Eugenol (4-allyl-2-methoxyphenol) is a phenol derivative and generally used in dental treatment. A few investigator reported that eugen이-induced C)πoto잉city by apopto디c pathway, but it is not yet well understood In the present study, to investigate the eugenol-induced cytoto잉city by apoptosis, we have examined the apoptotic molecules and pathway in primary human gingival fibroblast (HGF) and human salivary gland cells (HSG). To identify apoptotic cell death, 3-(4,5-dimethylthiazol-2-yl)-2 ,5-diphenyl tetrazolium bromide (MTT) reduction assay with or without N-acetylcysteine (NAC), and the morphological study by propidium iodide (pI) staining were screened. And to investigate the apoptotic pathway, reverse transcriptase-polymerase chain reaction (RT-PCR) for apoptotic molecules and caspase aαivity assay were performed. Both M1T reduction assay and an addition of NAC showed that eugenol act as a pro-oxidant led to cell death. With the morphological study, both cells showed apoptotic change by nuclear fragmentation and/or chromatin condensations. With the apoptotic machinery study, the Bax and Bcl-2 mRNA expression were not detected in HGF. But, for HSG, the increased expression of Bax with decreased of Bcl-2 was observed. And the expression of Apaf-l was not detected or nα significantly increased in HGF and HSG, respectively. With measure of caspase activity, there was no change of caspase activities in HGF. But, for HSG, there was decrease of caspase 9 activity and increased caspase 3 activity. We suggest eugenol-treated HGF underwent apoptosis independent of Bcl family and caspase. However, for eugenol-πeated HSG, apoptosis occurred via Bcl famiIy and caspase pathway.

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.

본 연구에서는 한우 태아의 시기별로 35일령, 50일령, 70일령 및 90일령의 fetal fibroblast cell line을 생산하였고, bovine-specific primer와 Y chromosome-specific primer를 이용하여 PCR에 의해 성을 판별하여 각각 암수 2 line의 한우 fetal fibroblast cell line을 확립하였다. 이들 cell line을 계대배양하여 passage number가 10 이상에서 염색체 분석을 실시하였는데 모두에서 80%이상의 세포가 60개의 정상 염색체수의 나타내어 계대배양이 karyotype에 영향을 미치지 않는 것으로 나타났다. Serum starvation과 confluent 배양 방법을 이용하여 Go 상태로 유도되었는지 확인하기 위해 PCNA antibody를 이용하여 Western blotting 분석을 실시하였는데 PCNA 발현이 현저히 감소되는 것을 확인할 수 있었고, 다시 정상 medium으로 환원시켰을 때 세포분열이 재개되어 Go상태로 유도되었음을 확인할 수 있었다. 또한 serum stravation 방법이 conflent한 배양방법보다 PCNA 발현양이 적은 것으로 나타나 좀더 효율적인 Go 상태 세포 주기 조절방법으로 판명되었다.