WO2012020566A1 - Ape1遺伝子導入による高機能化幹細胞・前駆細胞 - Google Patents
Ape1遺伝子導入による高機能化幹細胞・前駆細胞 Download PDFInfo
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Definitions
- the present invention relates to highly functionalized stem cells / progenitor cells. More specifically, the present invention relates to highly functional stem cells / progenitor cells such as vascular endothelial progenitor cells and mesenchymal stem cells whose functions such as angiogenesis have been improved by enhancing expression of Ape1.
- Endothelial progenitor cells are a group of CD34 positive cells derived from peripheral blood and bone marrow, which ultimately have the ability to differentiate into vascular endothelial cells to promote angiogenesis and revascularize Contribute (Non-Patent Document 1).
- Non-patent Document 2 It has been reported that the number of vascular endothelial progenitor cells in peripheral blood is inversely correlated with the degree of cardiovascular disease risk (Non-patent Document 2) and inversely correlated with the mortality of coronary artery disease patients (Non-patent Document 3). ing. This suggests that vascular endothelial progenitor cells play an important role in angiogenesis at the ischemic site.
- the CD34 positive cell content is about 100 times higher in bone marrow mononuclear cells than in peripheral blood mononuclear cells.
- revascularization (neoplastic) therapy using autologous bone marrow cells for peripheral arterial disease patients human ischemic limbs associated with ASO / Burger disease
- good results have been reported (Non-patent Document 4).
- transplantation using vascular endothelial progenitor cells derived from peripheral blood mononuclear cells is preferable to bone marrow cells. Therefore, a method forcibly mobilizing vascular endothelial progenitor cells from bone marrow to peripheral blood using VEGF, SDF, G-CSF and the like has also been attempted.
- Patent Document 1 transplantation of vascular endothelial progenitor cells suppresses the growth of cancer cells and brings about a significant change in the construction of tumor blood vessels, and has reported anticancer therapy using vascular endothelial progenitor cells.
- Non-patent Documents 5 to 7 vascular endothelial progenitor cells are decreased in patients with diabetes and chronic renal failure who are at high risk of cardiovascular disease and the elderly (Non-patent Documents 5 to 7), and the amount of cells necessary for transplantation Cannot be prepared.
- Patent Document 2 a method of proliferating vascular endothelial progenitor cells in vitro
- Patent Document 3 a method of inducing differentiation into immature stem cells by causing Notch ligand to act on vascular endothelial progenitor cells.
- vascular endothelial progenitor cell functions derived from reduced patients and prepare highly functional vascular endothelial progenitor cells.
- vascular endothelial progenitor cells there is a method of enhancing the function of vascular endothelial progenitor cells by stimulating with statin or prostacyclin, there is a problem that the effect is temporary and does not last.
- a method to increase the function of vascular endothelial progenitor cells by introducing genes such as VEGF and TERT is also considered, but only the partial function of EPC is improved, and the overall function improvement effect as EPC is not achieved. It is not an improvement method linked to clinical application.
- Ape1 (Apurinic / Apyrimidinic Endonuclease 1), also known as HAP1 or Ref-1, cleaves the phosphodiester backbone immediately 5 'to the AP site by hydrolysis to produce 3'-hydroxyl and 5'-deoxyribose AP endonuclease that produces single-stranded DNA fragments with phosphate ends.
- Non-patent Document 8 it has been reported that the growth and function of HUVEC and EPC are suppressed by a small molecule inhibitor of Ape1 (E3330) (Non-patent Document 8). It has also been reported that introduction of Tat-tagged Ape1 into endothelial cells suppresses the expression of cell adhesion molecule VCAM-1 induced by TNF- ⁇ and suppresses mononuclear cell adhesion (non- Patent Document 9).
- An object of the present invention is to improve the function of stem cells / progenitor cells, particularly vascular endothelial progenitor cells and mesenchymal stem cells, thereby using more limited stem cells / progenitor cells derived from patients to more effectively revascularize ( To realize neonatal therapy.
- the inventors have conducted various studies aiming at enhancement of the function of vascular endothelial progenitor cells (Endothelial Progenitor Cell: EPC) and found that the function of vascular endothelial progenitor cells is linked to the expression of Ape1 gene. Furthermore, it was confirmed that the function of vascular endothelial progenitor cells was remarkably enhanced by forced expression of the Ape1 gene, and that the vascular regeneration effect was increased when this highly functional vascular endothelial progenitor cell was administered to an animal experimental model.
- EPC Endothelial Progenitor Cell
- the inventors have also confirmed that cell damage due to oxidative stress is alleviated by introducing the Ape1 gene into mesenchymal stem cell lines and cardiac stem cells, as in the case of vascular endothelial progenitor cells.
- stem / progenitor cells such as vascular endothelial progenitor cells, mesenchymal stem cells, and myocardial stem cells highly functionalized by Ape1 are useful for improving tissue and organ regeneration therapy.
- the present invention relates to a stem cell / progenitor cell characterized by enhanced expression of Ape1 (Apurinic / Apyrimidinic Endonuclease 1).
- stem cells / progenitor cells have been described, but the stem cells / progenitor cells of the present invention are not limited thereto.
- Ape1 can be enhanced by, for example, Ape1 expression induction, Ape1 gene introduction, or Ape1 protein introduction.
- Ape1 expression induction can be achieved, for example, by applying an Ape1 expression inducer.
- Ape1 expression inducers that can be used include TNF- ⁇ , IL-1b, IF- ⁇ , and the like.
- the angiogenesis ability is improved by the enhanced expression of Ape1.
- impaired vascular repair action re-endothelialization action
- oxidative stress resistance are also improved.
- the stem cells / progenitor cells of the present invention can be suitably used in various regenerative medicines including angiogenesis / vascular repair.
- the present invention also provides a cell preparation containing the stem cells / progenitor cells described above.
- the stem cell / progenitor cell used is preferably derived from a patient in need of treatment.
- the cell preparation of the present invention is suitably used in regenerative medicine such as vascular regeneration, organ regeneration, cancer prevention / treatment, prevention / treatment of ischemic diseases including lower limb ischemia, myocardial infarction, and cerebral infarction.
- the application method of the cell preparation of the present invention is not particularly limited.
- it can be directly administered to the affected area by intravenous administration or intramuscular administration, or directly applied to the tissue by processing into a sheet shape.
- the form of the cell preparation of the present invention is not particularly limited, but as described above, it may be processed into a sheet that can be directly applied to the tissue.
- the present invention also provides a revascularization therapy characterized by administering the cell preparation of the present invention to a patient.
- the present invention relates to a method for evaluating the function of stem cells / progenitor cells using the expression level of Ape1 (Apurinic / Apyrimidinic Endonuclease 1) or Ape1 gene in stem cells / progenitor cells as an index, and a kit therefor (for evaluating stem cell / progenitor cell functions) Kit).
- the kit method is configured, for example, as including at least one of the following (a) to (c): (A) anti-Ape1 antibody, (B) an oligonucleotide primer that specifically binds to and amplifies the Ape1 gene; (C) A polynucleotide probe that specifically binds to and detects the Ape1 gene.
- the ability to regenerate tissue and organs of stem cells and progenitor cells such as impaired vascular repair (re-endothelialization), angiogenesis, resistance to oxidative stress, and new performance are improved, and regeneration using the cells is performed.
- the therapeutic effect can be improved.
- the function of stem cells / progenitor cells can be easily evaluated using Ape1 expression as an index. The results can be applied to preoperative evaluation of vascular regenerative cell therapy and diagnosis of vascular disorder diseases.
- FIG. 1 shows the adhesion ability of vascular endothelial progenitor cells derived from diabetic mice (DM) and aging mice (Aged).
- FIG. 2 shows the results of comparison of the expression level of Ape1 gene in vascular endothelial progenitor cells of aging mice (June 1 year: Aged-EPC) with young mice (12-14 weeks old: normal-EPC).
- FIG. 3 shows the expression of Ape1 in normal vascular endothelial progenitor cells without gene transfer (A: Non-transfected EPCs) and vascular endothelial progenitor cells into which Ape1 gene was introduced (B: Standard Method, C: Magnet TransfectionMethod).
- A Non-transfected EPCs
- B Standard Method
- C Magnet TransfectionMethod
- FIG. 4 shows the vascular remodeling effect by Ape1-introduced vascular endothelial progenitor cells after mouse femoral artery injury.
- A HE-stained image: From left, untreated (None), confluent EPC (ctEPC) administration, Ape1 gene-introduced EPC administration (Ape-EPC),
- B I / M ratio from left, untreated (None) , EPC in confluent state (ctEPC) administration, Ape1 gene introduction EPC administration (Ape-EPC)
- FIG. 5 shows the mitigation of cell damage caused by oxidative stress in pericyte-derived mesenchymal stem cells by introducing Ape1 ( ⁇ humanApe1-infected cells, ⁇ LacZ-expressing adenovirus-infected cells as controls).
- FIG. 6 shows the results of measuring the amount of ROS / Superoxide in human-derived cardiac muscle stem cells by flow cytometry.
- the present invention relates to a stem cell / progenitor cell having a function (for example, angiogenesis ability) superior to that of a normal stem cell / progenitor cell by enhancing Ape1 expression.
- the stem cells / progenitor cells of the present invention are referred to as “highly functionalized stem cells / progenitor cells”.
- the case of vascular endothelial progenitor cells is described as “highly functionalized vascular endothelial progenitor cells”
- mesenchymal stem cells is described as “highly functionalized mesenchymal stem cells”.
- Stem cell / progenitor cell The “stem cell” of the present invention is a cell having both the ability to differentiate into cells of multiple lines (multipotency) and the ability to maintain pluripotency even after cell division (self-replication ability). And various stem cells present in each tissue in vivo, for example, hematopoietic stem cells, neural stem cells, hepatic stem cells, skin stem cells, reproductive stem cells, ES cells, iPS and stem cells derived therefrom.
- Progenitor cells are cells that are in the process of being differentiated from the stem cells into specific somatic cells or germ cells, and include various progenitor cells as well as stem cells. In the present invention, vascular endothelial precursor cells and mesenchymal stem cells, which will be described later, can be mentioned as suitable examples.
- Endothelial progenitor cells are a group of cells derived from bone marrow, peripheral blood, umbilical cord blood, etc., and have the ability to differentiate into vascular endothelial cells. This is a cell group that promotes angiogenesis and contributes to blood vessel regeneration.
- vascular endothelial progenitor cells are cells that have the ability to differentiate into vascular endothelial cells and cells that do not differentiate directly into vascular endothelial cells but promote the formation and formation of new blood vessels through the production of various cytokines. It is a mixed heterogeneous cell population.
- Vascular endothelial progenitor cells can be isolated and collected from bone marrow as well as peripheral blood and umbilical cord blood.
- vascular endothelial progenitor cells When identifying vascular endothelial progenitor cells in peripheral blood, it is usually identified as a CD34 / KDR positive cell group for humans and as a cKit / Flk positive cell (mouse) for mice.
- CD34 + cells in the case of humans and Lin- / cKit + or Sca1 + cells (in the case of mice) mean the whole “undifferentiated cells” such as so-called hematopoietic stem cells.
- undifferentiated mononuclear cells a group of cells (fibronectin-adhesive and Acetyl) that adhere to the culture dish and grow to form colonies when cultured in a culture medium containing VEGF on a culture dish coated with fibronectin. It is considered that cells having LDL adsorbing ability and Lectin binding ability are a cell group containing a lot of vascular endothelial progenitor cells).
- Vascular endothelial progenitor cells can be separated from mononuclear cells by magnetic beads, flow cytometry, etc. using the surface markers described above, but as described above, the marker for accurately identifying vascular endothelial progenitor cells is currently unknown There are certain limitations with 2-3 types of markers.
- the inventors have isolated mouse vascular endothelial progenitor cells based on Lin negative, cKit positive, and Flk positive.
- vascular endothelial progenitor cells can be prepared by culturing mononuclear cells isolated from peripheral blood or bone marrow using a vascular endothelial differentiation promoting medium containing cytokines such as VEGF, and thereby attaching vascular endothelial progenitor cells as adherent cells. There is a way to recover.
- bone marrow undifferentiated cells and cells that highly express Notch ligands such as Jagged-1 or Delta-4 are co-cultured in an adjacent state to induce differentiation from bone marrow-derived differentiated cells to vascular endothelial progenitor conjugated cells.
- Notch ligands such as Jagged-1 or Delta-4
- vascular endothelial progenitor conjugated cells vascular endothelial progenitor conjugated cells.
- Japanese Unexamined Patent Publication No. 2007-89536 Japanese Unexamined Patent Publication No. 2007-89536
- a method of recovering vascular endothelial progenitor cells using a tissue-splitting filter Japanese Unexamined Patent Publication No. 2003-250820
- Progenitor cells can also be prepared.
- vascular endothelial precursor cells used here are CD34-positive mononuclear cells derived from bone marrow.
- vascular endothelial progenitor cells derived from induced pluripotent stem cells represented by iPS cells can also be suitably used as a source of highly functional vascular endothelial progenitor cells according to the present invention.
- Vascular endothelial progenitor cells are known to have functions such as angiogenic action, damaged vascular repair action (re-endothelialization action), oxidative stress resistance, etc. Therefore, in ischemic disease and arterial occlusive disease Used for revascularization cell therapy.
- vascular endothelial progenitor cells suppresses the proliferation of cancer cells and brings about a significant change in the construction of tumor blood vessels, and has reported anticancer therapy using vascular endothelial progenitor cells ( WO2008 / 142862). That is, vascular endothelial progenitor cells play an important function in cell transplantation therapy for various diseases associated with abnormal angiogenesis.
- vascular endothelial progenitor cells that can be obtained from patients are limited, and the number of vascular endothelial progenitor cells contained in peripheral blood that can be collected without any burden on the patient is particularly small. Furthermore, the function of vascular endothelial progenitor cells obtained from patients is often lower than usual, and there is a problem that sufficient effects cannot be obtained even after transplantation.
- vascular endothelial progenitor cells If the functions of limited vascular endothelial progenitor cells can be enhanced, it will contribute to the improvement of regenerative medicine using vascular endothelial progenitor cells.
- Mesenchymal stem cells are somatic stem cells derived from mesenchymal cells that have the ability to differentiate into cells belonging to the mesenchymal system. Application to is expected.
- Bone marrow mesenchymal stem cells are thought to exist in all tissues with mesenchymal tissue. Bone marrow mesenchymal stem cells are contained in bone marrow stromal cells. Bone marrow stromal cells are a type of cells that support hematopoietic cells, which are the main components in the bone marrow. Bone marrow stromal cells take a reticulated structure to support hematopoietic cells in the bone marrow. The bone marrow is the main organ of hematopoiesis that produces blood in the body.
- Pericytes are mesodermal cells also called Rouget cells.
- the effect of Ape1 forced expression was demonstrated using a mesenchymal stem cell line prepared from the pericytes.
- Ape1 (Apurinic / Apyrimidinic Endonuclease 1)
- Ape1 (Apurinic / Apyrimidinic Endonuclease 1), also known as APEX1 (Official Gene Symbol), HAP1 or Ref-1, cleaves the phosphodiester backbone immediately 5 'to the AP site by hydrolysis, 3'- AP endonuclease that produces single-stranded DNA fragments with hydroxyl and 5'-deoxyribose phosphate ends.
- Ape1 has been reported to have weak DNA 3′-diesterase activity, 3 ′-> 5 ′ exonuclease activity and RNaseH activity in addition to AP endonuclease activity. In addition to DNA repair activity, Ape1 also has a function to regulate many transcription factor DNA binding activities by redox mechanism in vitro. In other words, Ape1 is one of the molecules responsible for protecting cells against cellular oxidative stress.
- the base sequence of the Ape1 gene and the amino acid sequence of Ape1 are already known, and their sequences are published in public databases such as GenBank.
- the base sequence (mRNA) and amino acid sequence of human Ape1 are Accession Number NM_001641.2 and NP_001632.2 (variant 1), NM_080648.1 and NP_542379.1 (variant 2), or NM_080649. Published as 1 and NP_542380.1 (variant 3).
- the base sequence and amino acid sequence of mouse Ape1 are published as Accession Number NM_009687.1 and NP_033817.1.
- the Ape1 gene can be obtained by extracting RNA from cells and amplifying the target gene using a primer designed based on the above-described sequence.
- a primer designed based on the above-described sequence.
- An example of a primer is described below.
- FOREWARD PRIMER 5-GGA TTG GGT AAA GGA AGA AGC A-3 (mouse and rat shared: SEQ ID NO: 1) 5-GTG CCC ACT CAA AGT TTC TTA C-3 (for human: SEQ ID NO: 2)
- REVERSE PRIMER 5-CAA GGC GCC AAC CAA CAT TCT T-3 (human, mouse, rat shared: SEQ ID NO: 3)
- the inventors have found that the expression of Ape1 plays an important role in oxidative stress maintenance and angiogenic ability of vascular endothelial progenitor cells in search of genes involved in the function of vascular endothelial progenitor cells. Furthermore, the inventors confirmed the possibility that cell damage was alleviated by introducing Ape1 into mesenchymal stem cell lines and cardiac muscle stem cells.
- Ape1 increases resistance to oxidative stress, and promotes re-endothelialization and angiogenesis promotion in the damaged blood vessel wall. Therefore, by enhancing the expression of Ape1, the viability of stem cells and progenitor cells under oxidative stress environment is increased, and its “re-endothelialization” and “angiogenesis” action in the ischemic site and damaged blood vessels Can be fully exhibited.
- the stem cell / progenitor cell according to the present invention is a highly functional stem cell / progenitor cell characterized by enhanced expression of Ape1.
- “Ape1 expression is up-regulated” includes both direct Ape1 activity increase by increasing the expression level of Ape1 protein and indirect Ape1 activity increase by releasing related inhibitory system. To do.
- enhancement of Ape1 expression can be brought about by induction of Ape1 expression (induction of expression of Ape1 gene or Ape1 protein) by an external factor.
- methods for inducing Ape1 expression include Ape1 gene introduction, Ape1 protein introduction, and an Ape1 expression inducer.
- stem cells / progenitor cells with enhanced Ape1 expression can be obtained by selecting, isolating and amplifying Ape1 highly active cells from heterogeneous stem cell / progenitor cell populations regardless of external factors. You can also.
- Ape1 expression is“ enhanced ” means that Ape1 expression is higher in cells after induction of Ape1 expression than in intact stem cells / progenitor cells.
- Ape1 expression is up-regulated means that Ape1 expression in the selected and amplified cells is higher than the average Ape1 expression in the stem / progenitor cell population from which it was derived. .
- the expression level of Ape1 protein is not particularly limited, but it is desirable that the expression level is improved by 5 times or more, preferably 10 times or more compared to the reference cell.
- whole vascular endothelial progenitor cells or mesenchymal stem cells and whole myocardial stem cells are infected with Ape-Adenovirus, and the Ape expression level of the whole cells is about 2 to 5 times higher than that of control cells. Admits. Since the infection rate is estimated to be about 20%, it is estimated that the expression is about 10-20 times in the infected cells.
- “increased functionality” refers to organ regeneration ability, neoplastic ability (for example, vascular regeneration ability, neoplastic ability), resistance to oxidative stress, etc. of stem cells / progenitor cells. It means that the function and viability of have improved. As described above, in the present invention, enhancement of the function of stem cells / progenitor cells is brought about by enhancement of Ape1 expression.
- the cell when a patient-derived cell is used, the cell is used for treatment by enhancing Ape1 expression and improving angiogenic ability, resistance to oxidative stress, etc., compared to a cell whose function has been reduced.
- it is used with higher functionality than or equal to or higher than that of stem cells / progenitor cells derived from healthy subjects and normal controls (young people in their 30s, non-disease age, etc.).
- the highly functionalized stem cells / progenitor cells of the present invention can be prepared, for example, by inducing Ape1 expression.
- Ape1 gene introduction Ape1 expression can be induced by introducing the Ape1 gene into stem cells / progenitor cells.
- the Ape1 gene can be prepared based on the known sequence described above according to a conventional method.
- the cDNA of the target Ape1 gene can be prepared by extracting RNA from cells derived from bone marrow or peripheral blood, preparing primers based on known sequences, and cloning by PCR.
- Ape1 gene is introduced into cells by a method commonly used for transfection of animal cells, for example, a calcium phosphate method, a lipofection method, an electroporation method, a microinjection method, a method using a retrovirus or baculovirus as a vector.
- a method using an adenovirus, an adeno-associated virus or a retrovirus as a vector is preferable from the viewpoint of safety and introduction efficiency.
- the viral vector may be prepared based on, for example, the method of Miyake et al. (Miyake, S. et al, Proc. Natl. Acad. Sci. 93: 1320-1324, (1993)), but commercially available Adenovirus® Cre / 1oxP Kit (Takara Shuzo) can also be used.
- This kit is a recombinant adenovirus vector production kit using a new expression control system (Kanegae Y. et.al., 1995 Nucl. Acids Res. 23,3816) using Cre recombinase of P1 phage and its recognition sequence 1oxP
- a recombinant adenovirus vector incorporating the Ape1 gene can be easily prepared.
- MOI multiple of infection
- MOI 10 to 500 (more preferably around 100) is preferable for Ape1 gene recombinant adenovirus.
- Ape1 protein introduction Ape1 expression can be induced by introducing Ape1 protein into stem cells / progenitor cells.
- Ape1 protein can be introduced into a stem cell / progenitor cell together with a protein introduction reagent according to a known method, or a known protein transduction domain at the N-terminus of Ape1 protein (JP 2005-287418, JP 2005-110565). Etc.) are integrated and introduced.
- the “protein transduction domain” may be any peptide that can assist in protein transduction, such as HIV TAT (Green and Loewnstein, Cell, 56 (6): 1179-88 (1988); Frankel and Pabo, Cell, 55 (6): 1189-93 (1988)), Antenna Pedia Homeodomain: Vives et al., J. Biol.
- HSV VP22 Elliott and O'Hare, Cell, 88 (2): 223-33 (1997)
- CPP cell penetrating peptides
- a substance that binds to a receptor expressed specifically for the stem cell / progenitor cell to be introduced can also be used as the “protein transduction domain”.
- Ape1 expression can be induced by introducing an “Ape1 expression inducing agent” capable of externally inducing Ape1 expression into a target stem cell / progenitor cell.
- Ape1 expression inducer examples include TNF- ⁇ , IL1 ⁇ , IFN ⁇ and the like. These “Ape1 expression inducers” are added to a medium for culturing stem cells / progenitor cells described later, and the stem cells / progenitor cells are cultured by a usual method. The culture time is at least 6 hours, preferably 24 to 48 hours, but is not limited thereto. The amount of the Ape1 expression inducer added to the medium is appropriately set. For example, in the case of TNF- ⁇ , it is 1 to 100 ⁇ ng / ml, preferably about 5 ng / ml.
- the highly functionalized stem cells / progenitor cells of the present invention can be cultured and expanded in the same manner as normal stem cells / progenitor cells.
- the medium is not particularly limited as long as it is a medium suitable for mononuclear cell culture.
- MEM medium BME medium, DME medium, ⁇ -MEM medium, IMEM medium, ES medium, DM-160 medium, Fisher medium F12 medium, WE medium, RPMI medium, StemSpan medium, StemPro medium, and mixtures thereof can be used.
- lymphocyte culture media for example, GT-T medium (Takara Bio), AIM IV medium (Invitrogen), T lymphocyte culture medium (Cosmo Bio), X-VIVO medium (Lonza), ECM medium ECM-MV2 medium, commercially available medium for vascular endothelial cells: for example, EGM-2 medium, EBM-2 medium and the like may be used. It is necessary to add VEGF and heparin to the basic medium.
- nutrient sources necessary for maintenance and growth of cells and components necessary for induction of differentiation may be appropriately added to the medium.
- carbon sources such as glycerol, glucose, fructose, sucrose, lactose, honey, starch, dextrin, etc.
- hydrocarbons such as fatty acids, fats and oils, lecithin, alcohols, ammonium sulfate, ammonium nitrate, ammonium chloride
- Nitrogen sources such as urea and sodium nitrate, salt, potassium salt, phosphate, magnesium salt, calcium salt, iron salt, manganese salt and other inorganic salts, monopotassium phosphate, dipotassium phosphate, magnesium sulfate, sodium chloride , Ferrous sulfate, sodium molybdate, sodium tungstate and manganese sulfate, various vitamins, amino acids and the like.
- the medium is preferably a “serum-free medium” that does not contain animal serum such as FBS and FCS, but a serum substitute, KSR (Knockout Serum Replacement), etc. may be added.
- the pH of the medium obtained by blending these components is in the range of 5.5 to 9.0, preferably 6.0 to 8.0, more preferably 6.5 to 7.5.
- vascular endothelial progenitor cells and mesenchymal stem cells are adherent cells that have the property of growing by attaching to the extracellular matrix.
- the highly functionalized vascular endothelial progenitor cells of the present invention also have similar properties. Therefore, it is preferable to use an appropriate scaffold in the culture.
- the scaffold is not particularly limited as long as it is a matrix, substrate or carrier that can adhere and divide and proliferate, for example, fibronectin, vitronectin, collagen, proteoglycan, laminin, tenascin, entactin, elastin, fibrillin, hyaluronic acid, gelatin, Examples include poly-L-lysine and poly-D-lysine. In particular, it is desirable to use fibronectin as a matrix.
- Cultivation is performed at 36 ° C. to 38 ° C., preferably 36.5 ° C. to 37.5 ° C., under conditions of 1% to 25% O 2 and 1% to 15% CO 2 , with appropriate medium exchange.
- Cell preparations using highly functionalized stem cells / progenitor cells The highly functionalized stem cells / progenitor cells of the present invention have excellent angiogenic action, damaged vascular repair action (re-endothelialization action), resistance to oxidative stress, and ischemic properties. It can be suitably used for revascularization medicine such as diseases and arterial occlusive diseases. That is, the present invention provides a cell preparation containing highly functional stem cells / progenitor cells.
- the administration method of the cell preparation of the present invention is not particularly limited, and depending on the application site, local transplantation by surgical means, intravenous administration, lumbar puncture administration, local injection administration, subcutaneous administration, intradermal administration, intraperitoneal administration, Intramuscular administration, intracerebral administration, intraventricular administration, intravenous administration, and the like are possible.
- Vascular endothelial progenitor cells in the blood accumulate in damaged blood vessel sites due to their own characteristics. That is, vascular endothelial progenitor cells have a feature that even when intravenously administered to a site away from the affected area, they are gathered specifically in the ischemic site and promote blood vessel regeneration at the damaged site. Therefore, revascularization treatment of the ischemic site away from the administration site can be performed by simple intravenous administration without burden on the patient. However, regarding the angiogenic action on peripheral ischemic organs, if the blood flow for transporting vascular endothelial precursor cells is insufficient, intramuscular injection may be performed locally.
- the preparation of the present invention may be applied directly to the affected area in the form of a sheet.
- the sheet may contain not only cells but also a suitable support.
- the cell preparation of the present invention may contain scaffolding materials and components for assisting cell maintenance / proliferation and administration to the affected area, and other pharmaceutically acceptable carriers.
- Components necessary for cell maintenance / proliferation include media components such as carbon sources, nitrogen sources, vitamins, minerals, salts, various cytokines, and extracellular matrix preparations such as Matrigel TM .
- scaffold materials and components that assist administration to the affected area include biodegradable polymers; for example, collagen, polylactic acid, hyaluronic acid, cellulose, and derivatives thereof, and a complex composed of two or more thereof, an aqueous solution for injection;
- biodegradable polymers for example, collagen, polylactic acid, hyaluronic acid, cellulose, and derivatives thereof, and a complex composed of two or more thereof, an aqueous solution for injection
- examples include physiological saline, medium, physiological buffers such as PBS, isotonic solutions containing glucose and other adjuvants (eg D-sorbitol, D-mannose, D-mannitol, sodium chloride), etc.
- Adjuvants such as alcohols, specifically ethanol, polyalcohols such as propylene glycol, polyethylene glycol, nonionic surfactants such as polysorbate 80, HCO-50, etc. may be used in combination.
- organic solvents polyvinyl alcohol, polyvinyl pyrrolidone, carboxyvinyl polymer, sodium carboxymethylcellulose, sodium polyacrylate, sodium alginate, water-soluble dextran, sodium carboxymethyl starch, pectin, methylcellulose as necessary , Ethyl cellulose, xanthan gum, gum arabic, casein, agar, polyethylene glycol, diglycerin, glycerin, propylene glycol, petrolatum, paraffin, stearyl alcohol, stearic acid, mannitol, sorbitol, lactose, surfactants acceptable as pharmaceutical additives, It may contain a buffer, an emulsifier, a suspension, a soothing agent, a stabilizer and the like.
- a purified antibody is dissolved in a solvent such as physiological saline, buffer solution, glucose solution, etc., and an adsorption inhibitor such as Tween 80, Tween 20, gelatin or the like is added thereto.
- a solvent such as physiological saline, buffer solution, glucose solution, etc.
- an adsorption inhibitor such as Tween 80, Tween 20, gelatin or the like is added thereto.
- Tween 80, Tween 20, gelatin or the like is added thereto.
- diseases that can be the target of the cell preparation of the present invention include ischemic diseases such as myocardial infarction and cerebral infarction and peripheral arterial occlusive diseases.
- the inventors have found that transplantation of vascular endothelial progenitor cells suppresses the proliferation of cancer cells and brings about significant changes in the construction of tumor blood vessels, and reports on anticancer therapy using vascular endothelial progenitor cells (see above). . Therefore, the cell preparation of the present invention can be suitably used for such cancer prevention / treatment.
- the cell preparation of the present invention can be used for cell therapy of various diseases associated with abnormal angiogenesis.
- vascular endothelial progenitor cells Conventional methods for evaluating the function of vascular endothelial progenitor cells include the ability to repair damaged vessels (re-endothelialization) and peripheral tissue blood in vivo, such as proliferation and migration in culture. There is a flow improvement degree (angiogenesis). However, since these methods are very complicated, the function of vascular endothelial progenitor cells is simply “estimated” by measuring the expression level of genes closely related to the function of vascular endothelial progenitor cells. The method is being tried.
- the present invention provides a method for simply and accurately evaluating the function of stem cells / progenitor cells (vascular endothelial progenitor cells, mesenchymal stem cells, myocardial stem cells, etc.) by using Ape1 as one of the target genes. Specifically, the Ape1 gene expression level or Ape1 protein expression level in stem cells / progenitor cells is measured, and this is compared with the average Ape1 gene or Ape1 protein expression level in allogeneic cells. To evaluate.
- stem cells / progenitor cells vascular endothelial progenitor cells, mesenchymal stem cells, myocardial stem cells, etc.
- RNA is extracted from the collected cells, and the expression level of Ape1 gene (mRNA) in the total RNA is determined using any of the methods described below. taking measurement.
- the method for extracting total RNA is not particularly limited.
- guanidine thiocyanate / cesium chloride ultracentrifugation method guanidine thiocyanate / hot phenol method, guanidine hydrochloride method, acidic guanidine thiocyanate / phenol / chloroform method (Chomczynski, P. and) Sacchi, N., (1987) nal Anal. Biochem., 162, 156-159) and the like can be employed.
- the extracted total RNA may be further purified to mRNA alone if necessary.
- the expression level of the gene is determined by nucleic acid hybridization method, RT-PCR method, real-time PCR method, subtraction method, differential display method, differential hybridization method, and cross-hybridization method using solid phase samples such as gene chips and arrays. It can be measured using a known method such as a hybridization method.
- the Ape1 protein expression level can be measured using, for example, an immunological method utilizing an antigen-antibody reaction.
- immunological methods include immunoprecipitation methods, Western blotting methods, dot blotting methods, slot blotting methods, ELISA methods, solid phase immunization methods including RIA methods, and known modified methods in which these are modified ( Sandwich ELISA, the method described in US Pat. No. 4202875, the method of Meager et al. (Meager A., Clin Exp. Immunol. 2003 Apr, 132 (1), p128-36) and the like. That is, based on these methods, the expression level of Ape1 protein is measured using an antibody that specifically binds to Ape1 protein.
- the anti-Ape1 antibody may be labeled as necessary.
- the anti-Ape1 antibody is directly labeled, or the antibody is used as a primary antibody and used in cooperation with a labeled secondary antibody that specifically recognizes the primary antibody (recognizes an antibody derived from the animal that produced the antibody).
- Preferred examples of the type of label include an enzyme (alkaline phosphatase or horseradish peroxidase) or biotin (however, an operation for binding an enzyme-labeled streptavidin to biotin of a secondary antibody is added), but is not limited thereto.
- Various types of pre-labeled antibodies (or streptavidin) are commercially available as labeled secondary antibodies (or labeled streptavidin). In the case of RIA, an antibody labeled with a radioisotope such as 125 I is used, and the measurement is performed using a liquid scintillation counter or the like.
- the expression level of the antigen is measured.
- a substrate that develops color or a substrate that emits light by a catalyst of these enzymes is commercially available.
- a colored substrate When a colored substrate is used, it can be detected visually using Western blotting or dot / slot blotting.
- the ELISA method it is preferable to measure and quantify the absorbance (measurement wavelength varies depending on the substrate) of each well using a commercially available microplate reader.
- a commercially available microplate reader By preparing a dilution series of the antigen used for antibody production described above, using this as a standard antigen sample and performing detection simultaneously with other samples, creating a standard curve plotting the standard antigen concentration and measured values, the other It is also possible to quantify the antigen concentration in each sample.
- a substrate that emits light when used, it can be detected by autoradiography using an X-ray film or an imaging plate or by taking a picture using an instant camera in Western blotting or dot / slot blotting. . Further, quantification using a densitometry, a molecular imager Fx system (manufactured by Bio-Rad) or the like is also possible. Furthermore, when a luminescent substrate is used in the ELISA method, the enzyme activity is measured using a luminescent microplate reader (for example, manufactured by Bio-Rad).
- the “expression level” of the Ape1 gene or KIAA protein used as an index is not limited to its physical quantity, but includes activity and titer (antibody titer, etc.) indirectly indicating this.
- evaluation is performed by comparing the reference value with the expression level of Ape1 gene or Ape1 protein in the test cell, using the average expression level of Ape1 gene or Ape1 protein in normal stem cells / progenitor cells as a reference value. Can do.
- the measurement of the expression level of Ape1 gene or Ape1 protein may be performed after previously stimulating cells with the aforementioned Ape1 expression inducer. In this case, the function of stem cells / progenitor cells can be evaluated as “Ape1 expression inducing ability”.
- test cell When the expression level of the Ape1 gene or Ape1 protein in the test cell is lower than the above-described reference value, the test cell can be evaluated as having a reduced function of stem cells / progenitor cells.
- stem cells / progenitor cells include preoperative evaluation of regenerative medicine using stem cells / progenitor cells such as vascular endothelial progenitor cells, mesenchymal stem cells, myocardial stem cells, etc. It can also be used to evaluate the risk of developing diseases such as myocardial infarction and the degree of the disease.
- the present invention also provides a reagent / kit for evaluating the aforementioned stem cell / progenitor cell function.
- the kit includes at least one of the following (a) to (c) as essential components: (A) anti-Ape1 antibody, (B) an oligonucleotide primer for specifically binding to the Ape1 gene and amplifying the gene (c) a polynucleotide probe for specifically binding to the Ape1 gene and detecting the gene.
- Each component may be used alone as a reagent for evaluating stem cell / progenitor cell function.
- the anti-Ape1 antibody can be prepared according to a known method. That is, it can be obtained by immunizing an animal with an Ape1 protein serving as an antigen or any partial polypeptide thereof, and collecting and purifying the antibody produced in the animal body by a conventional method.
- the antibody may be polyclonal, but is preferably a monoclonal antibody.
- Monoclonal antibodies are specific according to known methods (for example, Kohler and Milstein, Nature 256, 495-497, 1975, Kennet, R. ed., Monoclonal Antibody p.365-367, 1980, Prenum Press, NY). Hybridomas can be established and obtained by fusing antibody-producing cells that produce antibodies and myeloma cells.
- Ape1 protein as an antigen or a partial polypeptide (epitope peptide) consisting of at least 6 consecutive partial amino acid sequences thereof, or any amino acid sequence or carrier (for example, N-terminal addition)
- epitopope peptide consisting of at least 6 consecutive partial amino acid sequences thereof, or any amino acid sequence or carrier (for example, N-terminal addition)
- keyhole limpet hemocyanin is added
- the antigen polypeptide can be obtained by causing a host cell to produce Ape1 protein or a partial polypeptide (epitope peptide) thereof by genetic manipulation.
- a vector capable of expressing the Ape1 gene or a part thereof may be prepared and introduced into a host cell to express the gene.
- the anti-Ape1 antibody may be labeled with an appropriate label (eg, enzyme label, radioactive label, fluorescent label, etc.), or may be appropriately modified with biotin or the like.
- the support may be immobilized on a suitable support, or a support may be separately included in the kit so that it can be immobilized.
- Such supports include synthetic resins capable of attaching proteins such as polyethylene, polypropylene, polybutylene, polystyrene, polymethacrylate, and polyacrylamide, glass, nitrocellulose, cellulose, and agarose supports, or gel-type supports. Can be used.
- the form of the support is not particularly limited, but is provided in the form of microspheres or microparticles such as beads (for example, “latex” beads), tubes (inner walls) such as microcentrifuge tubes, microtiter plates (wells), and the like.
- Ape1 gene amplification primer a 5-30 base long continuous oligonucleotide having a sequence complementary to at least part of the Ape1 gene.
- Primers can be easily designed and amplified in accordance with conventional methods such as using commercially available primer design software based on the base sequence for Ape1 gene amplification (SEQ ID NO: 1). Examples of such primers include oligonucleotides having the base sequences described in SEQ ID NOs: 1 to 5.
- the Ape1 gene amplification primer may be labeled with an appropriate label (for example, an enzyme label, a radioactive label, a fluorescent label, etc.), or may be modified with biotin, phosphate, amine, or the like.
- an appropriate label for example, an enzyme label, a radioactive label, a fluorescent label, etc.
- the Ape1 gene detection probe is a polynucleotide that specifically hybridizes to the Ape1 gene, and preferably has a length of about 20 to 1500 bases.
- a single-stranded oligonucleotide or double-stranded DNA having a length of about 20 bases is preferably used.
- a double-stranded DNA having a length of about 100 to 1500 bases or a single-stranded oligonucleotide having a length of about 20 to 100 bases is preferably used.
- a single-stranded oligo with a length of about 25 bases is preferred. These are preferably designed as probes that specifically hybridize to a portion having a high sequence specificity present in the 3 ′ untranslated region of the Ape1 gene.
- the Ape1 gene detection probe may be labeled with an appropriate label (for example, an enzyme label, a radioactive label, a fluorescent label, etc.), or may be modified with biotin, phosphate, amine, or the like.
- an appropriate label for example, an enzyme label, a radioactive label, a fluorescent label, etc.
- the Ape1 gene amplification primer and the Ape1 gene detection probe may be labeled with an appropriate label (eg, enzyme label, radioactive label, fluorescent label, etc.), and modified with biotin, phosphate, amine, etc. Also good.
- the Ape1 gene detection probe may be appropriately fixed, such as a glass plate, nylon membrane, microbead, or silicon chip.
- the kit of the present invention includes not only the above-described components, but also a (labeled) secondary antibody specific to the anti-Ape1 antibody, a reagent for detecting a labeled body, a reaction buffer, an enzyme, a substrate, etc. Other elements necessary for detection may be included.
- Example 1 Vascular endothelial progenitor cells (EPC) 1. Decreased Vascular Endothelial Progenitor Cell (EPC) Function in Diabetic and Aged Mice After isolating mononuclear cells from bone marrow of diabetic (DM) and aging (aged: June 1) mice, a magnetic sorting system (MACS) ) To purify Lin-, cKit +, Flk + cells. The purified EPC was cultured for 48 hours in a fibronectin-coated medium containing ⁇ -MEM (GIBCO) (or Endothelial cell basal medium EBM (clontech)) (10% FCS, VEGF).
- GEBCO fibronectin-coated medium containing ⁇ -MEM
- EBM Endothelial cell basal medium
- Acetyl-LDL adsorption and Lectin binding positive cells were used as adhesion EPC, and the ability to adhere to Fibronectin, one of the cultured EPC functions, was evaluated (calculated as a percentage of the total number of cultured cells). .
- EPCs derived from diabetic (DM) mice and aged mice have lower adhesion ability and further reduced colony formation ability compared to normal mice (12-14 weeks old). It was suggested that That is, the adhered cells grow to form colonies, but the degree of colony formation (the number of cells per colony) is small. This can also be evaluated as a decrease in proliferation ability.
- Inflammatory cytokine-induced Ape1 expression ability in aging mouse-derived EPC EPCs were prepared from 12-14 week-old mice and aging mice (June 1 year) in the same manner as described above. The prepared cells are cultured in the same manner as in the previous section.However, after the culture period has been extended for another week to confluence, the cells are replaced with a medium containing the inflammatory cytokine TNF ⁇ 5-10 ng / ml, and 16 hours later. Was recovered. RNA was purified from the collected cells, and the amount of mRNA was measured by RT-PCR. The primer sequences used are shown below. Forward; atg ccg aag cgt ggg aaaag (SEQ ID NO: 4) Reverse; cag tgc tag gta tag ggt g (SEQ ID NO: 5)
- the expression level of Ape1 gene was significantly decreased in EPC derived from aged mice compared to EPC derived from 12-14 weeks old mice (FIG. 2). It was also confirmed that the response to the expression induction by TNF ⁇ was low. In EPC under normal culture, the expression level of Ape1 is not significantly different between aging mice and young mice. Therefore, it was confirmed that the response to the expression induction by TNF ⁇ was significantly reduced in aging EPC as compared with normal EPC.
- Adenovirus was introduced into two types, a Magnet Transfection Method using a magnet infection kit (Magnetofection TM -AdenoMag) and a standard method. Specifically, 20 ⁇ l of Ape1-introduced virus (AdenoApe) and 2 ⁇ l of AdenoMag were mixed, incubated at room temperature for 20 minutes, and then added to a culture plate containing EPC in a subconfluent state. The plate was placed on a magnetic plate, allowed to stand for 60 minutes, and then cultured at 37 ° C. for infection. Infected cells were immunoreacted with anti-Ape1 antibody and visualized (red) with TRITC secondary antibody.
- Ape1-introduced EPC cures vascular lesions
- the prepared EPC (1 ⁇ 10 4 / mouse) was introduced from the tail vein.
- the femoral artery was removed and fixed, and the short-axis section of the blood vessel was observed by HE staining (FIG. 4 (A)).
- the areas of the media (M) and thickened intima (I) were measured, and the neointimal thickening (I / M ratio) was calculated (FIG. 4 (B)).
- the normal EPC group (ct EPC) group Compared to the non-cell-introduced group (none), the normal EPC group (ct EPC) group also showed a decrease in neointimal thickening, but in the Ape1-introduced EPC (Ape-EPC) group, a marked neointimal The cost altitude improvement effect was confirmed.
- Example 2 Mesenchymal stem cells (MSC) 1. Establishment and preparation of mesenchymal stem cell line The mesenchymal stem cell line is derived from the peripheral capillary tissue of temperature-sensitive SV40T antigen-expressing transgenic mice (purchased from Fact Co., Ltd.) as an NG2-positive cell using anti-NG2 antibody. A cell line was established by isolation by the sorting method.
- the established cell line expresses genes such as NG2, CD146, PDGFR, and CD90 that are characteristic of pericytes and mesenchymal stem cells, as well as vascular cells and adipocytes (specific FABP4 expression, lipid droplet identification (oil red staining)) and osteoblasts (Osteopontin expression, Ca deposition (Alizarin Red staining)) are maintained, and are subcultured at 33 °C in DMEM (10% FCS) on a collagen-coated culture dish. Thus, the immortal state of the cells could be maintained.
- DMEM (10% FCS) fetal limbalcholine
- the degree of injury was evaluated by the WST assay method.
- the WST assay was performed using WST-1 (Roche Applied Science) based on the instructions for use.
- the survival rate was calculated by setting the H 2 O 2 non-treated group as 100% and the group dead by SDS as 0%.
- Example 3 Myocardial stem cells A human heart-derived myocardial stem cell line (CSC03) was established from micromyocardial tissue collected from the right ventricle of a patient with chronic myocardial infarction by subculture using an enzymatic degradation method and bFGF addition.
- CSC03 human heart-derived myocardial stem cell line
- the human Ape1-cDNA was incorporated into the retroviral vector pRetro-IRES-DsRed, and pRetro-Ape-IRES-DsRed, which expresses the fluorescent protein DsRed together with Ape1 by IRES, was introduced into CSC03.
- Ape1 gene-introduced cells were selectively purified by purifying DsRed positive cells by FACS.
- pRetro-IRES-DsRed was introduced, and DsRed positive cells were prepared as a control control.
- ROS reactive oxygen species
- the amount of ROS and superoxide that affects the activity of CSCO 3 is ROS (H 2 O 2 , ONOO-, HO ⁇ , NO, ROO ⁇ ) and superoxide (O) in the control cells (DsRed-CSC03).
- the expression (production) of 2 ) was 11.3%.
- Ape1 gene-introduced CSC Ape1: Ape1-DsRed-CSC03
- production of ROS and superoxide was suppressed to 5.1%.
- stem cells / progenitor cells such as angiogenesis action, damaged blood vessel repair action (re-endothelialization action), oxidative stress resistance, etc. can be enhanced, and more effective revascularization treatment can be performed.
- simple function evaluation of stem cells and progenitor cells can be performed using Ape1 expression as an index, thereby enabling preoperative evaluation of revascularization cell therapy and diagnosis of vascular disorder diseases.
- SEQ ID NO: 1 Ape1 amplification primer (forward: mouse, rat)
- SEQ ID NO: 2 Ape1 amplification primer (forward: human)
- SEQ ID NO: 3 Ape1 amplification primer (reverse: human, mouse, rat)
- SEQ ID NO: 4 Ape1 RT-PCR primer (forward: for mouse)
- Sequence number 5 Primer for Ape1 RT-PCR (reverse: for mice)
Abstract
Description
こうした幹細胞・前駆細胞機能の高機能化により、本発明の幹細胞・前駆細胞は、血管新生・血管修復を含む、種々の再生医療において好適に利用することができる。
本発明の細胞製剤において、用いられる幹細胞・前駆細胞は治療を必要とする患者由来のものであることが好ましい。
前記キット方法は、たとえば、以下の(a)~(c)の少なくとも1つを含むものとして構成される:
(a)抗Ape1抗体、
(b)Ape1遺伝子に特異的に結合し、該遺伝子を増幅するためのオリゴヌクレオチドプライマー、
(c)Ape1遺伝子に特異的に結合し、該遺伝子を検出するためのポリヌクレオチドプローブ。
本発明の「幹細胞」は、複数系統の細胞に分化できる能力(多分化能)と、細胞分裂を経ても多分化能を維持できる能力(自己複製能)を併せ持つ細胞であって、生体内各組織に存在する種々の幹細胞、例えば、造血幹細胞、神経幹細胞、肝幹細胞、皮膚幹細胞、生殖幹細胞、ならびにES細胞やiPS及びこれらから誘導された幹細胞を包含する。「前駆細胞」とは、前記幹細胞から特定の体細胞や生殖細胞に分化する途中の段階にある細胞であって、幹細胞と同様に種々の前駆細胞を包含する。本発明においては、後述する血管内皮前駆細胞及び間葉系幹細胞を好適な例として挙げることができる。
「血管内皮前駆細胞(endothelial progenitor cell; EPC)」とは、骨髄や末梢血、臍帯血などに由来する細胞群で、最終的に血管内皮細胞への分化能を有することで血管新生を促し、血管再生に貢献する細胞群である。
血管内皮前駆細胞は、骨髄のほか末梢血や臍帯血などからも分離・採取することができる。
間葉系幹細胞(mesenchymal stem cell)は、間葉に由来する体性幹細胞で、間葉系に属する細胞への分化能を有するため、骨や血管、心筋の再生医療への応用が期待されている。
Ape1(Apurinic/Apyrimidinic Endonuclease 1)は、APEX1(Official Gene Symbol)、HAP1又はRef-1としても知られ、AP部位のすぐ5'側にあるホスホジエステル・バックボーンを加水分解によって切断し、3'-ヒドロキシル及び5'-デオキシリボースリン酸末端をもつ一本鎖DNA断片を産生するAPエンドヌクレアーゼである。
FOREWARD PRIMER:5-GGA TTG GGT AAA GGA AGA AGC A-3 (マウス、ラット共用:配列番号1)
5-GTG CCC ACT CAA AGT TTC TTA C-3(ヒト用:配列番号2)
REVERSE PRIMER:5-CAA GGC GCC AAC CAA CAT TCT T-3(ヒト、マウス、ラット共用:配列番号3)
3.1 Ape1発現の亢進
本発明にかかる幹細胞・前駆細胞は、Ape1の発現が亢進していることを特徴とする高機能化幹細胞・前駆細胞である。ここで、「Ape1の発現が亢進している」とは、Ape1タンパクの発現量の増加による直接的なApe1活性亢進と、関連する抑制系の解除等による間接的なApe1活性亢進の両方を包含する。
本発明において「高機能化」とは、幹細胞・前駆細胞の有する臓器再生能、新生能(例えば、血管再生能、新生能)、酸化ストレス抵抗性等の機能やviabilityが向上していることを意味する。前述のとおり、本発明において幹細胞・前駆細胞の高機能化は、Ape1発現の亢進によってもたらされる。
本発明の高機能化幹細胞・前駆細胞は、たとえばApe1発現誘導により調製することができる。
4.1 Ape1遺伝子導入
Ape1遺伝子を幹細胞・前駆細胞に導入することにより、Ape1発現誘導を行うことができる。
Ape1遺伝子は、常法に従い、前述した公知の配列を基に調製することができる。たとえば、骨髄や末梢血由来の細胞からRNAを抽出し、公知の配列を元にプライマーを作製し、PCR法でクローニングすることにより目的とするApe1遺伝子のcDNAが調製できる。
Ape1タンパク質を幹細胞・前駆細胞に導入することにより、Ape1発現誘導を行うことも可能である。
Ape1発現を外的に誘導することのできる「Ape1発現誘導剤」を目的とする幹細胞・前駆細胞に導入することにより、Ape1発現誘導を行うことができる。
本発明の高機能化幹細胞・前駆細胞は、通常の幹細胞・前駆細胞と同様の方法で培養・増殖させることができる。
これらの成分を配合して得られる培地のpHは5.5~9.0、好ましくは6.0~8.0、より好ましくは6.5~7.5の範囲である。
本発明の高機能化幹細胞・前駆細胞は、優れた血管新生作用、障害血管修復作用(再内皮化作用)、酸化ストレス耐性を有し、虚血性疾患や動脈閉塞性疾患等の血管再生医療に好適に利用できる。すなわち、本発明は高機能化幹細胞・前駆細胞を含む細胞製剤を提供する。
従来、血管内皮前駆細胞の機能を評価する方法としては、培養系での増殖能や遊走能など、in vivoでは、障害血管修復能(再内皮化)や末梢組織血流改善度(血管新生)などがある。しかし、これらの方法は非常に煩雑であるため、簡易的に血管内皮前駆細胞の機能と密接に関連している遺伝子群の発現量を測定することにより、血管内皮前駆細胞機能を「推測」する方法が試行されている。
Ape1遺伝子の発現量は、回収した細胞からまず全RNAを抽出し、この全RNA中におけるApe1遺伝子(mRNA)の発現量を後述するいずれかの方法を用いて測定する。
Ape1タンパク発現量は、たとえば抗原抗体反応を利用した免疫学的方法を用いて測定することができる。
評価は、たとえば、正常幹細胞・前駆細胞における平均的Ape1遺伝子又はApe1タンパク発現量を基準値として、その基準値と被験細胞におけるApe1遺伝子又はApe1タンパク発現量を比較することにより行うことができる。Ape1遺伝子又はApe1タンパク発現量の測定は、前述したApe1発現誘導剤であらかじめ細胞を刺激してから行ってもよい。この場合、「Ape1発現誘導能」として幹細胞・前駆細胞の機能を評価できる。
本発明は、前記した幹細胞・前駆細胞機能評価のための試薬・キットも提供する。キットは、必須の構成要素として、以下の(a)~(c)の少なくとも1つを含む:
(a)抗Ape1抗体、
(b)Ape1遺伝子に特異的に結合し、該遺伝子を増幅するためのオリゴヌクレオチドプライマー
(c)Ape1遺伝子に特異的に結合し、該遺伝子を検出するためのポリヌクレオチドプローブ。
抗Ape1抗体は、公知の方法にしたがって調製することができる。すなわち、常法により、抗原となるApe1タンパク質、あるいはその任意の部分ポリペプチドを用いて動物を免疫し、該動物生体内に産生される抗体を採取、精製することによって得ることができる。
Ape1遺伝子増幅用プライマー、Ape1遺伝子の少なくとも一部に相補的な配列を有する5~30塩基長の連続したオリゴヌクレオチドである。プライマーはApe1遺伝子増幅用の塩基配列(配列番号1)に基づき、市販のプライマー設計ソフトを用いるなど、常法にしたがい容易に設計し、増幅させて調製することができる。このようなプライマーの例としては、たとえば配列番号1~5に記載の塩基配列を有するオリゴヌクレオチドを挙げることができる。
Ape1遺伝子検出用プローブは、Ape1遺伝子に特異的にハイブリダイズするポリヌクレオチドであって、20~1500塩基長程度のものが好ましい。具体的には、ノーザンハイブリダイゼーション法であれば、20塩基長程度の1本鎖オリゴヌクレオチドか2本鎖DNAが好適に用いられる。また、マイクロアレイであれば、100~1500塩基長程度の2本鎖DNA、又は20~100塩基長程度の1本鎖オリゴヌクレオチドが好適に用いられる。Affimetrix社のGene Chipシステムの場合は、25塩基長程度の1本鎖オリゴがよい。これらは、特にApe1遺伝子の3’非翻訳領域に存在する配列特異性が高い部分に特異的にハイブリダイズするプローブとして設計することが好ましい。
1.糖尿病、加齢マウスにおける血管内皮前駆細胞(EPC)機能の低下
糖尿病(DM)マウス及び加齢(aged:1年6月)マウス骨髄から単核球細胞を単離した後、マグネットソーティングシステム(MACS)により、Lin-, cKit+, Flk+ の細胞を精製した。精製したEPCは、α-MEM (GIBCO)(あるいはEndothelial cell basal medium EBM (clontech))(10% FCS, VEGF)を含むfibronectinコーティングした培地で、48時間培養した。接着している細胞のうち、Acetyl-LDL吸着かつLectin結合陽性細胞を接着EPCとして、培養EPC機能の一つであるFibronectinへの接着能を評価した(全培養細胞数の中の割合で算出)。
12~14週齢のマウス及び加齢マウス(1年6月)から、前項と同様にしてEPCを調製した。調製した細胞は、前項と同様の方法で培養するが、培養期間をさらに1週間延ばしてコンフルエントにしたのち、炎症性サイトカインTNFα 5-10 ng/mlを含む培地に交換して、16時間後に細胞を回収した。回収した細胞よりRNAを精製し、RT-PCR法によりmRNA量を測定した。使用したプライマー配列を下記に示す。
Forward; atg ccg aag cgt ggg aaa aag(配列番号4)
Reverse; cag tgc tag gta tag ggt g(配列番号5)
Adenovirusを用いてマウス血管内皮前駆細胞にヒトApe1遺伝子を導入した。導入後のEPCにおけるApe1発現量(赤)を測定した。Adenovirusへの導入は、マグネット感染キット(MagnetofectionTM-AdenoMag)を利用したMagnet Transfection Methodと通常の方法(Standard Method)の2種類で行った。すなわち、Ape1導入ウイルス(AdenoApe)20μlとAdenoMag2μlを混合し、20分間室温でインキュベートした後、サブコンフルエント状態のEPCが入っている培養プレートに添加した。このプレートを、magnetic plateにのせ、60分間静置した後、通常の37℃で培養して感染させた。感染細胞は、抗Ape1抗体で免疫反応させ、TRITC二次抗体で可視化(赤色)した。
12~14週齢のマウス大腿動脈障害モデルに、前項で作製したApe1導入EPCを経静脈より導入し、4週間後に血管リモデリング(新生内膜肥厚度=I/M比)を評価した。
1.間葉系幹細胞株の樹立、調製
間葉系幹細胞株は、温度感受性SV40T抗原発現トランスジェニックマウス(株式会社ファクトより購入)の末梢毛細血管組織由来より、抗NG2抗体を用いてNG2陽性細胞としてマグネットソーティング法にて単離して細胞株を樹立した。
コラーゲンコーティング培養皿に培養した上記間葉系幹細胞にヒトApe1(hApe1)発現用組み換えアデノウイルスを感染させることにより、間葉系幹細胞内にhuman Ape1 (hApe1)を強制発現させた。コントロール(Ct) として、LacZ発現アデノウイルス感染細胞を用いた。
ヒト心臓由来心筋幹細胞株(CSC03)は慢性心筋梗塞患者右室より採取した微小心筋組織から酵素分解法及びbFGF添加による継代培養にて樹立した。
配列番号2:Ape1増幅用プライマー(フォワード:ヒト用)
配列番号3:Ape1増幅用プライマー(リバース:ヒト、マウス、ラット用)
配列番号4:Ape1 RT-PCR用プライマー(フォワード:マウス用)
配列番号5:Ape1 RT-PCR用プライマー(リバース:マウス用)
Claims (22)
- Ape1(Apurinic/Apyrimidinic Endonuclease 1)の発現が亢進していることを特徴とする幹細胞・前駆細胞。
- Ape1の発現の亢進がApe1発現誘導に基づくものである、請求項1に記載の幹細胞・前駆細胞。
- Ape1発現誘導がApe1遺伝子導入に基づくものである、請求項2に記載の幹細胞・前駆細胞。
- Ape1発現誘導がApe1タンパク質導入に基づくものである、請求項2に記載の幹細胞・前駆細胞。
- Ape1発現誘導がApe1発現誘導剤に基づくものである、請求項2に記載の幹細胞・前駆細胞。
- Ape1発現誘導剤がTNF-α、IL1β、及びIFγから選ばれるいずれか1又は2以上である、請求項5に記載の幹細胞・前駆細胞。
- 血管新生能が向上していることを特徴とする、請求項1~6のいずれか1項に記載の幹細胞・前駆細胞。
- 障害血管修復作用及び/又は酸化ストレス抵抗性が向上していることを特徴とする、請求項1~6のいずれか1項に記載の幹細胞・前駆細胞。
- 幹細胞・前駆細胞が血管内皮前駆細胞である、請求項1~8記載の幹細胞・前駆細胞。
- 幹細胞・前駆細胞が間葉系幹細胞又は心筋幹細胞である、請求項1~8記載の幹細胞・前駆細胞。
- 請求項1~10のいずれか1項に記載の幹細胞・前駆細胞を含む細胞製剤。
- 細胞が前記細胞製剤による治療を必要とする患者由来のものである、請求項11記載の細胞製剤。
- 血管再生、臓器再生、癌の予防・治療、ならびに、下肢虚血、心筋梗塞、及び脳梗塞を含む虚血疾患の予防・治療用である、請求項11又は12記載の細胞製剤。
- 静脈投与、筋肉内投与、又は組織に直接適用されることを特徴とする、請求項11~13のいずれか1項に記載の細胞製剤。
- 幹細胞・前駆細胞が血管内皮前駆細胞である、請求項11~13記載の細胞製剤。
- 幹細胞・前駆細胞が間葉系幹細胞又は心筋幹細胞である、請求項11~13記載の細胞製剤。
- シート状である、請求項11~16のいずれか1項に記載の細胞製剤。
- 請求項11~17のいずれか1項に記載の細胞製剤を患者に投与することを特徴とする血管再生療法。
- 幹細胞・前駆細胞におけるApe1(Apurinic/Apyrimidinic Endonuclease 1)又はApe1遺伝子の発現量を指標として、当該幹細胞・前駆細胞の機能評価を行う方法。
- 以下の(a)~(c)の少なくとも1つを含む幹細胞・前駆細胞機能評価用キット。
(a)抗Ape1抗体、
(b)Ape1遺伝子に特異的に結合し、該遺伝子を増幅するためのオリゴヌクレオチドプライマー、
(c)Ape1遺伝子に特異的に結合し、該遺伝子を検出するためのポリヌクレオチドプローブ - 幹細胞・前駆細胞において、Ape1(Apurinic/Apyrimidinic Endonuclease 1)の発現を亢進させることを特徴とする、当該細胞の臓器再生能又は新生能を向上させる方法。
- 幹細胞・前駆細胞において、Ape1(Apurinic/Apyrimidinic Endonuclease 1)の発現を亢進させることを特徴とする、当該細胞の血管再生能又は新生能を向上させる方法。
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US13/816,107 US20140030239A1 (en) | 2010-08-10 | 2011-08-09 | Highly functionalized stem cell/progenitor cell by ape1 gene transfection |
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JP2019218271A (ja) * | 2018-06-15 | 2019-12-26 | 有限会社大阪空気機械サービス | 組織治癒剤 |
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