• Asian Pacific Journal of Cancer Prevention
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• 제13권12호
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• pp.6349-6355
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• 2012

Previous studies showed that Math1 homologous to human Hath1 can cause mouse goblet cells to differentiate. In this context it is important that the majority of colon cancers have few goblet cells. In the present study, the potential role of Hath1 in colon carcinogenesis was investigated. Sections of paraffin-embedded tissues were used to investigate the goblet cell population of normal colon mucosa, mucosa adjacent colon cancer and colon cancer samples from 48 patients. Hath1 and Muc2 expression in these samples were tested by immunohistochemistry, quantitative real-time reverse transcription -PCR and Western blotting. After the recombinant plasmid, pcDNA3.1(+)-Hath1 had been transfected into HT29 colon cancer cells, three clones were selected randomly to test the levels of Hath1 mRNA, Muc2 mRNA, Hath1, Muc2, cyclin D1 and p27 by quantitative real-time reverse transcription-PCR and Western blotting. Moreover, the proliferative ability of HT29 cells introduced with Hath1 was assessed by means of colony formation assay and xenografting. Expression of Hath1, Muc2, cyclin D1 and p27 in the xenograft tumors was also detected by Western blotting. No goblet cells were to be found in colon cancer and levels of Hath1 mRNA and Hath1, Muc2 mRNA and Muc2 were significantly down-regulated. Hath1 could decrease cyclin D1, increase p27 and Muc2 in HT29 cells and inhibit their proliferation. Hath1 may be an anti-oncogene in colon carcinogenesis.

• 한국수정란이식학회지
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• 제17권2호
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• pp.101-108
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• 2002

Human Prourokinase (proUK) offers potential as a novel agent with improved fibrin specificity and, as such, may offer advantages as an attractive alternative to urokinase that is associated with clinical benefits in patients with acute peripheral arterial occlusion. For production of transgenic cow as human proUK bioreacotor, we conducted this study to establish efficient production system for bovine transgenic embryos by somatic cell nuclear transfer (NT) using human prourokinase gene transfected donor cell. An expression plasmid for human prourokinase was constructed by inserting a bovine beta-casein promoter, a green fluorescent protein (GFP) marker gene, and human prourokinase target gene into a pcDNA3 plasmid. Cumulus cells were used as donor cell and transfected with the expression plasmid using the Fugene 6 as a carrier. To increase the efficiency for the production of transgenic NT, development rates were compared between non-transfected and transfected cell in experiment 1, and in experiment 2, development rates were compared according to level of GFP expression in donor cells. In experiment 1, development rates of non-transgenic NT embryos were significantly higher than transgenic NT embryos (43.3 vs. 28.4％). In experiment 2, there were no significant differences in fusion rates (85.4 vs. 78.9％) and cleavage rates (78.7 vs. 84.4％) between low and high expressed cells. However, development rates to blastocyst were higher in low expressed cells (17.0 vs. 33.3％), and GFP expression rates in blastocyst were higher in high expressed cells (75.0 vs. 43.3％), significantly.

• Asian Pacific Journal of Cancer Prevention
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• 제13권4호
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• pp.1425-1430
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• 2012

Cyclin L2 is a novel member of the cyclin family, recently implicated in the regulation of cell cycle progression and/or transcriptional regulation. The present study was undertaken to investigate the effects of overexpression on tumor cell growth and chemosensitivity in human gastric cells in vitro. Cyclin L2 was transfected into human gastric cancer cell line BCG823 and expressed with a mammalian expression vector pcDNA3.1. The effects and mechanisms of cyclin L2 on cell growth, cell cycling and apoptosis were studied. Compared to control vectors, overexpression of cyclin L2 inhibited the growth of BCG823 cells and enhance their chemosensitivity to fluorouracil, docetaxel and cisplatin. The anti-proliferative effects of cyclin L2 could be due to G0/G1 arrest and apoptosis. Cyclin L2 induced G0/G1 arrest and apoptosis involved upregulation of caspase-3 and down regulation Bcl-2 and survivin. The results indicated that overexpression of cyclin L2 protein may promote efficient growth inhibition and enhance chemosensitivity to chemotherapeutic agents in human gastric cancer cells by inducing G0/G1 cell cycle arrest and apoptosis.

• 대한임상검사과학회지
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• 제38권3호
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• pp.189-195
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• 2006

Behcet's disease (BD) is a chronic, multisystemic disorder which is more frequently seen in the Mediterranean basin, Middle East, and Far East. The causes and pathogenesis of BD are unknown although many possibilities are being investigated. The diagnosis of BD is based on clinical manifestations because there are no pathognomonic laboratory tests. So, the purpose of this study was to examine the prevalence of HLA-B51 in patients with BD. We used the whole blood of 33 patients diagnosed with BD at Chosun University Hospital from August 2003 to January 2006. For the HLA-B51 test, we extracted the DNA from the whole blood of 33 BD patients, and we investigated it through the nested PCR method. Data were analyzed using the SPSS/PC 10.0. The frequencies of gender of the 33 cases diagnosed as BD were male 13 (39.4%) and female 20 (60.6%). The frequencies of age group of the 33 cases diagnosed as BD were 20 yrs 8 (24.2%), 30 yrs 12 (36.4%), 40 yrs 8 (24.2%), 50 yrs 1 (3.0%), and 60 yrs and 70 yrs 2 (6.1%), respectively. The frequencies of HLA-B51 of the 33 cases diagnosed as BD were HLA-B51-negative 18 (54.5%) and HLA-B51-positive 15 (45.5%). In conclusion, BD occurred more often in women than men (1: 1.53), and the mean age of the BD patients was 39.8 years old. HLA-B51 was positive in 45.5% of patients with BD, and was statistically significant in age (p<0.05).

• Journal of Yeungnam Medical Science
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• 제38권4호
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• pp.326-336
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• 2021

Background: Sulfation of heparan sulfate proteoglycans (HSPGs) is critical for the binding and signaling of ligands that mediate inflammation. Extracellular 6-O-endosulfatases regulate posttranslational sulfation levels and patterns of HSPGs. In this study, extracellular 6-O-endosulfatases, sulfatase (Sulf)-1 and Sulf-2, were evaluated for their expression and function in inflammatory cells and tissues. Methods: Harvested human peripheral blood mononuclear cells were treated with phytohemagglutinin and lipopolysaccharide, and murine peritoneal macrophages were stimulated with interleukin (IL)-1β for the evaluation of Sulf-1 and Sulf-2 expression. Sulf expression in inflammatory cells was examined in the human rheumatoid arthritis (RA) synovium by immunofluorescence staining. The antigen presentation and phagocytic activities of macrophages were compared according to the expression state of Sulfs. Sulfs-knockdown macrophages and Sulfs-overexpressing macrophages were generated using small interfering RNAs and pcDNA3.1 plasmids for Sulf-1 and Sulf-2, respectively. Results: Lymphocytes and monocytes showed weak Sulf expression, which remained unaffected by IL-1β. However, peritoneal macrophages showed increased expression of Sulfs upon stimulation with IL-1β. In human RA synovium, two-colored double immunofluorescent staining of Sulfs and CD68 revealed active upregulation of Sulfs in macrophages of inflamed tissues, but not in lymphocytes of lymphoid follicles. Macrophages are professional antigen-presenting cells. The antigen presentation and phagocytic activities of macrophages were dependent on the level of Sulf expression, suppressed in Sulfs-knockdown macrophages, and enhanced in Sulfs-overexpressing macrophages. Conclusion: The results demonstrate that upregulation of Sulfs in macrophages occurs in response to inflammation, and Sulfs actively regulate the antigen presentation and phagocytic activities of macrophages as novel immune regulators.

• 대한약침학회지
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• 제18권3호
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• pp.32-41
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• 2015

Objectives: Condurango (Gonolobus condurango) extract is used by complementary and alternative medicine (CAM) practitioners as a traditional medicine, including homeopathy, mainly for the treatment of syphilis. Condurango bark extract is also known to reduce tumor volume, but the underlying molecular mechanisms still remain unclear. Methods: Using a cervical cancer cell line (HeLa) as our model, the molecular events behind condurango extract's (CE's) anticancer effect were investigated by using flow cytometry, immunoblotting and reverse transcriptase-polymerase chain reaction (RT-PCR). Other included cell types were prostate cancer cells (PC3), transformed liver cells (WRL-68), and peripheral blood mononuclear cells (PBMCs). Results: Condurango extract (CE) was found to be cytotoxic against target cells, and this was significantly deactivated in the presence of N-acetyl cysteine (NAC), a scavenger of reactive oxygen species (ROS), suggesting that its action could be mediated through ROS generation. CE caused an increase in the HeLa cell population containing deoxyribonucleic acid (DNA) damage at the G zero/Growth 1 (G0/G1) stage. Further, CE increased the tumor necrosis factor alpha ($TNF-{\alpha}$) and the fas receptor (FasR) levels both at the ribonucleic acid (RNA) and the protein levels, indicating that CE might have a cytotoxic mechanism of action. CE also triggered a sharp decrease in the expression of nuclear factor kappa-light-chain-enhancer of activated B cells ($NF-{\kappa}B$) both at the RNA and the protein levels, a possible route to attenuation of B-cell lymphoma 2 (Bcl-2), and caused an opening of the mitochondrial membrane's permeability transition (MPT) pores, thus enhancing caspase activities. Conclusion: Overall, our results suggest possible pathways for CE mediated cytotoxicity in model cancer cells.

• Maxillofacial Plastic and Reconstructive Surgery
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• 제28권5호
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• pp.375-395
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• 2006

Purpose: Lingual nerve (LN) damage may be caused by either tumor resection or injury such as wisdom tooth extraction, Although autologous nerve graft is sometimes used to repair the damaged nerve, it has the disadvantage of necessity of another operation for nerve harvesting. Moreover, the results of nerve grafting is not satisfactory. The nerve growth factor (NGF) is well-known to play a critical role in peripheral nerve regeneration and its local delivery to the injured nerve has been continuously tried to enhance nerve regeneration. However, its application has limitations like repeated administration due to short half life of 30 minutes and an in vivo delivery model must allow for direct and local delivery. The aim of this study was to construct a well-functioning $rhNGF-{\beta}$ adenovirus for the ultimate development of improved method to promote peripheral nerve regeneration with enhanced and extended secretion of hNGF from the injured nerve by injecting $rhNGF-{\beta}$ gene directly into crush-injured LN in rat model. Materials and Methods: $hNGF-{\beta}$ gene was prepared from fetal brain cDNA library and cloned into E1/E3 deleted adenoviral vector which contains green fluorescence protein (GFP) gene as a reporter. After large scale production and purification of $rhNGF-{\beta}$ adenovirus, transfection efficiency and its expression at various cells (primary cultured Schwann cells, HEK293 cells, Schwann cell lines, NIH3T3 and CRH cells) were evaluated by fluorescent microscopy, RT-PCR, ELISA, immunocytochemistry. Furthermore, the function of rhNGF-beta, which was secreted from various cells infected with $rhNGF-{\beta}$ adenovirus, was evaluated using neuritogenesis of PC-12 cells. For in vivo evaluation of efficacy of $rhNGF-{\beta}$ adenovirus, the LNs of 8-week old rats were exposed and crush-injured with a small hemostat for 10 seconds. After the injury, $rhNGF-{\beta}$ adenovirus($2{\mu}l,\;1.5{\times}10^{11}pfu$) or saline was administered into the crushed site in the experimental (n=24) and the control group (n=24), respectively. Sham operation of another group of rats (n=9) was performed without administration of either saline or adenovirus. The taste recovery and the change of fungiform papilla were studied at 1, 2, 3 and 4 weeks. Each of the 6 animals was tested with different solutions (0.1M NaCl, 0.1M sucrose, 0.01M QHCl, or 0.01M HCl) by two-bottle test paradigm and the number of papilla was counted using SEM picture of tongue dorsum. LN was explored at the same interval as taste study and evaluated electro-physiologically (peak voltage and nerve conduction velocity) and histomorphometrically (axon count, myelin thickness). Results: The recombinant adenovirus vector carrying $rhNGF-{\beta}$ was constructed and confirmed by restriction endonuclease analysis and DNA sequence analysis. GFP expression was observed in 90% of $rhNGF-{\beta}$ adenovirus infected cells compared with uninfected cells. Total mRNA isolated from $rhNGF-{\beta}$ adenovirus infected cells showed strong RT-PCR band, however uninfected or LacZ recombinant adenovirus infected cells did not. NGF quantification by ELISA showed a maximal release of $18865.4{\pm}310.9pg/ml$ NGF at the 4th day and stably continued till 14 days by $rhNGF-{\beta}$ adenovirus infected Schwann cells. PC-12 cells exposed to media with $rhNGF-{\beta}$ adenovirus infected Schwann cell revealed at the same level of neurite-extension as the commercial NGF did. $rhNGF-{\beta}$ adenovirus injected experimental groups in comparison to the control group exhibited different taste preference ratio. Salty, sweet and sour taste preference ratio were significantly different after 2 weeks from the beginning of the experiment, which were similar to the sham group, but not to the control group.

• Journal of Korean Neurosurgical Society
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• 제30권7호
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• pp.861-869
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• 2001

Objective : In this study, we investigated the relationship between the histologic grading of meningiomas and proliferative potentials determined by the Ki-67, proliferating cell nuclear antigen(PCNA) and flow cytometry (FCM) with the aim of determining whether these potentials can be used as a parameter to the proliferative activity, in particular of atypical and malignant meningiomas. Methods : This study consisted of 47 meningiomas(6 malignant, 14 atypical, and random sampled 27 benign meningiomas). By immunohistochemical staining of Ki-67 and PCNA on formalin-fixed, paraffin-embedded sections, the anti-human rabbit polyclonal antibody against Ki-67 antigen and anti-PCNA monoclonal antibody(PC10) scores were counted. FCM was also performed on paraffin-embedded tissue using a selective staining technique for DNA. DNA ploidy, S-phase fraction, and proliferative index(PI)) were determined. Results : The results are summarized as follows ; 1) Proliferation rates as assessed by Ki-67 and PCNA closely correlated with the degree of anaplastic histologic features. 2) Proliferative potentials determined by FCM(S-phase fraction and PI) were not able to distinguish between benign and atypical/malignant meningiomas. 3) DNA ploidy was not a useful indicator of histologic grade in these tumors. 4) Proliferative potentials such as Ki-67 staining index(SI) and PCNA SI did not correlate with the ploidy pattern. 5) There was a linear correlation between Ki-67 SI and PCNA SI, but we could not find a correlation between Ki-67 SI and S-phase fraction or PI. Our results also did not show a statistically signficant correlation between PCNA SI and S-phse fraction or PI. Conclusions : We conclude that evaluation of the proliferative potentials with Ki-67 and PCNA is important as an additional factor for the prediction of malignancy in meningiomas. A dual study of Ki-67 and PCNA SIs on the same tissue might improve the accuracy with which the proliferative potential of a tumor can be predicted. We demonstrated that FCM in meningiomas is not valuable in predicting the behavior of these neoplasms, but we did observe a trend toward more malignancy with higher percent S-phase fraction and higher PI. Analysis of the S-phase fraction and PI might therefore be a useful tool to discriminate among histologic grades of meningiomas.

• 한국수정란이식학회:학술대회논문집
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• 한국수정란이식학회 2002년도 국제심포지엄
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• pp.75-75
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• 2002

The present study was conducted for the production of transgenic cloned cows by somatic cell nuclear transfer (SCNT) that secrete human prourokinase into milk. To establish an efficient production system for bovine transgenic SCNT embryos, the offset was examined of various conditions of donor cells including cell type, size, and passage number on the developmental competence of transgenic SCNT embryos. An expression plasmid far human prourokinase (pbeta-ProU) was constructed by inserting a bovine beta-casein promoter, a green fluorescent protein (GFP) marker gene, and a human prourokinase target gene into a pcDNA3 plasmid. Three types of bovine somatic cells including two adult cells (cumulus cells and ear fibroblasts) and fetal fibroblasts were prepared and transfected using a lipid-meidated method. In Experiment 1, developmental competence and rates of GFP expression in bovine transgenic SCNT embryos reconstructed with cumulus cells were significantly higher than those from fetal and ear fibroblasts. In Experiment 2, the effect of cellular senescence in early (2 to 4) and late (8 to 12) passages was investigated. No significant differences in the development of transgenic SCNT embryos were observed. In Experient 3, different sizes of GFP-expressing transfected cumulus cells [large (>30 ${\mu}{\textrm}{m}$) or small cell (<30 ${\mu}{\textrm}{m}$)] were used for SCNT. A significant improvement in embryo development and GFP expression was observed when small cumulus cells were used for SCNT. Taken together, these results demonstrate that (1) adult somatic cells could serve as donor cells in transgenic SCNT embryo production and cumulus cells with small size at early passage were the optimal cell type, and (2) transgenic SCNT embryos derived from adult somatic cells have embryonic development potential.

• KSBB Journal
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• 제13권4호
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• pp.418-423
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• 1998

Cellular transfections with cationic liposomes are widely empolyed for gene and oligonucleotide transfer in vitro because of their safety and ease of use. However, they still suffer from the low transfection efficiency comparing with viral vectors. Substantial effort shave been focused on increasing transfection efficiency by supplementing the liposome/DNA complexes(lipoplex) with various components. In this work, we tired three kinds of supplements, Poly-L-lysine(PLL), transferrin and a mixture of anionic lipids(PS/PE/PC), to study their effects on gene transfer yield and gene expression efficiency. PLL, a polycationic polymer, enhanced gene transfer yield by 3 times but the gene expression efficiency was increased only by 1.5 times. this result implies that PLL can enhance the transfection efficiency mainly by increasing the rate of outermembrane transport of lipoplex into the cells. On the other hand, transferrin which can facilitate the gene transfer via ligand-receptor interaction gave not only increased gene transfer yield but also enhanced gen expression efficiency by 2.8 times. Transferrin seems to contribute to the escape of plasmid from endosomes through ligand-receptor recycle mechanism. When the cells were treated with a mixture of anionic lipids for 3 hours before the transfection, gene transfer yield was slightly decreased but the gene expression efficiency was enhanced by 1.9 times. This is presumably due to the accelerated liposome-plasmid dissociation by the anionic lipids, and the increased delivery of plasmid to the nucleus. According to these results, it is clear that the supplementation to ameliorate transfection efficiency with cationic liposomes should be contrived in the direction of increasing delivery of plasmid.