It has been reported that light-emitting diodes(LED) can be used in the treatment of oral diseases. Although bio-stimulatory effects of LED irradiation such as promotes stimulation of wound healing have been well known, there are few reports about molecular mechanism associated with cell cycle by LED irradiation. The purpose of present study was to examine the molecular event in cell cycle of LED irradiation on primary human gingival fibroblast(hGF) in vitro. The source of light for irradiation was a continuous-wave LED emitting at a wavelength of 635nm, and manufactured that energy density was 5mW/cm2 on sample surface. The hGF were irradiated for 1 hour at 37℃ in 5% CO2 humidified chamber. Experimental samples were acquired at 0 (right after irradiation), 8 and 24 hour after irradiation. To investigate the molecular mechanisms associated with cell cycle, growth phase was determined by flow cytometry and mRNA expression of cyclin A, cyclin B, cyclin D1, cyclin E, cdc2, PCNA, p18, p27, p21, and p53 were determined by real time RT-PCR. Flow cytometric analysis demonstrated the percentage of cells in the G1 and S phase were decreased, but the G2 phase increased, which showed cells irradiated by LED were transitioned from S to G2 phase. For mRNA expression, cyclin B, cdc2, PCNA and p53 were increased at 0 hour after irradiation, and most of cell cycle molecules were increased at 8 hour after irradiation. At 24 hour after irradiation, cyclin A, cyclin E, PCNA and p18 were increased. Taken together, LED irradiation induced proliferation of hGF cells through transition from S to G2 phase.

본 연구는 demecolcine 처리에 의한 탈핵과 수핵란 세포질의 세포 주기가 소 체세포 핵이식란의 발육에 미치는 영향을 검토하였다. 체외에서 16~20시간 성숙배양된 난자를 극체 방출 유무 및 MI, MII기 난자로 구분하여 0.4㎕/mL demecolcine으로 40분간 처리 후 염색체 부위가 돌출된 난자는 탈핵 후 핵이식에 공시하였다. 소의 귀 피부 세포를 탈핵란에 이식하여 전기융합과 활성화 처리(Ca-ionophore+DMAP)를 거쳐 체외 배양하였다. Demecolcine처리 후 86.2%의 난자가 염색체 부위의 돌출을 보여 이 중 98.8%가 탈핵에 성공하였다. Demecolcine은 핵이식란의 발육에 영향을 주지 않았다. 제1극체 방출란 유래 핵이식란의 배반포 발육율은 극체 미방출란 유래 핵이식란에 비하여 유의적으로 높았다(18.2% vs.4.6%, P<0.05). 한편, MI 난자 유래 핵이식란의 분할율 및 배반포 발육율은(69.4%와 5.9%) MII 난자 유래 핵이식란에 비하여 유의적으로 낮았다(96.7%와 23.9%, P<0.05). 본 연구의 결과는 demecolcine 처리가 소 난자의 탈핵에 매우 효과적이며 MII기 난자가 MI기 난자에 비하여 수핵란 세포질로 더 적절하나 극체 미방출란 및 MI기 난자도 비록 제한적이기는 하지만 핵이식란의 배반포 발육을 지원할 수 있음을 보여준다.

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.

To improve the efficiency of production of cloned embryos and animals by nuclear transplantation in the rabbit, the effect of cell cycle of donor nuclei and type of recipient cytoplasm on the in vitro developmental potential and production efficiency of offspring was determined. The embryos of 16-cell stage were collected from the mated does at 48h post-hCG injection and they were synchronized to G phase of 32-cell stage. The oocytes collected at 14h post-hCG injection were freed from cumulus cells and then enucleated. One group of the enucleated cytoplasms was activated by electrical stimulation prior to injection of donor nucleus, and the other group was not pre-activated. The separated Gphase blastomeres of 32-cell stage embryos were injected into the perivitelline space of recipient cytoplasms. After culture for 20h post-hCG injection, the nuclear transplant oocytes were electrofused and activated by electrical stimulation and the fused nuclear transplant embryos were co-cultured for 120h and the nuclear transplant embryos developed to blastocyst stage were stained with Hoechst 33342 dye and their blastomeres were counted. Some of the nuclear transplant embryos developed in vitro to 2- to 4-cell stage were transferred into the oviducts of synchronized recipient does. The electrofusion rate was similar between the types of donor nuclei and recipient cytoplasms used. However, the nuclear transplant embryos using G phase donor nuclei were developed to blastocyst at higher rate(60.3%) than those using S phase ones(24.7%). Also, when non-preactivated oocytes were used as recipient cytplasms, the develop-mental rates of nuclear transplant embryos to blastocysts were significantly(P< 0.05) higher(57.1%) than those using preactivated ones(20.8%). The cell counts of nuclear transplant embryos developed to blastosyst stage were increased signficantly(P<0.05) more in the non-preactivated recipient cytoplasm(163.7 cells), as compared whit the preactivated recipient cytoplasm(85.4 cells), A total of 49 nuclear transplant embryos were tranferrid into 5 recipient does, of which two offsprings were produced from a foster mother 31 days after embryo transfer. these results showed that the blastomeres of G1 phase and non-preactivated oocytes might be utillzed efficiently as donor nuclei and recipient cytoplasms in the nuclear transplant procedure, thought the offspring production remained still low.

The objectives of the present study were improvements in the efficiency of developmental rates to morula and blastocyst stages to produce a large number of genetically identical nuclear transplant embryos. The oocytes collected from slaughterhouse ovaries were matured for 24 h and then enucleated and cultured to allow cytoplasmic maturation and gain activation competence. And then the donor embryos were treated for 12 h with 10 g /ml nocodazole and 7.5 g /ml cytochalasin B to synchronize the cell cycle stage at 26 h after the onset of culture. The blastomeres were transferred into the perivitelline space of the enucleated nocytes and blastomeres and oocytes were fused by electrofusion. The cloned embryos were then cultured in various conditions to allow further development. The age of the recipient(30 vs 40 h) had no significant effect on the fusion rates(82.4 vs 82.1%) and the developmental rates to morula /blastocyst(9.8 vs 11.0%). Effect of Nocodazole treatment on the donor cell cyle synchronization to improve the developmental rates of bovine nuclear transplant embryos was significantly higher than control group(21.4 vs 10.1%, p<0.05). Significant differences were in the percentage of fusion rates(72.9,77.1vs 61.9%) in three types of fusion medium(PBS(+), mannitol and sucrose, p<0.01). The developmental rates of bovine nuclear transplant embryos appeared to be highest in mSOF medium under 5% 0 condition, but no significant differences were found when compared with TCM199-BOEC and mSOF under two different oxygen ratio(5 and 20%).

BACKGROUND Ca2+ oscillations during fertilization induce eggs activation and embryonic development in mammalian eggs.. The type 1 inositol 1,4,5-trisphosphate receptor (IP3R1) is in charge of Ca2+ oscillations for the release of stored Ca2+ from the endoplasmic reticulum. The capacity of this oscillation is obtained during egg maturation and corresponds with an increase in the sensitivity of the IP3R1 and their localization in cytoplasm. Cluster formation of IP3R1 in the egg cortex is important to initiation of Ca2+ oscillations during egg and sperm fusion. In this study, we investigated that cell cycle–coupled redistribution of IP3R1 and Ca2+- oscillatory activity in mouse zygotes. MATERIALS AND METHODS Metaphase II arrested eggs were collected from ICR female mouse after super ovulation induction. At 14 hr post hCG, MII eggs were collected, and artificially activated in Ca2+ free CZB medium with 10 mM SrCl2 for 2 hrs. Pronuclear zygotes (PN) were collected from Strontium activated eggs at 8 hr post activation, and the first mitotic eggs were collected at 16~17 hr post activation. To identify cell cycle coupled IP3R1 redistribution, MII eggs, zygotes, and first mitotic eggs were collected, and fixed for immunostaining with anti-IP3R1antibody (CT-1) and observed on CLSM. Ca2+-oscillatory activity was monitored with fluorescence microscope mounted SimplePCI program (Hamamatsu) after injection of cRNA of mouse phospholipase C zeta (mPLCZ). RESULT IP3R1 were shown clusters, 1~2 um in diameter, in cortex of ovulated MII eggs with high Ca2+ oscillatory activity by mPLCZ injection. These eggs represent more than 6 spikes per 60 min. However, IP3R1 clusters were disappeared in PN eggs and these eggs showed very low Ca2+- oscillatory activity by mPLCZ. In mitosis I stage eggs, clusters of IP3R1 were appeared and Ca2+-oscillatory activity was reactivated slightly (2 spikes per 60 min). CONCLUSIOINS This study introduced the redistribution of IP3R1 clusters were occurred in egg activation according to cell cycle dependent manner. Also, functional modification of IP3R1 including protein phosphorylation was associated with cortical clustering of IP3R1 in cell cycle coupled Ca2+ oscillatory activity.