초록 ▼

효과적인 간경화 세포치료제의 개발을 위해 간세포성장인자(hepatocyte growth factor; HGF)를 과발현하는 사람중간엽줄기세포(MSC/HGF)를 제작하였음. MSC/HGF를 DMN으로 유도한 흰쥐 간경화동물모델에 비장이식하고 세포이식후 12일후에 희생하여 collagen의 감소, 조직손상의 감소, 몸무게의 증가를 확인했음. 특히 세포이식전보다 희생후의 collagen의 양이 감소하여 치료적 효과를 확인할 수 있었음. 더불어 간 및 담관조직의 손상을 감소하였음을 확인하였고, 간기능을 회복시켰음을 확인함. 치료효과에 대한

효과적인 간경화 세포치료제의 개발을 위해 간세포성장인자(hepatocyte growth factor; HGF)를 과발현하는 사람중간엽줄기세포(MSC/HGF)를 제작하였음. MSC/HGF를 DMN으로 유도한 흰쥐 간경화동물모델에 비장이식하고 세포이식후 12일후에 희생하여 collagen의 감소, 조직손상의 감소, 몸무게의 증가를 확인했음. 특히 세포이식전보다 희생후의 collagen의 양이 감소하여 치료적 효과를 확인할 수 있었음. 더불어 간 및 담관조직의 손상을 감소하였음을 확인하였고, 간기능을 회복시켰음을 확인함. 치료효과에 대한 기전연구결과 fibrogenic cytokine들의 감소, 간경화시 세포외기질의 주요생산자인 Ito cells의 활성화 억제, 세포외 기질 조절자인 MMP들의 발현 증가, TIMP의 발현감소를 확인하였음. 이식세포를 indium표지하여 추적한 결과 손상된 간조직으로의 MSC와 MSC/HGF의 이동성을 확인할 수 있었음. QC 지표의 개발을 위한 세포 표면항원 FACS analysis의 결과 다수의 표면항원에 대한 발현 정도 profile을 확보할 수 있었음. 한편, nude mouse tumorigenesis실험에서는 6개월 경과한 현재 전혀 암을 발생하지 않음을 확인함.

Abstract ▼

Hepatocyte growth factor(HGF) is known to be a promising therapeutic cytokine to target fibrotic diseases such as liver fibrosis. As a `proof of concept` experiment, we addressed whether mesenchymal stem cell(MSC) might be useful as a vehicle to carry HGF into fibrotic liver, thus results in a bette

Hepatocyte growth factor(HGF) is known to be a promising therapeutic cytokine to target fibrotic diseases such as liver fibrosis. As a `proof of concept` experiment, we addressed whether mesenchymal stem cell(MSC) might be useful as a vehicle to carry HGF into fibrotic liver, thus results in a better therapeutic effect than MSC alone. Liver fibrosis was induced by the intraperitoneal injection of dimethylnitrosamine. Adenovirus with HGF cDNA was infected to MSC to provide HGF-secreting MSC(MSC/HGF). Normal or fibrotic rats were intra-splenically injected with the cells, and sacrificed at 12 d after the injection. As expected, HGF mRNA in liver and protein in portal vein were higher in MSC/HGF group than in MSC group. Evidently, tissue fibrosis measured histochemically was reduced by MSC, and even more by MSC/HGF, corroborating with the HGF level. Surprisingly, collagen level from liver extract measured by Sirius red dye revealed actual decrease comparing to that measured at 1 d prior to the injection of the cells, suggesting the resolution of the fibrosis. Both mRNA and protein contents of collagen followed the same pattern with histochemistry, evidencing the better therapeutic effect of MSC/HGF than MSC alone, furthermore, mRNA levels of the fibrogenic cytokines, POGF-bb and TGF-b1, were significantly decreased by HGF/MSC comparing to MSC. In relation to the decrease of collagen, expression of MMP-9 and 13 was increased by MSC/HGF, whereas that of Timp-1 was decreased. We next checked the cell trafficking of MSC and MSC/HGF in rats with normal or fibrotic liver. Indium labeled human MSC or MSC/HGF was transplanted into spleen of rats with normal or fibrotic liver, respectively, and then was monitored by g-camera for 12 days without immunosuppression. Surprisingly, even without immunosuppressant, transplanted cells could survive in a considerable amount for 12 days in both animal groups. Moreover, the survival ratio was higher in fibrosis group than in normal group during the whole period. Both cells were present mainly in the spleen and the liver of both animal groups and additionally, in the lung of fibrosis group at 0 day. Astonishingly, while the relative amount of both cells in uninjured tissue was gradually decreased in both groups, those in the liver and the spleen was stably maintained only in fibrotic group, which suggests that both MSC and MSC/HGF have the tendency to gather into injured tissue. Finally, nude mouse tumorigenesis assay revealed no tumor formation by MSC/HGF at least for six months.

목차 Contents

• II. 총괄연구과제 연구결과...12
• 1. 연구개발과제의 배경 및 필요성...13
• 1.1. 연구필요성 및 배경...13
• 1.2. 연구개발의 최종목표 및 성격...16
• 2. 국내외 기술개발 현황...17
• 2.1. 연구개발대상 기술의 국내.외 현황...17
• 3. 연구개발과제의 추진체계...19
• 3.1. 연차별 연구개발의 목표 및 내용...20
• 3.2. 연도별 연구개발의 추진일정...22
• 4. 연구개발수행 내용 및 결과...23
• 4.1. 간경화 동물모델에서 MSC/HGF의 유효성 입증...23
• 4.2. 유효성의 기전 연구...30
• 4.3. 이식 세포의 tracking...33
• 4.4. MSC 생산시 QC를 위한 표준지표 개발...36
• 4.5. 종양 발생 가능성 탐색...37
• 5. 목표달성도 및 관련분야 기여도...39
• 5.1. 총괄연구개발과제의 목표달성도...39
• 5.2. 관련분야 기여도...39
• 6. 향후 연구성과 추진 계획...40
• 7. 연구개발결과의 파급효과...40
• 7.1. 기술적 파급효과...40
• 7.2. 경제.산업적 파급효과...40
• 8. 연구개발결과의 활용계획...41
• 8.1. 연구개발결과의 활용계획 및 추가연구의 필요성...41
• 8.2. 사업화(기업화) 목표 달성도 및 계획...41
• 8.3. 사업화 수준(계획 및 완료 포함)...41
• IV. 첨부서류...42
• 1. 자체평가의견서...44
• 2. 실적 증빙자료...45
• 3. 기타 첨부서류...45