In this study, the apoptotic effects of the actin disruption agent, latrunculin B(LB) have been investigated on p53 deficient chronic myeloid leukemia cell line K562. A dose-dependent decrease in K562 cell proliferation was observed after LB treatment with maximum decrease in cell proliferation being at 1.5μM where the percent inhibition was 66.53%. F-actin stained with TRITC-phalloidin was shown as a peripheral ring or appeared diffusely distributed throughout the cytoplasm in untreated cells, this actin ring was decreased following LB treatment, and even large focal actin aggregates were formed. Treatment of K562 with LB(1.5μM) generated ROS substantially. LB activated expression in a dose-dependent manner. Therefore it can be concluded that LB, depolymerising agent of actin, induces apoptosis by producing ROS and up-regulating NF-kB and COX-2 activation.

Low energy photon irradiation by light in the far red to near infrared spectral range(630~1000nm) using low energy lasers or light emitting diode arrays has been found to modulate various biological processes in cell culture and animal models. The purpose of this study was to examine the light emitting diode irradiation effect on activity of normal human osteoblast on titanium plate in vitro by various energy density, and to observe morphologic change of NHost on titanium plate and to analysis concentration of Ca++, IP and ALP. NHost were cultured in DMEM containing 10% FBS, and observed by inverted microscope for attatchment to the surface of titanium plate. Ca++, I.P., and alkaline phosphatase( ALP) concentration in medium was calculated during 4 weeks, which was treated with Wilcoxon rank, Anova test and linear regression. Morphologic changes showed LED produced in vitro increases of cell growth of 144~256% in NHost. During a culture period, Ca++ concentration was decreased. LED treatment(>3J/cm2) stimulate calcium consumption in NHost. Statistically, a significant difference was not found between LED power density. LED treated group(>3J/cm2) had higher total inorganic phosphate concentrations than control group in NHost. Statistically, a significant difference was not found between LED power density. No significant changes were observed between ALP acitivity and LED treatment. In spite of LED power density, there were rapid growth rate of NHost and no significant of Ca++, IP and P concentration but these concentration showed predominant change than that of control.

The prominent side effect of cyclosporin A, immunosuppressive agent, in oral tissues is gingival overgrowth(GO). It is characterized by the gingival enlargement with epithelial thickening, a large number of proliferating fibroblasts and overproduction of ECM components. Fibroblast accumulation in Cs A induced GO is caused by the Inhibition of apoptosis. But CsA effect on normal human oral keratinocyte(NHOK) remains unclear. Transglutaminase 2(TGase 2) which is expressed and activated in tissue where epithelial cells undergo apoptosis has been used as a marker for apoptotic cells. The purpose of this study were to study the effect of CsA on the proliferation and apoptosis of cultured NHOK by TGase 2 expression. After primary cultured NHOK was treated by 0.1, 1.0 and 10ug/ml Cs A, growth curve, MTT assay for succinyl dehydrogenase activity and RT-PCR for TGase 2 mRNA expression were done. The obtained results were as follows. MTT assay showed about 65% cell viability at 1.0μg/ml and 40% at 10μg/ml CsA. Growth curve showed normal S curve on control & DMSO, while decreased growth rate after 3 days of higher CsA tx. TGase 2 mRNA expression of cultured NHOK was the highest at 10μg/ml Cs A. TEM showed chromosome margination, and vacuole formation and clustered mitochodria in cytoplasm of cultured NHOK after CsA tx. It suggested that higher CsA might induce apoptosis of NHOK correlated with increased TGase 2 mRNA expression

We attempted to control the maturation promoting factors (MPF) activity and nuclear remodeling of somatic cell nuclear transfer (NT) bovine embryos. Bovine ear skin fibroblasts were fused to enucleated oocytes treated with either 5 mM caffeine for 2.5 h or 0.5 mM vanadate for 0.5 h and activated. The nuclear remodeling type of the reconstituted embryos was evaluated 1.5 h after activation. MPF activity was assessed in enucleated and chemical treated oocytes before the injection of a donor cell. Effect of chemicals on the embryonic development was evaluated with parthenogenetic embryos. MPF activity increased significantly by caffeine treatment, but decreased by vanadate treatment (p<0.05). Caffeine or vanadate had no deleterious effect on the parthenogenetic embryo development. In caffeine treated group, premature chromosome condensation (PCC) was occurred in 72.2% of NT embryos (p<0.05). In contrast, vanadate induced the formation of a pronucleus-like structure (PN) in a high frequency (68.9%, p<0.05) without PCC (NPCC). Blastocyst development of NT embryos increased by treating with caffeine (30.3%), whereas decreased by treating with vanadate (11.4%) compared to control (22.1%, p<0.05). The results indicate that caffeine or vanadate can control of MPF activity and remodeling type of NT embryos, resulting in the increased or decreased in vitro development.

곰피추출물의 CCD-986sk cell line monolayer (human fibroblast, KCBL-21947)에 대한 피부세포 생리활성효과를 측정하고, 또한 곰피추출물의 Clone M-3 mouse melano-cyte cell line에 대한 melanin formation 저해효과를 측정하기 위해 in vitro레벨에서 실험을 실시하였다. 곰피는 다년생 갈조류의 일종으로 이 종은 한국 연안해역에서 중요한 1차생산자의 역할을 담당하고 있는

본 연구는 체세포 복제란 이식우의 분만에 있어서 혈중 스테로이드호르몬, TGF-β1 농도와 분만지연의 상관 관계를 살펴보고자 실시하였다. 대조군으로는 인공수정(AI)을 통하여 임신한 암소(cow)들을 사용하였다(AI-R). 모든 AI-R들은 자연분만(n=5, 임신 284+-0.71일)을 하였다. 분만징후를 보이지 않는 체세포 복제란 이식우(n=5, SCNT-R)들은 분만 예정일보다 10일 정도 지난 임신 292일째에 제왕절개(Caesarean section, C-sec)를 실시하여 분만하였다. 혈액 및 태반 샘플을 분만 전.후에 채취하여 형태 및 중량 등을 측정하였다. 혈장호르몬인 Progesterone(P4)와 Estradiol-17β (E2) 농도는 방사선동위원소 면역 분석 시험(RIA) 방법을 이용하여 측정하였다. 혈장 및 태반분엽의 TGF-β1 농도는 ELISA 방법으로 측정하였다. SCNT-R에서 회수한 태반의 무게는 AI-R의 것과 비교하여 유의적으로 무거웠다(p<0.05). 분만 직전 SCNT-R들의 혈장 내 P4 농도는 AI-R들의 그것과 비교하여 유의하게 높았다(p<0.01). 하지만 SCNT-R들의 혈장 내 E2 농도는 AI-R과 비교하여 상대적으로 낮게 나타났다(p<0.01). 한편, 분만 전.후 SCNT-R들에서 혈장 또는 태반분엽의 TGF-β1 단백질 발현 수준은 AI-R들과 비교하여 각각 유의적으로 높은 수준을 유지하였다(p<0.01). 이상의 결과를 종합하여 보면, 분만 시 P4 및 E2의 이상 발현과 높은 수준의 혈장 및 태반 내 TGF-β1 단백질은 체세포 복제태아의 분만지연을 야기하는 중요한 요인들 중의 하나일 것이라 사료된다.

Vaginal cytology with behavioral observation was performed in 34 estrous cycles in 16 Miniature Schnauzer dogs to evaluate the its usefulness. The mean duration of proestrus and estrus in Miniature Schnauzer based on behavioral observation and vaginal cytology was (Mean S.D., range: ) and days for proestrus, and and days for estrus, respectively. The duration of each phase of the estrous cycle was not significantly different based on between behavioral observation and vaginal cytology. The gestational length from the first day of male acceptance was days, days from the first male refusal, and days from the onset of cytologic diestrus, respectively. Vaginal cytology during the estrous cycle were significantly characteristic of large intermediate cells in proestrus, anuclear cells in estrus, small intermediate cells in diestrus, and parabasal cells and small intermediate cells in anestrus (p<0.001), respectively. Cornification index (CI) by vaginal cytology was higher in proestrus () and estrus (), then it was decreased in diestrus () and anestrus ().

조혈 줄기 세포에의 효과적인 유전자 전달은 유전자 치료의 새로운 가능성을 제시할 수 있다. 레트로바이러스를 이용한 유전자 전달 기술은 많은 기초 연구와 임상 시도가 이루어진 대표적인 바이러스이다. 그러나 현재 사용되고 있는 in vitro에서의 조혈 줄기 세포에의 유전자 도입은 조혈 줄기 세포의 분화 유도, 자기 복제 능력과homing 능력의 저하 등 많은 문제점이 있다. 본 연구는 이러한 문제점을 극복하기 위한 방법으로서 마우스의 대퇴골에 직접 레트로바이러스를 이식하는 IBM (Intra-Bone Marrow) 방법을 이용하여 조혈 줄기 세포에의 효과적인 유전자 도입을 시도하였다. IBM 이식 2주 후 마우스의 각 조직을 분석한 결과, 골수뿐 아니라 림파절, 비장, 간장 세포 등에서 유전자가 안정적으로 발현하는 것을 관찰하였다. 또한, 6.4+-2.7%의 골수조직 존재 조혈줄기/전구세포에서 도입된 유전자가 안정적으로 발현하고 있는 사실을 확인하였다. 본 연구의 결과를 바탕으로 IBM 이식 방법을 이용한 생체 조직 내 레트로바이러스의 유전자 도입은 조혈 줄기 세포를 이용한 유전자 치료에 매우 효과적인 방법이라는 사실을 시사해주고 있다.

본 연구는 돼지 골수에서 존재하는 조혈 줄기 세포 및 전구 세포를 이용해 이종 동물 모델인 태아 마우스 복강 생체 이식을 통하여 돼지 조혈 세포의 이종 조혈 조직에서의 증식과 분화 특성을 규명하였다. 선천성 면역 부전 마우스인 NOD/SCID 마우스 태아 조혈 환경에 돼지 골수 유래 조혈 줄기 세포 및 전구 세포를 이식하고, 이식 후 5주령에 마우스 조혈기관에서의 돼지 조혈 세포의 증식과 분화 특성을 돼지 특이적 항체 면역 염색으로 유세포 분석을 실시한 결과, 마우스 조혈 조직인 골수, 흉선, 간장, 비장 및 림파절에서 돼지 조혈 세포의 분화 및 증식이 관찰되었다. 특히 돼지의 T 면역세포가 골수계 세포에 비해서 높은 chimerism이 관찰되어 태생 초기의 NOD/SCID 조혈 환경에 의한 특이적 T 면역세포의 증식에 적합한 조혈 환경을 제공하고 있다는 사실이 밝혀졌다. 본 마우스 신생 NOSD/SCID 복강 이식 동물 모델을 이용해 돼지 T 면역세포의 분화 발달 연구 및 이종 장기 이식 기전 연구에 좋은 모델로서 활용이 기대된다.

Hydroxyapatite (HAp) and biphasic calcium phosphate (BCP) nano powders were synthesized using the microwave-assisted synthesis process dependent on pH and microwave irradiation time. The average size of a powder was less than 100 nm in diameter. Through in-vitro cytotoxicity tests by an extract dilution method, the HAp and BCP nano powders have shown to be cytocompatible for L-929 fibroblast cells, osteoblastlike MG-63 cells and osteoclast-like Raw 264.7 cells. The activation of osteoblast was estimated by alkaline phosphatase (ALP) activity. When the HAp and BCP were treated to MG-63 cells, alkaline phosphatase activities increased on day 3, compared with those of the untreated cells. Also, the collagen fibers increased when the HAp and BCP powders suspension were treated to MG-63 cells, compared to those of the untreated cells. Quantitative alizarin red S mineralization assays showed a trend toward increasing mineralization in osteoblast cultured with powder suspension. In conclusion, hydroxyapatite and biphasic calcium phosphate appeared to be a bone graft substitute material with optimal biocompatibility and could be further applied to clinical use as an artificial bone graft substitute.

Cyto kinc production by epiclerma l keratinocytes has been investiga ted ext ensive ly cluring the past decacle in the skm Furthermore‘ cyto kines produced by pidermal kerat inocytes may be regar decl as important regulators in inflammation, 1 nunune responses‘ and during wound healing, The associa tion of specific cytokine patterns in proliferative a ncl/or infl ammatory related cha nges in the s kin suggests a role in the pathogenesis of certain skin diseases, Although it is conceiva b1e that the pa ttern of cytokine express ion in o1'al kera tinocytes might be sirnilar t o tha t of epidermal origin, the1'e a re only sparse reports that have s hown t his experi menta lly, In addition, there is s ome evidence that there may be differe n ces in the proliferative capacity of oral versus epidermal keratinocytes, Since t his may be crucial for better un clerstanding the biologica l processes in the oral mucosa and how they may differ from the e pidennis, we a na lyzed the cyto kine expression pat tern of human oral kera tinocy tes, The purpose of this study were to investigate mRNA & protein express ion 0 1' va rious cy tokines between NHOK and NHEK by RT-PCR & immunoslot blotting, and to apply its results 1‘0 1' bet ter understancling t he pathological processes in the o1'al mucosal d1seases Cultured NHEK showecl larger a rea of cel lula r s tratil'icat ion tha n cul tu ++ 1'ed NHOK in 0 05mM Ca concentra tion, 1L - 1α , IL- 6 mRNA expression 0 1' cult ured NHOK we1'e hi gher than that of NHEK, TNF- mRNA expression of NHEK was about 1, 2 folds than that 0 1' NHOK, ICAM- 1 mRNA expression of NHOK was a bout 13 folds t han that of NHOK, while NHEK was weakly detected, 1L- 1a , IL-6 pro tein expression of cul tured NHOK were hi gher t han t ha t of NHEK TNF-a protein expression of NHEK was about 1, 2 fo lds than that of NHOK, 1CAM- 1 protein expression of NHOK was about 40 folcls than that of NHOK, whil e NHEK was weakly detectecl , mRNA express ion was associa ted wi th prot ein expression in cul tured NHOK ancl NHEK, It s uggestecl that lL- 1a ‘ 11-6 and ICAM-1 mRNA and protein be highl y expressecl in cultured NHOK, Especially, ICAM- 1 would be a useful ma rker for the pathogenesis of oral mucosal di sease,

AJthough salivary gland adenocarcinoma accounts for third prevalence rate of all salivary gland tumors. it is one of the most aggressive solid tumors. Current therapy does not s ignificantly improve survival rates. Thus‘ investigating new therape utic modali t ies aga inst sali va ry gland adenocarcinoma is necessary. Manumycin A. a natural product o{ Streptα7Jyces parvuJus‘ inhi bits farn esy l- transferase by competition with farnesyl pyrophosphate groups . Manumycin has shown antitumor activity in several ex per‘ imental systems through identifying the regulatory pathway of apoptosis. The hi erarchical relationship of caspase-8 to caspase-3 and caspase-9 in the drug-induced a poptosis pathway in antitumol effect is not clear. The hi erarchical relations hip between cytochrome c and the caspases and provided evidence to support the hypothesis that the release of cytochrome c was upstream of caspase activation in the enhanced apoptosis induced by manumycin A Manumycin A has not been examined extensively in human salivary gland tumor and has not yet been clarified. The purpose of this study were to investigate mRNA and protein expression of Bc l- 2 、Bax, Cytochrome C‘ caspase- 3 , 一8 and -9 in SGT cell line by RT-PCR and immunoslot blotting, and to a pply its results to exami ne iLs chemoprevention for salivary gland adenocarcinoma. MTI assay showed about 50% cellular viability of SGT cell line treated by 50μM manunycin A Bcl-2. Bax‘ and caspase-8 mRNA expression in SGT cell line were unchangeable after 50μM manu nycin A Cytochrome C‘ caspase-3 and -9 showed about 1.5-5 folds higher mRNA expression in SGT cell line than that of control a nd DMSO- t reated group a fter 50M manunycin A. Bcl-2, Bax, and caspase-8 protein expression in SGT ce ll line were unchangcable after 50μM manunycin A. Cy Lochrorne C, caspase-3 and -9 showed about 2-7 fo lds higher protein express ion in SGT cell line than that of control and DMSO-treated group after 50μM manunycin A. mRNA expression was assoc iated with protein expression in SGT cell line after 50μM manunycin A. It suggested that manumycin A would induce poptotic effect on SGT cell line by caspase-3 and - 9 activation through cytochrorne c release. And man umycin A will be a useful chemoprevention drug for human salivary gland carcinoma in future.

The human ELAV(embryonic lethal abnormal vision)-like protein HuR stabilizes a certain group of cellul ar mHNAs that contain AU- rich elements in their 3’ - untranslated region , To test the significance of HuR in carcinogenesis of head and neck squamous cell carcinomas(HNSCCs), we have investigated HuH expression from 32 benign epithelial lesions , 14 prema lignant epitheli al lesions and 80 HNSCCs, There were two different staining patterns of HuR in HNSCCs : nuclear expression was seen in 78 7% (63 of 80) 01' cases; and an additional cyto plasmic expression was seen in 28, 7%(23 of 80) 01 cases, Nuclear expression of HuR was s ignificantly increased in premalignant lesions and HNSCCs, whereas increased cytoplasπli c expression of HuR was only observed in HNSCCs Cytoplasmic HuR expression was significantly increased in pa tients of HNSCC younger than 60 yea rs , Al though there was no significant correlation between a natomic s ites of HNSCCs and HuR express ion , cyto plasmic HuR expression was highly increased in HNSCCs of larynx, There was no significant co rrela tion between HuR expression and other clinicopathological parameters such as histological type‘ tumor s ize‘ 0 1' n odal s tatus , ln conclusion, this study s uggests that overexpression of HuR in HNSCCs may be part of a regula tory pathway tha t co ntro ls the mHNA stability 0 1' several important targets in carcinogenesis of HNSCCs

본 연구에서는 내분비계 장애물질으로 알려져 있고, 플라스틱 제품의 가소제로 사용되고 있는 di-(2-ethylhexyl) phthalate (DEHP)가 흰쥐의 간세포 미세구조와 간조직내 metallothionein (MT)의 발현 양상에 미치는 영향을 조사하였다. DEHP는 흰쥐 간세포의 미세구조와 MT 발현에 영향을 주었다. 실험군의 경우 조면소포체가 발달하고, 미토콘드리아가 증가하며 리소솜 혹은 퍼옥시좀들이 증가하는 경향을 나타냈다. 한편, MT