心筋再生医療を目的とした心筋に内在する心筋組織幹細胞・心筋前駆細胞同定の試み

尝试鉴定心肌固有的心肌组织干细胞和心肌祖细胞，用于心肌再生医学的目的

• 批准号: 13877213
• 负责人: 大村 健二
• 金额: $ 1.41万
• 依托单位: Kanazawa University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Exploratory Research
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-13877213/
• 关键词: 心筋組織幹細胞・前駆細胞; ラット心筋梗塞モデル; Nestin; BrdU; 心筋組織幹細胞,前駆細胞

ラット心筋梗塞モデルで、BrdUラベリングによりDNA合成能を有する心筋細胞を同定し、リン酸化HistoneH3(増殖細胞マーカー),Nestin(未分化細胞マーカー)に対する特異抗体を用いて免疫染色を施行した。また心筋組織を左室自由壁(梗塞境界〜梗塞領域),心室中壁(非梗塞領域)に分け,コラゲナーゼ処理にて心筋細胞を単離し塗抹標本を作製,核の数及び心筋細胞の大きさを計測した。実験動物は生後8週体重250gの雄ウィスターラットを使用,左小開胸下に左冠動脈を結紮し心筋梗塞モデルを作成,冠動脈結紮群(MI群:n=4),非結紮群(N群:n=4)とした.1週間後BrdUを腹腔内投与(50mg/kgを4時間毎24時間)し心摘出.摘出心は輪状に切除し病理組織切片を作成した.結果はBrdUの免疫染色では,MI群の梗塞境界領域で心筋細胞の核陽性像を一組織切片に平均4.8個認めた.N群では心筋細胞の核陽性像は認めなかった.Nestinの免疫染色ではMI群梗塞境界領域の心筋細胞で瘢痕組織を取り囲むように陽性像が認められた.連続切片にてBrdU陽性の心筋細胞はリン酸化HistoneH3及びNestinに陽性であることが確認できた.塗抹標本では48%が単核,50%が二核の心筋細胞で,両群とも左室自由壁,心室中壁でその割合に差は認めなかった.心筋細胞の大きさは心室中隔の単核細胞ではN/MI群:1695±692/1974±689μm2.二核細胞ではN/MI群:2104±924/2387±809μm2.左室自由壁の単核細胞ではN/MI群:1893±588/1723±608μm2,二核細胞ではN/MI群:2091±609/2326±679μm2であった.心室中壁で生じる梗塞後の単核心筋細胞の肥大化は左室自由壁では認めず,大きさは小さいまま維持された.以上より,ラットを用いた実験で心筋梗塞境界領域にBrdUにラベルされるDNA合成能を有する心筋細胞の存在を確認した.また,成体心筋組織中に内在する心筋組織幹細胞,前駆細胞の存在可能性を示唆する所見として梗塞境界〜梗塞領域では単核で小さい心筋細胞が維持されていることを確認した.

Immunostaining was performed with specific antibodies against myocardial infarction, BrdU, and DNA synthesis. The number of nuclei and the size of myocardial cells were measured in the left ventricular free wall (infarct area ~ infarct area) and the middle ventricular wall (non-infarct area). After 8 weeks of life, the animals weighed 250g and were used as male. Left coronary artery was ligated under the left small thoracotomy. Coronary artery ligation group (MI group:n=4) and non-ligation group (N group:n= 4) were established. BrdU was injected intraperitoneally (50mg/kg, 4 times every 24 times) after 1 week. The heart was excised and pathological sections were prepared. Results The results showed that there were 4.8 positive nuclear images in MI group in infarct area and 4.8 positive nuclear images in N group in MI group in infarct area. Even though the sections were BrdU positive, the cardiac muscle cells were acidified by HistoneH3 and Nestin positive. 48% of them are single nuclei, 50% of them are binucleated cardiac muscle cells, and the left ventricular free wall and the ventricular middle wall are divided into two groups. The N/MI ratio of cardiac muscle cells to ventricular septal mononuclear cells was 1695±692/1974±689μm2. The N/MI ratio of binucleated cells to ventricular septal mononuclear cells was 2104±924/2387±809μm2. The N/MI ratio of left ventricular free wall mononuclear cells to ventricular septal mononuclear cells was 1893±588/1723±608μm2. The N/MI ratio of binucleated cells to ventricular septal mononuclear cells was 2091±609/2326±679μm2. The hypertrophy of the left ventricular free wall after infarction was observed in the middle wall of the ventricle, and the large and small ones were maintained. The above results confirm the existence of cardiac muscle cells in the field of myocardial infarction. In addition, the possibility of the existence of intrinsic cardiac muscle stem cells and anterior muscle cells in adult cardiac muscle tissue was confirmed.