초록

Ⅳ. 연구개발결과
- 알파갈 적중 세포주에 인간 면역억제 유전자 (CD47, TFPI)가 동시에 발현하는 형질전환 복제 수정란의 생산
- 다중 형질전환 세포주와 선천성 면역세포와의 세포독성 검사를 통한 면역거부반응 억제 발견 및 그 기작 연구
- 알파갈 넉아웃 형질전환 복제 수정란 생산 및 체세포 핵치환 기술을 이용한 복합형질전환 복제 수정란 이식

Abstract ▼

In xenotransplantation from pig- to- human, the transgenic porcine expressing optimal human gene are required to overcome the immune response barrier. But, between the expressing human genes, some combinations of genes showed the effect of proceeding immune response. In order to overcome these effec

In xenotransplantation from pig- to- human, the transgenic porcine expressing optimal human gene are required to overcome the immune response barrier. But, between the expressing human genes, some combinations of genes showed the effect of proceeding immune response. In order to overcome these effect, we combined the human CD47(hCD47) and human TFPI(hTFPI). hCD47 and hTFPI co-expressing vector was constructed, and then alpha-gal knock-out and hCD47/hTFPI co-expressing porcine cell was established. The hCD47 and hTFPi co-expressing pocine cells showed the synergic effect to impede the phagocytosis by human macrophage in vitro. The level of cytotoxicity dicreased in hCD47 and hTFPi co expressing cells than only hCD47 or hTFPI expressing cells. And the synergetic effect between hCD47 and hTFPI expression was regulated by thrombosopondin-1(TSP-1) derived from human macrophage.
While TSP-1 blocking antibody treatment or TSP-1 siRNA treatment, the level of cytotoxicity using hCD47/hTFPI co-expressing porcine cells test increased than no treatment contol.And then, we conducted the somatic cell nuclear transfer(SCNT) using alpha-gal knock-out porcine fibroblast(pFF), hCD47/hTFPI co-expressing porcine kidney cells(PK15) and normal fibroblast. The differentiation level to blastocyst stage in alpha-gal knock-out pFF was lower than in normal pFF. But, using hCD47/hTFPI co-expressing PK15, differentiation to blastocytst was observed. Then, we conducted the embryo transfer using hCD47/hTFPI co-expressing PK15. However, we could not implant the embryos in the germ-free porcine uterus.We confirmed the down regulation effect of immune response using multiple immune related genes and alph-gal knockout in vitro.
As the alpha-gal knockout and multiple human related gene expressing porcine is established in germ-free facility, we might cirfirm the synergic effect in vivo and immune response down-regulation experiment.