JP5271705B2 - 生体組織のヘパリンコーティング - Google Patents
生体組織のヘパリンコーティング Download PDFInfo
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- JP5271705B2 JP5271705B2 JP2008520221A JP2008520221A JP5271705B2 JP 5271705 B2 JP5271705 B2 JP 5271705B2 JP 2008520221 A JP2008520221 A JP 2008520221A JP 2008520221 A JP2008520221 A JP 2008520221A JP 5271705 B2 JP5271705 B2 JP 5271705B2
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- heparin
- avidin
- biotin
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- biological tissue
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- HTTJABKRGRZYRN-UHFFFAOYSA-N Heparin Chemical compound OC1C(NC(=O)C)C(O)OC(COS(O)(=O)=O)C1OC1C(OS(O)(=O)=O)C(O)C(OC2C(C(OS(O)(=O)=O)C(OC3C(C(O)C(O)C(O3)C(O)=O)OS(O)(=O)=O)C(CO)O2)NS(O)(=O)=O)C(C(O)=O)O1 HTTJABKRGRZYRN-UHFFFAOYSA-N 0.000 title claims description 71
- 229920000669 heparin Polymers 0.000 title claims description 70
- 229960002897 heparin Drugs 0.000 title claims description 70
- 239000011248 coating agent Substances 0.000 title claims description 21
- 238000000576 coating method Methods 0.000 title claims description 21
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 claims description 68
- 108090001008 Avidin Proteins 0.000 claims description 43
- 229960002685 biotin Drugs 0.000 claims description 38
- 239000011616 biotin Substances 0.000 claims description 38
- 235000020958 biotin Nutrition 0.000 claims description 34
- 239000003153 chemical reaction reagent Substances 0.000 claims description 26
- 238000000034 method Methods 0.000 claims description 17
- 125000003396 thiol group Chemical class [H]S* 0.000 claims description 10
- 102000004169 proteins and genes Human genes 0.000 claims description 9
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- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims description 4
- 125000000524 functional group Chemical group 0.000 claims description 4
- 238000000338 in vitro Methods 0.000 claims description 3
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- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims 2
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- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 7
- 230000008827 biological function Effects 0.000 description 7
- 230000008859 change Effects 0.000 description 7
- 239000011780 sodium chloride Substances 0.000 description 7
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- 238000003380 quartz crystal microbalance Methods 0.000 description 6
- JWDFQMWEFLOOED-UHFFFAOYSA-N (2,5-dioxopyrrolidin-1-yl) 3-(pyridin-2-yldisulfanyl)propanoate Chemical compound O=C1CCC(=O)N1OC(=O)CCSSC1=CC=CC=N1 JWDFQMWEFLOOED-UHFFFAOYSA-N 0.000 description 5
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- JJGWLCLUQNFDIS-GTSONSFRSA-M sodium;1-[6-[5-[(3as,4s,6ar)-2-oxo-1,3,3a,4,6,6a-hexahydrothieno[3,4-d]imidazol-4-yl]pentanoylamino]hexanoyloxy]-2,5-dioxopyrrolidine-3-sulfonate Chemical compound [Na+].O=C1C(S(=O)(=O)[O-])CC(=O)N1OC(=O)CCCCCNC(=O)CCCC[C@H]1[C@H]2NC(=O)N[C@H]2CS1 JJGWLCLUQNFDIS-GTSONSFRSA-M 0.000 description 4
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- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical compound SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 description 2
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- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 2
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- UVGHPGOONBRLCX-NJSLBKSFSA-N (2,5-dioxopyrrolidin-1-yl) 6-[5-[(3as,4s,6ar)-2-oxo-1,3,3a,4,6,6a-hexahydrothieno[3,4-d]imidazol-4-yl]pentanoylamino]hexanoate Chemical compound C([C@H]1[C@H]2NC(=O)N[C@H]2CS1)CCCC(=O)NCCCCCC(=O)ON1C(=O)CCC1=O UVGHPGOONBRLCX-NJSLBKSFSA-N 0.000 description 1
- 206010053567 Coagulopathies Diseases 0.000 description 1
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- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
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- -1 heparin sulphates Chemical class 0.000 description 1
- 230000005661 hydrophobic surface Effects 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 208000028867 ischemia Diseases 0.000 description 1
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- 230000017423 tissue regeneration Effects 0.000 description 1
- 229950003937 tolonium Drugs 0.000 description 1
- HNONEKILPDHFOL-UHFFFAOYSA-M tolonium chloride Chemical compound [Cl-].C1=C(C)C(N)=CC2=[S+]C3=CC(N(C)C)=CC=C3N=C21 HNONEKILPDHFOL-UHFFFAOYSA-M 0.000 description 1
- 230000002792 vascular Effects 0.000 description 1
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Description
本発明は、ヘパリンコーティングを有する生体組織を提供するin vitroにおける方法に関する。
哺乳動物の先天性免疫系は、内因性構造物と外因性構造物とを区別するように設計される。外因性構造物が露呈されるとすぐに、それらは先天性免疫系によって認識され、そして多くの防御系が誘導、開始され、これらは凝固および炎症の活性化の促進をもたらす。血管は最外層でヘパラン硫酸(ヘパリン様物質)を発現する内皮細胞で覆われている。ヘパリンと同様に、ヘパラン硫酸はアンチトロンビンの阻害作用を促進することができる糖質領域を提示する。例えば、酸素の欠乏(虚血)、機能的損傷等によって開始する病理過程により、病変組織の化学構造および生物学的構造が変化し、その組織が外因性と認識され得る。組織工学の領域において、細胞(例えば、ランゲルハンス島)の単離および処理は、そのような細胞が血液に輸液される場合に外因性構造物の暴露をもたらし、最終的には、即時性血液媒介炎症反応(IBMIR)のために、細胞の生物学的機能の損失および崩壊さえももたらす(Diabetes, vol. 48, 1907-1914, 1999)。
特許出願PCT/SE00/00223は、負電荷のヘパリン複合体と膵島表面の正電荷との間のイオン相互作用に基本的に依存した1ステップの手順によって、ヘパリン複合体で表面がコーティングされたランゲルハンス島を提供する方法を開示する。1ステップの手順は魅力的ではあるが、最近の評価は、この比較的単純なアプローチで再現性のある結果を得ることが困難であるだろうことを指摘しており、それはおそらく、単離した膵島の表面特性が調製物によって変化し得るという事実による。従って、再現性が向上し、幅広い生体組織に適用可能であるような性質の手順が非常に望まれている。
外因性の生体構造物の投与(つまり、ランゲルハンス島を血流へ)後の先天性免疫系の不要な活性化についての上記の危険性を考慮して、先天性免疫系を活性化することなく生体機能を保持する膵島のコーティングが望ましい。
本出願の目的のために、以下の定義を定める。生体組織は、単一細胞、細胞の集積、細胞群、臓器の一部および臓器全体を指す。用語「層」は、1分子の厚さである単一の連続した層または膜を含む分子単層と定義する。
以下の実施例は、コーティングプロトコルにおいてアビジンを含むことの重要性を説明するために計画された。PVC管によって例示される人工表面を、生体基質を再現するためにアルブミンでコーティングした。
ヒトランゲルハンス島を、標準的な手順に従って単離し、そして培養液中に保持した。まず、膵島の一部分をビオチン(EZ-Link(商標)Sulfo-NHS-LC-Biotin, Perbio, 1 mg/ml)により60分間室温でインキュベーションし、そして次に、3倍容の新規の培地で洗浄した。次に、膵島をアビジン(1 mg/ml)中で30分間室温でインキュベーションし、次いで再び3倍容の培地で洗浄した。最後のインキュベーションは、Corline(Sweden)のヘパリン複合体(1 mg/ml)を用いて、60分間室温で実施し、そして3倍容の培地で最後の洗浄を行った。蛍光標識化アンチトロンビンで染色した後、共焦点顕微鏡を用いてヘパリン修飾化膵島を非修飾化膵島と比較した。蛍光標識化アンチトロンビンの密着した層が、図2に示すように、ヘパリン修飾化膵島において観察された。
ヒトランゲルハンス島は、標準的な手順に従って単離し、そして培養液中に保持し、先に実施例2に記載したようにして、3つの異なるタイプのビオチン試薬(EZ-Link(商標)Sulfo-NHS-LC-BiotinまたはEZ-Link(商標)Sulfo-NHS-LC-LC-BiotinまたはEZ-Link(商標)TFP-PEO-Biotin)を用い、Corline(Sweden)のヘパリン複合体を用いて修飾した。ヘパリン修飾化膵島を、別に記載される(Diabetes, 48, 1907-1914, 1999)Chandlerループモデルを用いて非修飾化膵島と比較した。つまり、抗凝血剤の非存在下ではあるが非修飾化膵島またはヘパリン修飾化膵島を添加したヒト血液を、37℃で最長1時間、ヘパリンでコーティングした管ループ内で振動させた。非修飾化膵島は肉眼で見える凝固を引き起こし、そして血小板数はChandlerループモデルのベースラインレベルの5%未満に減少した。使用したビオチン試薬に関係なく、へパリン修飾化膵島による肉眼で見える凝固は無く、IBMIRの程度を測定するためのパラメーターとして用いた血小板数およびトロンビン-アンチトロンビン複合体(TAT)の生成は、顕著に上昇した。
成体ブタ膵島を、標準的な手順に従って単離し、そして培養液中に保持し、先に実施例3に記載したように、Corline(Sweden)のヘパリン複合体を用いて修飾した。
提供された膵臓からヒト血管を切開して、生理食塩水中に保持した。血管を小さな内腔管に接続して、血管のかん流を促進した。これらをまずビオチン(EZ-Link(商標)Sulfo-NHS-LC-Biotin; 1 mg/ml)で30分間室温でインキュベーションし、次に3倍容の生理食塩水で洗浄した。次に血管をアビジン(1 mg/ml)で60分間室温でインキュベーションし、次に再び3倍容の生理食塩水で洗浄した。最後のインキュベーションは、Corline(Sweden)のヘパリン複合体(0.1 mg/ml)を用いて60分間室温で行い、3倍容の生理食塩水で最後の洗浄を行った。トルイジンブルーによる染色および共焦点顕微鏡による検査は、血管内腔表面におけるヘパリンの存在を確認した。
Claims (9)
- (a)ビオチンを生体組織へ結合させるステップであって、前記ビオチンが生体組織表面上の適切なタンパク質残基と化学結合を形成することができる官能基を含み、前記適切なタンパク質残基がチオール基、1級アミン基、およびカルボキシル基からなる群より選択されるステップと、
(b)アビジンをビオチン化生体組織へ結合させることにより、生体組織上にアビジン層を形成するステップと、
(c)アンチトロンビンと相互作用しアンチトロンビンの阻害作用を促進する能力を有するヘパリン試薬を、生体組織上に形成されたアビジン層に結合させて、前記組織にヘパリンコーティングを形成するステップと、
(d)洗浄して過剰のヘパリン試薬を除去するステップ
とを含む、ヘパリンコーティングを有する生体組織を提供するin vitroの方法。 - ビオチン、アビジンおよびヘパリンからなる群より選択される試薬の結合が、20〜37℃の温度で実施される、請求項1に記載の方法。
- ビオチン化ステップで使用するビオチンの濃度が0.01〜1 mg/mlの範囲内である、請求項1または2に記載の方法。
- アビジン結合ステップで使用するアビジンの濃度が0.01〜1 mg/mlの範囲内である、請求項1〜3のいずれか1項に記載の方法。
- ヘパリン結合ステップで使用するヘパリン試薬の濃度が0.01〜1 mg/mlの範囲内である、請求項1〜4のいずれか1項に記載の方法。
- ステップa)、b)およびc)の1つ以上の後に、過剰な試薬を除去するために洗浄ステップが実施される、請求項1〜5のいずれか1項に記載の方法。
- ビオチン層、アビジン層およびヘパリン層を含む、in vitroで培養された生体組織をコーティングするためのヘパリンコーティング用組成物であって、
ここで、ビオチン層が、生体組織表面上の適切なタンパク質残基と化学結合を形成することができる官能基を含むビオチンであって、前記適切なタンパク質残基がチオール基、1級アミン基、およびカルボキシル基からなる群より選択される、ビオチンを含み、
アビジン層が親和性によってビオチン層に結合したアビジンを含み、そして
ヘパリン層が親和性によってアビジン層に結合したヘパリン試薬を含む、
ヘパリンコーティング用組成物。 - ヘパリンでコーティングされたin vitroで培養された生体組織であって、ヘパリンがビオチンおよびアビジンを介して組織表面に結合した、生体組織。
- ヘパリンでコーティングされた移植片であって、ヘパリンがビオチンおよびアビジンを介して移植片表面に結合した、移植片。
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