• 한국수정란이식학회지
• /
• 제19권3호
• /
• pp.275-282
• /
• 2004

본 연구는 체외성숙/체외수정 유래의 돼지 난자를 이용하여 체외발달시 배양액의 종류나 교체에 따른 영향을 구명하고자 수행하였다. mNCSU-23에서 체외성숙시킨 다음 mTBM에서 체외수정시킨 난자를 목적에 따라 두 가지로 나누어 실험한 결과는 다음과 같다. 1. 체외성숙/체외수정란을 NCSU-23에서 배양액 교체없이 7일 동안 배양하거나 CZB에서 4일 배양한 다음 Pig-MEM으로 옮겨서 나머지 3일간 배양한 결과, 난분할율은 배양액간 차이를 보이지 않은 반면, 추정수정란대 배반포 발달을(P<0.05)과 분할란대 배반포 발달율은 NCSU-23에서 배양된 것이 유의적으로 높았다(P<0.01). 그러나 배 반포의 ICM 세포수, TE 세포수 및 총세포수에서는 모두 차이가 없었다. 2. 체외성숙/체외수정란를 NCSU-23에서 배양액 교체없이 7일 동안 배양하거나 체외배양 5일째에 신선한 동일 배양액이나 0.4％ BSA를 10％ FBS로 대체한 배양액(mNCSU-23F)으로 완전히 교체하여 배양한 결과, 난분할율, 배반포발달율, 배반포의 ICM 세포수, TE 세포수 및 총세포수 모두 처리간 유의적인 차이를 보이지 않았다. 결과적으로, NCSU-23이 CZB/Pig-MEM보다 체외성숙/체외수정 유래의 난자를 체외발달시키는데 적합한 것으로 사료되며, 체외발달배양 과정에 신선한 배양액이나 일부 변경된 배양액으로의 교체에 대해서는 더 많은 연구가 필요할 것으로 사료된다.

• 대한미생물학회지
• /
• 제35권3호
• /
• pp.251-261
• /
• 2000

V. vulnificus is an estuarine bacterium which causes septicemia and shock in susceptible patients. The organism produces a hemolytic cytolysin (VvH), which has a membrane damaging effect on erythrocytes. To clarify the mechanisms by which VvH might contribute to virulence, we examined its effect on macrophages. When mouse peritoneal macrophages were harvested and co-cultured with hemolysin-positive V. vulnificus strains (100 bacteria/cell), about 60% of the macrophages were killed; macrophages were not killed when co-cultured V. vulnificus strain CVD 707, a VvH-negative deletion mutant. Exposure of macrophages to filtered culture supernatants (2.5 HU/ml) and purified VvH (3 HU/ml) resulted in an increase in dead cells (80 and 90%, respectively), as determined by the trypan blue dye exclusion method and LDH release from macrophages was also increased (70 and 65.5%, respectively). The cytotoxic effect of VvH on macrophages was both the dose- and time-dependent. The VvH caused damage to the macrophage membrane and was blocked significantly by preincubation with cholesterol (p<0.01). Fetal bovine serum showed remarkable inhibition of VvH synthesis by V. vulnificus and inhibited VvH activity in culture supernatant. Cell viability was increased by 35% (p<0.01) and LDH release decreased by 28% (p<0.01) when macrophages were incubated with V. vulnificus (100 bacterial cell) in DMEM-10% FBS for 2 hr. Bacterial clearance activity of mice against V. vulnificus CVD 707 was decreased by pretreatment with 10 HU of VvH. This result suggests that the VvH can impair the membrane of macrophages and may playa role in the pathogenesis of V. vulnificus septicemia.

• 한국수정란이식학회지
• /
• 제16권2호
• /
• pp.91-98
• /
• 2001

본 연구는 phosphatidylinositol 대사의 기질 및 억제물질이 돼지 난모세포의 체외 성숙·수정에 미치는 영향에 대하여 실험을 하였다. 난모세를 inositol (250 mM) 첨가 또는 무첨가한 mTLP-PVA 배양액에서 46시간 체외배양하였다. 성숙이 완료된 난모세포는 신선 돼지정액을 이용하여 6시간 동안 mTALP-PVA 배양액에서 체외수정을 시켰다. 수정 후 6시간에 이들 난모세포를 10% 혈청을 첨가한 TCM-199 배양액에서 추가로 12시간 배양하였다. 제 2성숙분열중기로 성숙한 남모세포의 비율은 대조구 (67.3%)에 비하여 inositol (81.4%)을 첨가하여 46시간 체외성숙시킨 난모세포에서 더 높았다 (P<0.05). 체외수정 후 18시간에 웅성전핵 형성율은 대조구의 27.3%보다 inositol (42.0%)을 첨가한 배지에서 성숙시킨 난모세포에서 더 높게 나타났다 (P<0.05). 난모세포의 성숙에 대한 inositol의 작용을 조사하기 위하여 LiCl (10 mM) 또는 dbcAMP (0.5 mM)를 첨가한 배지에서 난모세포를 배양하였을 때, 이들 두 약제는 난모세포의 성숙을 억제하였다 (P<0.05). 그러나 이 두 약제의 억제작용은 inositol 첨가에 의해 해제되어 대조구 수준까지 성숙율이 개선되었다. DbcAMP와 verapamil은 상승작용을 하여 난모세포의 성숙을 억제하였다. Verapamil을 단독으로 첨가하였을 때, 난모세포의 성숙분열 개시에는 영향이 없었으나 제 2성숙분열중기로의 성숙은 억제되었다. 이상의 결과로부터, inositol 첨가에 의해 PI-Cycle이 활성화되어 간모세포의 핵 및 세포질 성숙과 막의 변화가 유도되어 난모세포의 성숙이 inositol 첨가에 의해 개선되었다는 것이 시사되었다.

• Imaging Science in Dentistry
• /
• 제38권3호
• /
• pp.125-132
• /
• 2008

Purpose : To investigate the effects of irradiation on transforming growth factor ${\beta}_1$ (TGF-${\beta}_1$) mRNA expression and calcific nodule formation in MC3T3-E1 osteoblastic cell line. Materials and Methods : Cells were cultured in alpha-minimum essential medium ($\alpha$-MEM) supplemented with 10% fetal bovine serum and antibiotics. When the cells reached the level of 70-80% confluence, culture media were changed with $\alpha$-MEM supplemented with 10% FBS, 5 mM $\beta$-glycerol phosphate, and $50\;{\mu}g/mL$ ascorbic acid. Thereafter the cells were irradiated with a single dose of 2, 4, 6, 8 Gy at a dose rate of 1.5 Gy/min. The expression pattern of TGF-${\beta}_1$ mRNA, calcium content and calcific nodule formation were examined on day 3, 7, 14, 21, 28, respectively, after the irradiation. Results : The amount of TGF-${\beta}_1$ mRNA expression decreased significantly on day 7 after irradiation of 4, 6, 8 Gy. It also decreased on day 14 after irradiation of 6, 8 Gy. and decreased on day 21 after irradiation of 8 Gy. The amount of calcium deposition decreased significantly on day 7 after irradiation of 4, 8 Gy (P < 0.01) and showed a decreased tendency on day 14, 21 after irradiation of 4, 6, 8 Gy. The number of calcific nodules was decreased on day 7 after irradiation of 4, 8 Gy. Conclusion: Irradiation with a single dose of 4, 6, 8 Gy influences negatively the bone formation at the molecular level by affecting the TGF-${\beta}_1$ mRNA expression that was associated with proliferation and the production of extracellular matrix in MC3T3-E1 osteoblastic cell line.

• Archives of Pharmacal Research
• /
• 제28권5호
• /
• pp.626-635
• /
• 2005

Liposome as a carrier of topotecan (TPT), a promising anticancer drug, has been reported in attempt to improve the stability and antitumor activity of TPT. However, the biodistr ibution pattern of TPT liposome in vivo and PEG-modified liposome containing TPT have not been studied systemically. In this paper, the in vitro stability and in vivo biodistribution behavior of several liposomes containing TPT with different lipid compositions and PEG-modification were studied. Compared with the 'fluid' liposome (S-Lip) composed of soybean phosphatidylcholine (SPC), the 'solid' liposome (H-Lip) composed of hydrogenated soybean phosphatidylcholine HSPC decreased the leaking efficiency of TPT from liposome and enhanced the stability of liposome in fetal bovine serum (FBS) or human blood plasma (HBP). The results of biodistribution studies in S$_{180}$ tumor-bearing mice showed that liposomal encapsulation increased the concentrations of total TPT and the ratio of lactone form in plasma. Compared with free TPT, S-Lip and H-Lip resulted in 5- and 19- fold increase in the area under the curve (AUC$_{0\rightarrow\propto}$), respectively. PEG- modified H-Lip (H-PEG) showed 3.7-fold increase in AUC$_{0\rightarrow\propto}$ compared with H-Lip, but there was no significant increase in t$_{1/2}$ and AUC$_{0\rightarrow\propto}$ for PEG-modified S-Lip (S-PEG) compared with S-Lip. Moreover, the liposomal encapsulation changed the biodistribution behavior, and H-Lip and H-PEG dramatically increased the accumulation of TPT in tumor, and the relative tumor uptake ratios were 3.4 and 4.3 compared with free drug, respectively. There was also a marked increase in the distribution of TPT in lung when the drug was encapsulated into H-Lip and H-PEG. Moreover, H-PEG decreased the accumulation of TPT in bore marrow compared with unmodified H-Lip. All these results indicated that the membrane fluidity of liposome has an important effect on in vitro stability and in vivo biodistribution pattern of liposomes containing TPT, and PEG-modified 'solid' liposome may be an efficient carrier of TPT.

• Journal of Periodontal and Implant Science
• /
• 제35권3호
• /
• pp.623-634
• /
• 2005

Human periodontal ligament fibroblast(hPDLF) is very important to cure periodontal tissue because it can be diverged into various cells. This study examined the expression of MMP-1, TIMP-1, periodontal ligament specific PDLs22, Type I collagen, Fibronectin, TIMP-2, telomerase mRNA in a replicative senescence of hPDLF. The periodontal ligament tissue was obtained from periodontally healthy and non-carious human teeth extracted for orthodontic reasons at the Chosun University Hospital of Dentistry with the donors' informed consent. The hPDLF cells were cultured in a medium containing Dulbecco's modified Eagle medium(DMEM, Gibco BRL, USA) supplemented with 10% fetal bovine serum(FBS, Gibco BRL, USA) at 37C in humidified air with 5% $CO_2$. For the reverse transcription-polymerase chain reaction(RT-PCR) analysis, the total RNA of the 2, 4, 8, 16, 18, and 21 passage cells was extracted using a Trizol Reagent(Invitrogen, USA) in replicative hPDL cells. Two passage cells, i.e. young cells, served as the control, and ${\beta}-actin$ served as the internal control for RT-PCR The results of this study about cell morphology and gene expression according to aging of hPDLF using RT-PCR method are as follows: 1. The size of hPDLF was increased with aging and it was showed that the hPDLF was dying in the final passage. 2. PDLs22 mRNA was expressed in young hPDLF of the two, four, and six passage. 3. TIMP-1 mRNA was expressed in young hPDLF of the two and four passage. 4. There was a tendency that MMP-1 mRNA was weakly expressed over eighteen. 5. Type 1 collagen mRNA was expressed in almost all passages, but it was not expressed in the final passage. 6. Fibronectin mRNA was observed in all passages and it was weakly expressed in the final passage. 7. TIMP-2 and telomerase mRNA were not expressed in this study. Based on above results, it was observed that PDLs22, Type 1 collagen, Fibronectin, MMP-1. and TIMP-1 mRNA in hPDLF were expressed differently with aging. The study using the hPDLF that is collected from healthy patients and periodontitis patients needs in further study.

• Archives of Pharmacal Research
• /
• 제19권2호
• /
• pp.116-121
• /
• 1996

Antisense oligonucleotides seem to provide a promising new tool for the therapy. Choi et al. (1995) reported antisense phosphorothioate oligonucleotides (PS-ODN, 25 mer) complementary to TGF-.betha. mRNA designed for scar formation inhibitor to eliminate scars, which was caused by undesired collagen deposition due to overexpression of TGF-.betha., in wounded skin. PS-ODN were evaluated in vitro for skin penetration using normal and tape-stripped damaged rat skin. The in vitro skin transports were carried out with partially modified PS-ODN (6S) and fully modified PS-ODN (25S). The cumulative amount of PS-ODN (6S) penetrated through normal rat skin was $0.234{\pm}0.041{\mu}g/cm^2$ and that of tape-stripped damaged rat skin was $1.077{\pm}0.301{\mu}g/cm^2$ over 8 hrs. PS-ODN (25S) can not be found in receptor medium through normal skin due to high molecular weight (Mol.Wt.=8,000) and polyanionic charge. However, the cumulative amount of PS-ODN (25S) penetrated across damaged rat skin in PBS was $0.340{\pm}0.296{\mu}g/cm^2$ over 8 hrs. The absense of dermis raised the cumulative amount of PS-ODN (6S) penetrated through rat skin. And the fluxes of PS-ODN (6S) and PSODN (25S) at 8hrs across damaged rat skin were $134.63{\pm}37.67{\mu}g/cm^2$ h, and $42.50{\pm}36.95ng/cm^2$ h, respectively. While PS-ODN (25S) was stable in 10% heat inactivated fetal bovine serum (FBS) during 24 hrs, PS-ODN (6S) was less stable than PS-ODN (25S), but was markedly stable than unmodified phosphodiester. It is suggested that the cumulative amount of PS-ODN (6S) penetrated through damaged rat skin is larger than that of PS-ODN (25S) since the former is easier to degrade by nuclease than the latter and then is apt to penetrate into skin. Thus, PS-ODN represents a logical candidate for further evaluation due to the potential for delivery into the wounded skin.

• 대한약침학회지
• /
• 제22권3호
• /
• pp.176-183
• /
• 2019

Objective: In the current work, we investigated the cytotoxic and apoptotic effects of Thymoquinone (TQ), an active compound of Nigella sativa (N. sativa) and Cis-platinum, on normal renal epithelial (GP-293) and human renal adenocarcinoma cell lines (ACHN). Methods: GP-293 and ACHN cell lines were cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% Fetal bovine serum (FBS) and 1% penicillin plus streptomycin antibiotic. The MTT assay was used for cellular viability assessment. Viability of cells was observed using inverted light microscope 24, 48 and 72 h after exposure of the cells to various concentrations of TQ (1, 2.5, 5, 10, 50 and $100{\mu}g/ml$) and Cis-platinum (0.5, 1, 1.5, 2, 3, 6 and $12.5{\mu}g/ml$). Moreover, apoptosis was analyzed with a flow-cytometry method. The untreated cells were considered as control group. Results: Morphological changes such as reduced cell number and increased intercellular distance and reduced cell viability in ACHN and GP-293cell lines were observed in both TQ and Cis- platinum groups; however, Cis-platinum had greater effect on ACHN cell line than GP-293 cell line. In addition, GP-293 cell line was more sensitive to TQ compared to ACHN cell line. Furthermore, TQ and Cis-platinum had apoptotic effects on both ACHN and GP-293 cell lines. Conclusion: Our findings demonstrated that TQ and Cis-platinum had cytotoxic and apoptotic effects on both cell lines, However, GP-293 cell line was more sensitive to TQ. Additionally, Cis-platinum was more effective on ACHN cell line than on GP-293 cell line.

• 한국가축번식학회지
• /
• 제21권2호
• /
• pp.167-176
• /
• 1997

본 연구는 수정란을 수란우에 이식하기 이전에 형질전환가능 수정란을 선발할 수 있다면 형질전환동물의 생산에 크게 도움이 되므로 3.2 kb $\beta$-actin promoter (lacZ/n대) DNA를 미세주입하여 배반포기배에서의 발현을 확인하여 이들을 선발할 수 있는가를 규명하고자 하엿다. 채란된 난포란은 10%FBS, 5$\mu\textrm{g}$/ml LH, 0.5$\mu\textrm{g}$/ml FSH, 100 unit/ml penicillin 및 100 $\mu\textrm{g}$/ml streptomycin이 함유된 TCM199에 22~24 시간동안 체외성숙을 유도후 5$\mu\textrm{g}$/ml heparin으로 수정능획득을 유도한 1$\times$106 sperm/ml의 정자로 체외수정을 시켰다. 체외수정후 18~20시간째에 vortexing에 의해 과립막세포를 제거하고 원심분리시켜 자/웅전핵이 확인되는 수정란의 핵에 3~4 ng/${\mu}\ell$ lacZ/neo DNA를 미세주입하였다. 모든 수정란의 배양은 3 mg/ml BSA, 20${\mu}\ell$/ml NEM AMINO acids 및 40${\mu}\ell$/ml BME amino acids가 함유되어 있는 CR1aa 배양액에 neo/DNA로 transfected 된 BRL 단층에서 실시하였다. G418에 대한 적정농도를 찾기 위하여 정상적인 수정란에 0, 50, 100 및 200 $\mu\textrm{g}$/ml G418를 첨가하여 배양한 결과 8일째에 30.3%(44/145), 8.7%(13/150), 0.7%(1/151) 및 0% (0/134)의 수정란이 배반포기까지 발달하였다. 그래서 본 실험에서는 일정하게 100$\mu\textrm{g}$/ml G418을 첨가하여 배양하였다. 총 1,127개의 수정란을 미세주입후 G418 없는 배양액에서 710개 (63.0%)가 분할하였다. 미세주입후 48시간째에 2-세포기이상 분할된 수정란을 대조구 및 100$\mu\textrm{g}$/ml G418처리구를 무작위로 할당하여 배양하였으며, 또한 740개의 정상수정란도 같은 반복수로 배양을 실시하였다. 미세주입한 수정란은 8일 후 11.6%(26/255) 및 5.2% (14/267)가 대조구 및 G418 처리구에서 배반포기까지 발달하였으며 정상수정란은 27.2% (151/740)가 배반포기 배까지 발달하였다. 미세주입후 대조구에서는 23.1$\pm$2.6/70.7$\pm$4.7 (32.7%)의 할구가 $\beta$-Gal 활력을 보였고, 반면에 100$\mu\textrm{g}$/ml G418 처리구에서는 40.3$\pm$4.1/48.8$\pm$7.5 (82.6%)가 $\beta$-Gal 활력을 보였다. 비록 mosaic 형태로 외래유전자가 발현되었지만 대조구에서 87.0% (26/30개) 배반포기가 $\beta$-Gal 활력을 보인 반면, G418 처리구에서는 모든 배반포기가 $\beta$-Gal 활력을 보였다 (P<0.05). 그러나 대조구 및 G418 처리구의 ICM colony에서는 영양배엽과 내배엽을 제외한 epiblast에서는 확인되지 않았다. 그러나 이 결과로부터 $\beta$-actin promoter/lacZ gene이 integration되지 않는 것인지 또는 다만 염색 확인이 되지 않는 것인지를 판단할 수는 없다. 이상의 결과는 미세주입후 G418에서 배양한 배반포기배에서는 대부분의 할구에서 주입된 gene을 발현하고 있었으나 ICM colony에서는 특히 epiblast에서는 발현되지 않거나 침묵하고 있었다. 비록 G418 처리구에서 훨씬 더 높은 비율로 주입된 gene이 발현되고 있으나 총세포수는 유의적으로 감소하여 이후 형질전환동물의 생산과 ES like-cell의 설립에는 감소될 것으로 사려된다. 그러나 형질전환 수정란의 선발 및 형질전환동물의 생산능력에 관해서는 더 많은 연구가 필요하다고 사려된다.

• 한국수정란이식학회지
• /
• 제21권1호
• /
• pp.21-27
• /
• 2006

고양이의 연령에 따른 난포의 분포를 알아보고 난포의 배양과 난자 생산의 가능성을 알아보고자 0.3세부터 5세까지의 총 41 마리 고양이를 난소 적출술 후 사용하였다. 고양이 난소의 무게와 크기를 측정하고 난포의 분포를 알아보기 위해 난소를 10% formalin에 보관한 후 고정된 난소를 $3{\mu}m$-sections으로 자른 후 조직 슬라이드를 준비하여 hematoxylin와 eosin으로 염색하였다. 난포의 분포를 200 배율와 400 배율 현미경하에서 평가하였으며 난포를 원시 난포(primordial), 일차 난포(primary), 이행성 난포(transitional), 전동난포(preantral), 동난포(antral)로 분류하여 관찰하였다. 단순 기계적인 방법에 의해 전동난포(preantral follicles)를 분리하여 배양배지가 담긴 96 microliter plates well로 옮겨 배양하였다. 난포의 배양액은 Medium 199에 1% ITS(insulin, transferrin, selenium)를 첨가하고 10% FBS나 10% PVA를 첨가하여 사용하였으며 배양배지위에 mineral oil를 덮고 16일 동안 난포를 배양하였다. 난포의 크기는 4일마다 측정하였다. 0.3세부터 5세까지 고양이 난소의 무게는 0.1g에서 0.3g으로 증가하는 양상을 보이기는 했으나 유의적인 차이가 없었다. 난포의 분포는 고양이의 연령에 관계없이 원시난포의 분포가 그 외 난포들의 분포보다 높게 나타났다(p<0.05). 난포를 4일 이상 배양하였을 때 배양액의 조성성분과 관계없이 난포의 크기가 감소하였으며, 체외 배양된 난포로부터 적은 수의 난자만을 회수할 수 있었다. 많은 수의 원시 난포 등을 분리하기 위한 유용한 난포 분리법과 난포의 배양에 필요한 기타 성분들의 비교 연구가 이루어져야 할 것으로 생각되며 이러한 집 고양이를 이용한 기초 번식 기술은 미래에 멸종위기에 처한 고양이 과 동물을 보존하기 위한 중요한 방법이 될 것으로 기대된다.