JP2011015609A - Method for induction of proliferation/differentiation of endothelial progenitor cell (epc) - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an endothelial progenitor cell (EPC) proliferation/differentiation method which has higher proliferation efficiency of an EPC and higher differentiation efficiency of an EPC into an endothelial cell, which is safer, and which can be effected at a lower cost.SOLUTION: The proliferation efficiency of an endothelial progenitor cell (EPC) and the differentiation efficiency of an EPC into an endothelial cell can be improved by culturing the EPC in vitro in the presence of prostacyclin or a prostacyclin analogue and heparin.

Description

  The present invention relates to a method for inducing proliferation / differentiation of endothelial progenitor cells (EPC), a prophylactic / therapeutic agent for ischemic diseases, and an EPC proliferation promoter / differentiation inducing agent.

  Endothelial progenitor cells (EPC) are cells that act on angiogenesis or repair of damaged blood vessels, and are known to play an important role in the pathogenesis of organ ischemic disease, organ regeneration, and the like (Non-patent Document 1). In addition, in an ischemic disease refractory to existing treatment methods, it has been shown that introduction of self-derived EPC into the body has a certain therapeutic effect without causing rejection (Non-patent Document 2). . However, the therapeutic effect is still not fully satisfactory. The main reasons are (1) that the amount of EPC cannot be sufficiently secured, and (2) that EPC with high differentiation efficiency into vascular endothelium cannot be obtained.

  Regarding the above point (1), an attempt has been made to secure a sufficient amount of EPC by introducing a telomerase gene into EPC in order to increase the number of EPC's limited cell division (Non-patent Document 3). There remains a problem of safety against EPC phenotypic changes (particularly canceration) due to gene transfer. On the other hand, regarding the above point (2), various growth factors (VEGF, HGF, etc.) have been clarified so far as substances that promote the proliferation and differentiation of EPC, and the genes of these growth factors have been identified as organs or EPCs. Attempts have been made to increase the differentiation efficiency of EPC by introducing it into itself (Non-Patent Document 4). However, these growth factor introduction methods cannot be said to be sufficiently effective, and technical problems such as the efficiency of gene introduction of the growth factor and safety issues of the gene introduction itself are pointed out. Has been. Under the circumstances as described above, there has been a strong demand for an efficient EPC proliferation method and a method for producing EPC with high efficiency of differentiation into vascular endothelium.

  On the other hand, prostacyclin is a kind of prostaglandins and is known as a local hormone produced mainly in vascular endothelium in vivo. Since prostacyclin has physiological activities such as vasodilatory action, blood pressure lowering action, and platelet aggregation inhibitory action, prostacyclin and its analogs are obstructive arteriosclerosis, pulmonary hypertension, ischemic heart disease, diabetic It is used for the treatment of macrovascular disorders, diabetic nephropathy, diabetic retinopathy and the like (for example, Patent Document 1).

  Heparin (heparinic acid) is a highly sulfated dextrorotary mucopolysaccharide having specific anticoagulant properties, and is widely used to prevent thrombosis after surgery. This substance is present in many mammalian species as a natural component of various tissues, particularly the liver and lungs, and products isolated from various sources can be isolated in free or pharmaceutically acceptable salt forms, such as various Are commercially available in the form of alkali metal salts or alkaline earth metal salts.

  However, it has never been known that the combination of prostacyclin and heparin can improve the proliferation efficiency of EPC and the differentiation efficiency from EPC to vascular endothelium.

Special table 2007-519604 gazette Angiogenesis and vasculogenesis as therapeutic strategies for postnatal neovascularization. J. Clin. Invest. 103; 1231-1236, 1999. Therapeutic stem and progenitor cell transplantation for organ vascularization and regeneration.Nature Med. 9: 702-712, 2003. Constitutive human telomerase reverse transcriptase expression enhances regenerative properties of endothelial progenitor cells. Circulation, 106: 1133-1139, 2002 Endothelial progenitor cell vascular endothelial growth factor gene transfer for vascular regeneration. Circulation, 105: 732-738, 2002

  An object of the present invention is to provide a method for inducing proliferation / differentiation of EPC that has higher proliferation efficiency of endothelial progenitor cells (EPC) and differentiation efficiency into endothelial cells, and is safer and less costly.

  As described in the background art above, the conventional method has practical problems in terms of safety and cost, including EPC proliferation efficiency and differentiation efficiency into endothelial cells. Therefore, as a result of intensive studies, the inventors of the present application have used prostacyclin (or prostacyclin analog), which is not known at all in relation to the proliferation efficiency of EPC and the efficiency of differentiation into endothelial cells, and heparin in combination. It has been found that the above problems can be solved, and the present invention has been completed.

  That is, the present invention includes (1) a method of proliferating EPC, characterized in that it comprises a step of in vitro culturing endothelial progenitor cells (EPC) in the presence of prostacyclin or a prostacyclin analog and heparin; The prostacyclin analog is one or more substances selected from Carbaprostacyclin, Beraprost, Taprostene, Nileprost, Iloprost, Cicaprost, Ciprostene, Treprostinil, Bonsentan, and the proliferation of EPC as described in (1) above Regarding the method.

  The present invention also includes (3) a method for inducing differentiation of EPC, comprising a step of in vitro culturing endothelial progenitor cells (EPC) in the presence of prostacyclin or a prostacyclin analog and heparin; ) The EPC of (3) above, wherein the prostacyclin analog is one or more substances selected from Carbaprostacyclin, Beraprost, Taprostene, Nileprost, Iloprost, Cicaprost, Ciprostene, Treprostinil, Bonsentan The present invention relates to a differentiation induction method.

  Furthermore, the present invention comprises (5) prostacyclin or prostacyclin analog, and EPC obtained by in vitro culture of endothelial progenitor cells (EPC) in the presence of heparin, preventing ischemic disease -The therapeutic agent or (6) the prostacyclin analog is one or more substances selected from Carbaprostacyclin, Beraprost, Taprostene, Nileprost, Iloprost, Cicaprost, Ciprostene, Treprostinil, Bonsentan It relates to a preventive / therapeutic agent for ischemic disease according to 5).

  Furthermore, the present invention provides (7) a prostacyclin or prostacyclin analog and an EPC growth promoter characterized by containing heparin, and (8) a prostacyclin analog comprising Carbaprostacyclin, Beraprost, Taprostene, Nileprost, The EPC proliferation promoter according to (7) above, which is one or more substances selected from Iloprost, Cicaprost, Ciprostene, Treprostinil and Bonsentan.

  The present invention also relates to (9) EPC differentiation inducer characterized by containing prostacyclin or a prostacyclin analog and heparin, and (10) a prostacyclin analog comprising Carbaprostacyclin, Beraprost, Taprostene, Nileprost, Iloprost The EPC differentiation inducer according to (9) above, which is one or more substances selected from Cicaprost, Ciprostene, Treprostinil and Bonsentan.

  According to the method for proliferating endothelial progenitor cells (EPC) of the present invention, EPC can be proliferated more safely and remarkably efficiently. In addition, EPC obtained by the proliferation method of the present invention is considered to have a high degree of differentiation and a markedly high differentiation efficiency into endothelial cells. Furthermore, according to the EPC proliferation / differentiation induction method of the present invention, a sufficient amount and quality (e.g., differentiation efficiency into endothelial cells) of EPC can be proliferated / differentiated from a small amount of EPC. For example, regenerative treatment that is more effective and less burdensome on the patient becomes possible.

  The endothelial progenitor cell (EPC) proliferation method and differentiation induction method of the present invention (hereinafter also referred to as “proliferation / differentiation induction method of the present invention”) are referred to as prostacyclin or prostacyclin analog (hereinafter referred to as “prostacyclin etc.”). As long as it has a step of culturing endothelial progenitor cells (EPC) in vitro in the presence of heparin. Here, prostacyclin and the like, and the presence of heparin mean adding a prostacyclin or a composition containing prostacyclin and a heparin or heparin-containing composition to a medium used for in vitro culture. To do. The concentration of prostacyclin and the like in the medium is not particularly limited, but is preferably 0.1 to 100 nM, more preferably 1 to 10 nM, and the concentration of heparin in the medium is not particularly limited, Preferably it is 0.1-100 U / ml, More preferably, it can be set to 1-10 U / ml.

  The origin of the above-mentioned endothelial progenitor cells (EPC) is not particularly limited, such as mouse, rat, dog, pig, monkey, human, etc., but humans can be preferably exemplified, and in the case of human EPC, even if it is derived from a donor Although it is good, it is particularly preferable that it is self-derived because there is no problem such as rejection. These EPCs can be isolated from bone marrow and peripheral blood of the animal from which they originated by conventional methods. The method for isolating EPC is not particularly limited. For example, concentration gradient centrifugation is performed on bone marrow and peripheral blood to obtain mononuclear cells (MNC), and then using a magnetic sorting system. EPCs can be isolated from mononuclear cells. Although it does not restrict | limit especially as EPC, Lineage negative and cKit / Sca1 positive EPC can be illustrated suitably.

  The prostacyclin may be natural or synthesized. The prostacyclin analog includes (1) the property of interacting with the prostacyclin receptor, and (2) the activity of promoting the proliferation and differentiation of EPC when EPC is cultured in a medium containing heparin. In addition to pharmaceutically acceptable salts or derivatives of prostacyclin, Carbaprostacyclin, Beraprost, Taprostene, Nileprost, Iloprost, Cicaprost, Ciprostene, Treprostinil, Bonsentan (respectively JP 2005-120069) No.), ONO 54918 (15-cis- (4-n-propylcyclohexyl) -16,17,18,19,20-pentanor-9-deoxy-6,9-alpha-nitriloprostaglandin F1 (Brain Research Bulletin 60, 2003, 275 -281); manufactured by Ono Pharmaceutical Co., Ltd.) and the like, and pharmaceutically acceptable salts or derivatives thereof can be preferably exemplified. In the proliferation / differentiation induction method of the present invention, only one type may be used as prostacyclin or the like, or two or more types may be used in combination. The activity of promoting the proliferation and differentiation of EPC can be easily confirmed by the assay described in the examples described later.

  The heparin may be natural or synthesized, but when EPC is cultured in a medium containing prostacyclin or the like, EPC proliferation and differentiation As long as it has the activity to promote the above, pharmaceutically acceptable salts or derivatives of heparin are also included in the heparin in the present invention for convenience.

The method for in vitro culture in the method for inducing proliferation / differentiation of the present invention is not particularly limited as long as endothelial progenitor cells (EPC) are cultured in vitro in the presence of prostacyclin or the like and heparin, but on a plastic plate coated with fibronectin. A method of culturing EPC under conditions of 37 ° C. and 5% CO _{2 in} the presence of prostacyclin or the like and heparin, and replacing with a fresh medium every 3 to 4 days can be preferably exemplified. The medium for the in vitro culture is not particularly limited as long as it is a medium containing prostacyclin and the like and heparin and containing an appropriate carbon source and nitrogen source, but an α-MEM (manufactured by GIBCO) medium [ 10% by weight fetal bovine serum, 100 μg / ml endothelial cell growth factor (Sigma), 100 ng / ml mouse recombinant VEGF (Sigma), 10 U / ml heparin sulfate (Sigma), 100 U / ml penicillin, It contains 100 g / ml streptomycin and contains a prostacyclin receptor stimulant (ONO54918; manufactured by Ono Pharmaceutical Co., Ltd.) at a predetermined concentration].

  Examples of the prophylactic / therapeutic agent for ischemic disease of the present invention (hereinafter, also simply referred to as “prophylactic / therapeutic agent of the present invention”) include endothelial progenitor cells in the presence of prostacyclin or prostacyclin analog and heparin ( It is characterized by containing EPC obtained by in vitro culture of EPC (hereinafter also referred to as “EPC in the present invention”). The EPC in the present invention is an EPC obtained by the method for inducing proliferation / differentiation of the present invention, and has a higher degree of differentiation compared to the EPC obtained by the conventional method. Can be used. When the prophylactic / therapeutic agent of the present invention is administered into a patient's blood vessel, the EPC in the present invention differentiates into endothelial cells at the site of vascular injury or ischemia in the body, and promotes vascular repair and angiogenesis, thereby causing ischemic disease. Can be prevented and treated.

  The disease to be prevented or treated by the preventive / therapeutic agent of the present invention is not particularly limited as long as it is a disease that can be prevented or treated by administration of EPC, but it is obstructive arteriosclerosis, pulmonary hypertension, ischemic heart disease, Preferred examples include ischemic diseases such as diabetic macrovascular disorder, diabetic nephropathy, diabetic retinopathy, ischemic brain diseases (cerebral infarction, cerebral hemorrhage, etc.).

The concentration of EPC in the prophylactic / therapeutic agent of the present invention is determined depending on the target patient and the type of the disease, etc., using EPC proliferation efficiency, differentiation induction efficiency, or therapeutic effect obtained by administering to the patient as an index. It can be adjusted accordingly.
Moreover, the preventive / therapeutic agent of the present invention may have an optional component in addition to the above components as long as the EPC proliferation and differentiation are not inhibited. As said arbitrary component, the carrier normally used as an administration agent in a blood vessel, the prostacyclin etc., and EPC proliferation promotion or differentiation promotion components other than heparin can be illustrated.

  The dosage form of the prophylactic / therapeutic agent of the present invention is not particularly limited, but is preferably an injection administered into a blood vessel. In addition, the preventive / therapeutic agent in the present invention means a prophylactic or therapeutic agent.

  The method for preventing / treating ischemic disease in the present invention is not particularly limited as long as it is a method for administering the preventive / therapeutic agent of the present invention to an administration subject (animal such as a mammal). The administration target may be an animal suffering from an ischemic disease or an animal not yet suffering from an ischemic disease. The dosage, administration interval, etc. of the preventive / therapeutic agent of the present invention can be adjusted as appropriate.

  The EPC growth promoter or differentiation inducer of the present invention (hereinafter also referred to as “the growth promoter or differentiation inducer of the present invention”) is not particularly limited as long as it contains prostacyclin and the like and heparin. Other optional components may be included as long as they do not interfere with the proliferation and differentiation of EPC. As said arbitrary component, a component required for the proliferation and differentiation of EPC can be illustrated suitably. The concentration of prostacyclin or the like or heparin contained in the above growth promoter or differentiation inducer can be appropriately adjusted.

  The growth promoter or differentiation inducer of the present invention can be used by adding it to a medium for in vitro culture of EPC and culturing EPC in vitro in that medium. The amount of the growth promoter or differentiation inducer added to the medium can be appropriately adjusted based on the EPC proliferation efficiency, differentiation induction efficiency, etc. in the subsequent culture.

  EXAMPLES Hereinafter, although an Example demonstrates this invention more concretely, the technical scope of this invention is not limited to these illustrations.

[Isolation of endothelial progenitor cells (EPC)]
Bone marrow collected from wild-type mice was subjected to concentration gradient centrifugation using Histopaque 1083 (registered trademark) (manufactured by Sigma) to obtain mononuclear cells. Then, lineage-negative and cKit / Sca1-positive cells were isolated from mononuclear cells using a magnetic sorting system (Lineage deletion kit and Multi-sorting system; manufactured by Miltenyi Biotec) according to the manufacturing protocol. The obtained Lin− / cKit + / Sca1 + cells were used as EPC in subsequent experiments.

[EPC proliferation assay]
Α-MEM (GIBCO; # 12571-063) medium [10 mass% fetal bovine serum, 100 μg / ml endothelial cell growth factor E2759 (Sigma) on the plate coated with fibronectin from the EPC obtained in Example 1 ), 100 ng / ml mouse recombinant VEGF (manufactured by Sigma), 10 U / ml heparin sulfate (manufactured by Sigma), 100 U / ml penicillin, 100 μg / ml streptomycin, and a prostacyclin receptor stimulant ( ONO 54918; Ono Pharmaceutical Co., Ltd.) is contained at a predetermined concentration] and cultured at 37 ° C. for 7 days in the presence of 5 mass% CO ₂ . The mitogenic activity of the cultured cells was examined by a colony formation assay in which the number of cells formed per colony was evaluated as an indicator of the degree of proliferation of the cells. That is, EPC was identified by staining with DiI-acLDL and FITC-lectin, and the number of cells in each colony was counted (wild / heparin). The result is shown in FIG.

  Further, in the proliferation assay method described in Example 2 above, the proliferation assay was performed in the same manner except that a conventional medium not containing heparin sulfate and a prostacyclin receptor stimulant was used instead of the medium. Went (wild / cont). As a conventional medium, α-MEM (manufactured by GIBCO; # 12571-063) medium [10% by mass fetal calf serum, 100 μg / ml endothelial cell growth factor E2759 (manufactured by Sigma) on a plate coated with fibronectin; 100 ng / ml mouse recombinant VEGF (manufactured by Sigma), 10 U / ml heparin sulfate (manufactured by Sigma), 100 U / ml penicillin, containing 100 μg / ml streptomycin]. Further, in the proliferation assay method described in Example 2 above, proliferation assay was performed in the same manner as that except that EPC (IP − / −) lacking prostacyclin receptor was used instead of EPC ( IP-/-/ heparin). Furthermore, in the proliferation assay described in Example 2 above, it was the same except that (IP − / −) lacking the prostacyclin receptor was used instead of EPC, and the above-mentioned conventional medium was used. Proliferation assay was performed by the method of (IP-/-/ cont). Following these proliferation assays, the colony formation assay described in Example 2 above was performed. The results are shown in FIG. As can be seen from the results in FIG. 1, the prostacyclin receptor stimulant is used only when normal EPC (wild) that does not lack the prostacyclin receptor is used and in the presence of heparin (wild / heparin). EPC proliferation was promoted in a concentration-dependent manner. In contrast, if heparin coexistence and / or the lack of either or both of prostacyclin receptors, even if the concentration of the prostacyclin receptor stimulant changes, there is no significant change in the degree of EPC proliferation. I couldn't.

[EPC differentiation assay]
The EPC obtained in Example 1 was mixed with α-MEM (GIBCO) medium [10% by mass fetal bovine serum, 100 μg / ml endothelial cell growth factor (Sigma), 100 ng / ml on a plate coated with fibronectin. Mouse recombinant VEGF (manufactured by Sigma), 10 U / ml heparin sulfate (manufactured by Sigma), 100 U / ml penicillin, 100 g / ml streptomycin, and prostacyclin receptor stimulant (ONO54918; Ono Pharmaceutical Co., Ltd.) (Made by company) or not contained at a predetermined concentration] and cultured at 37 ° C. for 21 to 28 days in the presence of 5 mass% CO ₂ . The medium was replaced with fresh medium every 3-4 days. This cell obtained by culture was used as a sample cell (RTC (IP agonist)) for RT-PCR described later.

As other sample cells for RT-PCR described later, cells obtained by the following methods (1) to (4) were used.
(1) The EPC obtained in Example 1 was inoculated on the above-mentioned conventional medium and cultured in the presence of 5% by mass CO ₂ for 10 to 14 days. The medium was replaced with fresh medium every 3-4 days. By this culture, sample cells (EPC (control)) were obtained.
(2) Lineage positive mature lymphocytes were isolated from the mononuclear cells obtained in Example 1 using a magnetic sorting system (Lineage deletion kit and Multi-sorting system; manufactured by Miltenyi Biotec). The mature lymphocyte cells were cultured by the same method as in Example 4 to obtain sample cells (Lin +).
(3) Lin + cells were isolated from the mononuclear cells (MNC) obtained in Example 1 by a magnetic sorting system to obtain sample cells (MNC).

Each sample cell obtained in Example 4 and Example 5 above was subjected to RT-PCR by the following method to examine the degree of differentiation such as EPC.
RNA was isolated from each sample cell using RNeasy kit (Qiagen), and the contaminating DNA was removed with DNase. The RT-PCR sample was prepared using a superscript one-step RT-PCR kit (manufactured by Invitrogen), and a cycle consisting of an annealing reaction at 55 ° C. for 30 seconds and an extension reaction at 72 ° C. for 35 seconds was repeated. An extension reaction was carried out at 5 ° C. for 5 minutes.
In the RT-PCR, the following primers were used depending on the gene to be detected. IP (prostacyclin receptor) gene; IP forward (TTT CTG CTG CCT CTT CTG CTC ACT; SEQ ID NO: 1), IP reverse (ACC AGA ACT TGA GGC GTT GGA AGA; SEQ ID NO: 2). Flk (VEGFR2) gene; Flk forward (AGC GGA GAC GCT CTT CAT AA; SEQ ID NO: 3), Flk reverse (GCC CCT TTG CTC TTA TAG GG; SEQ ID NO: 4). eNOS gene; eNOS forward (AGG CAT CAC CAG GAA GAA GA; SEQ ID NO: 5), eNOS reverse (CAC AGA AGT GGG GGT ATG CT; SEQ ID NO: 6). G6PDH gene; G6PDH forward (GCC ATC AAC GAC CCC TTC ATT G; SEQ ID NO: 7), G6PDH reverse (TGC CAG TGA GCT TCC CGT TC; SEQ ID NO: 8). The IP gene is a prostacyclin receptor gene, the Flk gene is a marker gene indicating EPC, the eNOS gene is a marker gene indicating the degree of EPC differentiation, and the same amount of sample is used for the G6PDH gene. It is a control gene to confirm that

  FIG. 2 shows the result of electrophoresis of the PCR product obtained by the RT-PCR described above on an agarose gel. As can be seen from the results in FIG. 2, even when MNC or mature lymphocyte cells (Lin +) are cultured in the presence of heparin and a prostacyclin receptor stimulant, prostacyclin receptor expression, EPC presence, and EPC differentiation In contrast, EPC (IP agonist) and EPC (control) confirmed the expression of prostacyclin receptor, the presence of EPC, and the differentiation of EPC. However, in EPC (IP agonist) cultured in the presence of a prostacyclin receptor stimulator and heparin, the expression level of eNOS was increased as compared to EPC (control) cultured in the absence thereof. From this, it was shown that EPC differentiation is promoted when cultured in the presence of a prostacyclin receptor stimulant and heparin.

It is a figure which shows the effect | action which the combination of a prostacyclin etc. and heparin exerts on the proliferation of EPC. It is a figure which shows the effect | action which the combination of a prostacyclin etc. and heparin exerts on the differentiation of EPC.

Claims (10)

A method of proliferating EPC comprising the step of in vitro culturing endothelial progenitor cells (EPC) in the presence of prostacyclin or a prostacyclin analog and heparin. The method for proliferating EPC according to claim 1, wherein the prostacyclin analog is one or more substances selected from Carbaprostacyclin, Beraprost, Taprostene, Nileprost, Iloprost, Cicaprost, Ciprostene, Treprostinil and Bonsentan. . A method for inducing differentiation of EPC, comprising a step of in vitro culturing endothelial progenitor cells (EPC) in the presence of prostacyclin or a prostacyclin analog and heparin. The differentiation induction of EPC according to claim 3, wherein the prostacyclin analog is one or more substances selected from Carbaprostacyclin, Beraprost, Taprostene, Nileprost, Iloprost, Cicaprost, Ciprostene, Treprostinil and Bonsentan. Method. A prophylactic / therapeutic agent for ischemic disease, comprising EPC obtained by in vitro culture of endothelial progenitor cells (EPC) in the presence of prostacyclin or a prostacyclin analog and heparin. 6. The ischemic disease according to claim 5, wherein the prostacyclin analog is one or more substances selected from Carbaprostacyclin, Beraprost, Taprostene, Nileprost, Iloprost, Cicaprost, Ciprostene, Treprostinil, Bonsentan. Prophylactic / therapeutic agent. An EPC growth promoter comprising prostacyclin or a prostacyclin analog and heparin. The prostacyclin analog is one or more substances selected from Carbaprostacyclin, Beraprost, Taprostene, Nileprost, Iloprost, Cicaprost, Ciprostene, Treprostinil, Bonsentan, and promoting proliferation of EPC according to claim 7 Agent. A differentiation inducer for EPC, comprising prostacyclin or a prostacyclin analog, and heparin. The differentiation induction of EPC according to claim 9, wherein the prostacyclin analog is one or more substances selected from Carbaprostacyclin, Beraprost, Taprostene, Nileprost, Iloprost, Cicaprost, Ciprostene, Treprostinil and Bonsentan. Agent.

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