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.

To examine the differential expression of proteins during the cycling (70～80% confluences) and G0/G1 (full confluences) phases in porcine fetal fibroblast cells, we used a global proteomics approach by 2‐D gel electrophoresis (2‐DE) and MALDI‐TOF‐MS. Cycling cell were harvested at approximately 70% to 80% confluent state while cells in G0/G1 phase were recovered after maintenance of a confluent state for 48 hr. Cellular proteins with isoelectric points ranging between 3.0～10.0, were analyzed by 2‐DE with 2 replicates of each sample. A total of approximately 700 spots were detected by 2‐D gels stained with Coomassie brilliant blue. On comparing the cell samples obtained from the cycling and G0/G1 phases, a total of 13 spots were identified as differentially expressed proteins, of which 8 spots were up‐regulated in the cycling cell and 5 were up‐regulated in the G0/G1 phase. Differentially expressed proteins included K3 keratin, similar to serine protease 23 precursor, protein disulfide‐isomerase A3, microsomal protease ER‐60, alpha‐actinin‐2, and heat‐shock protein 90 beta. The identified proteins were grouped on the basis of their basic functions such as molecular binding, catabolic, cell growth, and transcription regulatory proteins. Our results show expression profiles of key proteins in porcine fetal fibroblast cells during different cell cycle status.

본 연구에서는 clonal cell lines을 효율적으로 확립할 수 있는 방법을 제시하기 위하여 배양액 내에 catalase와 ME 첨가가 clonal cell line 확립 효율에 미치는 영향을 검토하였다. 임신 50일령의 암퇘지에서 얻은 태아섬유아세포를 2회 passage한 후 동결 보관하였다가 실험에 이용하였다. 단일세포를 catalase나 ME가 첨가된 배양액이 들어 있는 96-well dish로 옮겨 배양하였다. 단층이 형성된 세포는 4-we

This study was performed to establish the effective culture condition for the establishment of clonal lines from porcine fetal fibroblast cells. Fibroblasts derived from a pig fetus (Day 50) were cultured and passaged two times before use. A single cell was seeded in 96-well plates, cultured in medium supplemented with different concentrations of FBS, catalase or β-mercaptoethanol (βME), and classified by cell size and morphology. Cells were passaged two times into 4-well dish before freezing. The establishment efficiencies were not different among different concentrations of FBS (0.3 to 5.1%). However, population doubling time (PDT) was significantly decreased by increasing the FBS concentration (P＜0.05). The establishment efficiency of βME-added group (10.4%) was significantly higher than those of catalase-added and control groups (3.5%, and 3.5%, respectively, p＜0.05), and PDT was significantly decreased (23.6 vs 28.1, and 25.5 h, respectively, p＜0.05). However, catalase did not show a positive effect on the establishment efficiency. Cell size and morphology did not affect the establishment efficiency and PDT of clonal lines. The result of present study shows that the establishment efficiency of clonal cell lines can be enhanced by the culture in media supplemented with 30% FBS and βME.