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In contrast to cerebral ischemia, there have been few experimental sdudies on intracerebral hemorrhage (ICH), alth6ugh ICH is one of the most important cerebral vascular diseases, as much so as cerebral infarction. In 1990, Rosenberg and coworkers developed an elegant rat model in which intrastriatal injection of bacterial collagenase causes bleeding into the brain tissue. This model is considered to be a reproducible method to induce a hematoma and to regulate its size depending on the amount of collagenase injected. But this reproducibility has never been discussed ; we made a modified collagenase induced ICH model in order to verify the reliability of this model. A total 56 of adult male rats were studied. 2μl artificial cerebropinal fluid, which was adjusted to a pH 7.4 by buffer, containing 0.25 unit bacterial collagenase was stereo- tactically injected into the left caudoputamen in 28 rats. The remaining 28 rats were injected with 0.5 unit bacterial collagenase under the same procedure. Volumetry was performed at 2, 4, 12, 24, 48, 168, and 720 hours after injection to compare the difference between groups and observe the natural history of the hematoma. At 4 hours after injection there was a marked hematoma within the caudoputamen which stopped developing in size by 24 hours. At 24 hours, 0.25 unit collagenase caused 60μl hematoma and 0.5 unit collagenase caused 100μl hematoma. At 1 week, the hematoma was resolving and reduced the size to 35% of the maximum value. At 1 month, the hematoma represented a slit-like appearance accompanying the atrophy of ipsilateral hemisphere. The hematoma of 0.5 unit collagenase was significantly larger than that of 0.25 unit collagenase for the entire experimental period. But there was no difference between the groups concerning the time course of hematoma. We conclude that our modified collagenase induced ICH model is quite reproducible and most suitable for the study of ICH. This model should therefore be used for further investigation of ICH.

ラット骨肉腫由来の3種の培養細胞株において, コラゲナーゼの活性レベルと組織接着を精査した。さらにそれらのデータを, 各培養細胞細胞株を近交系F344ラットに注入して得られた腫瘍の遠隔転移率と比較検討した。1)in vivoにおいて, 培養細胞のコラゲナーゼ活性レベルと臓器接着の間には明らかな関連が認められた。すなわち, 酵素活性が高い細胞株は, ラットの肝切片に対する高い接着を示した。2)これらの接着はEDTAおよびo-フェナントロリンによって濃度依存的に阻害された。このことから, コラゲナーゼはラット肝切片に対する接着に重要な役割を果たしていると考えられた。3)コラゲナーゼ活性のレベルが高い株によって形成された戻し移植腫瘍は, その活性のレベルが低いものによって形成された腫瘍に比較して, 高率に遠隔転移がみられた。これらの結果は, 骨肉腫の遠隔転移のメカニズムに関して, コラゲナーゼが重要な役割を持つことを示唆している。さらに, 同一の腫瘍から得られ, しかもそれぞれ異なる性格を持つ3種の培養株は, 今後のコラゲナーゼと腫瘍の転移に関する研究において有用であると考えられた。, Collagenase activity and organ adhesion potentiality of three culture cell lines derived from rat osteosarcoma were examined. Moreover metastatic ratios of tumors produced by injection of each cell line into isogenetic F344 rats were analyzed. The results are summarized as follows. 1. There was definite relation between collagenase activities and organ adhesion of culture cells in vitro. A cell line with high enzyme activity showed the high adhesiveness to cryostat sections of rat liver. 2. These adhesive potentialities were inhibited by EDTA and o-phenanthroline with a dose-dependent manner. This result indicated that collagenase played important role on the cell adhesion to liver section. 3. Reproducing tumors by cell line with high collagenase activity metastasized more frequently to the rat lung as compared with low activity cell line. These results demonstrated that collagenase activity is closely related to metastatic mechanism of osteosarcoma activity cells. In conculusion, we expect that the phenotypic heterogeneity among these line derived from same osteosarcoma will proved a foothold for future studies of collagenase and tumor metastasis.

ラット骨肉腫由来の3種の培養細胞株において, コラゲナーゼの活性レベルと組織接着を精査した。さらにそれらのデータを, 各培養細胞細胞株を近交系F344ラットに注入して得られた腫瘍の遠隔転移率と比較検討した。1)in vivoにおいて, 培養細胞のコラゲナーゼ活性レベルと臓器接着の間には明らかな関連が認められた。すなわち, 酵素活性が高い細胞株は, ラットの肝切片に対する高い接着を示した。2)これらの接着はEDTAおよびo-フェナントロリンによって濃度依存的に阻害された。このことから, コラゲナーゼはラット肝切片に対する接着に重要な役割を果たしていると考えられた。3)コラゲナーゼ活性のレベルが高い株によって形成された戻し移植腫瘍は, その活性のレベルが低いものによって形成された腫瘍に比較して, 高率に遠隔転移がみられた。これらの結果は, 骨肉腫の遠隔転移のメカニズムに関して, コラゲナーゼが重要な役割を持つことを示唆している。さらに, 同一の腫瘍から得られ, しかもそれぞれ異なる性格を持つ3種の培養株は, 今後のコラゲナーゼと腫瘍の転移に関する研究において有用であると考えられた。, Collagenase activity and organ adhesion potentiality of three culture cell lines derived from rat osteosarcoma were examined. Moreover metastatic ratios of tumors produced by injection of each cell line into isogenetic F344 rats were analyzed. The results are summarized as follows. 1. There was definite relation between collagenase activities and organ adhesion of culture cells in vitro. A cell line with high enzyme activity showed the high adhesiveness to cryostat sections of rat liver. 2. These adhesive potentialities were inhibited by EDTA and o-phenanthroline with a dose-dependent manner. This result indicated that collagenase played important role on the cell adhesion to liver section. 3. Reproducing tumors by cell line with high collagenase activity metastasized more frequently to the rat lung as compared with low activity cell line. These results demonstrated that collagenase activity is closely related to metastatic mechanism of osteosarcoma activity cells. In conculusion, we expect that the phenotypic heterogeneity among these line derived from same osteosarcoma will proved a foothold for future studies of collagenase and tumor metastasis.

血圧降下作用を有する機能性食品素材の提供を目的として，ハーブ水抽出物のin vitro反応系におけるアンジオテンシンI変換酵素（ACE）に対する阻害能を測定した。さらに，細菌コラゲナーゼに対する阻害能も同時に測定した。その結果，in vitroにおいてACEを特異的に強く阻害し，in vivoにおける作用が期待できるハーブとして13種，(アブラナ花，マスタード葉，ウド葉，コーンフラワー花，タラゴン葉，タンポポ葉，シシウド葉，セリモドキ葉，ディル葉，イタリアンパセリ葉，クズ花葉，フジ花及びシャロット葉)，コラゲナーゼに対してin vivoで阻害作用が期待できるハーブとして５種，(アブラチャン実，柘榴実，キンミズヒキ花，ゲンノショウコ葉及びサルトリイバラ実）が得られた。さらに，キレートの形成など他のメタルプロテアーゼに対して阻害作用が期待でき，口腔中のコラゲナーゼ阻害（歯周病予防）に有効と思われるハーブが多数存在することが明らかになった。 これら結果は，今後の血圧降下作用及びリューマチ，歯周病予防などに有効な機能性食品や医薬品の創製に対して有用な情報を与えるものであろう。, Article, 信州大学農学部紀要. 31(2): 97-107

[ABSTRACT] Excessive degradation of stromal collagen fibrils occurs in corneal ulceration. Keratocytes (corneal fibroblasts) seem to play a primary role in collagen degradation in corneal ulceration. However, the mechanisms of collagen degradation in corneal ulceration have not been fully understood. Tounderstand the pathobiology of corneal ulceration, I developed a new in-vitro model in which keratocytes were cultured in a collagen matrix threedimensionally. Using this model, I searched for any factors which might regulate collagen degradation by keratocytes. Collagen degradation wasmeasured by the amount of hydroxyproline in the hydrolysate. Collagen degradation by keratocytes depended on the number of cells, the concentration of collagen and the period of incubation. Among various cytokines and growth factors such as epidermal growth factor ( EGF ), fibroblast growth factor ( FGF ) , insulin-like growth factor 1 ( IGF-1) , interleukin 1 (IL- 1) and interleukin 6 (IL- 6), only IL- 1 was found to enhance collagen degradation by keratocytes in a dose dependent manner. Interleukin 1 receptor antagonist (IL- 1 ra) antagonized the stimulatory effect of IL-1 on collagen degradation by keratocytes. IL- 1 also stimulated collagenase synthesis by keratocytes. When phagocytic activity or intracellular lysosomal enzyme activities were inhibited by the addition of cytochalasin B or chloroquine, collagen degradation showed a decrease in the presence of IL- 1 . These findings suggested that both extracellular and intracellular collagen degradation played important roles in the stimulatory effect of IL- 1 on collagen degradation by keratocytes., 学位の種類：医学 学位授与年月日：1994/3/24 指導： 大鳥, 利文 教授(Director: Prof. Otori, Toshifumi) 報告番号：甲第278号 学内授与番号：医甲225 NDL書誌ID：000000278319

胃癌が発育進展するとき,あるいは潰瘍が進行,治癒するときには病巣及び病巣周囲の間質結合織はそれに伴い崩壊する一方,増生もしてくる.この間質の変化は,結合織崩壊に働く因子と増生に働く因子の均衡によっている.結合織崩壊の因子の中で最も重要なものの一つとして,生理的条件下でcollagen分子を特異的に分解するcollagenaseがあげられる.浸潤しつつある癌の先進部では,癌細胞より分泌されたcollagenaseを主とする蛋白分解酵素が間質を破壊し,その間隙を癌細胞が拡がって行くと考えるのが妥当であろう.すでに胃癌浸潤とcollagenaseの関連について,胃癌組織各部のcollagenase活性を計測し,癌先進部で最も活性が高く,胃癌の進展にcollagenaseが関与しているとの報告がある.しかし,そのcollagenaseが胃癌細胞から分泌されているのかという最も興味ある疑問に答えることはできていない.著者はcollagenase抗体を作製し,免疫組織学的方法(酵素抗体法)を用いて,胃癌組織内におけるcollagenaseの局在を検索した.その結果collagenaseは間質側に多く存在するが,癌細胞内にも存在することを確認した.また胃癌を組織型別,深達度別,浸潤様式別等に分類し,各組織でのcollagenaseの局在を検索し,これらの結果や他の報告をも含めて,胃癌の進展にcollagenaseがどのように関与しているのか考察した.更には対照組織として正常ヒト皮膚,胃潰瘍,大腸癌を検討し,各組織におけるcollagenaseの局在と免疫学的組織特異性の有無も検討した., Collagenase plays an important role in the invasion of gastric cancer. However, it is not known if gastric cancer cells produce collagenase. In this study, anti-collagenase antibodies were generated by inoculation of collagenase, which was isolated and purified from normal human cultured skin tissue. The distribution of collagenase in gastric cancer tissue from 52 gastric cancer patients was analysed by the peroxidase-labeled antibody method. Collagenase existed on the surface of collagen fibers and in cell plasma of fibroblasts. Moreover, cancer cells themselves produced collagenase, as the cell plasma of cancer cells stained. The relation of the histological type and depth of invasion to positive staining of cancer cells was then analysed. Cancer cells of the differentiated type stained frequently, while positive staining was low in cases of early gastric cancer. The percentage of positive staining increased according to the advance of gastric cancer. The infiltrative growth pattern also changed the distribution of parechymal collagenase.