Previous ly we have s hown that fï brob last• growth factor-2 (FGF-2) and dexamethasone (Dex) in combination strongly stimulate both p l 이 i fe rati o n a nd differe nt iation of mesenchymal stem cells (MSCs) into osteoblasts and adipocytes, In the present s tudy we invesL igaLed whether inhibition 01' FGF-2 and Dex-induced adipogenic differentiation of bone marrow derived s Lem cells (BMSCs) by GW9662, an antagoni s t of proxisome proliferators-activated receptol γ (PPARy) which plays a key role in ad ipogenic differentiation , enhances proliferation and osteoblastic differentiation of BMSCs Proliferation 01' BMSCs t reated wi 네 FGF-2 a nd Dex was further increased by GW9662 up to 9,7, 10,6, and 7,2% at 3, 5, and 7 days of cul Lu re , Expansion of BMSCs with FGF-2, Dex and GW9662 followed by osteoblastic different iation showed that osteoblas tic differentiation 01' BMSCs was in creased by 37 % (p=O, 01) compared to those expanded with FGF-2 and Dex, ln contrast , ad i pogenic di fferenti a tion of FGF-2 and Dex-expanded BMSCs was substantially reduced to 14% (p=O, 036) by GW9662, Taken toget her , these resul ts demonstrate that FGF-2 and Dex in combination with GW9662 f ur t her stimu late proliferation 01' BMSCs and those cells expanded with these factors acquire enhanced potentiaIs to be dif ferentiated i n to osteoblas ts

Matrix metalloproLeases(MMPs) a re a family of zinc dependent enzymes with the capacity to degrade extracellular matrix prote ins. MMPs express ion correlates with cancer cell invasiveness and metas taslS The purpose of our sωdy was t。 determine the role 0 1' stroma l fïbrob lasts in ca ncer cell invasi on by examining the expression and activity of MMP-2 and -9 We used a YD- lOB squalllous ca rcinoma cell line that was est a blished in Yonsei Univer s ity ‘ College 0 1' Dentistry. We then appli ed two types of s lrolllal fibroblastsdel‘ ived from normal gingival tissue and cancer supporting ti ss ue Morphologic examination of fïbroblas ls was carried out by rnicroscopy and doubling time was measured by MTI assay . YD-lOB cells were cultured by lh ree-d imensional culture using collagen gel with two types of flbroblasts To examine the stromal - mesenchyma l inleraclion. we used a direct co-cul tu re system between YD-IOB cells and fibroblasts. Gelatin zymography was performed to exa llline MMP-2 a ncl 9 activit ies. We found that cancer-derived fibroblasts di splay stel late-shaped cells wi th many cyLopl asmic processes. whereas normal gingi val fibroblasts were spindle shaped. The c1oubling time of bOLh lïbroblasLs was not statistically different. Three-dimensional co-culture 0 1' ca ncer cells with ca n cer- c1erived fïbroblasts induced the formation 0 1' multi - Iayered atypical cells, as compared to culturing with normal gin gival f lbrobl asts Both three-c1 imens ional culture ti ss ues inclucecl the invasive gro￦th of cancer cells into the dermal eq uival enLs . Gelatin zymography s howecl that gelatinolytic activity of MMP-2 was activaterl in both co-culture models. However , MMP-9 ac li vity was not a lterecl in YD-lOB carcinoma cells In conclusion. enhanced MMP-2 activity was inc1 uced by boLh cance r- c1eri vecl a ncl norlllal gingival fibroblasts. suggesting that the potential to invacle by stromal fibroblasts was noL l imilecl to ca ncer- c1 eri ved lïbroblasts

Extensive oral mucosa loss from a variety of conditions is associated with significant functional morbidity and mortality. Although it is known that keratinocytes are a rich source of wound healing promoting factors such as transforming growth factor-β1(TGF-β1), it is not clear whether differentiated keratinocytes in a multi-layer form release this multi-functional growth factor. This study examined the hypothesis that keratinocytes in mono- and multi-layer forms expressed different levels of TGF-β1. When NHOK reached confluency in serum free medium(KBM), in test medium containing 1.2 mM Ca++ KBM NHOK were allowed to form multi-layers and differentiate. The purpose of this study were to investigate the mRNA level of TGF-β1, FGF-2, and TIMP-1 by RT-PCR analysis and also to evaluate the expression of TGF-β1 and involucrin in keratinocytes at different times of the onset of differentiation. The numbers and sizes of these nodules were increased as the process of keratinocyte differentiation proceed. Cultured NHOK in preconfluency under KBM medium expressed a significantly higher level of TGF-β1 relative to those grown in multi-layer forms, while the level of TGF-β1 mRNA gradually reduced to its lowest level at 7 days of growing cells in test medium. Cultured NHOK in preconfluency of KBM medium expressed a lower level of FGF-2 and TIMP-1 relative to those grown in multi-layer forms, while the level of FGF-2 and TIMP-1 mRNA showed the highest level at 3 days at gradually reduced to its lowest level at 7 days of growing cells in test medium. As a differentiation marker for keratinocytes at different time points, the highest level of involucrin mRNA expression was found at the later stage of cell differentiation. It suggested that the expression of TGF-β1 mRNA be consistent with the expression of FGF-2 and TIMP-1 mRNA in NHOK grown in high calcium medium during the terminal differentiation. But differentiated NHOK expressing higher involucrin mRNA could show constant espression of TGF-β1, FGF-2 and TIMP-1.

본 연구는 소 난관 상피 세포를 채취 체외 배양을 실시하고, 이에 착상과 관련이 있은 IL-4를 첨가하여 배양액내의 임신에 관련된 호르몬들(P4, E2, TGF-)의 변화를 관찰함으로써, 소 난관 상피 세포와 착상과의 관계를 구명하고자 실시하였으며, 그에 따른 결과는 다음과 같다. 소 난관 상피 세포의 체외 배양시 IL-4 첨가에 의한 배양액내의 P4의 생산은 0.001 ng/ml의 IL-4를 첨가한 배양액의 P4의 농도는 배양 시간이 경과할수록 증가하

The aim of this study was to investigate the cytotoxic effect and its mechanism on Radix Aconiti(RA) extract in lung cancer cell lines. RA extract treatment decreased the cell viability in a dose-dependent fashions in lung cancer cells including A549, H460, H23 and H157 cells. Many investigators reported that A549 and H460 cells expressed wild-type p53, but H23 and H157 cells preserved mutated p53. After treatment with RA extract in A549 and H460 cells, we measured the expression of p53 protein levels using Western blot. analysis. In both cells treated with RA extracts, p53 protein expressions were increased in a dose-dependent manner. In our experiments, RA extracts also have cytotoxic effects in H23 and H157, which have mutated p53. Treatment with RA extract decreased bcl-2 protein expressions in both cells. These results suggest that RA extracts have cytotoxic effects via p53 expression increase and bcl-2 inhibitable pathways in A549, H460 cells and H23, H157 cells, respectively.

Many naturally occurring plant extracts are studied for their beneficial effects for health and particularly on cancer. Apoptosis, or programmed cell death, occurs in both normal and pathological conditions, including cancer Dysregulation of apoptosis allows transformed cells to continually and uninhibitedly enter the cell cycle, thus perpetuating the sequence of mutation, genomic instability and, finally, oncogenesis. To investigate the apoptosis-inducing effect of the extract of Fructus Trichosanthis (EFT) on leukemic HL-60 cells and its mechanism, HL-60 cells in vitro in culture medium were given different doses of the extract. The inhibitory rate of cells were measured by microculture tetrazolium assay, cell apoptotic rate was detected by flow cytometry, morphology of cell apoptosis was observed by DAPI fluorescence staining, and the activations of caspases and PARP were detected using Western blotting analysis. The extract could activate the caspase-3 and caspase-8, induce PARP cleavage, inhibit growth of HL-60 cells, and cause apoptosis significantly The suppression was in dose-dependent manner. Marked morphological changes of cell apoptosis including condensation of chromatin and nuclear fragmentation were observed clearly by DAPI fluorescence staining especially. These results will provide strong laboratory evidence of EFT for clinical treatment of acute leukemia.

Annexin I plays an important role in the process of keratinization as a compont of the cornified envelope of skin epithelium. The effect of annexin I on the terminal ifferentiation of normal human oral keratinocyte(NHOK) have remained to be defined. To understand the role of annexin I on the terminal differentiaiton of NHOK, NHOK and NHEK cells were primarily cultured in KBM bullet kit. When the cells reached confluence, terminal differentiation was induced by switching the medium to KGM bullet kit containing 1.2mM Ca2+. Preconfluency of NHOK under 0.05mM Ca++ conc as control group was used. The cells was examined with inverted microscope. Under 0.05mM Ca++ conc(Precon, Postcon), and 1.2mM Ca++ conc(Postcon), RT-PCR for annexin I mRNA measurement, and immunoblotting for annexin I protein measurements in triplicate, respectively. The purpose of this study were to study differential mRNA & protein expression of annexin I between NHOK & NHEK by using RT-PCR & immunoslot blotting during terminal differentiation, and to apply these results to study a role of annexin I on epithelial differentiation of oral mucosal diseases in the future. Cultured NHEK showed larger area of cellular stratification than cultured NHOK in 1.2mM Ca ++ concentration. Annexin I mRNA and protein expression of cultured NHOK showed higher than that of cultured NHEK in higher calcium concentration. Annexin I mRNA and protein expression of cultured NHOK showed about 2-2.7 fold higher in 1.2mM Ca++ conc. than in 0.05mM Ca++ conc. Although annexin I was involved in the terminal differentiation of cultured NHOK & NHEK in higher calcium concentration, annexin I play an important role in the terminal differentiation of cultured NHOK in higher calcium concentration. From the aboving results, It was suggested that annexin I would play an important role in the terminal differentiation of NHOK in higher calcium, which be helpful to study epithelial differentiation of oral mucosal diseases.

In order to perform the protein analysis using the paraffin sections previous fixed with formalin, we applied the ImmunoMemBlot (IMB) method1) to detect the epitopes of target proteins with specific antibodies. In this study the protein extracts were obtained from the paraffin sections of each representative case of ameloblastoma, adenomatoid odontogenic tumor (AOT), and normal gingiva, and more a protein extract from fresh tissue of ameloblastoma was also compared to evaluate the IMB results used with 24 different antibodies. First of all, in the comparison between the paraffin section extract and fresh tissue extract of ameloblastoma, the latter consistently showed more positive IMB reaction than the former. Meanwhile, the paraffin section extract of ameloblastoma was more comparable with that of normal gingival, disclosing that most of proliferating genes, oncogenes, and apoptosis related genes, i.e., PCNA, CDK4, c-erbB2, CEA, p53, Bax, Bad, FLIP, FAS, Bcl-2, p21, N-ras, MMP-2, MMP-9, caspase-3, -8, -9, were highly expressed in ameloblastoma, but EGFR, HGF, and VEGF were similarly expressed both in the ameloblastoma and in normal human gingiva. On the other hand, the comparison between ameloblastoma and AOT both in the immunohistochemistry and IMB using their paraffin section extracts clearly demonstrated that the ameloblastoma showed more expression of proliferating genes and oncogenes while the AOT showed more expression of apoptosis related genes, i.e., Bax, Bad, FLIP, and caspase-9. Taken together, these data suggest that the IMB can be used for the primary screening of quantitative protein analysis using the paraffin section extract, and that the IMB results could be evaluated in conjunction with the immunohistochemical observation.

Polo-Like Kinase(PLK) is a cell cycle-regulated, cyclin-independent serine/threonine protein kinase. Recent reports have shown a critical role for PLK during tumorigenesis. To explore whether PLK plays a general role as a tumor marker of oral squamous cell carcinomas, we examined the expression of PLK mRNA and protein in oral squamous cell carcinoma cells and immortalized normal oral keratinocytes(INOK). We also investigated that PLK mRNA was expressed in specimens from 4NQO-induced SD rat tongue carcinomas using in situ RT-PCR methods. Immunocytochemically, most of the PLK was highly expressed in the nucleus of carcinoma cells, but not INOK. RT-PCR revealed PLK1 mRNA was detected in the FaDu and Hep2 cancer cells, but no detected in the INOK. In situ RT-PCR revealed PLK1 mRNA expression increased sequentially from hyperplasia to dysplasia, and squamous cell carcinoma during the malignant progression. PLK1 expression could reflect the degree of malignancy and proliferation in oral squamous cell carcinomas. Thus, in addition to being of diagnostic value, modulation of PLK1 activity in the tumors by chemotherapeutic agents or gene therapy may prove to be of therapeutic value.

The tumor suppressor gene, phosphate and tensin homologue(PTEN) has been shown to dephosphorylate the phosphatidylinositol 3-kinase(PI 3-K)-generated phosphatidylinositol(3-5)-triphosphate in vivo, thus interfering with the potentially oncogenic signals emanating from PI 3-K. Promoter hypermethylation of CpG islands has recently been shown to be an epigenetic change resulting in loss of function in some genes involved in cell cycle regulation and DNA repair. Immunohistochemal staining for monoclonal antibody 6H2.1 was performed from paraffin embedded blocks of 20 benign epithelial lesions and 40 head and neck squamous cell carcinomas(HNSCCs). Immunoreactivity was graded semiquantitatively by considering the percentage and intensity of the staining of the tumor cells. Also, this study tried to identify PTEN methylation in benign epithelial lesions(24 cases) and HNSCCs(44 cases of paraffin embedded blocks, 4 cases of frozen tissues) using methylation-specific PCR(MSP). In HNSCCs, immunoreactive scores of stage 1 and 2(12 cases, average score 85.2) were higher than those of stage 3 and 4(15 cases, 41.9) and statistically significant(P=0.017). Immunoreactive scores of moderate and poorly differentiated carcinomas(22 cases, 61.6) are more or less lower than those of well differentiated carcinoma(15 cases, 87.0) but not significant(P=0.361). Among 24 cases of benign epithelial lesions, 12 cases showed unmethylated PTEN but none methylated. In HNSCCs, 22 of 44 paraffin embedded blocks showed unmethylated PTEN but none methylated, and all 4 frozen tissue revealed unmethylated PTEN, one of which(25%) methylated. We consider that the loss of PTEN protein expression may be associated with the progression of HNSCCs and the other alteration rather than methylation may be important in the inactivation of PTEN in HNSCCs.

Shit-tzu견에서 발정 주기, 교배 적기 및 배란 시기를 정확하게 추정하기 위한 질 세포 검사를 확립하기 위하여 Shih-tzu 견 12두를 대상으로 발정 주기동안 질 세포 검사 및 estradiol-와 progesterone 농도를 측정하여 다음과 같은 결과를 얻었다. 발정주기별 질 세포상의 특징적인 변화로서 발정 전기에는 무핵 세포와 적혈구, 발정기에는 표층 세포와 무핵 세포 및 적혈구가 주종을 이루는 세포였으며 발정 휴지기에는 소형 중간 세포와

본 연구는 융합 전 수핵란의 활성화 처리 유무와 활성화 처리 시간이 소 체세포 유래 핵이식란의 체외 발육능에 미치는 영향을 검토하기 위해 수행하였다. 소 체외성숙란의 탈핵 후 체세포 핵을 이식하고 일부는 전기 융합 후 활성화를 유기하였고(pre-AC), 일부는 먼저 활성화 처리 후 융합을 실시(post-AC)하였다. 난자의 활성화는 Ca2+-ionophore(A23187)로 처리 후 DMAP로 4시간 배양하는 방법으로 유기하였다. 핵이식란은 CR1aa액에서 9일간 배양하여 발육율을 검토하였으며, 활성화 후 30분～2.5시간에 고정하여 confocal microscope 하에서 핵형 변화를 검사하였다. 배반포기까지 발육율은 post-AC구(20.6%)가 pre-AC(15.3%)보다 다소 높게 나타났다. 또한 활성화 처리를 하여 핵이식란의 배 발달을 비교한 결과 post-AC구가 더 늦은 배 발달 속도를 나타내었다. Post-AC구를 활성화 후 30분, 2시간, 4시간으로 나누어 융합하여 발육율을 검토한 결과 발육율에 차이가 없었다. 본 연구의 결과는 수핵란의 활성화 시간에 따라 핵이식란의 발육 및 핵형이 영향을 받을 수 있음을 시사한다.