JP2002020298A - Neovascularization promoter - Google Patents
Neovascularization promoterInfo
- Publication number
- JP2002020298A JP2002020298A JP2000207800A JP2000207800A JP2002020298A JP 2002020298 A JP2002020298 A JP 2002020298A JP 2000207800 A JP2000207800 A JP 2000207800A JP 2000207800 A JP2000207800 A JP 2000207800A JP 2002020298 A JP2002020298 A JP 2002020298A
- Authority
- JP
- Japan
- Prior art keywords
- growth factor
- vascular
- neovascularization
- vascular endothelial
- cells
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 206010029113 Neovascularisation Diseases 0.000 title abstract 5
- 239000003102 growth factor Substances 0.000 claims abstract description 15
- SHGAZHPCJJPHSC-YCNIQYBTSA-N all-trans-retinoic acid Chemical compound OC(=O)\C=C(/C)\C=C\C=C(/C)\C=C\C1=C(C)CCCC1(C)C SHGAZHPCJJPHSC-YCNIQYBTSA-N 0.000 claims abstract description 13
- 229930002330 retinoic acid Natural products 0.000 claims abstract description 13
- 229960001727 tretinoin Drugs 0.000 claims abstract description 13
- 208000023589 ischemic disease Diseases 0.000 claims abstract description 12
- 230000006444 vascular growth Effects 0.000 claims abstract description 12
- 239000003814 drug Substances 0.000 claims abstract description 8
- 229940124597 therapeutic agent Drugs 0.000 claims abstract description 6
- 210000003556 vascular endothelial cell Anatomy 0.000 claims description 15
- 210000001185 bone marrow Anatomy 0.000 claims description 13
- 239000003795 chemical substances by application Substances 0.000 claims description 13
- 230000001737 promoting effect Effects 0.000 claims description 13
- 210000005087 mononuclear cell Anatomy 0.000 claims description 12
- 230000033115 angiogenesis Effects 0.000 claims description 11
- 210000004204 blood vessel Anatomy 0.000 claims description 9
- 102000018233 Fibroblast Growth Factor Human genes 0.000 claims description 6
- 108050007372 Fibroblast Growth Factor Proteins 0.000 claims description 6
- 229940126864 fibroblast growth factor Drugs 0.000 claims description 6
- 108090000386 Fibroblast Growth Factor 1 Proteins 0.000 claims description 4
- 102100031706 Fibroblast growth factor 1 Human genes 0.000 claims description 4
- 102000003745 Hepatocyte Growth Factor Human genes 0.000 claims description 4
- 108090000100 Hepatocyte Growth Factor Proteins 0.000 claims description 4
- 102000010780 Platelet-Derived Growth Factor Human genes 0.000 claims description 4
- 108010038512 Platelet-Derived Growth Factor Proteins 0.000 claims description 4
- 239000000122 growth hormone Substances 0.000 claims description 4
- 102000009840 Angiopoietins Human genes 0.000 claims description 3
- 108010009906 Angiopoietins Proteins 0.000 claims description 3
- 108090000723 Insulin-Like Growth Factor I Proteins 0.000 claims description 3
- 102000005604 Myosin Heavy Chains Human genes 0.000 claims description 3
- 108010084498 Myosin Heavy Chains Proteins 0.000 claims description 3
- 102000013275 Somatomedins Human genes 0.000 claims description 3
- 230000001605 fetal effect Effects 0.000 claims description 3
- 210000002460 smooth muscle Anatomy 0.000 claims description 3
- 210000002565 arteriole Anatomy 0.000 abstract description 9
- 230000000302 ischemic effect Effects 0.000 abstract description 7
- 210000004369 blood Anatomy 0.000 abstract description 6
- 239000008280 blood Substances 0.000 abstract description 6
- 206010002383 Angina Pectoris Diseases 0.000 abstract description 4
- 206010061216 Infarction Diseases 0.000 abstract description 4
- 201000004810 Vascular dementia Diseases 0.000 abstract description 4
- 206010008118 cerebral infarction Diseases 0.000 abstract description 4
- 208000026106 cerebrovascular disease Diseases 0.000 abstract description 4
- 230000007574 infarction Effects 0.000 abstract description 4
- 210000002889 endothelial cell Anatomy 0.000 abstract description 3
- 206010003210 Arteriosclerosis Diseases 0.000 abstract 1
- 208000011775 arteriosclerosis disease Diseases 0.000 abstract 1
- 230000000747 cardiac effect Effects 0.000 abstract 1
- 210000001616 monocyte Anatomy 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 9
- 239000002953 phosphate buffered saline Substances 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- 108090000379 Fibroblast growth factor 2 Proteins 0.000 description 4
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 4
- 239000012530 fluid Substances 0.000 description 4
- 208000010125 myocardial infarction Diseases 0.000 description 4
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- 208000030831 Peripheral arterial occlusive disease Diseases 0.000 description 3
- 239000006285 cell suspension Substances 0.000 description 3
- 239000007924 injection Substances 0.000 description 3
- 238000002347 injection Methods 0.000 description 3
- 210000004731 jugular vein Anatomy 0.000 description 3
- 230000009707 neogenesis Effects 0.000 description 3
- 239000002504 physiological saline solution Substances 0.000 description 3
- 210000003462 vein Anatomy 0.000 description 3
- LPXQRXLUHJKZIE-UHFFFAOYSA-N 8-azaguanine Chemical compound NC1=NC(O)=C2NN=NC2=N1 LPXQRXLUHJKZIE-UHFFFAOYSA-N 0.000 description 2
- 241000282472 Canis lupus familiaris Species 0.000 description 2
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 2
- 102000003974 Fibroblast growth factor 2 Human genes 0.000 description 2
- 102100024785 Fibroblast growth factor 2 Human genes 0.000 description 2
- 108010051696 Growth Hormone Proteins 0.000 description 2
- 102100038803 Somatotropin Human genes 0.000 description 2
- 102000004142 Trypsin Human genes 0.000 description 2
- 108090000631 Trypsin Proteins 0.000 description 2
- 230000002491 angiogenic effect Effects 0.000 description 2
- 210000001367 artery Anatomy 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 230000010261 cell growth Effects 0.000 description 2
- 210000004748 cultured cell Anatomy 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 239000011259 mixed solution Substances 0.000 description 2
- 210000004197 pelvis Anatomy 0.000 description 2
- 238000011282 treatment Methods 0.000 description 2
- 239000012588 trypsin Substances 0.000 description 2
- 102000009088 Angiopoietin-1 Human genes 0.000 description 1
- 108010048154 Angiopoietin-1 Proteins 0.000 description 1
- 102000009075 Angiopoietin-2 Human genes 0.000 description 1
- 108010048036 Angiopoietin-2 Proteins 0.000 description 1
- 208000000575 Arteriosclerosis Obliterans Diseases 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 229920001917 Ficoll Polymers 0.000 description 1
- 101001052035 Homo sapiens Fibroblast growth factor 2 Proteins 0.000 description 1
- 101000835998 Homo sapiens SRA stem-loop-interacting RNA-binding protein, mitochondrial Proteins 0.000 description 1
- 101000808011 Homo sapiens Vascular endothelial growth factor A Proteins 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 1
- 102100025491 SRA stem-loop-interacting RNA-binding protein, mitochondrial Human genes 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 206010000891 acute myocardial infarction Diseases 0.000 description 1
- 210000000709 aorta Anatomy 0.000 description 1
- 210000002798 bone marrow cell Anatomy 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 229910002092 carbon dioxide Inorganic materials 0.000 description 1
- 239000001569 carbon dioxide Substances 0.000 description 1
- 238000003320 cell separation method Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 210000004351 coronary vessel Anatomy 0.000 description 1
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 1
- 235000015220 hamburgers Nutrition 0.000 description 1
- 102000058223 human VEGFA Human genes 0.000 description 1
- 208000028867 ischemia Diseases 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 238000000034 method Methods 0.000 description 1
- 238000001000 micrograph Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 230000002107 myocardial effect Effects 0.000 description 1
- 210000004165 myocardium Anatomy 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 102220240796 rs553605556 Human genes 0.000 description 1
- 210000002027 skeletal muscle Anatomy 0.000 description 1
- 210000001562 sternum Anatomy 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 210000003606 umbilical vein Anatomy 0.000 description 1
- 210000000689 upper leg Anatomy 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Landscapes
- Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
Description
【０００１】[0001]
【発明の属する技術分野】本発明は、種々の虚血性疾患
の治療に有用な血管新生促進剤に関する。[0001] The present invention relates to an angiogenesis promoting agent useful for treating various ischemic diseases.
【０００２】[0002]
【従来の技術及び発明が解決しようとする課題】心筋梗
塞、狭心症、脳梗塞、血管性痴呆、閉塞性動脈硬化症、
バージャー病などに代表される心臓、脳及び末梢におけ
る虚血性疾患の重症例では、現存する治療法はほとんど
無効である。虚血部位で新しく血管を生成させれば、虚
血を改善させることができるとの考えから、血管新生剤
として各種の細胞増殖因子が検討されている。これら従
来の血管新生剤では、毛細血管の新生は計れるものの、
細動脈以上の血管の充分な新生を計れないため、虚血部
位に充分な血液量を送ることができなかった。従って、
本発明は、毛細血管だけでなく細動脈を著明に新生し、
虚血部位に充分な血液を供給し得る虚血性疾患に有効な
血管新生促進剤を提供することにある。2. Description of the Related Art Myocardial infarction, angina pectoris, cerebral infarction, vascular dementia, arteriosclerosis obliterans,
In severe cases of ischemic disease of the heart, brain and periphery, such as Burger's disease, existing treatments are almost ineffective. Various cell growth factors have been studied as angiogenic agents from the viewpoint that ischemia can be improved by generating new blood vessels at the ischemic site. Although these conventional angiogenic agents can measure the formation of capillaries,
A sufficient amount of blood could not be sent to the ischemic site because sufficient neogenesis of blood vessels beyond arterioles could not be measured. Therefore,
The present invention remarkably renews not only capillaries but also arterioles,
An object of the present invention is to provide an angiogenesis promoting agent effective for ischemic diseases capable of supplying sufficient blood to an ischemic site.
【０００３】[0003]
【課題を解決するための手段】そこで本発明者は、各種
血管成長因子だけでは充分な新生ができない細動脈の新
生促進作用について種々検討してきた結果、全く意外に
も血管内皮細胞又は骨髄単核球細胞と血管成長因子類又
はレチノイン酸とを組み合せて投与すれば、細動脈が顕
著に新生されることを見出し、本発明を完成するに至っ
た。The present inventors have conducted various studies on the effect of promoting arteriolar neogenesis that cannot be sufficiently generated by various vascular growth factors alone. The present inventors have found that arteriolar arteries are remarkably regenerated by administering a combination of sphere cells and vascular growth factors or retinoic acid, thereby completing the present invention.
【０００４】すなわち、本発明は、（Ａ）血管内皮細
胞、並びに（Ｂ）血管成長因子類及びレチノイン酸から
選ばれる１種又は２種以上を含有する血管新生促進剤及
び虚血性疾患治療剤を提供するものである。That is, the present invention provides an angiogenesis promoting agent and a therapeutic agent for ischemic disease containing (A) vascular endothelial cells and (B) one or more selected from vascular growth factors and retinoic acid. To provide.
【０００５】[0005]
【発明の実施の形態】本発明に用いられる（Ａ）血管内
皮細胞及び骨髄単核球細胞は、培養細胞及び非培養細胞
のいずれも使用することができる。血管内皮細胞は、動
脈、大動脈、静脈及び臍帯静脈のいずれの血管の内皮細
胞でもよい。血管から内皮細胞を分離するには、例えば
血管内壁をトリプシン等のプロテアーゼ処理して遊離す
る細胞を採取すればよい。骨髄単核球細胞は骨髄液から
常法に従って分離できる。骨髄液は、胸骨又は骨盤から
採取する。また、分離した細胞は、必要に応じて培養し
て用いてもよい。なお、これらの血管内皮細胞や骨髄単
核球細胞は、患者本人由来又は移植適合性のある他人由
来のいずれでもよいが、患者本人由来のものが好まし
い。BEST MODE FOR CARRYING OUT THE INVENTION As (A) vascular endothelial cells and bone marrow mononuclear cells used in the present invention, both cultured cells and non-cultured cells can be used. The vascular endothelial cells may be endothelial cells of any blood vessels, such as arteries, aorta, veins and umbilical veins. In order to separate the endothelial cells from the blood vessel, for example, cells that are released by treating the inner wall of the blood vessel with a protease such as trypsin may be collected. Bone marrow mononuclear cells can be separated from bone marrow fluid by a conventional method. Bone marrow fluid is collected from the sternum or pelvis. Further, the separated cells may be cultured and used as needed. In addition, these vascular endothelial cells and bone marrow mononuclear cells may be derived from the patient himself or from a transplant-compatible other person, but are preferably derived from the patient himself.
【０００６】また、本発明に用いられる（Ｂ）血管成長
因子類としては、繊維芽細胞成長因子（ＦＧＦ）〔塩基
性ＦＧＦ及び酸性ＦＧＦを含む〕、血管内皮細胞成長因
子（ＶＥＧＦ）〔血小板由来が好ましい〕、肝細胞成長
因子（ＨＧＦ）、アンギオポエチン（アンギオポエチン
−１及びアンギオポエチン−２を含む）、血小板由来成
長因子（ＰＤＧＦ）、インシュリン様成長因子（ＩＧ
Ｆ）、胎児型平滑筋ミオシン重鎖（ＳＭｅｍｂ）、成長
ホルモン（ＧＨ）もしくはその類縁物質、その他の細胞
増殖促進因子等が挙げられる。これらの血管成長因子類
は１種を用いてもよいし、２種以上を混合して用いても
よい。更に、レチノイン酸とこれらの血管成長因子類と
を組み合せて投与すると、血管新生促進効果が更に増強
されるので、特に好ましい。The (B) vascular growth factors used in the present invention include fibroblast growth factor (FGF) (including basic FGF and acidic FGF), vascular endothelial cell growth factor (VEGF) [platelet-derived Is preferred), hepatocyte growth factor (HGF), angiopoietin (including angiopoietin-1 and angiopoietin-2), platelet-derived growth factor (PDGF), insulin-like growth factor (IG
F), fetal smooth muscle myosin heavy chain (SMemb), growth hormone (GH) or a related substance thereof, and other cell growth promoting factors. These vascular growth factors may be used alone or as a mixture of two or more. Furthermore, it is particularly preferable to administer retinoic acid in combination with these vascular growth factors, because the effect of promoting angiogenesis is further enhanced.
【０００７】本発明においては、前記成分（Ａ）と
（Ｂ）とを予め混合するか、又は投与直前に混合して投
与することにより、虚血疾患の虚血部位に充分な血液を
供給するのに必要な細動脈の新生を顕著に促進すること
ができる。従って、本発明の血管新生促進剤は、心筋梗
塞、狭心症、脳梗塞、血管性痴呆、閉塞性動脈硬化症な
どの虚血性疾患の治療剤として有用である。In the present invention, sufficient blood is supplied to the ischemic site of ischemic disease by pre-mixing the components (A) and (B) or mixing them immediately before administration and administering them. Can significantly promote the neogenesis of the necessary arterioles. Therefore, the angiogenesis promoting agent of the present invention is useful as a therapeutic agent for ischemic diseases such as myocardial infarction, angina pectoris, cerebral infarction, vascular dementia, and atherosclerosis obliterans.
【０００８】本発明の医薬は、血管内投与、筋肉内投与
あるいは局所投与するのが好ましく、更には心筋内投
与、心膜腔内投与、脳硬膜下投与、クモ膜下投与、脳実
質内投与、四肢骨格筋内投与等の虚血部への局所投与が
特に好ましい。これら注射剤は、前記有効成分を注射
用、生理食塩水、緩衝液、リン酸緩衝生理食塩水（ＰＢ
Ｓ）等に添加混合することにより調製するのが好まし
い。The medicament of the present invention is preferably administered intravascularly, intramuscularly or locally, and further administered intracardiacly, intrapericardially, subdurally, subarachnoidally, intrathecally. Local administration to the ischemic area, such as administration and intralimb skeletal muscle administration, is particularly preferred. These injections contain the active ingredient for injection, physiological saline, buffer, phosphate buffered saline (PB
It is preferably prepared by adding and mixing to S) and the like.
【０００９】本発明の医薬の投与量は、疾患、投与経
路、処置期間及びその他の要因によって左右されるが、
一般に前記成分（Ａ）として１日当り約１万〜５００万
cells、特に約５０万〜１００万cellsが好ましい。ま
た、成分（Ｂ）のうち、血管成長因子の投与量は１日当
たり約１〜１０００μｇ、特に１０〜５００μｇが好ま
しい。また成分（Ｂ）のうち、レチノイン酸の投与量は
１日当り０．１〜５００mg、特に１〜１００mgが好まし
い。[0009] The dose of the medicament of the present invention depends on the disease, administration route, treatment period and other factors.
Generally, about 10,000 to 5,000,000 per day as the component (A)
Cells, especially about 500,000 to 1,000,000 cells are preferred. Further, among the components (B), the dose of vascular growth factor is preferably about 1 to 1000 μg, particularly preferably 10 to 500 μg per day. Further, among the component (B), the dose of retinoic acid is preferably 0.1 to 500 mg, particularly preferably 1 to 100 mg per day.
【００１０】[0010]
【実施例】次に実施例を挙げて本発明を更に詳細に説明
するが、本発明はこれに何ら限定されるものではない。Next, the present invention will be described in more detail with reference to examples, but the present invention is not limited thereto.
【００１１】実施例１ （１）血管内皮細胞分離法 １）頸静脈の分枝を結札し、両端を縛り、無菌的に摘出
する。 ２）ＰＢＳ１mLを注入し、残存血液を洗い出す。 ３）０．１％トリプシン／０．０２％ＥＤＴＡを頸静脈
内に注入し、炭酸ガスインキュベーターで３７℃で１０
分間反応させる。 ４）頸静脈内液を採取し、８０％Ｍ１９９メジウム、２
０％ＰＢＳ２０mLに懸濁し、１０００Ｇで５分間遠心す
る。 ５）洗浄を２回繰り返し、１mLの血管内皮細胞懸濁液
（約１０5〜１０6cells／mL）を得る。Example 1 (1) Isolation method of vascular endothelial cells 1) Branches of the jugular vein are ligated, both ends are tied, and aseptically removed. 2) Inject 1 mL of PBS and wash out remaining blood. 3) 0.1% trypsin / 0.02% EDTA was injected into the jugular vein, and 10% at 37 ° C. in a carbon dioxide incubator.
Let react for minutes. 4) The fluid in the jugular vein was collected, and 80% M199 medium, 2%
Suspend in 20 mL of 0% PBS and centrifuge at 1000 G for 5 minutes. 5) Repeat washing twice to obtain 1 mL of vascular endothelial cell suspension (about 10 5 to 10 6 cells / mL).
【００１２】（２）骨髄細胞からの単核球分離法 骨髄穿刺針を用い、麻酔ビーグル犬の骨盤ないしは大腿
部を穿刺し、２０％ＰＢＳを加えたＨＡＮＫＳ−ＢＢＳ
液１０mLをあらかじめ入れた２０mL注射筒で骨髄を１０
mL吸引する。ついで、試験管にＦｉｃｏｌｌ液を半量入
れておき、そのうえに骨髄液を重層する。これを、１５
００〜３０００rpmで２０分遠心分離する。これによ
り、単核球層が分離されるので、それを吸引する。吸引
した層を２０％ＰＢＳを加えたＨＡＮＫＳ−ＢＢＳ液に
入れ、同様に遠心分離する。上清を吸引する。これを、
２回くりかえすと単核球が得られる。１mLの栄養液に浮
遊させると１０5〜３×１０5個／mLの生きた単核球が得
られる。(2) Mononuclear Cell Separation Method from Bone Marrow Cells Hanks-BBS with 20% PBS added thereto by puncturing the pelvis or thigh of an anesthetized beagle dog using a bone marrow puncture needle.
10 ml of bone marrow with a 20 mL syringe containing 10 mL of liquid
Aspirate mL. Next, half of the Ficoll solution is placed in a test tube, and the bone marrow fluid is overlaid thereon. This is 15
Centrifuge at 00-3000 rpm for 20 minutes. As a result, the mononuclear cell layer is separated and sucked. The aspirated layer is placed in a HANKS-BBS solution containing 20% PBS, and centrifuged similarly. Aspirate the supernatant. this,
Repeat twice to obtain mononuclear cells. When suspended in 1 mL of nutrient solution, 10 5 to 3 × 10 5 cells / mL of living mononuclear cells are obtained.
【００１３】（３）塩基性線維芽細胞成長因子（ｂＦＧ
Ｆ）：１００μｇヒト型ｂＦＧＦ含有生理塩水溶液１mL （４）血管内皮細胞成長因子（ＶＥＧＦ）：１０μｇヒ
ト型ＶＥＧＦ含有生理食塩水１mL （５）レチノイン酸：レチノイン酸１mg含有の０．１％
ＤＭＳＯ溶液１mL 上記１）の血管内皮細胞懸濁液又は（２）の骨髄単核球
懸濁液に（３）、（４）及び／又は（５）を混合し、本
発明血管新生促進剤を得る。(3) Basic fibroblast growth factor (bFG)
F): 1 mL of a physiological saline solution containing 100 μg human bFGF (4) Vascular endothelial cell growth factor (VEGF): 1 mL of physiological saline containing 10 μg human VEGF (5) Retinoic acid: 0.1% containing 1 mg of retinoic acid
1 mL of DMSO solution The above (1) vascular endothelial cell suspension or (2) bone marrow mononuclear cell suspension was mixed with (3), (4) and / or (5), and the angiogenesis promoting agent of the present invention was added. obtain.
【００１４】実施例２ （１）心筋梗塞モデルの作製法と投与法 麻酔ビーグル犬を用い、経カテーテル的に冠動脈前下行
枝を閉塞して急性心筋梗塞を作製した。ついで、実施例
１で得た混合液を梗塞領域の心筋内に注入した。覚醒
後、２週間飼育し、再度造影を行い、屠殺し、心臓をホ
ルマリン固定し、梗塞部位をアザン染色し、血管数を調
べた。Example 2 (1) Method for Preparing and Administering Myocardial Infarction Model Using an anesthetized beagle dog, the anterior descending coronary artery was occluded transcatheterly to produce acute myocardial infarction. Next, the mixed solution obtained in Example 1 was injected into the myocardium in the infarct region. After awakening, they were reared for 2 weeks, imaged again, sacrificed, the heart was fixed in formalin, the infarct site was stained with Azan, and the number of blood vessels was examined.
【００１５】（２）血管数の計測 ２週間後、屠殺し、梗塞部位をホルマリン固定し、アザ
ン染色を行い、単位面積当りの細胞脈数を数えた。(2) Measurement of Number of Blood Vessels Two weeks later, the animals were sacrificed, the infarct site was fixed in formalin, stained with Azan, and the number of cell veins per unit area was counted.
【００１６】（３）結果 図１に、血管内皮細胞、ｂＦＧＦ、ＶＥＧＦ及びレチノ
イン酸混合液投与の実例を示す（左：２００倍、右：４
００倍）。その結果、明らかに著名な細動脈の増加が見
られた。(3) Results FIG. 1 shows an example of administration of a mixed solution of vascular endothelial cells, bFGF, VEGF and retinoic acid (left: 200 times, right: 4 times).
00 times). As a result, a marked increase in arterioles was clearly observed.
【００１７】また、単位面積当りの細胞脈数を表１に示
す。Table 1 shows the number of cell veins per unit area.
【００１８】[0018]
【表１】 [Table 1]
【００１９】表１より、血管内皮細胞と血管成長因子類
及び／又はレチノイン酸を組み合せて用いると、細動脈
が顕著に新生されることがわかる。From Table 1, it can be seen that arterioles are significantly regenerated when vascular endothelial cells are used in combination with vascular growth factors and / or retinoic acid.
【００２０】実施例３ 骨髄単核球細胞にｂＦＧＦ、ＶＥＧＦ、レチノイン酸を
添加した液を用いて、実施例２と同様にして細動脈の増
加を観察した。結果を表２に示す。Example 3 An increase in arterioles was observed in the same manner as in Example 2 using a solution obtained by adding bFGF, VEGF and retinoic acid to bone marrow mononuclear cells. Table 2 shows the results.
【００２１】[0021]
【表２】 [Table 2]
【００２２】表２より、骨髄単核球細胞と血管成長因子
類及び／又はレチノイン酸を組み合せて用いると、細動
脈が顕著に新生されることがわかる。From Table 2, it can be seen that arterioles are remarkably regenerated when bone marrow mononuclear cells are used in combination with vascular growth factors and / or retinoic acid.
【００２３】[0023]
【発明の効果】本発明の血管新生促進剤を投与すると、
虚血性疾患の虚血部位に充分な血液を供給するのに必要
な細動脈の新生を顕著に促進することができる。従っ
て、本発明の血管新生促進剤は、心筋梗塞、狭心症、脳
梗塞、血管性痴呆、閉塞性動脈硬化症などの虚血性疾患
の治療剤として有用である。When the angiogenesis promoting agent of the present invention is administered,
It can significantly promote the formation of arterioles necessary to supply sufficient blood to the ischemic site of ischemic disease. Therefore, the angiogenesis promoting agent of the present invention is useful as a therapeutic agent for ischemic diseases such as myocardial infarction, angina pectoris, cerebral infarction, vascular dementia, and atherosclerosis obliterans.
【図１】本発明血管新生促進剤注入後の心筋組織の顕微
鏡写真を示す（左：２００倍、右：４００倍）。FIG. 1 shows a micrograph of myocardial tissue after injection of the angiogenesis promoting agent of the present invention (left: 200 ×, right: 400 ×).
───────────────────────────────────────────────────── フロントページの続き (51)Int.Cl.7 識別記号 ＦＩ テーマコート゛(参考） Ａ６１Ｐ 9/00 Ａ６１Ｋ 37/02 9/10 37/36 Ｆターム(参考） 4C084 AA02 BA44 DB22 DB52 DB54 DB55 DB58 DB62 DC50 MA02 NA05 ZA361 ZC032 ZC232 ZC752 4C087 AA01 AA02 BB63 CA04 MA02 NA05 ZA36 ZA54 ZC75 4C206 AA01 AA02 DA12 MA02 MA04 NA05 ZA36 ZA54 ZC75 ──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. 7 Identification symbol FI Theme coat ゛ (Reference) A61P 9/00 A61K 37/02 9/10 37/36 F-term (Reference) 4C084 AA02 BA44 DB22 DB52 DB54 DB55 DB58 DB62 DC50 MA02 NA05 ZA361 ZC032 ZC232 ZC752 4C087 AA01 AA02 BB63 CA04 MA02 NA05 ZA36 ZA54 ZC75 4C206 AA01 AA02 DA12 MA02 MA04 NA05 ZA36 ZA54 ZC75
Claims (4)
胞、並びに（Ｂ）血管成長因子類及びレチノイン酸から
選ばれる１種又は２種以上を含有する血管新生促進剤。1. An angiogenesis promoting agent comprising (A) vascular endothelial cells or bone marrow mononuclear cells, and (B) one or more selected from vascular growth factors and retinoic acid.
子、血管内皮細胞成長因子、肝細胞成長因子、血小板由
来成長因子、インシュリン様成長因子、胎児型平滑筋ミ
オシン重鎖、成長ホルモン類及びアンギオポエチンから
選ばれる１種又は２種以上である請求項１記載の血管新
生促進剤。2. A blood vessel growth factor comprising fibroblast growth factor, vascular endothelial cell growth factor, hepatocyte growth factor, platelet-derived growth factor, insulin-like growth factor, fetal smooth muscle myosin heavy chain, growth hormones and 2. The angiogenesis promoting agent according to claim 1, wherein the agent is one or more selected from angiopoietin.
胞、並びに（Ｂ）血管成長因子類及びレチノイン酸から
選ばれる１種又は２種以上を含有する虚血性疾患治療
剤。3. A therapeutic agent for ischemic disease containing (A) vascular endothelial cells or bone marrow mononuclear cells, and (B) one or more selected from vascular growth factors and retinoic acid.
子、血管内皮細胞成長因子、肝細胞成長因子、血小板由
来成長因子、インシュリン様成長因子、胎児型平滑筋ミ
オシン重鎖、成長ホルモン類及びアンギオポエチンから
選ばれる１種又は２種以上である請求項１記載の虚血性
疾患治療剤。4. A blood vessel growth factor comprising fibroblast growth factor, vascular endothelial cell growth factor, hepatocyte growth factor, platelet-derived growth factor, insulin-like growth factor, fetal smooth muscle myosin heavy chain, growth hormones, The therapeutic agent for ischemic disease according to claim 1, which is one or more selected from angiopoietin.
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| JP2000207800A JP2002020298A (en) | 2000-07-10 | 2000-07-10 | Neovascularization promoter |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1415662A1 (en) * | 2002-10-28 | 2004-05-06 | Biosense, Inc. | Homing autologous cells to a target zone in tissue for delivery of translocation stimulators |
| EP1415660A1 (en) * | 2002-10-28 | 2004-05-06 | Biosense, Inc. | Homing donor cells to a target zone in tissue for delivery of translocation stimulators |
| JP2004516242A (en) * | 2000-07-26 | 2004-06-03 | サイムド ライフ システムズ， インコーポレイテッド | Therapeutic angiogenesis by transplanting bone marrow-derived cells into ischemic tissue of the heart and skeletal muscle |
| WO2004100972A1 (en) * | 2003-05-16 | 2004-11-25 | Kyowa Hakko Kogyo Co., Ltd. | Preventive and/or remedy for diseases accompanied by tissue destruction |
| JP2006321740A (en) * | 2005-05-18 | 2006-11-30 | Kanazawa Univ | Post-ischemic angiogenesis promoter |
| WO2010116665A1 (en) * | 2009-04-07 | 2010-10-14 | 国立大学法人旭川医科大学 | New revascularization cells derived from mononuclear cells, and method of inducing differentiation thereof |
| JP2015071568A (en) * | 2013-10-03 | 2015-04-16 | 国立大学法人 千葉大学 | Preventive and / or therapeutic agent for cerebral circulation disorder |
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2000
- 2000-07-10 JP JP2000207800A patent/JP2002020298A/en active Pending
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2004516242A (en) * | 2000-07-26 | 2004-06-03 | サイムド ライフ システムズ， インコーポレイテッド | Therapeutic angiogenesis by transplanting bone marrow-derived cells into ischemic tissue of the heart and skeletal muscle |
| EP1415662A1 (en) * | 2002-10-28 | 2004-05-06 | Biosense, Inc. | Homing autologous cells to a target zone in tissue for delivery of translocation stimulators |
| EP1415660A1 (en) * | 2002-10-28 | 2004-05-06 | Biosense, Inc. | Homing donor cells to a target zone in tissue for delivery of translocation stimulators |
| JP2004149533A (en) * | 2002-10-28 | 2004-05-27 | Biosense Inc | Homing of donor cell to specified target zone in tissue by active therapeutic agent or substance |
| JP2004149532A (en) * | 2002-10-28 | 2004-05-27 | Biosense Inc | Homing of autologous cell to specified target zone in tissue by active therapeutic method or substance |
| WO2004100972A1 (en) * | 2003-05-16 | 2004-11-25 | Kyowa Hakko Kogyo Co., Ltd. | Preventive and/or remedy for diseases accompanied by tissue destruction |
| JP2006321740A (en) * | 2005-05-18 | 2006-11-30 | Kanazawa Univ | Post-ischemic angiogenesis promoter |
| WO2010116665A1 (en) * | 2009-04-07 | 2010-10-14 | 国立大学法人旭川医科大学 | New revascularization cells derived from mononuclear cells, and method of inducing differentiation thereof |
| US8951795B2 (en) | 2009-04-07 | 2015-02-10 | National University Corporation Asahikawa Medical University | Revascularization cells derived from mononuclear cells, and method of inducing differentiation thereof |
| JP2015071568A (en) * | 2013-10-03 | 2015-04-16 | 国立大学法人 千葉大学 | Preventive and / or therapeutic agent for cerebral circulation disorder |
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