Several human leukocyte subsets including natural killer (NK) cells, cytotoxic T lymphocytes (CTL), and polymorphonuclear neutrophils (PMN) participate in cellular immune responses directed against vascularized pig-to-human xenografts. As these leukocytes express the death receptor Fas either constitutively (PMN) or upon activation (NK, CTL), we explored in vitro whether the transgenic expression of membrane-bound human Fas ligand (mFasL) on porcine fetal fibroblasts is a valuable strategy to protect porcine xenografts. cDNA of mFasL carrying the deletion at the cleavage site with metalloproteinase and lacking the death domain in its cytoplasmic tail was subcloned into pCAGGS expression vector driven by the chicken β-actin promoter containing blastidin- resistance cassette. The mFasL expression vector was transfected into mini-pig fetal fibroblasts by lipofection method. Blastidin-resistant cells were screened by PCR and FISH. The expression of mFasL was confirmed by Western blot and FACS with the mouse anti-human FasL antibody. Interaction of two transgenic clonal cell lines with human leukocytes was analyzed using functional assay for cytotoxicity. mFasL expressed on porcine fetal fibroblasts protected porcine fetal fibroblasts against killing mediated by human NK cells. The rate of NK cell mediated cytotoxicity was significantly reduced in transgenic clonal cells (54±10.80%) compared to normal minipig fetal fibroblasts. This result indicated that grafts of transgenic pigs expressing mFasL could control the cellular immune response to xenografts, and create a window of opportunity to facilitate xenograft survival.

본 연구는 배양 각막 상피세포에 anti - F AS and anti - F AS ligand antibody를 노출시 킨 후 세포고사 메커니즘을 결정하기 위해 시행하였다. 배양각막 상피세포에 antihuman FAS(N-18) goat polyclonal IgG를 50, 200, 500, 1,000 ng/m~ 또는 anti-human FAS ligand(C• 20) goat polyclonal IgG를 500 ng/m~으로 2 일과 4 일 동안 처 리 하였다. 주어진 기간 동안 배양한 후 배양액에 부유한 세포와 부착된 세포를 원심분리와 트립 신 처리 원섬분리를 이용하여 수확하였다. 각막상피세포에 대한 anti - F AS and antiF AS ligand antibody 의 영향을 알아보기 위해 Hoechst 33342 staining과 TUNEL stammg 방법을 이용하여 세포 세포고사 유도를 확인하였다. 세포막 수용체인 FAS protein의 발현을 알아보기 위해 ìmmunocytochemistry 를 시행하였다. anti-FAS antìbody를 처리한 군에서는 대조군에 비해 시간과 농도에 비례하여 후기 세포고사 소 견이 증가하였다. 그러나 anti - F AS ligand antibody를 처리한 군에서는 대조군과 차이 가 거의 없었다. 본 연구의 결과 FAS-FAS ligand system 이 각막상피세포에 발현되었 으며 이는 정상 각막 상피 생리 즉， 세포 탈피에 중요한 기능을 갖는 것으로 사료된다

Prolactin (PRL) surge in cycling rats at proestrous afternoon has previously been reported as an inducer of apoptotic cell death of luteal cells. This death-inducing action of PRL seeins unusual, because PRL can he categorized as a cell-survival factor, if other known physiological functions of PRL are taken into account. In this study, the apoptotic action of PRL was assessed in cultured cells prepared from rat luteal tissue and underlying molecular /cellular mechanism of PRL-induced luteolysis was analyzed. The latest crop of corpora lutea (CLs) were enucleated from rat ovaries at 18:00 h on the proestrous day before the next ovulation. Donor rats were pretreated with CB154, a dopamine agonist, in order to he exempted from the endogenous PRL surge. The harvested GLs were dispersed and cultured with or without PRL (2g /ml) for 24 or 48 h. An addition of PRL to the culture medium changed the parameters indicative of cell death via apoptosis: a decrease in cell viability (MTT) and an increase in chromatin condensation. Most of the DNA breakdown in nuclei induced by PRL occurred in steroidogenic cells which were identified by 3-HSD activity staining, and the number of 3-HSD-positivecells were significantly decreased. Interestingly, most of the cells with an apoptotic nucleus adhered to one or more intact and seemingly non-steroidogenic cells. Because the expression of Fas has heen shown to be abundant in murine ovary, and Fas is known to have an exact physiological role in occurrence of apoptotic cell death, the membrane form-Fas ligand (rnFasL) was quantified in the cell lysate. An addition of PRL increased expression of mFasL. Moreover, an addition of concanavalin A (ConA), a T-cell specific activator, in place of PRL, enhanced the apoptotic parameters. Cumulatively, the apoptotic PRL action was addressed to cells unknown than steroidogenic lute~ cells. The most prohable candidate for the direct target cells is Tcells in the luteal tissue that can express mFasL in response to PRL.

Fas는 세포자연사를 유도하는 세포 표면 수용체 단백질로서 면역계에서 중요한 역할을 한다. 이러한 Fas mRNA는 림프조직뿐만 아니 라 비 림프조직에서도 발현한다. 한편 대다수의 난포들은 세포자연사와 연관된 기 작을 통해 난포폐쇄로 진행되는 것으로 알려지고 있으나, 난포폐쇄에 관한 기작은 아직 규명되지 않았다. 따라서 본 연구의 목적은 먼저 생쥐의 난소의 과립세포와 난자에서 Fas와 Fas ligand의 발현 여부를 알아보고, Fas와 Fas liga