu.g/ml puromycin) for 48 hours, and culturing for 7 days after replacement of the puromycin-free DMEM/F12.

2. Primary mouse pericyte culture

Isolation procedure was the same as that of microvascular endothelial cells, and after 48 hours of culture, the cells were replaced with DMEM/F12 containing only 10% FBS and cultured for two weeks.

3. Primary mouse astrocyte culture

The newborn mice were briefly soaked in 75% alcohol for sterilization, taken out and placed in a 10cm petri dish in an ice bath, the cortex was separated, and the vascular membrane attached to the tissue was thoroughly peeled off. Most of the anatomic fluid is aspirated, leaving a small amount just covering the tissue. The tissue was minced with ophthalmic scissors, papain and an appropriate amount of dnase were added, and digested in a incubator at 37 ℃ for 30min, with shaking every 5min. The digestion was terminated by adding an appropriate amount of inoculation medium, after which it was transferred to several Ep tubes and rapidly cooled to 0 ℃. The blue gun head is used for gently blowing the tissue block, the tissue block is kept at a low temperature for 2min after 10 times of blowing, the supernatant is transferred into a new 15ml centrifuge tube, and the residual block is continuously blown after being supplemented with new culture medium and a proper amount of DNase. Repeat 3 times and discard the un-blown pellet. Centrifugation at 1000rpm for 5min, the supernatant was discarded, and an appropriate amount of inoculum (DMEM/F12 in 10% FBS) was added and plated.

4. Primary three-dimensional blood brain barrier model establishment

(1) The Transwell chamber was coated with 15. Mu.g/ml collagenase and 30. Mu.g/ml fibronectin on both the upper and lower sides overnight at 37 ℃.

(2) The Transwell chamber was inverted to the midpoint of the dish, and 5000 cell suspensions per well were added dropwise at 37℃for 4 hours.

(3) Astrocytes, 3x 10A 4/well, were plated in 24-well plates.

(4) The Transwell chamber is placed in the corresponding well of the 24-well plate, and endothelial cell suspension is added to the upper chamber, 5x10 x 4/well. The culture medium of the upper and lower chambers is endothelial cell complete culture medium.

5. Statistical analysis of data

Data are expressed as mean ± standard error (mean ± SEM), statistical analysis is by single factor analysis of variance, "#" is compared to control group, wherein ^## P<0.01; ". Times." indicates that in comparison to the hypoxia +IL-1β injury group, wherein ^** P<0.01， ^* P<0.05。

6. Results

The inventor establishes an in-vitro brain trauma model in a primary three-dimensional blood brain barrier by using an anoxic cell and IL-1 beta, and detects the integrity of the barrier by using a luciferase leakage experiment. The ex vivo brain trauma model resulted in increased paracellular permeability of endothelial cells and increased fluorescein leakage. Exogenous administration of 3 μg/ml of TIMP2 protein and Ala+TIMP2 protein can obviously reduce fluorescein leakage, and Ala+TIMP2 protein can reduce paracellular permeability increase in an in vitro brain trauma model. The above results indicate that TIMP2 protein can enhance endothelial barrier integrity through non-MMP inhibitory activity (fig. 7).

EXAMPLE 5 TIMP2 reduces the impairment of intercellular junctions in ex vivo models of brain trauma by binding to the membrane receptor Intergrinα3β1

1. Co-immunoprecipitation

HBMEC cells were scraped with a cell scraper, washed once with pre-chilled PBS, and then lysed on ice for 30min with the addition of lysis solution (50 mM Tris-HCl,150mM NaCl,1%NP-40, pH 7.5), centrifuged at 13,000rpm at 4℃for 20min, and the supernatant was taken to determine the protein concentration, with the total amount of protein used in each experimental group controlled at 0.8-1mg. Mu.l of the corresponding antibody was added to 20. Mu.l of magnetic beads (DynabeadsTMProtein G), and incubated in 600. Mu.l of PBS for 15min at room temperature with rotation. The antibody-conjugated magnetic beads were incubated overnight at 4℃with each histone lysate. The next day, the column was washed with 1ml of cell lysate, 5 times each time, unbound proteins were washed away, 1 Xloading buffer was added, heating was performed at 70℃for 10min, the pole was left standing to take the supernatant, heating was performed at 99℃for 10min for denaturation, and Western blotting analysis was performed.

siRNA knockdown of Integrin alpha 3 or Integrin beta 1

The siRNA sequences of the HBMEC knockdown Intigrin beta 1 and Intigrin alpha 3 are respectively as follows:

si ITGB1：5’-CCGUAGCAAAGGAACAGCA)；si ITGA3：5’-GUGUACAUCUAUCACAGUA)。

transfection was performed in a 12-well plate, 200. Mu.l buffer was added to 3. Mu.l siRNA, vortexed for 10s, and then 4. Mu. l Polyplus interferin transfection reagent was added, and after vortexing for 10s, the mixture was allowed to stand at room temperature for 10min, and complete medium was added dropwise for 6 hours to change the solution for subsequent handling.

3. Statistical analysis of data

Data are expressed as mean ± standard error (mean ± SEM), statistical analysis is by single factor analysis of variance, "#" is compared to control group, wherein ^## P<0.01; ". Times." indicates that in comparison to the hypoxia +IL-1β injury group, wherein ^** P<0.01; ns represents and moduloThere was no statistical difference in the group ratios.

4. Results

TIMP2 can be combined with HBMEC cell membrane complex Intigrin alpha 3 beta 1

Immunoprecipitation with TIMP2 antibody in HBMEC cells, intelgrin α3 and intelgrin β1 were detected in TIMP2 affinity eluted fractions; TIMP2 was detected in the re-affinity eluted fractions by immunoprecipitation with antibodies to Intigrin. Alpha.3 or Intigrin. Beta.1. It was shown that TIMP2 can bind to HBMEC cell membrane complex interserin α3β1, and there is an interaction between the three (fig. 8).

TIMP2 regulates expression and paracellular permeability of the ligation complex through membrane receptor intelgrinα3β1

After the Integrinα3 or the Integrinβ1 is knocked down, the damage of hypoxia +IL-1β to cells cannot be reversed by exogenously adding TIMP2, including the down-expression of the closely connected protein and the increase of luciferase penetration (figure 9), which shows that the TIMP2 is used as a secretion protein and needs to be combined with a membrane receptor Integrinα3β1 so as to play a role in protecting the blood brain barrier. Therefore, the TIMP2 protein can be used as an Intergrin alpha 3 beta 1 ligand for preparing a therapeutic drug for treating central nervous system diseases caused by blood brain barrier disorder.

The above embodiments are only for illustrating the technical concept of the present invention, and are not intended to limit the scope of the present invention for those skilled in the art to understand the content of the present invention and implement the same. All equivalent changes or modifications made according to the spirit of the present invention should be construed to be included in the scope of the present invention.

Sequence listing

<110> institute of medicine at the national academy of medical science

<120> use of matrix metalloproteinase endogenous inhibitor-2 in the preparation of a medicament for preventing or treating traumatic brain injury

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Ala Gly Lys Ala Glu Gly Asp Gly Lys Met His Ile Thr Leu Cys Asp

115 120 125

Phe Ile Val Pro Trp Asp Thr Leu Ser Thr Thr Gln Lys Lys Ser Leu

130 135 140

Asn His Arg Tyr Gln Met Gly Cys Glu Cys Lys Ile Thr Arg Cys Pro

145 150 155 160

Met Ile Pro Cys Tyr Ile Ser Ser Pro Asp Glu Cys Leu Trp Met Asp

165 170 175

Trp Val Thr Glu Lys Asn Ile Asn Gly His Gln Ala Lys Phe Phe Ala

180 185 190

Cys Ile Lys Arg Ser Asp Gly Ser Cys Ala Trp Tyr Arg Gly Ala Ala

195 200 205

Pro Pro Lys Gln Glu Phe Leu Asp Ile Glu Asp Pro

210 215 220

<210> 4

<211> 221

<212> PRT

<213> endogenous inhibitor of human matrix metalloproteinase-2 mutant1 (Tissue inhibitor metalloproteinases-2 mutant1)

<400> 4

Met Gly Ala Ala Ala Arg Thr Leu Arg Leu Ala Leu Gly Leu Leu Leu

1 5 10 15

Leu Ala Thr Leu Leu Arg Pro Ala Asp Ala Ala Cys Ser Cys Ser Pro

20 25 30

Val His Pro Gln Gln Ala Phe Cys Asn Ala Asp Val Val Ile Arg Ala

35 40 45

Lys Ala Val Ser Glu Lys Glu Val Asp Ser Gly Asn Asp Ile Tyr Gly

50 55 60

Asn Pro Ile Lys Arg Ile Gln Tyr Glu Ile Lys Gln Ile Lys Met Phe

65 70 75 80

Lys Gly Pro Glu Lys Asp Ile Glu Phe Ile Tyr Thr Ala Pro Ser Ser

85 90 95

Ala Val Cys Gly Val Ser Leu Asp Val Gly Gly Lys Lys Glu Tyr Leu

100 105 110

Ile Ala Gly Lys Ala Glu Gly Asp Gly Lys Met His Ile Thr Leu Cys

115 120 125

Asp Phe Ile Val Pro Trp Asp Thr Leu Ser Thr Thr Gln Lys Lys Ser

130 135 140

Leu Asn His Arg Tyr Gln Met Gly Cys Glu Cys Lys Ile Thr Arg Cys

145 150 155 160

Pro Met Ile Pro Cys Tyr Ile Ser Ser Pro Asp Glu Cys Leu Trp Met

165 170 175

Asp Trp Val Thr Glu Lys Asn Ile Asn Gly His Gln Ala Lys Phe Phe

180 185 190

Ala Cys Ile Lys Arg Ser Asp Gly Ser Cys Ala Trp Tyr Arg Gly Ala

195 200 205

Ala Pro Pro Lys Gln Glu Phe Leu Asp Ile Glu Asp Pro

210 215 220

<210> 5

<211> 220

<212> PRT

<213> endogenous inhibitor of human matrix metalloproteinase-2 mutant2 (Tissue inhibitor metalloproteinases-2 human mutant 2)

<400> 5

Met Gly Ala Ala Ala Arg Thr Leu Arg Leu Ala Leu Gly Leu Leu Leu

1 5 10 15

Leu Ala Thr Leu Leu Arg Pro Ala Asp Ala Cys Ser Cys Ser Pro Val

20 25 30

His Pro Gln Gln Ala Phe Cys Asn Ala Asp Val Val Ile Arg Ala Lys

35 40 45

Ala Val Ser Glu Lys Glu Val Asp Ser Gly Asn Asp Ile Tyr Gly Asn

50 55 60

Pro Ile Lys Arg Ile Gln Tyr Glu Ile Lys Gln Ile Lys Met Phe Lys

65 70 75 80

Gly Pro Glu Lys Asp Ile Glu Phe Ile Tyr Thr Ala Pro Ser Ser Ala

85 90 95

Val Ser Gly Val Ser Leu Asp Val Gly Gly Lys Lys Glu Tyr Leu Ile

100 105 110

Ala Gly Lys Ala Glu Gly Asp Gly Lys Met His Ile Thr Leu Cys Asp

115 120 125

Phe Ile Val Pro Trp Asp Thr Leu Ser Thr Thr Gln Lys Lys Ser Leu

130 135 140

Asn His Arg Tyr Gln Met Gly Cys Glu Cys Lys Ile Thr Arg Cys Pro

145 150 155 160

Met Ile Pro Cys Tyr Ile Ser Ser Pro Asp Glu Cys Leu Trp Met Asp

165 170 175

Trp Val Thr Glu Lys Asn Ile Asn Gly His Gln Ala Lys Phe Phe Ala

180 185 190

Cys Ile Lys Arg Ser Asp Gly Ser Cys Ala Trp Tyr Arg Gly Ala Ala

195 200 205

Pro Pro Lys Gln Glu Phe Leu Asp Ile Glu Asp Pro

210 215 220

<210> 19

<211> 126

<212> PRT

<213> endogenous inhibitor of human matrix metalloproteinase-2N terminal (Tissue inhibitor metalloproteinases-2 human N-terminal)

<400> 19

Cys Ser Cys Ser Pro Val His Pro Gln Gln Ala Phe Cys Asn Ala Asp

1 5 10 15

Val Val Ile Arg Ala Lys Ala Val Ser Glu Lys Glu Val Asp Ser Gly

20 25 30

Asn Asp Ile Tyr Gly Asn Pro Ile Lys Arg Ile Gln Tyr Glu Ile Lys

35 40 45

Gln Ile Lys Met Phe Lys Gly Pro Glu Lys Asp Ile Glu Phe Ile Tyr

50 55 60

Thr Ala Pro Ser Ser Ala Val Ser Gly Val Ser Leu Asp Val Gly Gly

65 70 75 80

Lys Lys Glu Tyr Leu Ile Ala Gly Lys Ala Glu Gly Asp Gly Lys Met

85 90 95

His Ile Thr Leu Cys Asp Phe Ile Val Pro Trp Asp Thr Leu Ser Thr

100 105 110

Thr Gln Lys Lys Ser Leu Asn His Arg Tyr Gln Met Gly Cys

115 120 125

<210> 2

<211> 220

<212> PRT

<213> endogenous inhibitor of mouse matrix metalloproteinase-2 (Tissue inhibitor metalloproteinases-2 mouse)

<400> 2

Met Gly Ala Ala Ala Arg Ser Leu Arg Leu Ala Leu Gly Leu Leu Leu

1 5 10 15

Leu Ala Ser Leu Val Arg Pro Ala Asp Ala Cys Ser Cys Ser Pro Val

20 25 30

His Pro Gln Gln Ala Phe Cys Asn Ala Asp Val Val Ile Arg Ala Lys

35 40 45

Ala Val Ser Glu Lys Glu Val Asp Ser Gly Asn Asp Ile Tyr Gly Asn

50 55 60

Pro Ile Lys Arg Ile Gln Tyr Glu Ile Lys Gln Ile Lys Met Phe Lys

65 70 75 80

Gly Pro Asp Lys Asp Ile Glu Phe Ile Tyr Thr Ala Pro Ser Ser Ala

85 90 95

Val Cys Gly Val Ser Leu Asp Val Gly Gly Lys Lys Glu Tyr Leu Ile

100 105 110

Ala Gly Lys Ala Glu Gly Asp Gly Lys Met His Ile Thr Leu Cys Asp

115 120 125

Phe Ile Val Pro Trp Asp Thr Leu Ser Ile Thr Gln Lys Lys Ser Leu

130 135 140

Asn His Arg Tyr Gln Met Gly Cys Glu Cys Lys Ile Thr Arg Cys Pro

145 150 155 160

Met Ile Pro Cys Tyr Ile Ser Ser Pro Asp Glu Cys Leu Trp Met Asp

165 170 175

Trp Val Thr Glu Lys Ser Ile Asn Gly His Gln Ala Lys Phe Phe Ala

180 185 190

Cys Ile Lys Arg Ser Asp Gly Ser Cys Ala Trp Tyr Arg Gly Ala Ala

195 200 205

Pro Pro Lys Gln Glu Phe Leu Asp Ile Glu Asp Pro

210 215 220

<210> 3

<211> 221

<212> PRT

<213> endogenous inhibitor of mouse matrix metalloproteinase-2 mutant2 (Tissue inhibitor metalloproteinases-2 mutant2)

<400> 3

Met Gly Ala Ala Ala Arg Ser Leu Arg Leu Ala Leu Gly Leu Leu Leu

1 5 10 15

Leu Ala Ser Leu Val Arg Pro Ala Asp Ala Ala Cys Ser Cys Ser Pro

20 25 30

Val His Pro Gln Gln Ala Phe Cys Asn Ala Asp Val Val Ile Arg Ala

35 40 45

Lys Ala Val Ser Glu Lys Glu Val Asp Ser Gly Asn Asp Ile Tyr Gly

50 55 60

Asn Pro Ile Lys Arg Ile Gln Tyr Glu Ile Lys Gln Ile Lys Met Phe

65 70 75 80

Lys Gly Pro Asp Lys Asp Ile Glu Phe Ile Tyr Thr Ala Pro Ser Ser

85 90 95

Ala Val Cys Gly Val Ser Leu Asp Val Gly Gly Lys Lys Glu Tyr Leu

100 105 110

Ile Ala Gly Lys Ala Glu Gly Asp Gly Lys Met His Ile Thr Leu Cys

115 120 125

Asp Phe Ile Val Pro Trp Asp Thr Leu Ser Ile Thr Gln Lys Lys Ser

130 135 140

Leu Asn His Arg Tyr Gln Met Gly Cys Glu Cys Lys Ile Thr Arg Cys

145 150 155 160

Pro Met Ile Pro Cys Tyr Ile Ser Ser Pro Asp Glu Cys Leu Trp Met

165 170 175

Asp Trp Val Thr Glu Lys Ser Ile Asn Gly His Gln Ala Lys Phe Phe

180 185 190

Ala Cys Ile Lys Arg Ser Asp Gly Ser Cys Ala Trp Tyr Arg Gly Ala

195 200 205

Ala Pro Pro Lys Gln Glu Phe Leu Asp Ile Glu Asp Pro

210 215 220

<210> 6

<211> 220

<212> PRT

<213> endogenous inhibitor of mouse matrix metalloproteinase-2 mutant3 (Tissue inhibitor metalloproteinases-2 mutant3)

<400> 6

Met Gly Ala Ala Ala Arg Ser Leu Arg Leu Ala Leu Gly Leu Leu Leu

1 5 10 15

Leu Ala Ser Leu Val Arg Pro Ala Asp Ala Cys Ser Cys Ser Pro Val

20 25 30

His Pro Gln Gln Ala Phe Cys Asn Ala Asp Val Val Ile Arg Ala Lys

35 40 45

Ala Val Ser Glu Lys Glu Val Asp Ser Gly Asn Asp Ile Tyr Gly Asn

50 55 60

Pro Ile Lys Arg Ile Gln Tyr Glu Ile Lys Gln Ile Lys Met Phe Lys

65 70 75 80

Gly Pro Asp Lys Asp Ile Glu Phe Ile Tyr Thr Ala Pro Ser Ser Ala

85 90 95

Val Ser Gly Val Ser Leu Asp Val Gly Gly Lys Lys Glu Tyr Leu Ile

100 105 110

Ala Gly Lys Ala Glu Gly Asp Gly Lys Met His Ile Thr Leu Cys Asp

115 120 125

Phe Ile Val Pro Trp Asp Thr Leu Ser Ile Thr Gln Lys Lys Ser Leu

130 135 140

Asn His Arg Tyr Gln Met Gly Cys Glu Cys Lys Ile Thr Arg Cys Pro

145 150 155 160

Met Ile Pro Cys Tyr Ile Ser Ser Pro Asp Glu Cys Leu Trp Met Asp

165 170 175

Trp Val Thr Glu Lys Ser Ile Asn Gly His Gln Ala Lys Phe Phe Ala

180 185 190

Cys Ile Lys Arg Ser Asp Gly Ser Cys Ala Trp Tyr Arg Gly Ala Ala

195 200 205

Pro Pro Lys Gln Glu Phe Leu Asp Ile Glu Asp Pro

210 215 220

<210> 20

<211> 126

<212> PRT

<213> endogenous inhibitor of mouse matrix metalloproteinase-2N terminal (Tissue inhibitor metalloproteinases-2 mouse N-terminal)

<400> 20

Cys Ser Cys Ser Pro Val His Pro Gln Gln Ala Phe Cys Asn Ala Asp

1 5 10 15

Val Val Ile Arg Ala Lys Ala Val Ser Glu Lys Glu Val Asp Ser Gly

20 25 30

Asn Asp Ile Tyr Gly Asn Pro Ile Lys Arg Ile Gln Tyr Glu Ile Lys

35 40 45

Gln Ile Lys Met Phe Lys Gly Pro Asp Lys Asp Ile Glu Phe Ile Tyr

50 55 60

Thr Ala Pro Ser Ser Ala Val Cys Gly Val Ser Leu Asp Val Gly Gly

65 70 75 80

Lys Lys Glu Tyr Leu Ile Ala Gly Lys Ala Glu Gly Asp Gly Lys Met

85 90 95

His Ile Thr Leu Cys Asp Phe Ile Val Pro Trp Asp Thr Leu Ser Ile

100 105 110

Thr Gln Lys Lys Ser Leu Asn His Arg Tyr Gln Met Gly Cys

115 120 125

<210> 21

<211> 24

<212> PRT

<213> endogenous inhibitors of matrix metalloproteinases-2 Ile43-Ala66 (Tissue inhibitor metalloproteinases-2 Ile43-Ala 66)

<400> 21

Ile Gln Tyr Glu Ile Lys Gln Ile Lys Met Phe Lys Gly Pro Glu Lys

1 5 10 15

Asp Ile Glu Phe Ile Tyr Thr Ala

20

<210> 23

<211> 18

<212> PRT

<213> endogenous inhibitors of matrix metalloproteinases-2 Ile43-Ile60 (Tissue inhibitor metalloproteinases-2 Ile43-Ile 60)

<400> 23

Ile Gln Tyr Glu Ile Lys Gln Ile Lys Met Phe Lys Gly Pro Glu Lys

1 5 10 15

Asp Ile

<210> 22

<211> 18

<212> PRT

<213> endogenous inhibitors of matrix metalloproteinases-2 Gln49-Ala66 (Tissue inhibitor metalloproteinases-2 Gln49-Ala 66)

<400> 22

Gln Ile Lys Met Phe Lys Gly Pro Glu Lys Asp Ile Glu Phe Ile Tyr

1 5 10 15

Thr Ala

<210> 24

<211> 10

<212> PRT

<213> endogenous inhibitors of matrix metalloproteinases-2 Gln49-Lys58 (Tissue inhibitor metalloproteinases-2 Gln49-Lys 58)

<400> 24

Gln Ile Lys Met Phe Lys Gly Pro Glu Lys

1 5 10

<210> 8

<211> 663

<212> DNA/RNA

<213> endogenous inhibitor of human matrix metalloproteinase-2 (Tissue inhibitor metalloproteinases-2 human)

<400> 8

atgggcgccg cggcccgcac cctgcggctg gcgctcggcc tcctgctgct ggcgacgctg 60

cttcgcccgg ccgacgcctg cagctgctcc ccggtgcacc cgcaacaggc gttttgcaat 120

gcagatgtag tgatcagggc caaagcggtc agtgagaagg aagtggactc tggaaacgac 180

atttatggca accctatcaa gaggatccag tatgagatca agcagataaa gatgttcaaa 240

gggcctgaga aggatataga gtttatctac acggccccct cctcggcagt gtgtggggtc 300

tcgctggacg ttggaggaaa gaaggaatat ctcattgcag gaaaggccga gggggacggc 360

aagatgcaca tcaccctctg tgacttcatc gtgccctggg acaccctgag caccacccag 420

aagaagagcc tgaaccacag gtaccagatg ggctgcgagt gcaagatcac gcgctgcccc 480

atgatcccgt gctacatctc ctccccggac gagtgcctct ggatggactg ggtcacagag 540

aagaacatca acgggcacca ggccaagttc ttcgcctgca tcaagagaag tgacggctcc 600

tgtgcgtggt accgcggcgc ggcgcccccc aagcaggagt ttctcgacat cgaggaccca 660

taa 663

<210> 8

<211> 666

<212> DNA/RNA

<213> endogenous inhibitor of human matrix metalloproteinase-2 mutant1 (Tissue inhibitor metalloproteinases-2 human mutant 1)

<400> 8

atgggcgccg cggcccgcac cctgcggctg gcgctcggcc tcctgctgct ggcgacgctg 60

cttcgcccgg ccgacgccgc ttgcagctgc tccccggtgc acccgcaaca ggcgttttgc 120

aatgcagatg tagtgatcag ggccaaagcg gtcagtgaga aggaagtgga ctctggaaac 180

gacatttatg gcaaccctat caagaggatc cagtatgaga tcaagcagat aaagatgttc 240

aaagggcctg agaaggatat agagtttatc tacacggccc cctcctcggc agtgtgtggg 300

gtctcgctgg acgttggagg aaagaaggaa tatctcattg caggaaaggc cgagggggac 360

ggcaagatgc acatcaccct ctgtgacttc atcgtgccct gggacaccct gagcaccacc 420

cagaagaaga gcctgaacca caggtaccag atgggctgcg agtgcaagat cacgcgctgc 480

cccatgatcc cgtgctacat ctcctccccg gacgagtgcc tctggatgga ctgggtcaca 540

gagaagaaca tcaacgggca ccaggccaag ttcttcgcct gcatcaagag aagtgacggc 600

tcctgtgcgt ggtaccgcgg cgcggcgccc cccaagcagg agtttctcga catcgaggac 660

ccataa 666

<210> 9

<211> 663

<212> DNA/RNA

<213> endogenous inhibitor of human matrix metalloproteinase-2 mutant2 (Tissue inhibitor metalloproteinases-2 human mutant 2)

<400> 9

atgggcgccg cggcccgcac cctgcggctg gcgctcggcc tcctgctgct ggcgacgctg 60

cttcgcccgg ccgacgcctg cagctgctcc ccggtgcacc cgcaacaggc gttttgcaat 120

gcagatgtag tgatcagggc caaagcggtc agtgagaagg aagtggactc tggaaacgac 180

atttatggca accctatcaa gaggatccag tatgagatca agcagataaa gatgttcaaa 240

gggcctgaga aggatataga gtttatctac acggccccct cctcggcagt gtctggggtc 300

tcgctggacg ttggaggaaa gaaggaatat ctcattgcag gaaaggccga gggggacggc 360

aagatgcaca tcaccctctg tgacttcatc gtgccctggg acaccctgag caccacccag 420

aagaagagcc tgaaccacag gtaccagatg ggctgcgagt gcaagatcac gcgctgcccc 480

atgatcccgt gctacatctc ctccccggac gagtgcctct ggatggactg ggtcacagag 540

aagaacatca acgggcacca ggccaagttc ttcgcctgca tcaagagaag tgacggctcc 600

tgtgcgtggt accgcggcgc ggcgcccccc aagcaggagt ttctcgacat cgaggaccca 660

taa 663

<210> 10

<211> 663

<212> DNA/RNA

<213> endogenous inhibitor of human matrix metalloproteinase-2 mutant3 (Tissue inhibitor metalloproteinases-2 human mutant 3)

<400> 10

atgggcgccg cggcccgcac cctgcggctg gcgctcggcc tcctgctgct ggcgacgctg 60

cttcgcccgg ccgacgcctg cagctgctcc ccggtgcacc cgcaacaggc gttttgcaat 120

gcagatgtag tgatcagggc caaagcggtc agtgagaagg aagtggactc tggaaacgac 180

atttatggca accctatcaa gaggatccag tatgagatca agcagataaa gatgttcaaa 240

gggcctgaga aggatataga gtttatctac acggccccct cctcggcagt gtcaggggtc 300

tcgctggacg ttggaggaaa gaaggaatat ctcattgcag gaaaggccga gggggacggc 360

aagatgcaca tcaccctctg tgacttcatc gtgccctggg acaccctgag caccacccag 420

aagaagagcc tgaaccacag gtaccagatg ggctgcgagt gcaagatcac gcgctgcccc 480

atgatcccgt gctacatctc ctccccggac gagtgcctct ggatggactg ggtcacagag 540

aagaacatca acgggcacca ggccaagttc ttcgcctgca tcaagagaag tgacggctcc 600

tgtgcgtggt accgcggcgc ggcgcccccc aagcaggagt ttctcgacat cgaggaccca 660

taa 663

<210> 11

<211> 663

<212> DNA/RNA

<213> endogenous inhibitor of human matrix metalloproteinase-2 mutant4 (Tissue inhibitor metalloproteinases-2 human mutant 4)

<400> 11

atgggcgccg cggcccgcac cctgcggctg gcgctcggcc tcctgctgct ggcgacgctg 60

cttcgcccgg ccgacgcctg cagctgctcc ccggtgcacc cgcaacaggc gttttgcaat 120

gcagatgtag tgatcagggc caaagcggtc agtgagaagg aagtggactc tggaaacgac 180

atttatggca accctatcaa gaggatccag tatgagatca agcagataaa gatgttcaaa 240

gggcctgaga aggatataga gtttatctac acggccccct cctcggcagt gtcgggggtc 300

tcgctggacg ttggaggaaa gaaggaatat ctcattgcag gaaaggccga gggggacggc 360

aagatgcaca tcaccctctg tgacttcatc gtgccctggg acaccctgag caccacccag 420

aagaagagcc tgaaccacag gtaccagatg ggctgcgagt gcaagatcac gcgctgcccc 480

atgatcccgt gctacatctc ctccccggac gagtgcctct ggatggactg ggtcacagag 540

aagaacatca acgggcacca ggccaagttc ttcgcctgca tcaagagaag tgacggctcc 600

tgtgcgtggt accgcggcgc ggcgcccccc aagcaggagt ttctcgacat cgaggaccca 660

taa 663

<210> 12

<211> 663

<212> DNA/RNA

<213> endogenous inhibitor of human matrix metalloproteinase-2 mutant5 (Tissue inhibitor metalloproteinases-2 human mutant 5)

<400> 12

atgggcgccg cggcccgcac cctgcggctg gcgctcggcc tcctgctgct ggcgacgctg 60

cttcgcccgg ccgacgcctg cagctgctcc ccggtgcacc cgcaacaggc gttttgcaat 120

gcagatgtag tgatcagggc caaagcggtc agtgagaagg aagtggactc tggaaacgac 180

atttatggca accctatcaa gaggatccag tatgagatca agcagataaa gatgttcaaa 240

gggcctgaga aggatataga gtttatctac acggccccct cctcggcagt gtccggggtc 300

tcgctggacg ttggaggaaa gaaggaatat ctcattgcag gaaaggccga gggggacggc 360

aagatgcaca tcaccctctg tgacttcatc gtgccctggg acaccctgag caccacccag 420

aagaagagcc tgaaccacag gtaccagatg ggctgcgagt gcaagatcac gcgctgcccc 480

atgatcccgt gctacatctc ctccccggac gagtgcctct ggatggactg ggtcacagag 540

aagaacatca acgggcacca ggccaagttc ttcgcctgca tcaagagaag tgacggctcc 600

tgtgcgtggt accgcggcgc ggcgcccccc aagcaggagt ttctcgacat cgaggaccca 660

taa 663

<210> 25

<211> 378

<212> DNA/RNA

<213> endogenous inhibitor of human matrix metalloproteinase-2N terminal (Tissue inhibitor metalloproteinases-2 human N-terminal)

<400> 25

tgcagctgct ccccggtgca cccgcaacag gcgttttgca atgcagatgt agtgatcagg 60

gccaaagcgg tcagtgagaa ggaagtggac tctggaaacg acatttatgg caaccctatc 120

aagaggatcc agtatgagat caagcagata aagatgttca aagggcctga gaaggatata 180

gagtttatct acacggcccc ctcctcggca gtgtgtgggg tctcgctgga cgttggagga 240

aagaaggaat atctcattgc aggaaaggcc gagggggacg gcaagatgca catcaccctc 300

tgtgacttca tcgtgccctg ggacaccctg agcaccaccc agaagaagag cctgaaccac 360

aggtaccaga tgggctgc 378

<210> 13

<211> 663

<212> DNA/RNA

<213> endogenous inhibitor of mouse matrix metalloproteinase-2 (Tissue inhibitor metalloproteinases-2 mouse)

<400> 13

atgggcgccg cggcccgcag cctccggctg gcgctcggcc tcctgctgct agccacgctg 60

ctgcgcccgg ccgacgcctg cagctgctcc ccggtgcacc cgcaacaggc gttttgcaat 120

gcagacgtag tgatcagagc caaagcagtg agcgagaagg aggtggattc cgggaatgac 180

atctatggca accccatcaa gaggattcag tatgagatca agcagataaa gatgttcaaa 240

ggacctgaca aagacatcga gtttatctac acggccccct cttcagcagt gtgcggggtc 300

tcgctggacg ttggaggaaa gaaggagtat ctaattgcag gaaaggcaga aggagatggc 360

aagatgcaca ttaccctctg tgacttcatt gtgccctggg acacgcttag catcacccag 420

aagaagagcc tgaaccacag gtaccagatg ggctgtgagt gcaagatcac tcgctgtccc 480

atgatccctt gctacatctc ctccccggat gagtgcctct ggatggactg ggtcacagag 540

aagagcatca atgggcacca ggccaagttc ttcgcctgca tcaagagaag tgatggttct 600

tgcgcgtggt accgcggggc ggcacccccc aagcaagagt ttcttgacat cgaggacccg 660

taa 663

<210> 14

<211> 666

<212> DNA/RNA

<213> endogenous inhibitor of mouse matrix metalloproteinase-2 mutant1 (Tissue inhibitor metalloproteinases-2 mouse mutant1)

<400> 14

atgggcgccg cggcccgcag cctccggctg gcgctcggcc tcctgctgct agccacgctg 60

ctgcgcccgg ccgacgccgc ttgcagctgc tccccggtgc acccgcaaca ggcgttttgc 120

aatgcagacg tagtgatcag agccaaagca gtgagcgaga aggaggtgga ttccgggaat 180

gacatctatg gcaaccccat caagaggatt cagtatgaga tcaagcagat aaagatgttc 240

aaaggacctg acaaagacat cgagtttatc tacacggccc cctcttcagc agtgtgcggg 300

gtctcgctgg acgttggagg aaagaaggag tatctaattg caggaaaggc agaaggagat 360

ggcaagatgc acattaccct ctgtgacttc attgtgccct gggacacgct tagcatcacc 420

cagaagaaga gcctgaacca caggtaccag atgggctgtg agtgcaagat cactcgctgt 480

cccatgatcc cttgctacat ctcctccccg gatgagtgcc tctggatgga ctgggtcaca 540

gagaagagca tcaatgggca ccaggccaag ttcttcgcct gcatcaagag aagtgatggt 600

tcttgcgcgt ggtaccgcgg ggcggcaccc cccaagcaag agtttcttga catcgaggac 660

ccgtaa 666

<210> 15

<211> 663

<212> DNA/RNA

<213> endogenous inhibitor of mouse matrix metalloproteinase-2 mutant2 (Tissue inhibitor metalloproteinases-2 mouse mutant 2)

<400> 15

atgggcgccg cggcccgcag cctccggctg gcgctcggcc tcctgctgct agccacgctg 60

ctgcgcccgg ccgacgcctg cagctgctcc ccggtgcacc cgcaacaggc gttttgcaat 120

gcagacgtag tgatcagagc caaagcagtg agcgagaagg aggtggattc cgggaatgac 180

atctatggca accccatcaa gaggattcag tatgagatca agcagataaa gatgttcaaa 240

ggacctgaca aagacatcga gtttatctac acggccccct cttcagcagt gtccggggtc 300

tcgctggacg ttggaggaaa gaaggagtat ctaattgcag gaaaggcaga aggagatggc 360

aagatgcaca ttaccctctg tgacttcatt gtgccctggg acacgcttag catcacccag 420

aagaagagcc tgaaccacag gtaccagatg ggctgtgagt gcaagatcac tcgctgtccc 480

atgatccctt gctacatctc ctccccggat gagtgcctct ggatggactg ggtcacagag 540

aagagcatca atgggcacca ggccaagttc ttcgcctgca tcaagagaag tgatggttct 600

tgcgcgtggt accgcggggc ggcacccccc aagcaagagt ttcttgacat cgaggacccg 660

taa 663

<210> 16

<211> 663

<212> DNA/RNA

<213> endogenous inhibitor of mouse matrix metalloproteinase-2 mutant3 (Tissue inhibitor metalloproteinases-2 mouse mutant 3)

<400> 16

atgggcgccg cggcccgcag cctccggctg gcgctcggcc tcctgctgct agccacgctg 60

ctgcgcccgg ccgacgcctg cagctgctcc ccggtgcacc cgcaacaggc gttttgcaat 120

gcagacgtag tgatcagagc caaagcagtg agcgagaagg aggtggattc cgggaatgac 180

atctatggca accccatcaa gaggattcag tatgagatca agcagataaa gatgttcaaa 240

ggacctgaca aagacatcga gtttatctac acggccccct cttcagcagt gtctggggtc 300

tcgctggacg ttggaggaaa gaaggagtat ctaattgcag gaaaggcaga aggagatggc 360

aagatgcaca ttaccctctg tgacttcatt gtgccctggg acacgcttag catcacccag 420

aagaagagcc tgaaccacag gtaccagatg ggctgtgagt gcaagatcac tcgctgtccc 480

atgatccctt gctacatctc ctccccggat gagtgcctct ggatggactg ggtcacagag 540

aagagcatca atgggcacca ggccaagttc ttcgcctgca tcaagagaag tgatggttct 600

tgcgcgtggt accgcggggc ggcacccccc aagcaagagt ttcttgacat cgaggacccg 660

taa 663

<210> 17

<211> 663

<212> DNA/RNA

<213> endogenous inhibitor of mouse matrix metalloproteinase-2 mutant4 (Tissue inhibitor metalloproteinases-2 mouse mutant 4)

<400> 17

atgggcgccg cggcccgcag cctccggctg gcgctcggcc tcctgctgct agccacgctg 60

ctgcgcccgg ccgacgcctg cagctgctcc ccggtgcacc cgcaacaggc gttttgcaat 120

gcagacgtag tgatcagagc caaagcagtg agcgagaagg aggtggattc cgggaatgac 180

atctatggca accccatcaa gaggattcag tatgagatca agcagataaa gatgttcaaa 240

ggacctgaca aagacatcga gtttatctac acggccccct cttcagcagt gtcaggggtc 300

tcgctggacg ttggaggaaa gaaggagtat ctaattgcag gaaaggcaga aggagatggc 360

aagatgcaca ttaccctctg tgacttcatt gtgccctggg acacgcttag catcacccag 420

aagaagagcc tgaaccacag gtaccagatg ggctgtgagt gcaagatcac tcgctgtccc 480

atgatccctt gctacatctc ctccccggat gagtgcctct ggatggactg ggtcacagag 540

aagagcatca atgggcacca ggccaagttc ttcgcctgca tcaagagaag tgatggttct 600

tgcgcgtggt accgcggggc ggcacccccc aagcaagagt ttcttgacat cgaggacccg 660

taa 663

<210> 18

<211> 663

<212> DNA/RNA

<213> endogenous inhibitor of mouse matrix metalloproteinase-2 mutant5 (Tissue inhibitor metalloproteinases-2 mouse mutant 5)

<400> 18

atgggcgccg cggcccgcag cctccggctg gcgctcggcc tcctgctgct agccacgctg 60

ctgcgcccgg ccgacgcctg cagctgctcc ccggtgcacc cgcaacaggc gttttgcaat 120

gcagacgtag tgatcagagc caaagcagtg agcgagaagg aggtggattc cgggaatgac 180

atctatggca accccatcaa gaggattcag tatgagatca agcagataaa gatgttcaaa 240

ggacctgaca aagacatcga gtttatctac acggccccct cttcagcagt gtcgggggtc 300

tcgctggacg ttggaggaaa gaaggagtat ctaattgcag gaaaggcaga aggagatggc 360

aagatgcaca ttaccctctg tgacttcatt gtgccctggg acacgcttag catcacccag 420

aagaagagcc tgaaccacag gtaccagatg ggctgtgagt gcaagatcac tcgctgtccc 480

atgatccctt gctacatctc ctccccggat gagtgcctct ggatggactg ggtcacagag 540

aagagcatca atgggcacca ggccaagttc ttcgcctgca tcaagagaag tgatggttct 600

tgcgcgtggt accgcggggc ggcacccccc aagcaagagt ttcttgacat cgaggacccg 660

taa 663

<210> 26

<211> 378

<212> DNA/RNA

<213> endogenous inhibitor of mouse matrix metalloproteinase-2N terminal (Tissue inhibitor metalloproteinases-2 mouse N-terminal)

<400> 26

tgcagctgct ccccggtgca cccgcaacag gcgttttgca atgcagacgt agtgatcaga 60

gccaaagcag tgagcgagaa ggaggtggat tccgggaatg acatctatgg caaccccatc 120

aagaggattc agtatgagat caagcagata aagatgttca aaggacctga caaagacatc 180

gagtttatct acacggcccc ctcttcagca gtgtgcgggg tctcgctgga cgttggagga 240

aagaaggagt atctaattgc aggaaaggca gaaggagatg gcaagatgca cattaccctc 300

tgtgacttca ttgtgccctg ggacacgctt agcatcaccc agaagaagag cctgaaccac 360

aggtaccaga tgggctgt 378

<210> 27

<211> 72

<212> DNA/RNA

<213> endogenous inhibitor of mouse matrix metalloproteinase-2 Ile43-Ala66 (Tissue inhibitor metalloproteinases-2 mouse Ile43-Ala 66)

<400> 27

atccagtatg agatcaagca gataaagatg ttcaaagggc ctgagaagga tatagagttt 60

atctacacgg cc 72

<210> 28

<211> 54

<212> DNA/RNA

<213> endogenous inhibitors of mouse matrix metalloproteinases-2 Ile43-Ile60 (Tissue inhibitor metalloproteinases-2 mouse Ile43-Ile 60)

<400> 28

atccagtatg agatcaagca gataaagatg ttcaaagggc ctgagaagga tata 54

<210> 29

<211> 54

<212> DNA/RNA

<213> endogenous inhibitor of mouse matrix metalloproteinase-2 Gln49-Ala66 (Tissue inhibitor metalloproteinases-2 mouse Gln49-Ala 66)

<400> 29

cagataaaga tgttcaaagg gcctgagaag gatatagagt ttatctacac ggcc 54

<210> 30

<211> 30

<212> DNA/RNA

<213> endogenous inhibitor of mouse matrix metalloproteinase-2 Gln49-Lys58 (Tissue inhibitor metalloproteinases-2 mouse Gln49-Lys 58)

<400> 30

cagataaaga tgttcaaagg gcctgagaag 30

Claims (9)

1. Use of an endogenous inhibitor of matrix metalloproteinase-2 in the preparation of a medicament for preventing or treating traumatic brain injury.

2. The use according to claim 1, characterized in that the amino acid sequence of the endogenous inhibitor of matrix metalloproteinase-2 is the amino acid sequence shown in SEQ ID No.1-SEQ ID No.12 in the sequence listing.

3. Use of a nucleic acid molecule encoding an endogenous inhibitor of matrix metalloproteinase-2 in the preparation of a medicament for preventing or treating traumatic brain injury.

4. The use according to claim 3, wherein the nucleic acid molecule has the nucleotide sequence of SEQ ID NO.13-SEQ ID NO.30 or the nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO.13-SEQ ID NO.30 in the sequence Listing.

5. Use of an expression vector comprising the nucleic acid molecule of any one of claims 3-4 in the preparation of a medicament for preventing or treating traumatic brain injury.

6. The use according to claim 5, wherein said expression vector comprises an adeno-associated viral vector, an adenovirus vector, a retrovirus vector, an exosome, a liposome complex, a cationic polymer, a chitosan polymer, an inorganic nanoparticle.

7. Use of a host cell comprising the nucleic acid molecule of any one of claims 3-4 or the expression vector of any one of claims 5-6 in the manufacture of a medicament for preventing or treating traumatic brain injury.

8. The use according to claim 7, wherein the host cell is selected from the group consisting of bacteria, yeast, aspergillus, plant cells, insect cells, or mammalian cells.

9. The use according to any one of claims 1-8, wherein the traumatic brain injury comprises a blood brain barrier dysfunction caused by the traumatic brain injury, and a central nervous system disorder associated with the blood brain barrier dysfunction.

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