200301132 玖、發明說明 (發明說明應敘明：發明所屬之技術領域、先前技術、內容、實施方式及圖式簡單說明） [發明所屬之技術領域] 本發明爲關於以人羊膜所得之上皮細胞做爲有效成分的 糖尿病治療劑及將人羊膜所得之上皮細胞對糖尿病患者投 與爲其特徵的糖尿病治療法，又，關於以人羊膜所得之間 葉系細胞做爲有效成分的骨代謝異常症治療劑及將人羊膜 所得之間葉系細胞對骨代謝異常症患者投與爲其特徵的骨 代謝異常症治療法，更且，關於以人羊膜所得之間葉系細 ® 胞做爲有效成分的心臟疾病治療劑及將人羊膜所得之之間 葉系細胞對心臟疾病患者投與爲其特徵的心臟疾病治療法。 [先前技術] ¥ 近年，再生醫療性技術已顯露頭角。即，人類或其他生 m 物體，於一生期間因外傷和疾病而喪失一部組織，並且承 受大損害，而此時的再生能力爲根據動物種類、或根據組 織而異。自然上令無法再生的臟器和組織再生、並且恢復 0 機能，且由未分化的細胞分化出具有生物機能細胞的技術 已很發達。此類技術一般稱爲再生醫療。 但是，以往，供給此類技術的細胞，一般爲使用人類胎 兒所得的幹細胞，由倫理性方面而言被視爲有問題。 [發明內容] 本發明者爲了克服此類問題，於摸索可代替人類胎兒所 得之幹細胞時，發現若於一定條件下對生物體內投與人羊 膜所得之上皮細胞或間葉系細胞，則可改善生物體的病態， 200301132 並且完成本發明。 本發明第一爲關於以人羊膜所得之上皮細胞做爲有效成 分的醫藥組成物。 第二爲關於以人羊膜所得之上皮細胞做爲有效成分的糖 尿病治療劑。 第三爲關於將人羊膜所得之上皮細胞對糖尿病患者投與 爲其特徵的糖尿病治療法。 第四爲關於以人羊膜所得之間葉系細胞做爲有效成分的 醫藥組成物。 第五爲關於以人羊膜所得之間葉系細胞做爲有效成分的 骨代謝異常症治療劑。 第六爲關於將人羊膜所得之間葉系細胞對骨代謝異常症 患者投與爲其特徵的骨代謝異常症治療法。 第七爲關於以人羊膜所得之間葉系細胞做爲有效成分的 心臟疾病治療劑。 第八爲關於將人羊膜所得之間葉系細胞對心臟疾病患者 投與爲其特徵的心臟疾病治療法。 於本發明中，人羊膜細胞爲將取得同意（im formed consent)之孕婦予以剖腹，且以通常的外科性手法取得。 由羊膜以下列方法可取得上皮細胞。 將所得之羊膜，使用滅菌剪刀等予以斷片化，並且放入 D u 1 b e c c 〇 ’ s Μ 〇 d i f i e d E a g 1 e培養基等通常之動物細胞培養 中所用的培養液，並且振盪。此時，爲了令羊膜斷片以各 個細胞分散化，乃期望共存胰蛋白酶等之蛋白質分解酵素。 200301132 較佳的蛋白質分解酵素之濃度爲〇 . 1 %至〇 . 5 %。又，振盪 時之溫度期望爲2 5 t至4 0 °c之間。振盪之間隔期望爲 5 0rpm〜2 0 0rpm，且振盪時間期望爲20分鐘至60分鐘。 以離心操作收集細胞後，除去上淸液後，將該細胞以前 述同樣之條件下，於蛋白質分解酵素共存之培養液中再度 振盪。但，振盪之間隔期望爲4 0 0 rpm〜6 0 0 rpm，且振盪時 間期望爲2 0分鐘至6 0分鐘。 其後，將培養細胞以滅菌紗布等之篩網予以過篩，分離 出上皮細胞。上皮細胞爲以離心收集，並於含有1 〇 %血淸 之培養液中，於C 0 2培養箱中培養。培養溫度爲3 7 °C、C 0 2 濃度爲5 %爲佳。血淸以幼牛血淸或幼胎牛血淸爲佳。又， 於培養液中，共存濃度1 %左右的抗生素（例如，青黴素G 、1 00單位/毫升：鏈黴素、1 00微克/毫升：兩性黴素B、 〇 . 2 5微克/毫升）爲佳。於紗布上殘留的羊膜爲以上述同樣 之方法，再度斷片化，再予以細胞分散化。 所得之上皮細胞爲以離心收集，且同上述，於含有1 〇 % 血淸之培養液中，於C 0 2培養箱中培養。培養之細胞濃度 爲1 X 1 0 5〜1 X 1 0 6 /毫升爲佳。 通常由一枚羊膜可分離出5 X 1 0 7個上皮細胞。 羊膜間葉系之細胞可由完全移除上皮細胞之羊膜中分離 。於紗布等之篩網上殘留之羊膜以0 . 1〜1 . 0毫克/毫升之膠 原酶（和光純藥（株）製）等之蛋白質分解酵素及〇. 〇 1〜〇 · 1毫 克/毫升之D N A酶（S i g m a公司）等之核酸分解酵素予以振盪 、消化。振盪溫度以2 0〜4 0 °C爲佳，振盪時間以3 0分鐘 200301132 〜1 2 0分鐘爲佳，且振盪之間隔以1 Ο 0〜1 0 0 0 rp m爲佳。 將經消化之羊膜以滅菌紗布等之篩網予以過篩，且可分 離出間葉系細胞。所得之間葉系細胞以離心收集，並於含 有1 〇 %血淸之培養液中，於C 0 2培養箱中培養。培養溫度 爲3 7 °C、C 0 2濃度爲5 %爲佳。血淸以幼牛血淸或幼胎牛血 淸爲佳。又，於培養液中，共存濃度1 %左右之抗生素（同 前述）爲佳。細胞密度以1 〇 5〜1 〇6細胞/毫升爲佳。間葉系 細胞通常由一枚羊膜可分離出5 X 1 0 6個細胞。 肇 本發明之醫藥組成物爲含有上述方法所得之人羊膜所得 之上皮細胞或間葉系細胞爲其特徵。 將上皮細胞或間葉系細胞對患者投與時，爲了完全除去 共存抗生素和異種血淸，乃以生理食鹽水將細胞仔細洗淨 。於投與細胞之情形中，將細胞於生理食鹽水或培養液中 Λ 懸浮供使用。細胞濃度以1 〇 6〜1 〇1 ^細胞/毫升爲佳。爲了 輔助細胞，亦可混合存在增殖因子和細胞安定劑。 投與部位雖無特別限定，但可根據疾病，選擇胰臟、脾 ® 臟、風濕病疾病部位等之局部和各種血管內。 投與之總細胞數爲依據患者之病態、體重。 本發明之醫藥組成物或以治療法可治療的疾病爲如下。 高血脂症、高血糖症、耐糖能力不全（i m p a i r e d g 1 u c 〇 s e t ο 1 e r a n c e : I G T )狀態、糖尿病倂發症（例如網膜症、腎病、 白內障、冠狀動脈疾病等）、動脈硬化症、心血管性疾病 (例如缺血性心臟疾病等）。 初級骨質疏鬆症、內分泌骨質疏鬆症（甲狀腺機能亢進症、 200301132 副甲狀腺機能亢進症、庫興氏（C u s h ! n g s )症候群、及末端 肥大症）、遺傳性及先天性形態之骨質疏鬆症（骨形成不完 全、高胱胺酸尿症、M e n c a s e症候群、及L i 1 i d y症候群） 及因四肢固定所造成之骨質疏鬆症般的骨質疏鬆症。 青年及少年少女之骨骼的柏哲德氏（P a g e t ’ s )病（變形性 骨炎）。 來自固形腫瘤（乳房、肺及腎臟）之高鈣血症及血液學的 惡性疾病（多發性骨髓腫、淋巴囊腫及白血病）、特發性高 鈣血症、及甲狀腺機能亢進症及腎機能不全所關連的高鈣 血症。 外科手術後之類固醇投與所誘發之小腸及大腸損害所關 連的骨減少症、及慢性肝炎及腎臟病所關連之骨減少症。 外傷性負傷所關連之骨壞死、或高傑氏病、鎌形紅血球 性貧血、全身性紅斑性狼瘡及其他疾病所關連之非外傷性 壞死所關連的骨壞死、或細胞死滅。 慢性間接風濕病所造成之骨減少。 牙周骨喪失。 骨溶解性轉移。 變形性關節症、慢性間接風濕病或變形性脊椎症及來自 彼等之間接疼痛和頸肩腕痛、腰痛及麻痺。 缺血性心臟疾病（例如，狹心症、心肌梗塞、川崎病等） 、瓣膜症（例如，僧帽瓣狹窄症、.僧帽瓣閉鎖不全症、僧帽 瓣脫離症候群、大動脈瓣狹窄症、大動脈瓣閉鎖不全症等） 、心律不整（例如，心房細動、心房粗動、發作性上室性頻 200301132 拍、W P W症候群、房室結節回歸性頻拍、心室性期外收縮 、心室頻拍、心室細動、徐脈性心律不整、洞不全症候群 等）、大動脈-血管疾病（例如，上行部大動脈瘤、弓部大動 脈瘤、胸部大動脈瘤、胸部下行大動脈瘤、胸腹部大動脈 瘤、腹部大動脈瘤、閉塞性動脈硬化症、靜脈血栓、肺血 栓閉塞症等）、先天性心臟疾病（例如，心房中隔缺損症、 心室中隔缺損症、E i s e n m e n g e r症候群、肺動脈迴流異常 、動脈管開存症、心內膜床缺損症、完全大血管轉移症、 修正大血管轉移症、肺動脈狹窄症、F a 1 1 〇 t回徵症、大動 脈縮窄症、V a 1 s a 1 v a洞動脈瘤、E b s t e i η畸形等）、心肌症 (擴張型心肌症、肥大型心肌症、拘束型心肌症等）。 [實施方式] 以下列舉實施例再詳細說明本發明，但本發明並非被其 所限定。 實施例1 人類上皮細胞及間葉系細胞之分離 將取得通知同意之孕婦剖腹，取得羊膜。所得之羊膜以 減菌之手術用剪刀予以斷片化，並與含有〇 . 2 %胰蛋白酶之 細胞培養液（D u 1 b e c c 〇 ' s Μ 〇 d i f i e d E a g 1 e培養基：以下，稱 爲「D Μ E Μ」）共同放入5 0毫升的F ο 1 c ο n管中，且以3 7 °C 之振盪器以1 〇 〇 rp m振盪3 0分鐘。離心後除去上淸液，再 度以滅菌之手術用剪刀予以斷片化，且以3 7 t之振盪器以 4 0 0〜6 0 0 r p m振盪3 0分鐘。以0 . 2 %胰蛋白酶消化。將溶 液以滅菌紗布過篩，分離出上皮細胞。上皮細胞爲以離心 200301132 收集，並以含有培養液1 〇 %幼胎牛血淸及抗生素（青黴素G 、1 00單位/毫升：鏈黴素、1 00微克/毫升：兩性黴素B、 〇 . 2 5微克/毫升）之D Μ E Μ於3 7 °C、5 % C 0 2下培養。紗布上 殘留的羊膜爲再度以滅菌手術用剪刀斷片化，同樣以〇 . 2 % 胰蛋白酶消化3 0分鐘。將胰蛋白酶消化和細胞回收之操作 重覆3回，並以離心收集上皮細胞，以3 X 1 0 5細胞/毫升之 細胞密度，以含有1 〇 %幼胎牛血淸及上述同樣之抗生素之 D Μ E 於3 7 °C、5 % C 0 2下培養。上皮細胞爲通常由一枚羊 膜可分離出5 X 1 0 7個細胞。 羊膜間葉系之細胞爲由完全移除上皮細胞之羊膜中分離 。將紗布上殘留之羊膜以膠原酶（〇 . 7 5毫克/毫升、和光純 藥（株）製）、DNA酶（0.075毫克/毫升，Sigma公司製），於 3 7 °C以6 0 0 1· p m振盪、消化6 0分鐘。經消化之羊膜以紗布 過篩，分離出間葉系細胞。所得之間葉系細胞以離心收集 ，並以4 X 1 0 5細胞/毫升之密度以含有1 0 %幼胎牛血淸之 D Μ E Μ，於3 7 °C、5 % C 0 2下且抗生素（青黴素G、1 0 0單位/ 毫升：鏈黴素、1 〇 〇微克/毫升：兩性黴素B、0.2 5微克/ 毫升）存在下培養。間葉系細胞爲通常由一枚羊膜可分離出 5 X 1 〇 6個細胞。分離出之羊膜所得之上皮細胞和間葉系細 胞的照片示於第1圖。 試驗例1人羊膜所得之上皮細胞所造成的糖尿病治療效果 (1 )糖尿病鼠之作成 將 8 - 1 2 週之免疫不全 S C I D ( s e v e r c 〇 m b i n e d i m m u η o d e f i c i e n t)鼠使用做爲人羊膜所得之上皮細胞（以下， 200301132 單稱爲「上皮細胞」）或人羊膜所得之間葉系細胞（以下， 單稱爲「間葉系細胞」）之移植動物。對s C I D鼠以2 5 0毫 克/公斤之鏈脲黴素（streptozotocin :以下稱爲「STZ」）濃 度予以腹腔內投與誘發糖尿病，並且確認該鼠體重降低和 血糖値上升（> 3 0 0 m g / d 1)。S T Z投與1週後，麻醉下，對鼠 脾臟移植1 X 1 〇 6細胞/ 〇 . 1毫升的上皮細胞或間葉系細胞。 移植鼠之血糖値爲對於鼠尾部採血之上淸，歷一日使用 AventisPharma股份有限公司之Diasenser並且根據該公 司的實驗手則於上午9 - 1 1點之間測定。移植後經過2〜3 週後，血糖値恢復至正常範圍（<1 〇〇m g/dl)後，將鼠剖腹， 切除脾臟及胰臟，且供以後之解析。 (2)免疫染色 對於（1 )所得之鼠脾臟及胰臟，將1 〇 %甲醛固定之石蠟切 片薄切至4〜6微米，並於載玻片上貼附作用、供使用。 免疫染色或螢光免疫用之初次抗體爲使用以下之鼠所得 之單株抗體。抗人胰島素抗體（〇 n c 〇 g e n e公司製：A b - 1 / 1 ·· 4 0 0稀釋）、抗人β 2 -微球蛋白抗體（P h a ι· m i η o g e η公司製 :C ο 1 - 2 / 1 : 8 0 0稀釋）、及、抗人膠原11型抗體（S i g m a公 司製：Tu - 9 9 / 1 : 1 0 0 0稀釋）。二次抗體爲使用兔抗鼠抗體 ，且抗原檢測系統爲使用鏈黴抗生物素蛋白-生物素法 (S A B - Ρ Ο套件·· N i c h i r a y (株）製）。但，限於人膠原I I型檢 測之二次抗體爲使用F I T C標幟兔抗鼠抗體。於0 . 0 1 Μ -ΡΗ6.0檸檬酸緩衝液中以電爐進行15分鐘加熱處理，脫石 蠟後，進行內因性過氧化酶抑制操作（於〇 . 3 %過氧化氫添 200301132 加之甲醇中靜止3 0分鐘）。初次抗體結合反應爲於室溫下 進行1小時。生物素標幟二次抗體結合反應爲於室溫進行 3 〇分鐘，且令過氧化酶標幟鏈黴抗生物素蛋白（酵素試藥） 爲於室溫下結合3 0分鐘，其後使得D A Β (二胺基聯苯胺） 呈色。 (3 )以P C R檢討 爲了檢討人類細胞於（2 )所得之鼠脾臟及胰臟中之存在 ，乃由此些組織中萃取D N A，並以P C R法確認人類專一的 β 2 -微球蛋白基因是否存在。詳細記載於下。 使用 Molecular Research Center 公司的 DNAzol，由依 據該公司實驗方法於（2 )所得之鼠脾臟及胰臟中萃取D N A 。以所得之D N A 1微克爲基質，使用根據人β 2 -微球蛋白 基因中之人類專一核苷酸序列所設計的引子： 5'-gtgtctgggtttcatcaatc-3M；序列編號 1); 及 5'-ggcaggcata c t c a t c 1111 - 3 '(序歹丨」編號 2 ) 並且使用 Pharmacia 公司的 Ready -To-Go PCR Beads，依據 該公司的實驗手則，以9 4 °C 1分鐘、5 6 °C 1分鐘、7 2 °C 2 分鐘，進行P C R 3 0個循環。對於P C R後所得之D N A鏈， 使用2 %瓊脂糖膠，以1 0 0 V進行1小時電泳，於紫外線下 ，鑑定出相當於人β 2 -微球蛋白基因的1 6 3 b p帶。 (4 )結果 對8-12週之免疫不全SCID鼠投與STZ後第2日，可誘導 出血糖値爲超過3 0 0 m g / d 1的糖尿病。於S T Z投與後第一 200301132 週，對脾臟移植上皮細胞或間葉系細胞。血糖値於投與間 葉系細胞鼠之3例中3例爲依舊呈高値未降低，另一方面 ，於投與上皮細胞鼠之4例中雖1例爲依舊呈高値未降低 ，但3例爲恢復至正常範圍（< 1 0 0毫克/ d 1)之値（第2圖）。 更且於血糖値恢復至正常範圍之鼠中，於移植上皮細胞之 脾臟中，在免疫組織學上鑑定到人胰島素的表現（第3圖） 。更且於該脾臟中以免疫組織學上確認人β 2 -微球蛋白爲 有表現（第3圖）。更且，根據P C R可確認該脾臟中存在人 β 2 -微球蛋白基因。由此些結果，可確認由人羊膜上皮細胞 能誘導出β細胞。 試驗例2 人羊膜所得之間葉系細胞對於軟骨細胞的分化 將分離的間葉系細胞於生體外1 〇〇n g/ml之骨形態發生 蛋白質（Β Μ P )存在下，於約1個月以含有高分子乳酸-聚乙 二醇（分子量約9 5 0 0 )和1 0 %幼胎牛血淸之D Μ Ε Μ於3 7 °C、 5 % C Ο 2抗生素（青黴素G、1 0 0單位/毫升：鏈黴素、1 Ο 0微 克/毫升：兩性黴素Β、0 . 2 5微克/毫升）存在下共同培養後 ，以軟骨細胞專一性的膠原Π型抗體，如下述根據螢光免 疫染色法予以解析。將上述之培養間葉系細胞以磷酸緩衝 生理食鹽水（P B S )洗淨，且以1 0 0 %甲醇於-2 0 °C處理1 〇分 鐘、以1 0 0 %丙酮於-2 0 °C處理1分鐘將細胞固定。其次使 用抗人膠原Π型抗體（Sigma公司製：Tu- 9 9 / 1 : 1 0 0 0稀釋） 於室溫反應1小時並以PBS洗淨後，以二次抗體之FIT C 標幟兔抗鼠抗體於室溫反應30分鐘後，以PBS洗淨後， 以螢光顯微鏡觀察。其結果，僅於形態變化之細胞中察見 -14- 200301132 專一性的人膠原11型基因之表現（第4圖）。由此結果，顯 示可由人羊膜間葉系細胞誘導出軟骨細胞。 試驗例3 人羊膜所得之間葉系細胞對於心肌細胞的分化 (1 ) 將分離出的間葉系細胞於生體外1 〇 〇 n g / m 1之F G F或 5 μ Μ氮胞苷（5 - A Z A )存在下，於約1個月以含有1 0 %幼胎牛 血淸於3 7 °C 、5 % C 0 2抗生素（青黴素G、1 0 0單位/毫升： 鏈黴素、1 〇 〇微克/毫升：兩性黴素B、0 . 2 5微克/毫升）存 在下培養後，使用相對於心肌細胞專一性轉錄因子 N K X 2 · 5 ( K 〇 m u r ο，I .，I z u m 〇，S . ·· C s x ·· a murine homeobox-containing gene specifically expressed in the developing heart. Proc. Nat. Acad. Sci. 90: 8145-8149，1993)抗體之 螢光免疫染色法予以解析。將上述之培養間葉系細胞以磷 酸緩衝生理食鹽水（P B S )洗淨，且以1 0 0 %甲醇於-2 0 °C處理 1 〇分鐘、以1 〇 〇 %丙酮於-2 (TC處理1分鐘將細胞固定。其 次使用抗人類NXK2.5抗體（SANTA CRUZ公司製：將SC-14 0 3 3 稀釋 1 : 2 0 0 者） 於室 溫反應 1 小時 ，並以 P B S 洗淨後 ，以二次抗體之F I T C標幟山羊抗兔抗體於室溫反應3 0分 鐘後，以P B S洗淨，以螢光顯微鏡觀察。其結果，僅於F G F 處理之羊膜間葉系細胞中，察見NICX2.5蛋白質的表現（第 5圖）。由此結果，顯示可由人羊膜間葉系細胞誘導出心肌 細胞。 (2)以RT-PCR檢討 爲了檢討（1 )所得之細胞是否亢進心肌細胞專一性轉錄 因子NKX2.5基因的轉錄，乃由上述細胞中萃取全RNA， 200301132 並以RT-PCR法確認NKX2.5基因的表現。使用RNAzol (Μ ο 1 e c u 1 a r R e s e a ι· c h C e n t e r公司製）依據所附之手則，由（1 ) 所得之羊膜細胞萃取全RN A。以全RN A 1微克做爲基質， 並使用根據人類N K X 2 . 5基因中之人類專一性核苷酸序列 所設計之引子。 5’-cttcaagcca gaggcctacg-3’（序列表之序列編號 3); 及 5 ' - c c g c c t c t g t c 11 c 11 c a g c - 3 '(序列表之序列編號 4 ); 籲 進行RT-PCR。反應爲根據RT-PCR套件（TAKARA公司製） 所附之手則，以逆轉錄酶於4 2 t反應3 0分鐘，製作c D N A ，並重覆4 0回9 4 °C 3 0秒鐘、5 5 °C 3 0秒鐘、7 2 °C 3 0秒鐘之 溫度循環，進行P C R反應。所得之產物使用2 %。瓊脂糖 膠並以1 0 0伏特進行1小時電泳，且於紫外線下鑑定出相 當於來自人類NKX2.5 mRNA之放大產物的2 3 3 bp帶。其 結果，僅於F G F存在下培養之細胞中確認人類N K X 2 . 5專 -性的放大產物。 ^ [產業上之可利用性] 經由利用本發明，可不產生倫理上之問題地使用再生醫 療技術，提供糖尿病治療藥等，又，可治療糖尿病患者等。 [圖式簡單說明] 第1圖爲表示羊膜所得之上皮細胞及間葉系細胞圖。 第2圖爲表示羊膜所得之上皮細胞及間葉系細胞投與鼠 中之血糖値變動圖。圖中Μ爲表示間葉系細胞投與鼠，H A E 爲表示上皮細胞投與鼠之血糖値的變動。 -16- 200301132 第3圖爲表示上皮細胞移植脾臟中之人β 2 -微球蛋白及 人胰島素之表現的免疫組織染色圖。 第4圖爲培養羊膜所得之間葉系細胞中之人膠原11型基 因之表現圖。左圖爲位相差顯微鏡照片，觀察引起形態變 化的細胞。右圖爲抗人膠原Π型抗體的免疫染色圖，於引 起形態變化之細胞中觀察到人膠原Π型的表現。 第5圖爲表示將分離之間葉系細胞於1 OOng/ml之FGF 或5 μ Μ之5 -氮胞苷存在下培養約1個月，且以心肌專一性 的ΝΚΧ2.5抗體根據螢光免疫染色法予以染色之圖示。 第6圖爲將分離之間葉系細胞於1 00ng/ml之FGF或5μΜ 之5-氮胞苷存在下培養約1個月後萃取RN Α，並以RT-PC R 法確認人類NKX 2.5基因是否表現之圖示。 200301132 序歹ϋ表200301132 发明 Description of the invention (The description of the invention should state: the technical field to which the invention belongs, the prior art, the content, the embodiments, and the drawings are briefly explained. It is an active ingredient for treating diabetes and a method for treating diabetes by administering epithelial cells derived from human amniotic membrane to diabetics, and treatment of abnormal bone metabolism using mesenchymal cells derived from human amniotic membrane as an active ingredient. Agent and a method for treating osteodystrophy by administering mesenchymal cells derived from human amniotic membrane to patients with osteodystrophy, and regarding mesenchymal stem cells derived from human amniotic membrane as an active ingredient A heart disease treatment agent and a method for treating heart disease characterized by administering interstitial cells obtained from human amnion to patients with heart disease. [Prior technology] ¥ In recent years, regenerative medical technology has emerged. That is, human beings or other living organisms lose a tissue and suffer great damage due to trauma and disease during their lifetime, and the regenerative capacity at this time varies depending on the animal species or organization. Naturally, non-regenerating organs and tissues are regenerated, and 0 functions are restored, and biologically functional cells are differentiated from undifferentiated cells. Such technologies are commonly referred to as regenerative medicine. However, in the past, cells supplied with such technology were generally stem cells obtained from human fetuses, and were considered ethically problematic. [Summary of the Invention] In order to overcome such problems, the inventors have found that if epithelial cells or mesenchymal cells obtained from human amniotic membrane are administered to a living body under certain conditions when replacing stem cells obtained from a human fetus, it can be improved Morbidity of organisms, 200301132 and completed the present invention. The first aspect of the present invention relates to a medicinal composition that uses epithelial cells obtained from human amniotic membrane as an effective component. The second relates to a therapeutic agent for diabetic disease in which epithelial cells obtained from human amniotic membrane are used as an active ingredient. The third is a method of treating diabetes by administering epithelial cells derived from human amnion to patients with diabetes. The fourth is a medicinal composition using mesenchymal cells obtained from human amnion as an active ingredient. The fifth is a therapeutic agent for bone metabolism disorders using mesenchymal cells obtained from human amnion as an active ingredient. The sixth is about the method of administering mesenchymal cells obtained from human amnion to patients with abnormal bone metabolism. The seventh is a therapeutic agent for cardiac diseases which uses mesenchymal cells obtained from human amnion as an active ingredient. The eighth is a method of treating heart disease which is characterized by administering mesenchymal cells obtained from human amnion to patients with heart disease. In the present invention, human amniotic cells are obtained by laparotomy of a pregnant woman who has obtained consent (im formed consent), and are obtained by a usual surgical method. Epithelial cells can be obtained from the amniotic membrane in the following manner. The obtained amniotic membrane is fragmented using sterilized scissors or the like, and put into a culture medium used for ordinary animal cell culture, such as Du 1 b e c c 〇 's d i f i e d E ag 1 e medium, and shaken. At this time, in order to disperse the amniotic membrane fragments into individual cells, it is desirable to coexist a proteolytic enzyme such as trypsin. 200301132 A preferred proteolytic enzyme concentration is from 0.1% to 0.5%. The temperature at the time of oscillation is desirably between 2 5 t and 40 ° C. The interval between the oscillations is desirably 50 rpm to 200 rpm, and the oscillation time is desirably 20 minutes to 60 minutes. After the cells were collected by centrifugation, the supernatant solution was removed, and the cells were shaken again in a culture solution in which a proteolytic enzyme coexisted under the same conditions as described above. However, the interval between oscillations is expected to be from 400 rpm to 600 rpm, and the oscillation time is expected to be from 20 minutes to 60 minutes. Thereafter, the cultured cells are sieved with a sieve of sterilized gauze or the like to isolate epithelial cells. Epithelial cells were collected by centrifugation and cultured in a culture medium containing 10% blood limulus in a C02 incubator. The culture temperature is 37 ° C, and the concentration of C 0 2 is preferably 5%. The blood maggots are preferably young calf blood maggots or young calf blood maggots. In addition, in the culture medium, antibiotics with a concentration of about 1% (for example, penicillin G, 100 units / ml: streptomycin, 100 μg / ml: amphotericin B, 0.25 μg / ml) are good. The amniotic membrane remaining on the gauze was fragmented again in the same manner as described above, and the cells were dispersed. The obtained epithelial cells were collected by centrifugation, and cultured in a culture solution containing 10% of blood limulus in a C 2 incubator as described above. The cultured cell concentration is preferably 1 X 105 to 1 X 106 / ml. Usually 5 X 107 epithelial cells can be separated from one amnion. Amniotic mesenchymal cells can be isolated from amniotic membranes with epithelial cells completely removed. The amniotic membrane remaining on the gauze and other sieves is proteolytic enzymes such as collagenase (manufactured by Wako Pure Chemical Industries, Ltd.) at 0.1 to 1.0 mg / ml and 0.1 to 1.0 mg / ml Nucleases such as DNase (Sigma) are shaken and digested. The oscillation temperature is preferably 20 to 40 ° C, the oscillation time is preferably 30 minutes 200301132 to 120 minutes, and the interval between oscillations is preferably 100 to 100 rp m. The digested amniotic membrane is sieved with a screen of sterilized gauze and the like, and mesenchymal cells can be separated. The resulting mesenchymal cells were collected by centrifugation, and cultured in a culture solution containing 10% blood limulus in a C02 incubator. The incubation temperature is 37 ° C, and the concentration of C 0 2 is preferably 5%. The blood pupa is preferably calf blood pupa or young calf blood pupa. In the culture medium, it is preferable to co-exist antibiotics (same as above) at a concentration of about 1%. The cell density is preferably 105 to 106 cells / ml. Mesenchymal cells can usually be separated from a single amniotic membrane by 5 X 106 cells. The pharmaceutical composition of the present invention is characterized by containing epithelial cells or mesenchymal cells obtained from the human amniotic membrane obtained by the above method. When epithelial cells or mesenchymal cells are administered to a patient, the cells are carefully washed with physiological saline in order to completely remove coexisting antibiotics and heterogeneous blood pupa. In the case of administering the cells, the cells are suspended in physiological saline or culture medium for use. The cell concentration is preferably from 106 to 100 cells / ml. To assist the cells, a proliferation factor and a cell stabilizer may also be present. Although the site to be administered is not particularly limited, depending on the disease, local and intravascular vessels such as pancreas, spleen ®, and rheumatoid disease can be selected. The total number of cells administered is based on the patient's morbidity and weight. The medicinal composition of the present invention or a disease treatable by a therapeutic method is as follows. Hyperlipidemia, hyperglycemia, impaired glucose tolerance (impairedg 1 uc 〇set ο 1 erance: IGT) status, diabetic episodes (such as omental disease, kidney disease, cataract, coronary artery disease, etc.), arteriosclerosis, cardiovascular Disease (such as ischemic heart disease, etc.). Primary osteoporosis, endocrine osteoporosis (hyperthyroidism, 200301132 parathyroidism, Cush! Ngs syndrome, and terminal hypertrophy), hereditary and congenital osteoporosis ( Incomplete bone formation, homocystinuria, Mencase syndrome, and Li 1 idy syndrome) and osteoporosis-like osteoporosis caused by limb extremity fixation. Paige's disease (deformative osteitis) in the bones of young and adolescent girls. Hypercalcemia from solid tumors (breast, lung, and kidney) and hematological malignancies (multiple myeloma, lymphocyst, and leukemia), idiopathic hypercalcemia, and hyperthyroidism and renal insufficiency Associated Hypercalcemia. Osteopenia associated with small and large bowel damage induced by steroid administration after surgery, and osteopenia associated with chronic hepatitis and kidney disease. Osteonecrosis associated with traumatic injury, or osteonecrosis, or cell death associated with nontraumatic necrosis associated with Gaucher's disease, sickle cell anemia, systemic lupus erythematosus, and other diseases. Bone reduction caused by chronic indirect rheumatism. Periodontal bone loss. Osteolytic metastases. Deformed arthritis, chronic indirect rheumatism or deformed spondylosis and their indirect pain and neck, shoulder and wrist pain, low back pain and paralysis. Ischemic heart disease (e.g., asthma, myocardial infarction, Kawasaki disease, etc.), valvular disease (e.g., mitral valve stenosis, mitral valve atresia, mitral valve detachment syndrome, aortic valve stenosis, Aortic valve insufficiency, etc.), arrhythmia (for example, fine atrial motion, coarse atrial motion, paroxysmal supraventricular frequency 200301132, WPW syndrome, atrioventricular nodule recurrent frequency, ventricular extrasystole, ventricular frequency , Ventricular fine movements, venous arrhythmias, insomnia syndromes, etc.), aortic-vascular diseases (eg, ascending aortic aneurysm, arch aortic aneurysm, thoracic aortic aneurysm, descending thoracic aortic aneurysm, thoracoabdominal aortic aneurysm, abdominal aorta Neoplasms, occlusive arteriosclerosis, venous thrombosis, pulmonary thromboembolism, etc.), congenital heart diseases (eg, atrial septal defect, ventricular septal defect, Eismenmenger syndrome, abnormal pulmonary return, arterial ductsis , Endocardial Bed Defect, Complete Macrovascular Metastasis, Modified Macrovascular Metastasis, Pulmonary Artery Stenosis, Fa 1 1 〇 t-syndrome, vasoconstriction, Va 1 s a 1 v a cave aneurysm, E s t e i η malformation, etc.), cardiomyopathy (dilated cardiomyopathy, hypertrophic cardiomyopathy, restrained cardiomyopathy, etc.). [Embodiments] The present invention will be described in detail below with reference to examples, but the present invention is not limited thereto. Example 1 Isolation of Human Epithelial Cells and Mesenchymal Cells A pregnant woman who had obtained the consent to perform a laparotomy was obtained to obtain an amniotic membrane. The obtained amniotic membrane was fragmented with sterilizing surgical scissors, and was combined with a cell culture solution containing 0.2% trypsin (D u 1 becc 〇 ′ s Μ 〇dified E ag 1 e medium: hereinafter referred to as “D Μ E Μ ") were put together in a 50 ml F ο 1 c οn tube, and shaken at 1000 rpm for 30 minutes with a shaker at 37 ° C. After centrifugation, the supernatant liquid was removed, and the fragments were fragmented again with sterilized surgical scissors, and shaken at 40 to 600 r p m for 30 minutes with a 37 t shaker. Digested with 0.2% trypsin. The solution was sieved with sterile gauze to isolate epithelial cells. Epithelial cells were collected by centrifugation 200301132, and contained culture medium containing 10% of young fetal bovine blood pupa and antibiotics (penicillin G, 100 units / ml: streptomycin, 100 micrograms / ml: amphotericin B, 0. 25 μg / ml) of D M E M was cultured at 37 ° C., 5% C 0 2. The remaining amniotic membrane on the gauze was re-fragmented with sterile surgical scissors and similarly digested with 0.2% trypsin for 30 minutes. The operation of trypsin digestion and cell recovery was repeated 3 times, and the epithelial cells were collected by centrifugation, at a cell density of 3 X 105 cells / ml, with a concentration of 10% of young fetal bovine blood pupa and the same antibiotic as D M E was cultured at 37 ° C, 5% C02. Epithelial cells are usually 5 X 107 cells that can be separated from an amnion. The cells of the amniotic mesenchymal line are isolated from the amniotic membrane with the epithelial cells completely removed. The amniotic membrane remaining on the gauze was collagenase (0.75 mg / ml, manufactured by Wako Pure Chemical Industries, Ltd.) and DNase (0.075 mg / ml, manufactured by Sigma) at 3 7 ° C at 60 0 1 · Shake and digest for 60 minutes at pm. The digested amnion was sieved with gauze to isolate mesenchymal cells. The resulting mesenchymal cells were collected by centrifugation, and at a density of 4 X 105 cells / ml in D Μ E Μ containing 10% of young fetal bovine blood pupa at 37 ° C, 5% C 0 2 And cultured in the presence of antibiotics (penicillin G, 100 units / ml: streptomycin, 1000 μg / ml: amphotericin B, 0.2 5 μg / ml). Mesenchymal cells are usually 5 X 106 cells that can be separated from one amnion. The photographs of the epithelial cells and mesenchymal cells obtained from the separated amnion are shown in Fig. 1. Test Example 1 Diabetes treatment effect caused by epithelial cells obtained from human amnion (1) Preparation of diabetic rats 8-12 weeks of immunodeficiency SCID (severc ommbinedimmu η odeficient) mice were used as epithelial cells obtained from human amnion ( Hereinafter, 200301132 is referred to as "epithelial cell") or a mesenchymal cell derived from human amnion (hereinafter, referred to as "mesenchymal cell"). SID rats were administered intraperitoneally with a streptozotocin (streptozotocin: hereinafter referred to as "STZ") concentration of 250 mg / kg to induce diabetes, and the rats were confirmed to have reduced body weight and increased blood glucose (> 3 0 0 mg / d 1). One week after the STZ administration, the mouse spleen was transplanted with 1 × 10 6 cells / 0.1 ml of epithelial cells or mesenchymal cells under anesthesia. The blood glucose level of the transplanted mice was measured on the tail blood of the mice. Diasenser of AventisPharma Co., Ltd. was used and measured by the company's experimental hand at 9-11 hours. After 2 to 3 weeks after the transplantation, the blood glucose level returned to the normal range (< 1000 mg / dl), the mice were laparotomy, and the spleen and pancreas were removed for further analysis. (2) Immunostaining For the mouse spleen and pancreas obtained in (1), a 10% formaldehyde-fixed paraffin section was thinly cut to 4-6 microns, and attached to a glass slide for use. The primary antibodies used for immunostaining or fluorescent immunization are monoclonal antibodies obtained using the following mice. Anti-human insulin antibody (manufactured by 〇nc 〇gene: Ab-1/1 · · 4 0 0 dilution), anti-human β 2-microglobulin antibody (P ha ι · mi η oge η company: C ο 1 -2/1: 8 0 dilution), and anti-human collagen type 11 antibody (Sigma Corporation: Tu-9 9/1: 1 0 0 0 dilution). The secondary antibody was a rabbit anti-mouse antibody, and the antigen detection system was a streptavidin-biotin method (S A B-P 0 kit ·· N i c h i r a y Co., Ltd.). However, the secondary antibody limited to human collagen I type I detection is the use of F I T C flag rabbit anti-mouse antibody. Heated in an electric furnace for 15 minutes in a 0.01 M-P 6.0 citric acid buffer solution. After removing the paraffin, an endogenous peroxidase inhibition operation was performed (addition of 200301132 to 0.3% hydrogen peroxide and methanol to stand still) 30 minutes). The primary antibody binding reaction was performed at room temperature for 1 hour. The biotin-labeled secondary antibody binding reaction is performed for 30 minutes at room temperature, and the peroxidase-labeled streptavidin (enzyme reagent) is allowed to bind for 30 minutes at room temperature, and then the DA is allowed to Beta (diaminobenzidine) was colored. (3) Review by PCR In order to review the existence of human cells in the mouse spleen and pancreas obtained in (2), DNA was extracted from these tissues, and whether the human-specific β 2 -microglobulin gene was confirmed by PCR method presence. Details are described below. Using DNAzol from Molecular Research Center, D N A was extracted from mouse spleen and pancreas obtained according to the company's experimental method (2). Using 1 microgram of the obtained DNA as a matrix, using primers designed based on a human-specific nucleotide sequence in the human β 2 -microglobulin gene: 5'-gtgtctgggtttcatcaatc-3M; SEQ ID NO: 1); and 5'-ggcaggcata ctcatc 1111-3 '(SEQ ID No. 2) and using Ready-To-Go PCR Beads from Pharmacia, according to the company's experimental manual, at 9 4 ° C for 1 minute, 5 6 ° C for 1 minute, 7 2 ° C for 2 minutes, performing 30 cycles of PCR. The D N A chain obtained after PCR was electrophoresed at 100 V for 1 hour using 2% agarose gel. Under UV light, a 16 3 b p band corresponding to the human β 2 -microglobulin gene was identified. (4) Results: On the 2nd day after administration of STZ to immunodeficiency SCID mice at 8-12 weeks, diabetes with a blood glucose level of more than 300 mg / d1 can be induced. Epithelial cells or mesenchymal cells were transplanted into the spleen at the first 200301132 weeks after the administration of STZ. Blood glucose levels were still high in 3 of the 3 rats administered with mesenchymal cells and did not decrease. On the other hand, in 4 cases of mice administered with epithelial cells, 1 was still high and not decreased, but 3 In order to return to the normal range (< 100 mg / d 1) (Figure 2). Furthermore, in the rats whose blood glucose levels returned to the normal range, the expression of human insulin was immunohistologically identified in the spleen of epithelial cells transplanted (Figure 3). Furthermore, it was confirmed that human β 2 -microglobulin was expressed by immunohistology in this spleen (Figure 3). Furthermore, the presence of the human β 2 -microglobulin gene in the spleen was confirmed by PCR. From these results, it was confirmed that β-cells can be induced from human amnion epithelial cells. Test Example 2 Differentiation of chondrocytes from mesenchymal cells obtained from human amnion The isolated mesenchymal cells were isolated in the presence of 100 ng / ml of bone morphogenetic protein (BMP) in about 1 month. D M Ε Μ containing macromolecular lactic acid-polyethylene glycol (molecular weight of about 9500) and 10% of young fetal bovine blood at 37 ° C, 5% C 〇 2 antibiotics (penicillin G, 1 0 0 units / ml: streptomycin, 100 μg / ml: amphotericin B, 0.2 5 μg / ml), and co-cultured with chondrocyte-specific collagen type Ⅱ antibody in the presence of Light immunostaining was used for analysis. The above-mentioned cultured mesenchymal cells were washed with phosphate-buffered saline (PBS), and treated with 100% methanol at -20 ° C for 10 minutes, and 100% acetone at -20 ° C. The cells were fixed for 1 minute of treatment. Next, an anti-human collagen type Ⅱ antibody (manufactured by Sigma: Tu-9 9/1: 1 000) was used to react at room temperature for 1 hour and washed with PBS. The rabbit antibody was labeled with FIT C of the secondary antibody. After the mouse antibody reacted at room temperature for 30 minutes, it was washed with PBS and observed with a fluorescence microscope. As a result, the expression of -14-200301132 specific human collagen 11 gene was observed only in cells with morphological changes (Figure 4). As a result, it was shown that chondrocytes can be induced from human amnion cells. Test Example 3 Differentiation of Mesenchymal Cells Derived from Human Amniotic Membrane Cells to Cardiomyocytes (1) The isolated mesenchymal cells were isolated with 100 ng / m 1 FGF or 5 μM azacytidine (5-AZA) ) In the presence of 10% of young fetal bovine blood boiled at 37 ° C, 5% C 0 2 antibiotics (penicillin G, 100 units / ml: streptomycin, 1000 micrograms) in about 1 month. / Ml: amphotericin B, 0.25 μg / ml) was cultured in the presence of amphotericin B, and then a specific transcription factor NKX 2 · 5 (Komur ο, I., I zum 〇, S. · C sx ·· a murine homeobox-containing gene specifically expressed in the developing heart. Proc. Nat. Acad. Sci. 90: 8145-8149, 1993) The antibody was analyzed by fluorescent immunostaining. The above-mentioned cultured mesenchymal cells were washed with phosphate-buffered saline (PBS), and treated with 100% methanol at -20 ° C for 10 minutes, and 100% acetone at -2 (TC treatment). The cells were fixed for 1 minute. Next, an anti-human NXK2.5 antibody (manufactured by SANTA CRUZ: diluted SC-14 0 3 3 by 1: 2 0) was reacted at room temperature for 1 hour, and washed with PBS. The FITC-labeled goat anti-rabbit antibody of the secondary antibody was reacted at room temperature for 30 minutes, and then washed with PBS and observed with a fluorescent microscope. As a result, only in FGF-treated amniotic mesenchymal cells, NICX2 was observed. 5 protein expression (Figure 5). This result shows that cardiomyocytes can be induced from human amniotic mesenchymal cells. (2) Review by RT-PCR In order to examine whether the cells obtained (1) are hyper-specific cardiomyocyte-specific transcription The transcription of the factor NKX2.5 gene was obtained by extracting whole RNA from the above-mentioned cells, and 200301132, and the performance of the NKX2.5 gene was confirmed by RT-PCR. Using RNAzol (Μ ο 1 ecu 1 ar R esea ι · ch C enter company) ) According to the attached manual, the amniotic membrane obtained from (1) is fine Extraction of total RN A. 1 μg of total RN A was used as a substrate and primers designed based on the human-specific nucleotide sequence in the human NKX 2.5 gene were used. 5'-cttcaagcca gaggcctacg-3 '(Sequence Listing (Sequence number 3); and 5 '-ccgcctctgtc 11 c 11 cagc-3' (sequence number 4 of the sequence listing); RT-PCR is called. The reaction is based on the manual attached to the RT-PCR kit (manufactured by TAKARA), React with reverse transcriptase for 30 minutes at 4 2 t to make c DNA and repeat 40 times 9 4 ° C for 30 seconds, 5 5 ° C for 30 seconds, and 7 2 ° C for 30 seconds. Cycle and perform PCR reaction. 2% of the obtained product is used. Agarose gel was run at 100 volts for 1 hour, and a 2 3 3 bp band corresponding to the amplified product from human NKX2.5 mRNA was identified under ultraviolet light. As a result, human NKX 2.5 specific amplified products were confirmed only in cells cultured in the presence of FGF. ^ [Industrial Applicability] By using the present invention, regenerative medicine can be used without causing ethical problems. Technology, providing diabetes treatment drugs, etc., and can treat patients with diabetes, etc. [Brief Description of the drawings] The first graph represents cells obtained from the amniotic epithelial and mesenchymal cells of FIG. Fig. 2 is a graph showing changes in blood glucose levels in mice administered with epithelial cells and mesenchymal cells obtained from amniotic membrane. In the figure, M indicates that mesenchymal cells were administered to the rat, and H A E indicates the change in the blood glucose level of the rat when the epithelial cells were administered. -16- 200301132 Figure 3 is an immunohistochemical staining diagram showing the expression of human β 2 -microglobulin and human insulin in epithelial cell transplanted spleen. Fig. 4 shows the expression of human collagen type 11 gene in mesenchymal cells obtained by culturing amniotic membrane. The image on the left is a phase-contrast microscope photograph, observing the cells that cause morphological changes. The figure on the right is an immunostaining image of anti-human collagen type Ⅱ antibody. The expression of human collagen type Ⅱ was observed in cells that caused morphological changes. Figure 5 shows that isolated mesenchymal cells were cultured in the presence of 100 ng / ml FGF or 5 μM 5-azacytidine for about one month. Illustration of staining by immunostaining. Figure 6 shows the extraction of RN Α after culturing isolated mesenchymal cells in the presence of 100 ng / ml FGF or 5 μM 5-azacytidine for about one month, and confirming human NKX 2.5 gene by RT-PC R Whether to show performance. 200301132 Sequence List
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