초록 ▼

방법: 공여자 마우스 골수세포로부터 중간엽줄기세포를 분리배양하여 냉동보관한 후 필요할 때 배양하여 수를 증가시킴. 주 부 조직적합 불일치(B6, $H-2^b\rightarrow$B6D2F1, $H-2^{b/d}$) 마우스 동종 조혈모세포이식 후 초기(1-5일)에 급성 이식편대숙주질환이 발생하기 전 수혜자 마우스에 투여하여 중간엽줄기세포가 가지고 있는 면역억제 효과에 의해서 이식편대숙주질환의 발생을 감소시키는가를 확인하였음. 이식편대숙주질환의 발생을 감소시키는 중간엽줄기세포의 작용 기전을 규명하

방법: 공여자 마우스 골수세포로부터 중간엽줄기세포를 분리배양하여 냉동보관한 후 필요할 때 배양하여 수를 증가시킴. 주 부 조직적합 불일치(B6, $H-2^b\rightarrow$B6D2F1, $H-2^{b/d}$) 마우스 동종 조혈모세포이식 후 초기(1-5일)에 급성 이식편대숙주질환이 발생하기 전 수혜자 마우스에 투여하여 중간엽줄기세포가 가지고 있는 면역억제 효과에 의해서 이식편대숙주질환의 발생을 감소시키는가를 확인하였음. 이식편대숙주질환의 발생을 감소시키는 중간엽줄기세포의 작용 기전을 규명하기 위해서 (1) 이식편대숙주질환의 clinical severity 및 mortality을 측정하고, (2) 이식 후 혈액 또는 림프기관에서 공여자의 T 림프구수의 증가여부, (3) 혈청 생화학적 표지자 (TNF-$\alpha$, IFN-$\gamma$) 및 (4) 병리조직 검사를 시행하였음 동시에 여러 종류의 백혈병세포를 주입한 백혈병유도 마우스 모델에서 MSCs를 투여하였을 때 생존과 함께 육안/현미경 조직을 관찰하여 GVT 효과가 유지되는지를 확인하였음. IL-10 중간엽줄기세포를 이식 후 첫날 주입하고 동일한 방법으로 실험하여 이식편대숙주질환에 미치는 영향을 관찰함. 동일한 동종 이식모델에서 저용량 전처치(전신방사선조사, 400 cGy)이후에 동종조혈모세포 이식을 시행하여 혼합키메리즘을 유도한 후 2주 후에 종양세포주를 공여자 비장세포와 동시에 주입하여 공여자 림프구수혈에 의한 이식편대항종양 효과를 유도하면서 3일 간격으로 3회 중간엽줄기세포를 투여하여 종양크기 변화에 미치는 영향 대해서 관찰하고 실험실 내에서 기전을 규명함.

Abstract ▼

The immunosuppressive effect of mesenchymal stem cells (MSCs) in human beings, as a corollary to the immunosuppressive effect of MSCs in vitro and in preclinical animal models, suggests that MSCs may be useful for preventing and treating graft-versus-host disease (GVHB) in allogeneic hematopoietic s

The immunosuppressive effect of mesenchymal stem cells (MSCs) in human beings, as a corollary to the immunosuppressive effect of MSCs in vitro and in preclinical animal models, suggests that MSCs may be useful for preventing and treating graft-versus-host disease (GVHB) in allogeneic hematopoietic stem cell transplantation (allo-HSCT). However, the major focus on GVHD protection and the immunosuppression of MSCs has thus far been dominated by in vitro studies. Therefore, this study examined the effects of MSCs expanded ex vivo from the bone marrow of the donor-type on the occurrence of GVHD in a well-established murine alto-HSCT model of human GVHD. The GVHD and a graft-versus-tumor (GVT) effect are tightly linked, as demonstrated by the inverse correlation between the leukemia relapse rates and the severity of GVHD, We therefore want to determine whether MSCs reduce acute GVHD while preserving GVT effect following experimental alto-HSCT.
In a MHC-mismatched C57BL/6 $\rightarrow$ B6D2F1 model, the recipient animals were transplanted with $10\times10^6$ bone marrow (BM) cells and $20\times10^6$ splenocytes after 1100 cGy on day 0. Various doses ($1-3\times10^6$) of donor BM-derived MSCs were administered intravenously from day +1 to day +5, and the clinical and immunologic parameters were evaluated. The MSCs administered to the mice did not attenuate the severity of acute GVHD. The production of IL-10 has been shown to be one out of several mechanisms by which MSCs may suppress T cell respollses. Therefore, this study investigated whether or not the use of IL-10-transduced MSCs (IL-10 MSCs) could reduce the severity of acute GVHD. Lethally irradiated recipients were transplanted and injected with $2\times10^6$ IL-10 MSCs, the MSC expressing vector alone (vector MSCs), or the diluent (controls), respectively, on day +1. Compared with the vector MSCs or controls, there was a significantly lower mortality in the recipients of the IL-10 MSCs at day 50 after the transplant (percent survival, 0% or 10% vs 70%, p = 0.0004 or 0.0064 respectively).
The decrease in mortality was confirmed by the semi-quantitative GVHD score (P < 0.05), and was associated with decreased serum levels of the pro-inflammatory cytokines, IFN-$\gamma$, on day +7 (IL-10 MSCs vs. vector MSCs, 297 $\pm$ 116 pg/ml vs 681 $\pm$ 87 pg/ml, P = 0.015). Therefore, beneficial effects on GVHD were observed when MSCs were engineered to express the anti-inflammatory cytokine, TL-10.
Sublethally irradiated (total body irradiation=400cGy) B6D2F1 mice were reconstituted with $10\times10^6$ C57BL/6 bone marrow cells. All mice were found to be chimeric and seemed healthy, with no clinical signs of early GVHB. All mice were inoculated subcutneously with $1\times10^4$ P815 cells, to mimic a state of minimal residual disease on 14 days following allo-HSCT, Some mice received donor lymphocyte infusion (DLI, $30\times10^6$ spleen cells) for induction of GVT effects. The other groups received no donor splenocytes. Donor MSCs were infused intravenously on days 15, 18 and 21. Tumor growth was more rapid in mice injected MSCs than in the controls, indicating the immunosuppressive effect of MSCs may reduce the GVT effect after DLI. In contrast, mice without DLI showed the mixed chimerism. Injection of MSCs did not influence on the tumor growth.
In conclusion, the primary MSCs but not genetically modified MSCs expressing IL-10 failed to protect against GVHD in this experimental alto-SCT model. After the early injection of MSCs following DLI MSCs mifht reduce the GVT effect.

목차 Contents

• 표 지...1
• 제 출 문...3
• 목 차...4
• I. 연구개발결과 요약문...5
• 연구개발사업 최종보고서 요약문...5
• Project Summary...7
• II. 연구개발과제 연구결과...9
• 1. 연구개발과제의 최종 연구개발 목표...9
• 2. 연구개발과제의 연구대상 및 방법...10
• 3. 연구개발과제의 연구개발결과...12
• 4. 연구개발과제의 연구결과 고찰 및 결론...16
• 5. 연구개발과제의 연구성과 및 목표달성도...18
• 6. 연구개발과제의 활용계획...20
• 7. 참고문헌...21
• 8. 첨부서류...22