JP2018145114A - Vascular endothelial disorder preventive and/or therapeutic agent - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a preventive agent for vascular endothelial disorder, which uses a stem cell conditioned medium, is novel and has high efficacy, and/or a regeneration agent for vascular endothelium.SOLUTION: A vascular endothelial disorder preventive and/or therapeutic agent contains a stem cell conditioned medium.SELECTED DRAWING: Figure 3

Description

  The present invention relates to a preventive and / or therapeutic agent for vascular endothelial injury, and particularly relates to a preventive and / or therapeutic agent for vascular endothelial injury using a stem cell culture supernatant.

  Various clinical findings in recent years have revealed that vascular endothelial dysfunction causes various medical conditions such as arteriosclerosis, plaque accumulation, and arterial occlusion and the heart attack it causes. Vascular endothelial dysfunction is a disease caused by the vascular endothelium itself, and is thought to be triggered by an inflammatory reaction to damaged vascular endothelial cells.

  However, conventional therapies such as those that repair the damage caused by the rupture of plaque accumulated in the blood vessels (stents, drugs, angioplasty, coronary artery bypass surgery) are difficult to complete and temporary Therefore, not only is there a problem that damaged vascular endothelial cells cannot be completely treated, but it also delays the onset of plaque rupture accumulated in other sites. Thus, recent clinical findings have revealed that vascular endothelial dysfunction is the cause of various medical conditions and that there are problems with conventional therapies. Methods and treatments are being proposed.

  For example, Patent Document 1 includes roots, trunks, branches, stems and leaves of Salacia plants such as Salacia oblonga, and the roots, trunks, branches, stems and leaves of Salacia plants. The vasospasm inhibitor is obtained by extracting the shredded product with hot water and then intravenously injecting the vasospasm inhibitor to the blood vessels such as cerebral infarction after the onset of subarachnoid hemorrhage. It is disclosed to effectively treat a disease caused by spasm.

  In the invention proposed in Patent Document 1, since it can be extracted from an edible plant that can be stably supplied, it is highly safe and hardly suppresses the normal contraction action of blood vessels. ) And is capable of providing a water-soluble vasospasm inhibitor that can be used not only for oral administration but also as an immediate-acting injection.

  Patent Document 2 contains, as an active ingredient, a hydrolyzate of animal milk casein or an concentrate thereof containing Ile Pro Pro and / or Val Pro Pro as Xaa Pro Pro as an active ingredient. An agent having at least one of the effects of improving diastolic function and suppressing intimal thickening is disclosed.

  The invention according to Patent Document 2 can improve the function related to the vascular endothelium, prevent various diseases related to the vascular endothelial function, and suppress the thickening of the vascular intima, and can be expected to have an effect such as prevention of arteriosclerosis. It is said that an agent having at least one action of improving the vascular endothelial function and suppressing the thickening of the vascular intima, and an arteriosclerosis preventive agent can be provided.

JP, 2006-56138, A Japanese Patent No. 5642346

  As described above, it has been found from recent clinical findings that vascular endothelial dysfunction is a cause of various pathologies, and a novel and highly effective agent for preventing and / or treating vascular endothelial disorders. Development and provision are desired.

  In view of the above problems, an object of the present invention is to provide a novel and highly effective prophylactic and / or therapeutic agent for vascular endothelial injury containing a stem cell culture supernatant.

  The preventive and / or therapeutic agent for vascular endothelial injury of the first invention of the present application is characterized by containing a stem cell culture supernatant.

  The preventive and / or therapeutic agent for vascular endothelial injury of the second invention of the present application is an injection in the first invention of the present application.

  The preventive and / or therapeutic agent for vascular endothelial injury of the third invention of the present application is characterized in that, in the first invention of the present application or the second application of the present application, the stem cell is a stem cell derived from a deciduous dental pulp.

  According to the first invention of the present invention to the third invention of the present application, since endothelial cells of blood vessels are regenerated by cytokines, chemokines, exosome proteins, etc. contained in the stem cell culture supernatant, novel and highly effective prevention of vascular endothelial injury And / or a therapeutic agent can be provided.

  In particular, according to the second invention of the present application, since the stem cell culture supernatant can be directly administered into the blood vessel by injection into the blood vessel, the effect of regenerating endothelial cells of the blood vessel can be further enhanced.

  In particular, according to the third invention of the present application, since stem cells are derived from deciduous dental pulp, the stem cell collection does not involve severe biological invasion unlike the case of using other stem cells such as bone marrow or adipose-derived stem cells. In addition, cell proliferation ability and proliferation speed are higher than other stem cells. Moreover, since it is thought that differentiation potential is also higher, it is thought that a higher therapeutic effect can be exhibited.

FIG. 3 is a structural diagram of a blood vessel wall (arteries and veins). The figure which shows the effect | action of a vascular endothelial cell. The figure explaining creation of SHED-CM.

<SHED-CM>
Regenerative medicine using various stem cells including embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells), and somatic stem cells has attracted attention. Among somatic stem cells, mesenchymal stem cells (MSC) isolated from various tissues such as bone marrow, adipose tissue, skin, umbilical cord, and placenta are particularly used, but bone marrow puncture is invasive and painful for donors It is a sex technique. Furthermore, the number, proliferation, and differentiation ability of bone marrow stem cells (BMSC) are disadvantageous such as decreasing with age.

  Therefore, in recent years, human waste stem cells from human exfoliated deciduous teeth (SHED) and permanent pulp stem cells (DPSC) derived from wisdom are self-replicating and multipotent. As a new stem cell population having These cells are neural crest-derived cell populations that have properties similar to those of the neural lineage, and are known to be highly responsive to the induction of differentiation into nerve cells. It is excellent in that it has no painfulness. However, cell transplantation using stem cells as described above has problems such as immune rejection, risk of canceration, storage stability, culture method, etc., and many problems are piled up for the introduction of full-scale treatment. It has become.

  By the way, the supernatant (culture supernatant) produced when culturing stem cells as described above contains several hundred or more kinds of protein components, including cytokines, chemokines, exosome proteins, etc. It contains abundant information-transmitting substances called cell activity called Epidermal Growth Factor (EGF), Fibroblast Growth Factor (FGF), Platelet-Derived Growth Factor (PDGF), Hepatocyto Growth Factor (HGF) It is known to contain growth factors such as Trans Transforming growth factor (TGF) and vascular endothelial growth factor (VEGF), exosomes and the like.

  In addition, culture supernatants containing cytokines, chemokines, exosome proteins, etc. have functions to repair damaged tissues and protect tissues, and ultimately regenerate organs with their own stem cells. Has been proven from extensive research. In addition, since the cells themselves are not contained in the culture supernatant, immune rejection does not occur. For this reason, even culture supernatants prepared from other people's stem cells can be used, and the application range is very high. In addition, if the culture supernatant prepared in advance is stored, it is very convenient because it can be used immediately when necessary. For this reason, utilization of the culture supernatant is expected as a next-generation treatment method in the field of regenerative medicine, which is thought to take time.

  Here, the culture supernatant is produced using stem cells such as pulp of deciduous teeth or permanent teeth, bone marrow, fat, umbilical cord, etc., but the contained components differ depending on the cell type. Among them, the culture supernatant (deciduous dental pulp stem cell culture supernatant (SHED-CM)) made from deciduous dental pulp stem cells contains many protein components, including more than 500 types of cytokines, chemokines, exosome proteins, etc. Is known to be included.

  In this embodiment, the term “stem cell culture supernatant” is a culture solution obtained by culturing stem cells and does not contain cell components (ie, cells or stem cells). Therefore, for example, a culture supernatant that can be used in the present invention can be obtained by separating and removing cell components after culturing. A culture supernatant appropriately subjected to various treatments (for example, centrifugation, concentration, solvent replacement, dialysis, freezing, drying, lyophilization, dilution, desalting, storage, etc.) may be used.

  In addition, dental pulp stem cells can be selected as adherent cells in dental pulp cells. Therefore, the culture supernatant obtained by culturing the adherent cells in the pulp cells collected from the fallen deciduous teeth or permanent teeth or the passage cells thereof can be used as “culture supernatant of pulp stem cells”.

  “Culture supernatant of dental pulp stem cells” is defined as a culture solution that is obtained by culturing dental pulp stem cells and does not contain cell components. The prophylactic and / or therapeutic agent for vascular endothelial injury of the present invention contains “dental stem cell culture supernatant” as an active ingredient, but does not contain cells (regardless of cell type). The prophylactic and / or therapeutic agent (composition) for vascular endothelial damage of this embodiment is clearly distinguished from various compositions containing dental pulp stem cells, of course, due to the characteristics. A typical example of this embodiment is a composition composed only of the culture supernatant of dental pulp stem cells.

  In this embodiment, deciduous dental pulp stem cells (SHED) are preferably used. This is because the proliferation ability and proliferation speed of cells are higher than those of permanent dental pulp stem cells (DPSC). In addition, since it is considered that the differentiation potential is higher, it is an advantage of using SHED that a higher therapeutic effect can be exhibited. In addition, SHED has the advantage of being easy to collect.

  The stem cell culture supernatant preferably does not contain serum. This is because the safety is increased by not containing serum. For example, a culture supernatant without serum can be prepared by culturing dental pulp stem cells in a medium without serum (serum-free medium). A serum-free culture supernatant can also be obtained by performing subculture once or a plurality of times and culturing the last or last several subcultures in a serum-free medium. On the other hand, serum-free culture supernatant can also be obtained from the collected culture supernatant by removing the serum using dialysis or solvent replacement using a column.

<Vessel structure>
FIG. 1 is a structural diagram of a blood vessel wall. In FIG. 1, (a) is a diagram showing a configuration of an artery, and (b) is a diagram showing a configuration of a vein. A blood vessel is a tube that serves as a passage for sending blood to various parts of the body, and not only supplies the oxygen and nutrients taken into the entire body (cells) but also transports waste products discharged from the body (cells). To play a role. The blood vessels consist of three types: arteries, veins, and capillaries.As shown in Fig. 1, the arteries and veins are intimate, smooth muscle and elastic, whose inner peripheral surface is in direct contact with blood and covered with vascular endothelial cells. It has a three-layered structure consisting of a fiber media and outer membrane. Capillaries (not shown) are composed only of the intima.

<Vascular endothelial cells>
Traditionally, vascular endothelial cells only serve as a barrier to prevent substances contained in blood and blood cells from directly contacting smooth muscle and causing tissue changes, or blocking the passage of certain chemical substances. There was not much research done. However, in recent studies, when vascular endothelial cells are exposed to various stimuli in addition to changes in blood pressure and blood flow, they produce and release powerful vasodilators or vasoconstrictors in response to them. As a result, it has been clarified that it has various functions such as regulation of vascular tone, prevention of intravascular thrombus formation, and prevention of arteriosclerosis.

FIG. 2 is a diagram showing the function (function) of vascular endothelial cells. The main dilating factors derived from vascular endothelial cells include nitric oxide (hereinafter referred to as NO), prostacyclin, hyperpolarizing factor and the like. When NO synthase acts on L-arginine, NO is released and enters smooth muscle. NO activates soluble guanylate cyclase (GC) in smooth muscle. GC acts on GTP and promotes cGMP production. cGMP activates G- kinase, G- kinase by functional protein phosphorus oxidation result of sarcoplasmic reticulum Ca ₂ ⁺ uptake into, Ca ₂ ⁺ in emissions to the extracellular is promoted. It also results in inactivation of myosin light chain kinase, resulting in smooth muscle dilation (relaxation).

Further, when cyclooxygenase acts on arachidonic acid, PGH ₂ and G ₂ are produced, and prostacyclin synthase acts on PGH ₂ and G ₂ to produce PGI ₂ . PGI ₂ activates adenylate cyclase and results in smooth muscle relaxation through cAMP production. Furthermore, EDHF derived from endothelial cells opens K ⁺ channels and hyperpolarizes vascular smooth muscle, resulting in smooth muscle expansion, ie smooth muscle relaxation.

<Diseases caused by vascular endothelial cell injury>
As described above, vascular endothelial cells produce and release strong vasodilators in response to changes in blood pressure, blood flow, etc., as well as when exposed to strong vasoconstrictors. Protects the muscles. It also plays an important role in preventing intravascular thrombus formation, inhibiting the blood clotting system and activating the fibrinolytic system. Therefore, when vascular endothelial cells are exposed to damage, these effects are lost, resulting in an increase in cardiovascular diseases such as arteriosclerosis, peripheral blood flow disorder, hypertension, coronary and cerebrovascular spasm, thrombus and blood coagulation promotion To do.

  In recent years, not only aging due to aging but also westernization of eating habits (increased intake of animal fat, etc.), the decline of endothelial cells and blood vessels has become younger, which is a rapid increase in so-called lifestyle-related diseases. It is also connected to. In this way, endothelial cell disorders lead to an increase in cardiovascular diseases such as arteriosclerosis, peripheral blood circulation disorders, hypertension, coronary and cerebrovascular spasms, thrombosis and blood coagulation, etc. It is extremely important.

  The inventors of the present invention focus on the fact that the deciduous dental pulp stem cell culture supernatant (SHED-CM) is particularly useful among the culture supernatants using stem cells such as pulp, bone marrow, fat, and umbilical cord of deciduous teeth and permanent teeth. Then, using this deciduous dental pulp stem cell culture supernatant (SHED-CM), a novel and highly effective vascular endothelial disorder preventive and / or therapeutic agent that controls vascular endothelial disorders by repairing and regenerating vascular endothelial cells It is to provide.

<Create SHED-CM>
Next, with reference to FIG. 3, a method for preparing a deciduous dental pulp stem cell culture supernatant (SHED-CM) will be described. In addition, the preparation method demonstrated below is an example and you may produce a deciduous dental pulp stem cell culture supernatant (SHED-CM) with another method.

  As described above, the method for producing the deciduous dental pulp stem cell culture supernatant (SHED-CM) is not particularly limited, but the following steps (1) to (3), that is, (1) selecting adherent cells from dental pulp cells The production method preferably includes a step, (2) a step of culturing the adherent cells, and (3) a step of recovering the culture supernatant. Hereinafter, each step will be described.

  In step (1), dental pulp stem cells that are adherent cells are selected from dental pulp cells. Hereinafter, the dental pulp cells may be prepared by, for example, isolating from the living body in advance. This selection step may include preparation of dental pulp cells. A specific example of a series of operation procedures from preparation of dental pulp cells to selection of dental pulp stem cells is shown.

(a) Extraction of dental pulp After naturally deciduous deciduous teeth (or extracted deciduous teeth or permanent teeth) are sterilized with chlorohexidine or isodine solution, the crown portion is divided and the dental pulp tissue is collected with a dental reamer.

(b) Enzyme treatment The collected dental pulp tissue is suspended in a basic medium (10% bovine serum / antibiotic-containing Dulbecco's modified Eagle medium) and treated with 2 mg / ml collagenase and dispase at 37 ° C. for 1 hour. The pulp cells after enzyme treatment are collected by centrifugation for 5 minutes (5000 rpm). In principle, cell sorting with a cell strainer is not used because it reduces the collection efficiency of neural stem cell fractions of SHED and DPSC.

(c) Selection of adherent cells Cells are resuspended in 4 cc basal medium and seeded on 6 cm diameter adherent cell culture dishes. After adding a culture solution (for example, DMEM (Dulbecco's Modified Eagle's Medium) containing 10% FCS), the cells are cultured for about 2 weeks in an incubator adjusted to 5% CO ₂ and 37 ° C. After removing the culture solution, the cells are washed once or several times with PBS or the like. Instead of this operation (removal of culture solution and washing of cells), adhesive cells (dental pulp stem cells) that formed colonies may be collected. In this case, for example, the cells are treated with 0.05% trypsin / EDTA for 5 minutes at 37 ° C., and the detached cells are collected.

In step (2) following step (1), the selected adherent cells are cultured. For example, the cells are seeded in an adherent cell culture dish and cultured in an incubator adjusted to 5% CO ₂ and 37 ° C. Subculture as necessary. For example, when observing with the naked eye, it reaches the sub-confluent state (a state in which the cells occupy about 70% of the surface of the culture container) or confluence, and the cells are detached from the culture container and collected, and again the culture filled with the culture solution Seed in containers. Subculturing may be repeated. For example, the subculture is performed 1 to 8 times to grow to the required number of cells (for example, about 1 × 10 7 cells / ml). The cell can be detached from the culture vessel by a conventional method such as trypsin treatment. After the above culture, the cells may be collected and stored (storage conditions are, for example, -198 ° C.). Cells collected from various donors may be stored in the form of dental pulp stem cell banks.

  The culture medium can be a basic medium or a basic medium to which serum or the like is added. However, in order to prepare a “dental stem cell culture supernatant” that does not contain serum, a serum-free medium may be used throughout the entire process or for the last or several subcultures from the end. In addition to DMEM, Iscov modified Dulbecco medium (IMDM) (GIBCO, etc.), Ham F12 medium (HamF12) (SIGMA, GIBCO, etc.), RPMI1640 medium, etc. can be used as the basic medium. Two or more basic media may be used in combination. As an example of the mixed medium, a medium in which IMDM and HamF12 are mixed in equal amounts (for example, commercially available as trade name: IMDM / HamF12 (GIBCO)) can be mentioned. Examples of ingredients that can be added to the medium include serum (fetal calf serum, human serum, sheep serum, etc.), serum substitutes (Knockout serum replacement (KSR), etc.), bovine serum albumin (BSA), antibiotics, various Vitamins and various minerals can be mentioned.

  In step (3) following step (2), the culture supernatant of dental pulp stem cells selected and cultured by the above method is collected. For example, the culture solution can be collected by suction with a dropper or pipette. The collected culture supernatant is used as it is or after one or more treatments as an active ingredient of the composition of the present invention. Examples of the treatment here include centrifugation, concentration, solvent replacement, dialysis, freezing, drying, lyophilization, dilution, desalting, and storage (eg, 4 ° C., −80 ° C.).

<Concentration method of stem cell culture supernatant>
The physiologically active substance contained in the stem cell culture supernatant can be formulated. As a method for concentrating the cell culture supernatant for formulation, a conventional method for this purpose can be applied. Examples of the concentration method include the following two methods.

<Spin column concentration method>
The culture supernatant is concentrated using Amicon Ultra Centrifugal Filter Units-10K (Millipore) (maximum 75-fold concentration). The specific operation procedure is as follows.
(I) The culture supernatant (up to 15 ml) is put into Amicon Ultra Centrifugal Filter Units-10K, centrifuged at × 4000 g for about 60 minutes, and concentrated to 200 μl.
(Ii) Put the same amount of sterile PBS as the culture supernatant into the above tube, and centrifuge again at x4000g for about 60 minutes to replace the base solution with PBS.
(Iii) Collect 200 μl of the obtained solution into a micro test tube and use it as a concentrated stem cell culture supernatant.

<Ethanol precipitation concentration method>
The culture supernatant is concentrated using an ethanol precipitation method (up to 10-fold concentration). The specific operation procedure is as follows.
(I) Add 45 ml of 100% ethanol to 5 ml of culture supernatant, mix and leave at -20 ° C for 60 minutes.
(Ii) Centrifuge for 15 minutes at 4 ° C and 15000g.
(Iii) Remove the supernatant, add 10 ml of 90% ethanol and stir well.
(Iv) Centrifuge for 5 minutes at 4 ° C. and 15000 g.
(V) The supernatant is removed, and the resulting pellet is dissolved in 500 μl of sterilized water, collected in a micro test tube, and used as a concentrated stem cell culture supernatant.

<Method of freeze-drying stem cell culture supernatant>
The stem cell culture supernatant in the composition of the present invention can also be lyophilized. Thereby, good storage stability is obtained. As a freeze-drying method of the stem cell culture supernatant, a method usually performed for this purpose can be applied. Examples of the lyophilization method include the following methods.
(I) The stem cell culture supernatant or concentrated stem cell culture supernatant obtained by the above method is frozen at −80 ° C. for 2 hours to half a day.
(Ii) After freezing, the lid of the sample tube is opened and set in a freeze dryer.
(Iii) Lyophilize for 1-2 days.
(Iv) The obtained sample is used as a lyophilized stem cell culture supernatant (can be stored at −80 ° C.).

<Create SHED-CM>
The inventors have administered human milk dental pulp stem cell culture supernatant (SHED-CM) to 8 patients suspected of having vascular endothelial disorder or vascular endothelial disorder, and the symptoms of vascular endothelial disorder are improved. Carried out. Specifically, the above-mentioned patient was administered with the supernatant of deciduous dental pulp stem cell culture (SHED-CM) concentrated 10-fold by the above ethanol precipitation concentration method, and LOX-index (registered trademark) (hereinafter referred to as registered trademark). It was verified whether the value of the description is omitted).

  First, the preparation method of the deciduous dental pulp stem cell culture supernatant (SHED-CM) in the examples will be described. Basically, the preparation method is the same as that already described in the embodiment, and (1) dental pulp cells (2) a step of culturing the adherent cell, and (3) a step of collecting and concentrating the culture supernatant. Hereinafter, each step will be described.

step 1)
(a) Collection of dental pulp After naturally deciduous deciduous teeth were sterilized with chlorohexidine or isodine solution, the crown portion was divided and the pulp tissue was collected with a dental reamer.

(b) Enzyme treatment The collected dental pulp tissue was suspended in a basic medium (10% bovine serum / antibiotic-containing Dulbecco's modified Eagle medium) and treated with 2 mg / ml collagenase and dispase at 37 ° C. for 1 hour. Thereafter, the pulp cells after the enzyme treatment were collected by centrifugation for 5 minutes (5000 rpm). Cell sorting with a cell strainer was not used because it reduced the efficiency of SHED and DPSC neural stem cell fraction collection.

(c) Selection of Adherent Cells Cells were resuspended in 4 cc basal medium and seeded on 6 cm diameter adherent cell culture dishes. After adding a culture solution (for example, DMEM (Dulbecco's Modified Eagle's Medium) containing 10% FCS), incubate in an incubator adjusted to 5% CO ₂ and 37 ° C for 2 weeks, remove the culture solution, and then with PBS etc. Cells were washed several times.

Step (2)
Next, the selected adherent cells were seeded in an adherent cell culture dish and cultured in an incubator adjusted to 5% CO ₂ and 37 ° C. At this time, subculture was performed as necessary. In subculture, the cells are detached from the culture vessel and collected when sub-confluent (the cell occupies about 70% of the surface of the culture vessel) or confluence is reached by observation with the naked eye. Seeded in filled culture vessels. The cells were detached from the culture vessel by a conventional method such as trypsin treatment.

Step (3)
The supernatant of dental pulp stem cells selected and cultured by the above method is collected. For example, after collecting and collecting the culture supernatant with a dropper or pipette, the culture supernatant was concentrated 10 times using the ethanol precipitation method. The specific operation procedure is as follows.
(I) 45 ml of 100% ethanol was added to 5 ml of the culture supernatant, mixed, and left at −20 ° C. for 60 minutes.
(Ii) Centrifugation was performed at 4 ° C. and x15000 g for 15 minutes.
(Iii) The supernatant was removed and 10 ml of 90% ethanol was added and stirred well.
(Iv) Centrifugation was performed at 4 ° C. and x15000 g for 5 minutes.
(V) The supernatant was removed, and the resulting pellet was dissolved in 500 μl of sterilized water and collected in a micro test tube to obtain a 10-fold concentrated stem cell culture supernatant.

<Measurement of LOX-index>
Whether the vascular endothelial disorder was improved was evaluated based on the value of LOX-index. LOX-index is a blood test that determines the risk of future cerebral infarction and myocardial infarction from the progress of arteriosclerosis. SLOX-1 (soluble LOX-1: LOX-1 released into the blood) and LAB ( This is an index for measuring and calculating LOX-1 ligand containing ApoB). From the LOX-index value, it is possible to grasp the sclerosis status and sclerosis risk of the blood vessel wall. In addition, if the value of LOX-index is high, the incidence of cerebral infarction / myocardial infarction increases. In other words, a lower LOX-index value is better.

  Tables 1 to 8 below show the clinical test results of 8 patients (patients A to H). The values of “sLOX-1”, “LAB”, and “LOX-index” shown in Tables 1 to 8 are calculated from the measured values of blood collected before each treatment. For example, the values of “sLOX-1”, “LAB”, and “LOX-index” described in the first column are calculated from the measured values of blood collected before the first treatment. The values of “sLOX-1”, “LAB”, and “LOX-index” described in the second column are calculated from the measured values of blood collected before the second operation.

  In this example, 10-fold concentrated deciduous dental pulp stem cell culture supernatant (SHED-CM) prepared by the method described above was administered to each patient by intravenous infusion. That is, in Tables 1 to 8 below, “2 ml infusion” described in the item of treatment is a 10-fold concentrated deciduous dental pulp stem cell culture supernatant (SHED-CM) prepared as described above. ) It means that 2ml was administered to the patient by infusion. The term “5 ml drip” means that 5 ml of 10-fold concentrated pulp dental cell culture supernatant (SHED-CM) prepared as described above was administered to a patient by drip infusion. Further, “10 ml infusion” means that 10 ml of 10-fold concentrated supernatant of cultured dental pulp stem cells (SHED-CM) prepared as described above was administered to a patient by infusion.

  Table 1 is the data which shows the result of patient A (male: 62 years old). As shown in Table 1, patient A was calculated from sLOX-1 value 302, LAB value 3.4, sLOX-1 and LAB values before the first treatment (August 27, 2016) The value of LOX-index was 1027, but the value of sLOX-1 was 108, the value of LAB was 3.1, the value of sLOX-1 and LAB before the fourth treatment (December 21, 2016) The value of LOX-index calculated from the value is 335, and the value of LOX-index is about 1/3 after 3 treatments.

  Table 2 shows data indicating the results of patient B (male: 36 years old). As shown in Table 2, patient B was calculated from sLOX-1 value 383, LAB value 6.6, sLOX-1 and LAB values before the first treatment (October 1, 2016) The LOX-index value was 2528, but before the second treatment (November 19, 2016), the sLOX-1 value was 157, the LAB value was 3.4, the sLOX-1 and LAB values The value of LOX-index calculated from the value is 534, and the value of LOX-index is about 1/5 after only one treatment and a short period (about one and a half months), and a significant improvement is seen.

  Table 3 is the data which shows the result of the patient C (male: 67 years old). As shown in Table 3, patient C was calculated from the sLOX-1 value of 1219, LAB value of 3.2, sLOX-1 and LAB values before the first treatment (November 16, 2016) The LOX-index value was 3901, but before the third treatment (December 21, 2016), the sLOX-1 value was 567, the LAB value was 3.8, the sLOX-1 and LAB values The value of LOX-index calculated from the value is 2155, and the value of LOX-index is about ½, showing improvement. In the case of patient C, there is a significant improvement especially in the high value of sLOX-1.

  Table 4 shows data indicating the results of patient D (male: 53 years old). As shown in Table 4, patient D was calculated from sLOX-1 value 435, LAB value 4.5, sLOX-1 and LAB values before the first treatment (November 16, 2016) The LOX-index value was 1958, but before the second treatment (January 11, 2017), the sLOX-1 value was 696, the LAB value was 2.5, the sLOX-1 and LAB values The value of LOX-index calculated from the value is 1740, and there is a slight improvement in the value of LOX-index, so it is expected that the value of LOX-index will further improve by continuing treatment as it is .

  Table 5 is the data which shows the result of the patient E (male: 63 years old). As shown in Table 5, patient E was calculated from sLOX-1 value 544, LAB value 2.7, sLOX-1 and LAB values before the first treatment (April 20, 2016) The LOX-index value was 1469, but before the 7th treatment (November 19, 2016), the sLOX-1 value was 482, the LAB value was 1.8, the sLOX-1 and LAB values The value of LOX-index calculated from the value is 868, and the value of LOX-index is about 1/2. In the case of patient E, the LOX-index value improves as a whole while repeating the vertical movement.

  Table 6 is the data which shows the result of patient F (male: 69 years old). As shown in Table 6, patient F was calculated from sLOX-1 value 318, LAB value 4.0, sLOX-1 and LAB values before the first treatment (April 20, 2016) The LOX-index value was 1272, but before the second treatment (May 14, 2016), the sLOX-1 value was 222, the LAB value was 3.3, the sLOX-1 and LAB values were The LOX-index value calculated from the value was improved to 733. However, before the third treatment (July 16, 2016), the sLOX-1 value was 253, the LAB value was 4.8, and the LOX-index value calculated from the sLOX-1 and LAB values was 1214. And the value of LOX-index became high. In the case of patient F, it is considered that continuous monitoring is necessary to see if the LOX-index value will continue to improve.

  Table 7 is the data which shows the result of the patient G (male: 59 years old). As shown in Table 7, patient G was calculated from sLOX-1 value 567, LAB value 2.7, sLOX-1 and LAB values before the first treatment (April 20, 2016) The value of LOX-index was 1531, but before the sixth treatment (October 29, 2016), the value of sLOX-1 was 277, the value of LAB was 2.9, the value of sLOX-1 and LAB The LOX-index value calculated from the value is 1099, and the LOX-index value is about 2/3, showing improvement.

  Table 8 shows data indicating the results of patient H (male: 76 years old). As shown in Table 8, patient H is calculated from sLOX-1 value 283, LAB value 2.2, sLOX-1 and LAB values before the first treatment (August 20, 2016) The value of LOX-index was 623, but before the third treatment (September 17, 2016), the value of sLOX-1 was 253, the value of LAB was 1.9, the value of sLOX-1 and LAB The value of LOX-index calculated from the value is 481, and the value of LOX-index is about 3/4, showing improvement. In the case of patient H, considering that the LOX-index value is originally low, a significant improvement is expected.

  As described above, as a result of administration of 10-fold concentrated supernatant of deciduous dental pulp stem cells (SHED-CM) to 8 patients suspected of having vascular endothelial disorder or vascular endothelial disorder, LOX was observed in almost all patients. The improvement of -index value was seen. This was injured by the action of cytokines, chemokines, exosome proteins, etc. (especially VEGF (vascular endothelial growth factor)) contained in the dental pulp stem cell culture supernatant (SHED-CM) administered into the blood vessels of patients ( It is presumed that good results as in this example were obtained by repairing and regenerating damaged vascular endothelial cells. In the case of patients who have not improved much, other factors, for example, psychological factors such as stress may be the main cause, and the value of LOX-index may be high.

  That is, according to the present invention, the stem cell culture supernatant obtained by culturing stem cells contains a mixture of cytokines, chemokines, exosome proteins, and the like, and when administered into blood vessels, vascular endothelial cells Proliferate cells in damaged areas. As a result, the endothelial cell tissue is repaired, and vascular endothelial damage can be prevented or treated. When a mixture of cytokines, chemokines, exosome proteins, etc. in the stem cell culture supernatant used in the present invention acts as an induction signal for the endogenous stem cells of the target tissue, the endogenous stem cells can be differentiated and proliferated. Can be inferred. As a result, it is presumed that cells can be proliferated at the damaged part of the vascular endothelial cell, an extracellular matrix is generated, and the like, and can be repaired based on the regenerative ability of the endogenous stem cell of the vascular endothelial cell tissue.

<Other embodiments>
Examples of somatic stem cells used in the present invention include, but are not limited to, stem cells derived from the dermal system, digestive system, myeloid system, nervous system, and the like. Examples of dermal somatic stem cells include epithelial stem cells, hair follicle stem cells, and the like. Examples of digestive somatic stem cells include pancreatic (general) stem cells, liver stem cells, and the like. Examples of myeloid somatic stem cells include hematopoietic stem cells, mesenchymal stem cells, and the like. Examples of somatic stem cells of the nervous system include neural stem cells and retinal stem cells. Somatic stem cells used in the present invention may be natural or genetically modified as long as the desired treatment can be achieved.

  The origin of stem cells is classified as ectoderm, endoderm, or mesoderm. Stem cells derived from ectoderm are mainly present in the brain and include neural stem cells. Stem cells derived from endoderm are mainly present in bone marrow, and include vascular stem cells, hematopoietic stem cells, mesenchymal stem cells and the like. Stem cells derived from mesoderm are mainly present in organs, and include liver stem cells, pancreatic stem cells and the like.

  In the present invention, somatic stem cells that can be derived from any mesenchymal system are preferably used, more preferably somatic stem cells derived from dental pulp, and most preferably somatic stem cells derived from human deciduous deciduous teeth. Mesenchymal somatic stem cells are vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), insulin-like growth factor (IGF), platelet-derived growth factor (PDGF), transforming growth factor-beta (TGF) -β) -1 and -3, various cytokines such as TGF-α, KGF, HBEGF, and SPARC can be produced. In the present invention, the stem cell culture supernatant preferably contains at least two cytokines, vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), insulin-like growth factor (IGF), platelet-derived growth factor (PDGF) And a combination of two or more selected from the group consisting of transforming growth factor-beta (TGF-β).

  The mixture of cytokine, chemokine, exosome protein, etc. used in the present invention can be used as a part of the stem cell culture supernatant or as a mixture of cytokine, chemokine, exosome protein, etc. isolated from the stem cell culture supernatant. In a mixture of cytokines, chemokines, exosome proteins, etc. isolated from stem cell culture supernatant, one or more known cytokines, chemokines, exosome proteins, etc. are partly mixed with one or more known corresponding cytokines, chemokines, exosome proteins, etc. It may be replaced.

  The stem cell culture supernatant used in the present invention is not limited to the stem cell culture supernatant obtained from the culture of somatic stem cells. Embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells), embryonic carcinoma cells Stem cell culture supernatant obtained from culture such as (EC cells) may be included.

  The culture supernatant of somatic stem cells is a culture solution obtained by culturing somatic stem cells and does not contain the cells themselves. Therefore, for example, a culture supernatant that can be used in the present invention can be obtained by separating and removing cell components after culturing. A culture supernatant appropriately subjected to various treatments (for example, centrifugation, concentration, solvent replacement, dialysis, freezing, drying, lyophilization, dilution, desalting, storage, etc.) may be used.

  Stem cells for obtaining a stem cell culture supernatant can be selected by a conventional method, and can be selected based on the size and form of the cells or as adherent cells. In the case of dental pulp stem cells, as described later, it can be selected as adherent cells or their passage cells from dental pulp cells collected from fallen deciduous teeth or permanent teeth. The method for producing a composition for treating a central nervous disease, which will be described later, can be preferably applied as a method for producing the composition for treating an injury site. As the dental pulp stem cell culture supernatant, a culture supernatant obtained by culturing selected stem cells can be used. When other stem cells are used, a stem cell culture supernatant can be obtained by similarly obtaining stem cells from a tissue that may contain the target stem cells.

  The “stem cell culture supernatant” is defined as a culture solution that does not contain cells themselves obtained by culturing stem cells. The composition of the present invention contains “cultured supernatant of stem cells” as an active ingredient, and in one embodiment thereof, the composition as a whole does not contain cells (regardless of cell type). The composition of this embodiment is clearly distinguished from various compositions containing a stem cell, of course, by itself due to this characteristic. A typical example of this embodiment is a composition that does not contain stem cells and is composed only of culture supernatants of stem cells.

  The culture medium for stem cells can be a basic medium or a basic medium supplemented with serum or the like. However, in order to prepare a “dental stem cell culture supernatant” that does not contain serum, a serum-free medium may be used throughout the entire process or for the last or several subcultures from the end. In addition to DMEM, Iscov modified Dulbecco medium (IMDM) (GIBCO, etc.), Ham F12 medium (HamF12) (SIGMA, GIBCO, etc.), RPMI1640 medium, etc. can be used as the basic medium. Two or more basic media may be used in combination. As an example of the mixed medium, a medium in which IMDM and HamF12 are mixed in equal amounts (for example, commercially available as trade name: IMDM / HamF12 (GIBCO)) can be mentioned. Examples of ingredients that can be added to the medium include serum (fetal calf serum, human serum, sheep serum, etc.), serum substitutes (Knockout serum replacement (KSR), etc.), bovine serum albumin (BSA), antibiotics, various Vitamins and various minerals can be mentioned.

  Usually used conditions can be applied as they are to the culture of stem cells. The method for producing the stem cell culture supernatant may be the same as the method for producing the composition for treating a central nervous disease described later, except that the isolation and selection steps of the stem cells are appropriately adjusted according to the type of the stem cells. Those skilled in the art can appropriately perform isolation and selection of stem cells according to the type of stem cells.

Depending on the condition of the subject to which it is applied, it does not preclude the additional use of other components in the composition of the present invention, provided that the expected therapeutic effect is maintained. An example of components that can be additionally used in the present invention is as follows.
(I) Bioabsorbable material As the organic bioabsorbable material, hyaluronic acid, collagen, fibrinogen (for example, Bolheel (registered trademark)) or the like can be used.

(Ii) Gelling material As the gelling material, a material having high biocompatibility is preferably used, and hyaluronic acid, collagen, fibrin glue, or the like can be used. Various types of hyaluronic acid and collagen can be selected and used, but it is preferable to employ one suitable for the application purpose (application tissue) of the composition of the present invention. The collagen used is preferably soluble (acid-soluble collagen, alkali-soluble collagen, enzyme-soluble collagen, etc.).

(Iii) Others Other pharmaceutically acceptable components (for example, carriers, excipients, disintegrants, buffers, emulsifiers, suspensions, soothing agents, stabilizers, preservatives, preservatives, physiological saline, etc.) ) Can also be contained. As the excipient, lactose, starch, sorbitol, D-mannitol, sucrose and the like can be used. As the disintegrant, starch, carboxymethylcellulose, calcium carbonate and the like can be used. Phosphate, citrate, acetate, etc. can be used as the buffer. As the emulsifier, gum arabic, sodium alginate, tragacanth and the like can be used. As the suspending agent, glyceryl monostearate, aluminum monostearate, methyl cellulose, carboxymethyl cellulose, hydroxymethyl cellulose, sodium lauryl sulfate and the like can be used. As the soothing agent, benzyl alcohol, chlorobutanol, sorbitol and the like can be used. As the stabilizer, propylene glycol, ascorbic acid or the like can be used. As preservatives, phenol, benzalkonium chloride, benzyl alcohol, chlorobutanol, methylparaben, and the like can be used. As preservatives, benzalkonium chloride, paraoxybenzoic acid, chlorobutanol, and the like can be used. Antibiotics, pH adjusting agents, growth (growth) factors (for example, endothelial cell growth factor (VEGF), epidermal growth factor (EGF), nerve growth factor (NGF)) and the like may be contained.

The administration route of the agent for preventing and / or treating vascular endothelial damage of the present invention is not particularly limited, but is preferably parenteral administration, and parenteral administration is preferably direct administration into blood vessels. Examples of such administration include
Examples thereof include intravenous administration and intraarterial administration. Examples of other administration methods include local injection to the target site, application or spraying, intraportal administration, intradermal administration, subcutaneous administration, intramuscular administration, intraperitoneal administration, and intranasal administration. Can do. As the administration schedule, for example, once to several times a week can be adopted. In preparing the administration schedule, the gender, age, weight, disease state, etc. of the subject (recipient) can be considered. Moreover, you may administer in combination with another chemical | medical agent. In this case, it is preferable to combine a drug having an action mechanism different from that of the stem cell culture supernatant of the present invention. This is because the mechanism of action is different, so there is little risk of side effects and the effect can be expected to increase.

Claims (3)

  A preventive and / or therapeutic agent for vascular endothelial injury, comprising a stem cell culture supernatant.   The preventive and / or therapeutic agent for vascular endothelial injury according to claim 1, which is an injection. The agent for preventing and / or treating vascular endothelial damage according to claim 1 or 2, wherein the stem cell contained in the stem cell culture supernatant is derived from a deciduous dental pulp.

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