JP6129901B2 - 椎間板変性症の治療 - Google Patents
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Description
椎間板は、脊柱の長さの4分の1を構成する。環椎(C1)、軸椎(C2)及び尾骨の間には椎間板は存在しない。椎間板は血管を有しないため、必要な栄養素を拡散するのには終板に依存している。終板の軟骨層は、所定の位置で椎間板を固定している。
本発明は、プライミングした細胞を哺乳動物における椎間板領域に投与することであって、椎間板の変性を予防する又は遅延させることにより損傷した椎間板を治療する、投与することを包含する。プライミングした細胞は典型的には結合組織細胞であり、軟骨細胞又は線維芽細胞を含む。
一態様では、本発明は、対象のDNA配列を哺乳動物宿主の結合組織細胞に送達するex vivo及びin vivoでの技法を開示する。ex vivoの技法は、対象の遺伝子産物のin vivoでの発現を達成するための、標的結合組織細胞の培養、対象のDNA配列、DNAベクター又は他の送達媒体のin vitroでの結合組織細胞の中への形質移入、その後の改変した結合組織細胞の哺乳動物宿主の標的領域への移植を伴う。
プラスミドの構築
TGF−β1コード配列を含有する1.2−kb Bg1 II断片、及び3’末端の増殖ホルモンポリA部位をpMTMLVのBam HI部位中にサブクローニングすることにより、プラスミドpMTMLVβ1を作製した。pMTMLVベクターは、全gag配列及びenv配列並びに一部のΨパッケージ配列を欠失させることにより、レトロウイルスベクターMFGから得た。
穿刺後様々な週間隔で、ケタミン塩酸塩(25mg/kg)及びRompun(1mg/kg)の投与後に、放射線写真を取得した。各々の動物の放射線写真撮影時に一定レベルの麻酔を維持するのに、また各々の時点で同程度の筋弛緩を得るのに(これらは椎間板高さに影響を及ぼし得る)、細心の注意を払った。したがって、術前の放射線写真を、常にベースライン測定として使用した。脊椎をわずかに屈曲した位置に維持するようにも尽力した。脊椎の軸回転及びビーム広がり由来の誤差を減少させるために、放射線写真撮影は、側臥位にある各々の動物に対して少なくとも2回繰り返し、ビームをウサギ腸骨稜から4cmに集中させた。放射線写真は、画像取込ソフトウェアを使用してデジタル的にスキャンし、デジタル的に保存した。
デジタル化した放射線写真を使用して、椎体高さ及びIVD高さを含む測定値を、パブリック・ドメイン画像解析を使用して解析した。データをExcelソフトウェアにトランスポートし、IVD高さはLu et al. "Effects of chondroitinase ABC and chymopapain onspinal motion segment biomechanics. An in vivo biomechanical, radiologic, andhistologic canine study", Spine 1997;22:1828-34の方法を使用してDHIとして表現した。平均IVD高さ(DHI)は、IVDの前方部分、中間部分、及び後方部分から取得した測定値を平均し、それを隣接椎体高さの平均値で除算することにより算出した。注入した椎間板のDHIの変化は、%DHIとして表現し、術前のIVD高さの測定値に対して正規化した(%DHI=術後のDHI/術前のDHI×100)。被験体内標準偏差(Sw)は、以下の式を使用して算出した:
√(Σ(X1−X2)2/2n)
本研究では全てのウサギに対して、クワドラチャ四肢コイル受信器を有する0.3−T画像化装置(Airis II、4.0A版;Hitachi Medical System America, Inc.)を使用してMRI試験を行った。屠殺後、周辺軟部組織と共に脊柱を単離し、MRI解析に供した。サジタル面のT2強調セクションを以下の設定において取得した:TR(繰り返し時間)が4000ミリ秒でありTE(エコー時間)が120ミリ秒であるファストスピンエコー系列;256(h)×128(v)マトリクス;視野260;励起4。セクション厚みは2mmであり、ギャップは0mmであった。グレード1〜グレード4の信号強度の程度及び領域の変化に基づく修正トンプソン分類(1=正常、2=極めて小さい信号強度の減少、しかし高信号領域は明らかに狭くなっている、3=信号強度の中程度の減少、及び4=信号強度の重度の減少)を使用して、ブラインド状態の観察者が、MRIを評価した。コーヘンのカッパ相関係数により決定した、2つの評価に基づくMRIグレード評価の観察者内及び観察者間の信頼性の相関係数は、優れていた(それぞれK=0.98、0.90)。
損傷した椎間板の変性の予防
ニュージーランド白色雄性ウサギを使用した。開腹外科的技法を使用した。各々の動物において、腰椎における3つの椎間レベル:L2−3、L3−4、L4−5を実験的に処理し、又は対照として観察した。観察するウサギごとに、複数の部位/椎間板でバランスよくレベルに処理を割り当てた。被験体内デザイン、術前術後比較、椎間板レベルにわたる変化を対照として使用した。
非形質導入軟骨細胞を単独で、TGF−β1産生軟骨細胞を単独で使用する、又は混合細胞(ヒト軟骨細胞及びTGF−β1産生軟骨細胞)の注入による、ウサギにおける損傷した椎間板の変性の予防
実施例I〜実施例Vで使用した軟骨細胞の全ては、2歳未満の小児の指の硝子軟骨部分から取得された非椎間板軟骨細胞であり、幼若軟骨細胞である。
外科的前処置 注入処理
針穿刺 TGF−β1産生軟骨細胞(〜細胞数5×106)
針穿刺 混合:TGF−β1産生軟骨細胞
初代非形質導入ヒト軟骨細胞(比1:約3 5×106)
針穿刺 初代非形質導入ヒト軟骨細胞(〜5×106)
針穿刺 プライミングした非形質導入ヒト軟骨細胞(〜5×106) 針穿刺 DMEM
針穿刺 針穿刺のみ−注入なし
穿刺なし 非穿刺非処理対照
結果
結果は、本出願の図及び図の説明に示す通りである。単独の非形質導入軟骨細胞、単独の形質導入した軟骨細胞、単独のプライミングした軟骨細胞、又は形質導入した軟骨細胞と非形質導入軟骨細胞との混合物で処理した穿刺した椎間板により、媒体対照と比較して椎間板変性の予防又は遅延における有益な効果が示される。
混合細胞での処理は、ウサギに対して試験すると椎間変性抑制効果を有する。図1〜図4における様々な実験で効果が見られる。図1A〜図1Fは、損傷した椎間板の変性の緩徐化、遅延又は予防を示す。(A)は、外科処置前のウサギの脊椎のMRI放射線写真を示す。(B)は、外科処置4週間後のウサギの脊椎のMRI放射線写真を示し、ここで(i)L1/2の椎間板を損傷させてTGF−β1産生軟骨細胞を注入し、(ii)脊椎位置L2/3には穿刺及び処理は見られず、(iii)L3/4の椎間板を損傷させてTGF−β1産生軟骨細胞と非形質導入ヒト軟骨細胞との比1:3の混合物を注入した。矢印は、L1/2及びL3/4の椎間板領域を指す。(C)は、外科処置8週間後のウサギの脊椎のMRI放射線写真を示し、ここで(i)L1/2の椎間板を損傷させてTGF−β1産生軟骨細胞を注入し、(ii)脊椎位置L2/3は非穿刺及び非処理対照とし、(iii)L3/4の椎間板を損傷させてTGF−β1産生軟骨細胞と非形質導入ヒト軟骨細胞との比1:3の混合物を注入した。矢印は、L1/2及びL3/4の椎間板領域を指す。(D)は、上の(A)で説明したウサギのX線写真を示し、これは椎間板の形態、椎間板の変性又は再生のレベルを測定するための椎間板の椎間板高さ指数を取得するために使用される。(E)は、上の(B)で説明したウサギのX線写真を示し、これは椎間板の椎間板高さ指数を取得するために使用される。(F)は、上の(C)で説明したウサギのX線写真を示し、これは椎間板の椎間板高さ指数を取得するために使用される。
TGF−β1産生軟骨細胞での処理は、椎間変性抑制効果を有する。効果は図4A〜図4Dにおいて見られ、これらの図は損傷した椎間板の変性の緩徐化、遅延又は予防を示す。(A)は、外科処置前のウサギの脊椎のMRI放射線写真を示す。(B)は、外科処置4週間後のウサギの脊椎のMRI放射線写真を示し、ここで(i)L1/2の椎間板を損傷させてTGF−β1産生軟骨細胞と非形質導入ヒト軟骨細胞との比1:3の混合物を注入し、(ii)脊椎位置L2/3は非穿刺及び非処理対照とし、(iii)L3/4の椎間板を損傷させてTGF−β1産生軟骨細胞を注入した。矢印は、L1/2及びL3/4の椎間板領域を指す。(C)は、上の(A)で説明したウサギのX線写真を示し、これは椎間板の形態、椎間板の変性又は再生のレベルを測定するための椎間板の椎間板高さ指数を取得するために使用される。(D)は、上の(B)で説明したウサギのX線写真を示し、これは椎間板の椎間板高さ指数を取得するために使用される。
TGF−β1産生軟骨細胞での処理及び混合細胞での処理は、椎間変性抑制効果を有する。効果は図5A〜図5Dにおいて見られ、これらの図は損傷した椎間板の変性の緩徐化、遅延又は予防を示す。(A)は、外科処置前のウサギの脊椎のMRI放射線写真を示す。(B)は、外科処置4週間後のウサギの脊椎のMRI放射線写真を示し、ここで(i)L1/2の椎間板を損傷させてTGF−β1産生軟骨細胞と非形質導入ヒト軟骨細胞との比1:3の混合物を注入し、(ii)脊椎位置L2/3は非穿刺及び非処理対照とし、(iii)L3/4の椎間板を損傷させてTGF−β1産生軟骨細胞を注入した。矢印は、L1/2及びL3/4の椎間板領域を指す。(C)は、上の(A)で説明したウサギのX線写真を示し、これは椎間板の形態、椎間板の変性又は再生のレベルを測定するための椎間板の椎間板高さ指数を取得するために使用される。(D)は、上の(B)で説明したウサギのX線写真を示し、これは椎間板の椎間板高さ指数を取得するために使用される。
非形質導入軟骨細胞での処理は、椎間変性抑制効果を有する。効果は、図6〜図8の様々な実験において見られる。図6A〜図6Dは、損傷した椎間板の変性の緩徐化、遅延又は予防を示す。(A)は、外科処置前のウサギの脊椎のMRI放射線写真を示す。(B)は、外科処置4週間後のウサギの脊椎のMRI放射線写真を示し、ここで(i)L1/2の椎間板を損傷させて細胞培養培地DMEMを注入し、(ii)脊椎位置L2/3は非穿刺及び非処理対照とし、(iii)L3/4の椎間板を損傷させて非形質導入軟骨細胞を注入した。矢印は、L1/2及びL3/4の椎間板領域を指す。(C)は、上の(A)で説明したウサギのX線写真を示し、これは椎間板の形態、椎間板の変性又は再生のレベルを測定するための椎間板の椎間板高さ指数を取得するために使用される。(D)は、上の(B)で説明したウサギのX線写真を示し、これは椎間板の椎間板高さ指数を取得するために使用される。
プライミングした軟骨細胞での処理は、椎間変性抑制効果を有する。効果は、図9A〜図9Dにおいて見られ、これらの図は、損傷した椎間板の変性の緩徐化、遅延又は予防を示す。(A)は、外科処置前のウサギの脊椎のMRI放射線写真を示す。(B)は、外科処置8週間後のウサギの脊椎のMRI放射線写真を示し、ここで(i)L2/3の椎間板を損傷させて細胞培養培地DMEMを注入し、(ii)脊椎位置L3/4は非穿刺及び非処理対照とし、(iii)L4/5の椎間板を損傷させてプライミングした軟骨細胞を注入した。矢印は、L2/3及びL4/5の椎間板領域を指す。(C)は、上の(A)で説明したウサギのX線写真を示し、これは椎間板の形態、椎間板の変性又は再生のレベルを測定するための椎間板の椎間板高さ指数を取得するために使用される。(D)は、上の(B)で説明したウサギのX線写真を示し、これは椎間板の椎間板高さ指数を取得するために使用される。
ヒト軟骨細胞の供給源
初代ヒト軟骨細胞は、1歳の女児のヒトドナー由来の多指症の指の外科的切除から取得した軟骨組織から増殖させた。多指組織は、外科処置室で採取した。軟骨細胞の単離のための以下の手順を、バイオセーフティキャビネットで行った。軟骨組織を収容するプラスチックボトルをアルコールでスワイプし、ピペットを使用して無菌PBS(1X)で軟骨組織を洗浄した。7mgのコラゲナーゼ(Gibco BRL)を10mLのDMEM(10%FBSを含有する)に溶解し、0.2μmシリンジフィルター(Corning)により濾過することにより、コラゲナーゼ溶液を調製した。洗浄した軟骨組織を、37℃の振とうインキュベーターにおいてコラゲナーゼ溶液で17時間〜18時間処理した。翌日ボトルをアルコールで消毒した。コラゲナーゼで処理した材料をピペットで何回か出し入れして、組織塊から遊離の細胞を分離した。ピペッティングの後、70μmナイロン製cell strainer(Falcon)により上清を濾過した。一体性を喪失したコラゲナーゼで処理した組織(例えば遊離の細胞)は、フィルターを通過することができた。細胞濾液を50mLチューブ(Falcon)中に回収し、その後1500rpmで5分間遠心分離した。上清の3分の2を廃棄し、10mlの無菌PBS(1×)でペレットを洗浄した。再懸濁した細胞を再び1500rpmで5分間遠心分離し、上清の3分の2の除去後、10mlの無菌PBS(1×)で洗浄した。細胞を再度1500rpmで5分間遠心分離した後、DMEM(10%FBSを含有する)に再懸濁した。再懸濁した細胞を、その後4本のコーティングされていない25cm2のフラスコに移し、5%CO2を用いて37℃で4日間培養した。細胞をその後2本のコーティングされていない185cm2のフラスコ中に移した。細胞を2週間培養し、その後回収し、洗浄し、5:4:1の比でDMEM、FBS及びDMSOを有する凍結保存培地中に再懸濁した。細胞を凍結用バイアル中に分取し、4×105細胞/mLの細胞懸濁液1mLを含有するものとした。細胞を、気相液体窒素貯蔵庫中で保持した。
Claims (1)
- 椎間板欠損部位の中に注入される、該椎間板欠損部位での椎間板の変性を予防する又は遅延させるための薬剤であって、哺乳動物の軟骨細胞をTGF-β1とともにインキュベートして得られる、コラーゲン産生軟骨細胞に復帰した線維芽細胞性軟骨細胞であるプライミング軟骨細胞を有効成分として含む、薬剤。
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