Application of vascular adventitia stem cells in preparation of medicine for promoting thrombolysis

Abstract

The invention belongs to the technical field of thrombolysis drugs, and particularly relates to application of adventitia stem cells in preparation of drugs for promoting thrombolysis. The medicine prepared on the basis of the vascular adventitia stem cells can effectively dissolve thrombus (especially venous thrombus formed by vascular injury), thrombus dissolution is fast, and the technical difficulty is low. In addition, local medication can be achieved, and the risk of whole-body bleeding is avoided.

Description

Technical Field

[0001] The present invention belongs to the technical field of thrombolytic drugs, and particularly relates to the application of adventitial vascular stem cells in the preparation of drugs for promoting thrombolysis. Background Art

[0002] A thrombus is a solid substance formed by abnormal aggregation of blood components in blood vessels, which threatens human life and health. In particular, the pathological thrombus caused by vascular injury is the core pathological process of cardiovascular and cerebrovascular diseases. Such thrombi are likely to cause ischemic infarction (such as myocardial infarction) in the arterial system and embolism risk (such as pulmonary embolism) in the venous system, and the body's own fibrinolytic system often cannot remove abnormal thrombi in time.

[0003] Currently, the solutions for thrombolysis include drug thrombolysis and genetic engineering intervention, etc. However, the above solutions all have some defects, which limit their wide application in experiments and clinics. For example, the current drug thrombolysis (including fibrinolytic drugs (urokinase), anticoagulant drugs (low molecular weight heparin), and antiplatelet drugs (aspirin), etc.) belongs to the systemic use of thrombolytic drugs, and after use, it may cause systemic bleeding in the subjects. For example, excessive activation of the fibrinolytic system by fibrinolytic drugs and excessive inhibition of the coagulation process by anticoagulant drugs will both cause bleeding in the important organs of the subjects, and in severe cases, it will even endanger life; for another example, the technical requirements for the gene intervention technology to promote thrombolysis are high, and the gene editing operation is complex.

[0004] Therefore, it is necessary to develop a drug for promoting thrombolysis that is relatively safe and has lower technical requirements. Summary of the Invention

[0005] Based on this, the present invention has discovered and verified that the use of adventitial vascular stem cells in drugs for promoting thrombolysis can rapidly dissolve thrombi, and has lower technical requirements (for example, it is relatively easy to obtain adventitial vascular stem cells and the preparation method of the drug is simple).

[0006] In order to achieve the above object, the present invention can adopt the following technical solutions:

[0007] On the one hand, the present invention provides an application of adventitial vascular stem cells in the preparation of drugs for promoting thrombolysis.

[0008] Preferably, in the above application,

[0009] the thrombus is a thrombus formed after vascular injury; and / or

[0010] the adventitial vascular stem cells include adventitial vascular stem cells expressing Sca-1 antigen and / or adventitial vascular stem cells expressing CD34 antigen.

[0011] More preferably, the adventitial stem cells expressing Sca-1 antigen are derived from the thoracic / abdominal aorta.

[0012] Preferably, in the above application, the dosage forms of the drug include oral liquid, spray, tablet, powder, granule, capsule, patch, suppository, ointment or gel.

[0013] On the other hand, the present invention provides a gel product loaded with stem cells, which comprises adventitial stem cells and Pluronic F-127 gel.

[0014] Preferably, in the above gel product loaded with stem cells, the adventitial stem cells include stem cells expressing Sca-1 antigen and / or stem cells expressing CD34 antigen.

[0015] Preferably, in the above gel product loaded with stem cells, the concentration of adventitial stem cells in the gel is (1.8-2.2)×10 9 cells / L.

[0016] Preferably, in the above gel product loaded with stem cells, the preparation method of Pluronic F-127 gel includes: mixing Pluronic F-127 gel powder with cell culture medium to obtain Pluronic F-127 gel, and the mass-volume ratio of Pluronic F-127 gel powder in the cell culture medium is 20%-30%.

[0017] On the other hand, the present invention provides a drug for promoting thrombolysis, which comprises the gel product loaded with stem cells in the present invention.

[0018] On the other hand, the present invention provides the application of the gel product loaded with stem cells in the present invention in the preparation of a drug for promoting thrombolysis.

[0019] The beneficial effects of the present invention include:

[0020] (1) The drug prepared based on adventitial stem cells in the present invention can effectively dissolve the venous thrombus formed by vascular injury and achieve the treatment effect.

[0021] (2) The drug prepared based on adventitial stem cells in the present invention has better timeliness for thrombolysis than the existing gene knockout technology. It can be known through mouse verification that the thrombus begins to dissolve after 5 days of the action of the drug prepared from stem cells, while the thrombus of mice using the existing related gene knockdown / knockout technology begins to dissolve after 14 days.

[0022] (3) The adventitial stem cells in the present invention have a wide range of raw material sources, can be obtained by self-extraction, and a sufficient number of cells can be obtained through amplification technology; in addition, the drug preparation in the present invention is simple, and the overall solution is superior to the existing related gene knockdown technologies.

[0023] (4) In the present invention, adventitial stem cells are loaded on Pluronic F-127 hydrogel, and this hydrogel system can slowly release stem cells in 3-7 days, avoiding excessive proliferation of stem cells after vascular injury.

[0024] (5) Loading adventitial stem cells on Pluronic F-127 hydrogel in the present invention can achieve local drug administration, avoiding the risk of systemic bleeding. Description of the Drawings

[0025] Figure 1 Shows the situation of primary cells extracted;

[0026] Figure 2 Shows the identification situation of sorted Sca-1 + stem cells;

[0027] Figure 3 Shows the situation of vascular tissue and thrombus weight of mice in different groups;

[0028] Figure 4 Shows the pathological detection situation of mice in the model group after 7 days of feeding;

[0029] Figure 5 Shows the pathological detection situation of mice in the gene interference group after 7 days of feeding;

[0030] Figure 6 Shows the pathological detection situation of mice in the experimental group (stem cell transplantation group) after 7 days of feeding;

[0031] Figure 7 Shows the pathological detection situation of mice in the experimental group (stem cell transplantation group) after 5 days of feeding. Detailed Embodiments

[0032] The examples given are for better illustration of the present invention, but the content of the present invention is not limited only to the examples given. Therefore, those skilled in the art who make non-essential improvements and adjustments to the implementation solutions based on the above invention content still fall within the protection scope of the present invention.

[0033] The terms used in this document are only for describing specific embodiments and are not intended to limit the present disclosure. Unless there is an obviously different meaning in the context, singular expressions include plural expressions. As used herein, it should be understood that terms such as "including", "having", "containing" are intended to indicate the existence of features, numbers, operations, components, parts, elements, materials or combinations. Terms of the present invention are disclosed in the specification, and are not intended to exclude the possibility that one or more other features, numbers, operations, components, parts, elements, materials or combinations may exist or can be added. As used herein, depending on the circumstances, " / " can be interpreted as "and" or "or".

[0034] An embodiment of the present invention provides an application of adventitial stem cells in the preparation of a drug for promoting thrombolysis.

[0035] It should be noted that the thrombus in the present invention can be an arterial thrombus or a venous thrombus; in addition, it should be noted that adventitial stem cells can differentiate into cells such as vascular smooth muscle cells and fibroblasts to maintain the vascular wall structure, restore the normal function of blood vessels, and avoid secondary changes such as nutritional disorders and structural damage of the vascular wall caused by long-term blockage, so as to maintain the health state of blood vessels as a whole.

[0036] In some specific examples, in the above application, the thrombus is a thrombus formed after vascular injury.

[0037] It should be noted that drugs prepared based on stem cells are particularly suitable for thrombi formed after vascular injury. "Thrombi formed after vascular injury" refers to blood clots formed by the body through a series of physiological hemostasis mechanisms when the vascular wall is damaged.

[0038] In some specific examples, in the above application, adventitial stem cells include adventitial stem cells expressing Sca-1 antigen and / or adventitial stem cells expressing CD34 antigen.

[0039] It should be noted that adventitial stem cells expressing Sca-1 antigen can be called Sca-1+ stem cells, and adventitial stem cells expressing CD34 antigen can be called CD34+ stem cells; in addition, adventitial stem cells in the present invention can be adventitial stem cells that only contain one antigen expression, or adventitial stem cells that simultaneously contain multiple different antigen expressions. The present invention preferably uses Sca-1+ stem cells, and their thrombolytic effect is better than that of other stem cells.

[0040] In some specific examples, the above adventitial stem cells expressing Sca-1 antigen are derived from the thoracic / abdominal aorta.

[0041] It should be noted that the adventitial stem cells expressing Sca-1 antigen in the present invention are well-known in the art, such as the thoracic / abdominal aorta.

[0042] In some specific examples, in the above application, the dosage forms of the drug include oral liquid, spray, tablet, powder, granule, capsule, patch, suppository, ointment or gel.

[0043] It should be noted that the stem cells can be prepared into different dosage forms by combining with different excipients, such as oral liquid, spray, tablet, powder, granule, capsule, patch, suppository, ointment or gel, etc.; the above dosage forms can be comprehensively selected according to the tumor drug administration site and the patient's condition; in addition, the excipients of the above different dosage forms are well-known to those skilled in the art, and the preparation methods of drugs in different dosage forms are also well-known to those skilled in the art. For example, when preparing tablets, diluents (such as starch, dextrin, sucrose or mannitol, etc.), absorbents (calcium sulfate, calcium hydrogen phosphate or light magnesium oxide, etc.), binders (povidone, syrup or hydroxypropyl methylcellulose, etc.), wetting agents (water, etc.) or disintegrants (dry starch, sodium carboxymethyl starch or crospovidone, etc.) are mainly used; for example, when preparing oral liquid, solubilizers, suspending agents, emulsifiers or coloring agents are mainly used.

[0044] It should also be noted that in the present invention, it is preferably prepared into a gel or a patch, which can better achieve local drug administration, is more convenient for local drug administration, and has a better effect.

[0045] The embodiment of the present invention provides a gel product loaded with stem cells, which includes adventitial stem cells and Pluronic F-127 gel.

[0046] It should be noted that the adventitial stem cells and Pluronic F-127 gel can be prepared into a gel product, and the prepared gel product can slowly release adventitial stem cells for 3-7 days, avoiding the excessive proliferation of adventitial stem cells after vascular injury. In addition, adventitial stem cells can differentiate into cells such as vascular smooth muscle cells and fibroblasts, which are used to maintain the vascular wall structure, restore the normal function of blood vessels, avoid secondary changes such as vascular wall nutritional disorders and structural damage caused by long-term blockage, and overall maintain the health state of blood vessels.

[0047] In some specific examples, in the above gel product loaded with stem cells, the adventitial stem cells include adventitial stem cells expressing Sca-1 antigen and / or adventitial stem cells expressing CD34 antigen.

[0048] It should be noted that the adventitial stem cells in the present invention can preferably be adventitial stem cells expressing Sca-1 antigen and / or adventitial stem cells expressing CD34 antigen, and more preferably adventitial stem cells expressing Sca-1 antigen, and their therapeutic effects are superior to those of stem cells expressing other antigens.

[0049] In some specific examples, in the above-mentioned gel product loaded with stem cells, the concentration of adventitial stem cells in the gel is (1.8 - 2.2)×10 9 cells / L, such as 1.9×10 9 cells / L, 2×10 9 cells / L or 2.1×10 9 cells / L.

[0050] In some specific examples, in the above-mentioned gel product loaded with stem cells, the preparation method of Pluronic F-127 gel includes: mixing Pluronic F-127 gel powder with cell culture medium to obtain Pluronic F-127 gel, and the mass-volume ratio (w / v%) of Pluronic F-127 gel powder in the cell culture medium is 20% - 30%.

[0051] It should be noted that the above-mentioned "mass-volume ratio" refers to the ratio of the mass of Pluronic F-127 gel powder to the volume of cell culture medium. For example, a mass-volume ratio of 25% means "if the Pluronic F-127 gel powder is 1 g, the cell culture medium is 4 mL", and so on.

[0052] It should be noted that the preparation method of Pluronic F-127 gel is well-known in the art, such as the above-mentioned preparation method; in addition, in the above-mentioned preparation method, the cell culture medium is well-known in the art, such as DMEM culture medium, RPMI1640 culture medium or MEM culture medium; in addition, various additives can be added to the culture medium, such as fetal bovine serum, penicillin / streptomycin double antibody, leukemia inhibitory factor or 2-mercaptoethanol.

[0053] The embodiment of the present invention also provides a drug for promoting thrombolysis, which includes the gel product loaded with stem cells in the present invention.

[0054] It should be noted that the gel product loaded with stem cells in the present invention can not only be directly used as a drug itself, but also can be used to prepare drugs by adding other excipients, and other excipients are well-known in the art, such as humectants, preservatives, antioxidants, transdermal promoters or pH regulators, etc.

[0055] An embodiment of the present invention also provides an application of the gel product loaded with stem cells in the present invention in the preparation of a drug for promoting thrombolysis.

[0056] To better understand the present invention, the content of the present invention will be further clarified below in conjunction with specific examples, but the content of the present invention is not limited to the following examples only.

[0057] In the following examples, the unit "M" refers to the molar concentration unit "mol / L".

[0058] I. Obtaining Sca-1+ stem cells

[0059] In the following examples, the complete culture medium is 20% FBS + 1% triple antibiotics + 10 ng / mL lif + 0.1 mM β-mercaptoethanol

[0060] + 79% DMEM; 0.2% Triton-X-100 is 20 uL Triton-X-100 + PBS made up to 10 mL; in addition, the cell slides are soaked in absolute ethanol for 30 min, washed with PBS, air-dried in a laminar flow hood and then placed in a 12-well plate.

[0061] (1) Obtain the thoracic aorta segment and abdominal aorta segment of a mouse, and mechanically grind (high-throughput cryogenic tissue grinder SCIENTZ-48L,

[0062] grinding includes: at -20 °C, 65 Hz, grinding for 10 s and pausing for 10 s for a total of 15 cycles) into tissues of about 1 mm * 1 mm * 1 mm in size, and culturing in a cell incubator at 37 °C, 5% CO 2 for 7 days, changing the medium every 2 days (Gibco DMEM medium (C11995500BT) containing 15% FBS + 1% amphoteric antibiotics + 10 ng / mL LIF and 0.1 mM 2-mercaptoethanol) (changing the medium on the 3rd, 5th and 7th days) to obtain primary cells (as Figure 1 shown).

[0063] (2) After flow sorting of Sca-1+ stem cells, continue to amplify and culture; specifically including:

[0064] 1) When the cell density is above 90%, discard the original medium, digest with 2 mL of 0.25% trypsin for 2 min, and add 4 mL of FBS-containing medium to terminate digestion;

[0065] 2) After collecting the cells, centrifuge at 500 xg for 5 min and discard the supernatant;

[0066] 3) Then add PBS + 2% FBS to resuspend the cells, centrifuge, and repeat 2 times;

[0067] 4) Then add 5 μL FITC-Sca-1 antibody, dilute it with 100 μL PBS + 2% FBS, resuspend the cells, and let them stand on ice for 30 min;

[0068] 5) After standing, centrifuge at 500 x g for 5 min, and discard the supernatant;

[0069] 6) Resuspend the cells with PBS + 2% FBS, centrifuge, and repeat 2 times; Resuspend the cells with 2 mL PBS + 2% FBS in a sterile capped flow cytometry tube and sort them on the machine;

[0070] 7) Add 1 mL of complete medium to the cell collection tube, centrifuge, and repeat twice; Re-plate according to the number of cells; Change the medium the next day.

[0071] (3) Amplification and culture of Sca-1 + The stem cells are identified, specifically including:

[0072] 1) After flow cytometry sorting, when the density is above 90%, discard the old medium and wash the cells 3 times with PBS;

[0073] 2) Digest with 2 mL of trypsin for 2 min, and immediately add 4 mL of serum-containing medium to terminate the digestion;

[0074] 3) Centrifuge at 1000 rpm for 3 min, discard the supernatant, add 1 mL of complete medium to resuspend the cells, and passage them at a ratio of 1:3;

[0075] 4) Re-plate in a 12-well plate at about 50%, shake well, and add 1 mL of complete culture medium;

[0076] 5) After 24 h, discard the old medium, wash with PBS, add 1 mL of 4% paraformaldehyde to each well for 10 min at RT, and wash with PBS; Add 0.2% Triton-X-100 for 10 min at RT, and wash with PBS;

[0077] 6) Block with 3% BSA for 30 min;

[0078] 7) Add 50 μL of Sca-1 antibody (5 μg / mL), incubate overnight at 4°C, recover the primary antibody, and wash with PBS;

[0079] 8) Add the secondary antibody (1:500), incubate for 1 h at RT, and wash with PBS;

[0080] 9) Stain the nuclei with DAPI for 1 min at RT, and wash with PBS;

[0081] 10) Mount the slides with an anti-fluorescence quencher and store at 4°C for 1 week.

[0082] The identification results are as Figure 2 shown, and the results show that a large amount of Sca-1 is contained +Stem cells (the blue ones are Sca-1 + stem cells).

[0083] II. Preparation of F-127 hydrogel loaded with Sca-1 + stem cells

[0084] The amplified Sca-1 + stem cells were loaded into 25% Pluronic F-127 hydrogel to prepare F-127 hydrogel loaded with Sca-1 + stem cells, specifically including:

[0085] (1) 1 g of Pluronic F-127 (powder) was added to 4 mL of DMEM (Gibco) pre-cooled at 4°C to prepare a 25% (weight / volume fraction w / v%) mixed solution. After stirring evenly on a magnetic stirrer (1000 rpm, 30 min) under ice bath conditions, it was placed in a refrigerator at 4°C for 12 hours to fully mix the hydrogel to obtain a mixture;

[0086] (2) The mixture was filtered through a 0.22 μm filter to obtain Pluronic F-127 hydrogel, which was stored at 4°C;

[0087] (3) Pluronic F-127 hydrogel was resuspended with CM-Dil-labeled Sca-1+ stem cells, and the concentration of Sca-1+ stem cells was adjusted to 1×10 9 L -1 to obtain F-127 hydrogel loaded with Sca-1 + stem cells.

[0088] III. Thrombolysis promotion test

[0089] (I) Construction of vascular injury model

[0090] C57BL / 6J mice, 6 - 8 weeks old, male, were adaptively raised (in a SPF environment, free access to food) for 1 week and fasted for 8 hours before surgery; weighed, anesthetized by intraperitoneal injection of tribromoethanol (200 μL / 10 g), laparotomized under a stereomicroscope, and the infrarenal vena cava was fully exposed to the bifurcation of the common iliac vein; the vena cava and abdominal aorta were separated 1 mm below the infrarenal vena cava using micro forceps, and the vena cava was ligated with a 7-0 suture, and at the same time, the collateral veins on both sides were ligated (causing vascular injury). The abdominal organs were replaced, and the incision was sutured intermittently with a 5-0 suture; after the mice naturally woke up on a rewarming pad, they were normally raised for 7 days to obtain mice with a vascular injury model.

[0091] (II) Experimental design

[0092] The F-127 hydrogel loaded with Sca-1 +The F-127 hydrogel of stem cells was transplanted onto the adventitia of the injured blood vessels in mice with a blood vessel injury model, 0.2 mL per mouse, and a total of 5 mice were used as the experimental group;

[0093] In addition, a control group was set up, with a total of 5 mice: The mice were weighed, anesthetized by intraperitoneal injection of tribromoethanol (200 μL / 10 g), the abdomen was opened under a stereomicroscope, and the inferior vena cava was fully exposed to the bifurcation of the common iliac veins. The inferior vena cava and the abdominal aorta were separated 1 mm below the inferior vena cava using micro forceps without ligation; the abdominal organs were replaced, and interrupted sutures were made using 5-0 suture thread; after natural awakening on a rewarming pad, the mice were normally raised for 7 days;

[0094] In addition, mice with a blood vessel injury model were used as the model group, with a total of 5 mice;

[0095] In addition, a group of mice with gene interference (lentiviral gene interference of TSP1 gene, commissioned by Nanjing Core Bioscience and Technology Co., Ltd.) was also set up, with a total of 5 mice;

[0096] After raising the mice in the above groups for 5 days and 7 days, the thrombolysis situation was observed. The results showed that:

[0097] After 5 days of medication in the experimental group mice, the thrombus in the mice had begun to dissolve.

[0098] In addition, after raising the control group, model group, experimental group, and gene interference group mice for 7 days, the thrombus-containing venous segments of the inferior vena cava of each group of mice (from below the renal vein level to the bifurcation of the internal and external iliac veins at the bifurcation of the common iliac veins) were obtained for weighing (n = 7), and their weights (vascular tissue and thrombus weights) were analyzed. The results were as Figure 3 shown (in the figure, 7D is the model group). The results showed that the weight of the stem cell transplantation group (7D + Sca-1) was significantly reduced (p < 0.001). Compared with the gene interference group (7D + gene interference), although there was no statistical difference, there was still a difference in the mean value.

[0099] In addition, after raising the model group (stem cell transplantation group), gene interference group, and experimental group mice for 7 days, pathological examinations were respectively performed on the blood vessel lumens of each group of mice. The results were as Figure 4 , Figure 5 and Figure 6 shown. The results showed that the thrombus in the stem cell transplantation group was dissolved, the blood vessel wall was significantly thinned, and restenosis was not likely to occur, which was beneficial to blood vessel recanalization and beneficial to the pathological process after thrombus formation.

[0100] In addition, pathological examination was performed on the blood vessel lumen of the model group (stem cell transplantation group) mice after 5 days of raising. The results were as Figure 7 shown. The results showed that in the model group (stem cell transplantation group) mice on the 5th day, the thrombus began to dissolve.

[0101] In addition, thrombolysis in the gene interference group did not start until 14 days later.

[0102] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the spirit and scope of the technical solutions of the present invention, and they should all be covered by the scope of the claims of the present invention.

Claims

1. Application of vascular adventitial stem cells in the preparation of drugs for promoting thrombus dissolution.

2. The use according to claim 1, characterized in that: Thrombosis is a blood clot that forms after damage to a blood vessel; and / or The vascular adventitial stem cells include vascular adventitial stem cells expressing Sca-1 antigen and / or vascular adventitial stem cells expressing CD34 antigen.

3. The use according to claim 2, characterized in that: Vascular adventitial stem cells expressing Sca-1 antigen are derived from the thoracic / abdominal aorta.

4. The use according to any one of claims 1 to 3, characterized in that: The dosage forms of the drug include oral solution, spray, tablet, powder, granule, capsule, patch, suppository, ointment or gel.

5. A gel product loaded with stem cells, characterized in that: Includes vascular adventitial stem cells and Pluronic F-127 gel.

6. The gel product according to claim 5, characterized in that: The vascular adventitial stem cells include stem cells expressing the Sca-1 antigen and / or stem cells expressing the CD34 antigen.

7. The gel product according to claim 5 or 6, characterized in that: The concentration of adventitial stem cells in the gel is (1.8-2.2)×10 9 cells / L.

8. The gel product according to claim 5 or 6, characterized in that: The preparation method of Pluronic F-127 gel comprises: mixing Pluronic F-127 gel powder with cell culture medium to obtain Pluronic F-127 gel, wherein the mass volume proportion of Pluronic F-127 gel powder in the cell culture medium is 20%-30%.

9. A drug for promoting thrombolysis, characterized in that: A gel preparation loaded with stem cells comprising any one of claims 5 to 8.

10. Use of the gel preparation loaded with stem cells according to any one of claims 5 to 8 in the preparation of a drug for promoting thrombolysis.

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