CN114642722A - Use of plasminogen for preparing medicine for promoting angiogenesis - Google Patents
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- A—HUMAN NECESSITIES
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Abstract
The invention discloses an application of plasminogen in preparing a medicine for promoting angiogenesis, belonging to the field of medicines for promoting angiogenesis. The invention discloses the angiogenesis promoting effect of plasminogen for the first time, and the plasminogen can be prepared into a medicament, so that the cerebrovascular angiogenesis can be obviously promoted, and the cerebral infarction patients can be helped to recover the motor and sensory functions more quickly.
Description
Technical Field
The invention belongs to the field of angiogenesis promoting medicines.
Background
Stroke, commonly known as stroke, is the damage to brain tissue caused by the damage to blood vessels in the brain. In developed countries, stroke is the most common cause of disability and also a common cause of death. In China, stroke is the first cause of death and disability of adults, and has the characteristics of high morbidity, high disability rate, high death rate and high recurrence rate. Cerebral stroke is generally divided into two categories, ischemic stroke and hemorrhagic stroke. Ischemic stroke is also called cerebral infarction and accounts for about 70 percent of stroke in China.
The medicine can assist the angiogenesis of cerebral vessels, relieve ischemic injury, promote the recovery of nerve function in ischemic regions, and improve prognosis. The angiogenesis promoting drugs mainly include: statins, angiotensin receptor inhibitors, phosphodiesterases, vascular factors, and the like.
Plasminogen (Plasminogen, Plg) is an inactive precursor of plasma fibrinolytic enzymes. Activated by tissue activators t-PA, urokinase or various enzymes of the coagulation contact phase, exogenous activators such as streptokinase may also play an activating role. Plasmin degrades fibrin and fibrinogen, keeps vessels and glandular ducts unobstructed, and further researches show that the plasmin also has the functions of collagenase activity and plays an auxiliary role in nutrition and cell movement.
At present, reports on the angiogenesis promotion of plasminogen are not found, and reports on the cerebrovascular angiogenesis promotion of plasminogen are not found.
Disclosure of Invention
The invention aims to solve the problems that: provides a medicine for promoting the regeneration of blood vessels.
The technical scheme of the invention is as follows:
use of plasminogen for the manufacture of a medicament for promoting angiogenesis.
Further, the drug is a drug that promotes brain angiogenesis.
Further, the drug is a drug that inhibits the expression of TSP1 and TSP 2.
Further, the medicament is a medicament for treating cerebral infarction.
Further, the drug is a drug that accelerates recovery of sensory and motor functions after cerebral infarction.
A medicament for promoting angiogenesis, which is characterized in that: the drug is prepared by taking plasminogen as an active ingredient and adding pharmaceutically acceptable auxiliary ingredients.
Further, the drug is a drug that promotes brain angiogenesis.
Further, the drug is a drug that inhibits the expression of TSP1 and TSP 2.
Further, the medicament is a medicament for treating cerebral infarction.
Further, the drug is a drug that accelerates recovery of sensory and motor functions after cerebral infarction.
Has the advantages that:
the medicine of the invention can effectively promote the regeneration of cerebral vessels, accelerate the recovery of motor and sensory functions after cerebral infarction and play a certain role in treating cerebral infarction.
Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.
The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.
Drawings
FIG. 1: two mice had post-cerebral arterial infarction (post-ischemic stroke) behavioral deficits and recovery. (a) Step-staggered testing; (b) decal removal test.
FIG. 2: single-layer confocal microscopy imaging revealed cerebral infarction. (a) Microscope pictures, scale bar 100 μm; (b) and (5) counting the length of the blood vessel in unit area.
FIG. 3: the relationship between the density of cerebral vessels and the functional recovery of mice after cerebral infarction. (a) Step-staggered testing; (b) decal removal test.
FIG. 4: and (3) observing angiogenesis of primary brain capillary endothelial cells in vitro. (a) Normal mice; (b) a mutant mouse; (c) and (5) counting the length of the blood vessel-like structure in a unit area.
FIG. 5: western blot results of angiogenesis inhibitor (TSP 1/2). The abscissa is 1-Plg +/+ normal left hemisphere before molding, 2-Plg-/-normal left hemisphere before molding, 3-Plg +/+ ipsilateral (right) hemisphere after molding, 4-Plg-/-ipsilateral (right) hemisphere after molding, 5-Plg +/+ contralateral (left) hemisphere after molding, and 6-Plg-/-contralateral (left) hemisphere after molding.
Detailed Description
Example 1 cerebral infarction surgery experiment in Normal and plasminogen-knock-out mice
1. Method of producing a composite material
Constructing plasminogen gene knockout mouse, and performing middle cerebral artery infarction operation with normal mouse to perform cerebral infarction modeling.
The molding method comprises the following steps:
(1) mice of the corresponding genotype were continuously anesthetized with trifluorobromochloroethane gas. Sutures for ligation or occlusion of the artery are prepared in advance and infiltrated in saline.
(2) After several minutes, if the mouse can not turn over freely, the mouse is considered to reach the requirement of anesthesia depth, the mouse is bound on an anatomical table, the neck of the mouse is padded with a cotton swab, and iodophors disinfect the neck of the mouse and prepare the skin.
(3) The skin is cut along the center of the mouse neck, subcutaneous tissue and fat are separated bluntly by using bending forceps of ophthalmology and straight forceps of ophthalmology under the enlarged visual field of a stereomicroscope, superficial myofascium and platysma are scratched, and deep myofascium and anterior muscle of trachea are exposed.
(4) After separating to the anterior tracheal muscle, the deep cervical muscularis is torn at the front edge of the sternocleidomastoid muscle at the right side, the sternocleidomastoid muscle is exposed and pulled to the back side, and the separation is continued downwards along the sternocleidomoid muscle until the carotid artery sheath at the deeper position is exposed.
(5) While avoiding compression of the mouse trachea, the carotid sheath is dissected, the Common Carotid Artery (CCA) is isolated and threaded to stop blood flow with a slipknot, taking care not to injure the internal jugular vein and vagus nerve that accompany the sheath, and irritation or damage to the nerve may cause respiratory arrest in the mouse.
(6) It is separated along the common carotid artery and the External Carotid Artery (ECA) is located at the superior border of the thyroid cartilage, inside the superior carotid artery, near the trachea. The common carotid artery is divided into internal carotid artery and external carotid artery, and has carotid sinus and carotid glomerulus. The carotid sinus is an enlarged portion of the end of the common carotid artery and the beginning of the internal carotid artery.
(7) The small branch from the front inner side wall of the external carotid artery is the upper thyroid artery, which is electrically coagulated and burned off after separation. Continued separation up the external carotid artery revealed a second branch of the lingual artery, as well as the occipital and external maxillo-mandibular arteries. The double ligation is carried out by knitting threads at the branch parts close to the occipital artery and the extramaxillary artery, the external carotid artery is cut off at the middle of the two ligation parts, and the ligation thread at the proximal end is slightly kept long so as to facilitate the subsequent insertion of the suppository.
(8) The proximal end ligature of the external carotid artery is lifted slightly, exposing the inferior lateral Internal Carotid Artery (ICA). The internal carotid artery is divided into two branches before entering the cranium, namely a pterygopalatine artery positioned on the outer side and an internal carotid artery positioned on the inner side enter the cranium branch. To reduce the modeling procedure time and injury to the mouse, the pterygopalatine artery may not be isolated, and only threading under the internal carotid artery is needed to stop blood flow from the internal carotid artery with a catenary wire when the plug wire is inserted.
(9) A slipknot is additionally arranged at the proximal end of the external carotid artery, and is not tightened for the moment, and the thread is tightened after being inserted to a designated position so as to fix the thread. And slightly lifting the proximal ligature of the external carotid artery to make the external carotid artery and the internal carotid artery in the same direction. The braided wire under the internal carotid artery was suspended and temporarily clamped with a mini-arterial clamp to control blood flow. A small opening is cut at the proximal end of the external carotid artery by an ophthalmologic scissors, and a suppository thread with round thread end, smooth thread body and uniform thickness is selected to be gently inserted from the small opening, and the suppository thread can pass through the bifurcation from the external carotid artery and enter the internal carotid artery. Note that when the plug wire enters the internal carotid artery, it should be slightly depressed to raise the head of the plug wire, which avoids inserting the plug wire into the pterygopalatine artery. The reason why the pterygopalatine artery is not recommended to be separated or ligated is that the blocking of the pterygopalatine artery is prone to thrombus formation in the internal carotid artery, and the thrombus is wrapped around the thrombus line, which causes difficulty in thrombus removal and is not easy to be reflused in model perfusion.
(10) Slight resistance is encountered when the tether line is inserted around 9-10mm, indicating that the tether line has been inserted at the bifurcation of the Anterior Cerebral Artery (ACA) and the Middle Cerebral Artery (MCA), blocking the beginning of the main trunk of the middle cerebral artery. Without continuing the insertion until then, too deep insertion may cause the tether to puncture the ACA and induce subarachnoid hemorrhage (SAH) leading to modeling failure and even death of the mouse. If the insertion depth of the plug wire is about 6mm and the plug wire feels larger resistance, the plug wire can be too thick to enter the skull, or the plug wire can be inserted into the pterygopalatine artery by mistake, and the plug wire can be inserted inefficiently, and the plug wire is slightly withdrawn from the replacement angle and then is tried to be inserted into the skull-entering branch of the internal carotid artery.
(11) After inserting the plug thread to the designated position, the slipknot arranged on the proximal end of the external carotid artery is tightened to fix the plug thread. The slipknot blocking the common carotid artery was untied, the skin was sutured finally, the suture was disinfected with iodophor, and the mouse was returned to the cage.
1.1 Observation of mouse behavioral Performance
(1) Staggered step test
And detecting the percentage of the number of wrong steps of the left front paw on the damaged side of the cerebral infarction on the non-equidistant grid to the total number of steps, and evaluating the motor function of the left front paw on the damaged side of the mouse after the cerebral infarction.
(2) Sticker removal test
And (3) sticking a small quarter circle of adhesive paper on the front paw on the side with the cerebral infarction injury of the mouse, calculating the time for removing the adhesive paper by the mouse, and detecting the sensory disturbance and the dyskinesia of the mouse after the cerebral infarction.
1.2 vascular Density measurement
FITC is injected into the mice, and brain vessel imaging of brain tissues at different time points before and after cerebral infarction is displayed by using single-layer confocal micro-mirror imaging. The cerebral vascular density of the cortical areas surrounding the ipsilateral cortical infarct was calculated using ImageJ software.
1.3 measurement of the vascularization ability of cerebral vascular endothelial cells
Mice were sacrificed in plasminogen knockout mice and normal mice (without infarct modeling), primary cerebral capillary endothelial cells were isolated, suspended in DMEM complete medium (containing 20-30% FBS, 100ug/ml heparin sodium, 3ng/100ml bFGF, etc.), inoculated in 6-well or 24-well disposable plastic culture dishes coated with 1mg/ml type IV collagen and 0.1% fibronectin, placed at 37 ℃, and 5% CO2The incubator (2) is kept still for culture. Then using a tubule formation experiment kit, adding 2-4ml of endothelial cell culture medium into the cell particles, and blowing and beating up and down for several times to ensure the uniformity of the single cell suspension. Endothelial cells were obtained and counted at a coverage of 50% (WT), and 60% (plg-/-) as per this procedure. Cell suspension density of 75000/ml and 100000/ml; diluting the cells and plating the cells in previously prepared complete 8-well plates of Coating, 150 μ l cells (7500/well and 10000/well) per well; will eightThe plates were incubated at 37 ℃ for 2-3 hours with 5% CO2 and photographed to check for lumen formation every hour. And the photograph is recorded.
1.4 detection of angiogenesis inhibitors
A batch of mice were sacrificed at 14d before and after cerebral infarction, brain tissues were collected, and angiogenesis inhibitory factors TSP1 and TSP 2 were detected using Western blot.
2. Results
2.1 behavioral manifestations
As shown in figure 1, the day after the cerebral infarction, the mice had a clear behavioral deficit and then over time, the sensory and motor functions of the mice were continuously, progressively, but not completely restored. The improvement of motor and sensory functions after brain infarction was significantly worse in plasminogen knock-out mice (KO group) compared to normal mice (WT group) (n ═ 10/group;. p <0.05,. p <0.01,. p < 0.001).
This result indicates that plasminogen is beneficial for motor and sensory function recovery after cerebral infarction.
2.2 vascular Density
As shown in fig. 2a and b, plasminogen (Plg-/-) knockout mice had significantly lower cerebral vascular density on the stroke- impaired side 7, 14 and 28 days after cerebral infarction compared to normal mice (Plg +/+) (n ═ 10/group;. p < 0.05; one-way anova).
The results indicate that plasminogen is beneficial for cerebrovascular regeneration after cerebral infarction.
2.3 relationship between cerebrovascular Density and recovery of post-cerebral-infarction motor function
As shown in fig. 3, there was a significant correlation between the cerebral vascular density of the periinfarct cortical areas of normal mice (Plg +/+) and plasminogen knockout (Plg-/-) mice 28 days after the modeling of cerebral infarction and the results of the staggered test (fig. 3a) and the decal removal test (fig. 3 b).
The results show that the higher the density of the blood vessels, the better the motor function.
2.4 measurement of cerebral vascular Capacity
As shown in FIG. 4, normal (Plg +/+) mouse primary brain capillary endothelial cells can aggregate into a distinct network structure, have distinct migration traces, and can form a hollow lumen (FIG. 4 a). Dispersion of capillary-like tubes formed by plasminogen knock-out (Plg-/-) mouse primary capillary endothelial cells. Although there were some migration traces and voids, no significant network formation occurred (FIG. 4 b).
The ratio of the tube length to the unit area was used as an index of the tube density, and the capillary formation lengths of the two mice were calculated, respectively. Compared to normal (Plg +/+) mice (FIG. 4c), the angiogenic capacity of plasminogen knockout (Plg-/-) deficient mice was significantly reduced (N6/group;. p < 0.05; one-way anova).
This result indicates that plasminogen acts on capillary endothelial cells, promoting the latter to form new capillaries.
2.5 results of detection of angiogenesis inhibitor
As shown in figure 5, prior to cerebral infarction, the plasminogen gene knockout (Plg-/-) mice had higher expression of TSP1/2 (. + -. P <0.001vs WT) compared to normal mice. After cerebral infarction, the expression of TSP1/2 in the control group and the cerebral infarction group is obviously higher than that in the control group before cerebral infarction. In addition, plasminogen gene knockout (Plg-/-) group TSP1/2 expression was increased, especially in ipsilateral brain tissue (n ═ 3/group;. P <0.05,. P <0.05vs WT; # P <0.01,. # # P <0.01,. P # P <0.001vs WT), compared to normal post-infarct (Plg +/+) mice.
The results mainly indicate that the presence of plasminogen inhibits the level of angiogenesis inhibitors, which in turn can promote angiogenesis.
The results of this example show that plasminogen can promote angiogenesis, help cerebral infarction patients to restore cerebral vascular density in the peri-infarct cortical region, and help to restore motor and perception abilities.
Claims (10)
1. Use of plasminogen for the manufacture of a medicament for promoting angiogenesis.
2. Use according to claim 1, characterized in that: the medicine is used for promoting the angiogenesis of the brain.
3. Use according to claim 1 or 2, characterized in that: the drug is a drug that inhibits the expression of TSP1 and TSP 2.
4. Use according to claim 1 or 2, characterized in that: the medicine is used for treating cerebral infarction.
5. Use according to claim 1 or 2, characterized in that: the medicine is used for accelerating the recovery of feeling and motor function after cerebral infarction.
6. A medicament for promoting angiogenesis, which is characterized in that: the drug is prepared by taking plasminogen as an active ingredient and adding pharmaceutically acceptable auxiliary ingredients.
7. The medicament of claim 6, wherein: the medicine is used for promoting the angiogenesis of the brain.
8. The medicament of claim 6 or 7, wherein: the drug is a drug that inhibits the expression of TSP1 and TSP 2.
9. The medicament of claim 6 or 7, wherein: the medicine is used for treating cerebral infarction.
10. The medicament of claim 6 or 7, wherein: the medicine is used for accelerating the recovery of feeling and motor function after cerebral infarction.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5520912A (en) * | 1993-07-02 | 1996-05-28 | Immuno Aktiengesellschaft | Prevention and treatment of ischemic events and reperfusion injury resulting therefrom using lys-plasminogen |
| CN1911438A (en) * | 2002-10-23 | 2007-02-14 | 井上一知 | Angiogenesis inducer |
| US20190247472A1 (en) * | 2015-12-18 | 2019-08-15 | Talengen International Limited | Method for prevention or treatment of acute and chronic thrombosis |
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2020
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5520912A (en) * | 1993-07-02 | 1996-05-28 | Immuno Aktiengesellschaft | Prevention and treatment of ischemic events and reperfusion injury resulting therefrom using lys-plasminogen |
| CN1911438A (en) * | 2002-10-23 | 2007-02-14 | 井上一知 | Angiogenesis inducer |
| US20190247472A1 (en) * | 2015-12-18 | 2019-08-15 | Talengen International Limited | Method for prevention or treatment of acute and chronic thrombosis |
Non-Patent Citations (2)
| Title |
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| JINGHUAN FANG，等: "Plasminogen deficiency causes reduced angiogenesis and behavioral recovery after stroke in mice" * |
| 朱云波;邱海鹏;李佳佳;胡亚军;马征;高燕军;: "超时间窗静脉溶栓对大鼠血栓性大脑中动脉栓塞的治疗效果及机制探讨" * |
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