• Journal of Microbiology and Biotechnology
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• v.16 no.8
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• pp.1292-1300
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• 2006

Aeromonas encheleia, a potential human intestinal pathogen, was shown to infect a human intestinal epithelial cell line (Caco-2) in a noninvasive manner. The transcriptional profile of the Caco-2 cells after infection with the bacteria revealed an upregulated expression of genes involved in chloride secretion, including that of phospholipase A2 (PLA2) and platelet-activating factor (PAF) acetylhydrolase (PAFAH2). This was also confirmed by a real-time RT-PCR analysis. As expected from PLA2 induction, PAF was produced when the Caco-2 cells were infected with the bacteria, and PAF was also produced when the cells were treated with a bacterial culture supernatant including bacterial extracellular proteins, yet lacking lipopolysaccharides. Bacterial aerolysin was shown to induce the production of PAF.

• Clinical and Experimental Reproductive Medicine
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• v.36 no.1
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• pp.23-34
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• 2009

Objectives: Mesenchymal stem cells (MSC) comprise a promising tool for cellular therapy. It is known that long-term in vitro culture of human bone marrow and adipose tissue derived-MSCs lead to a reduction of life span and a change of stem-like characters. The aim of our study was to examine whether stem cell properties of human umbilical cord-derived stem cells (HUC) could be affected by in vitro expansion. Methods: HUC were isolated from human umbilical cord and cultured for 10 passages in vitro. Morphology and population doubling time (PDT) were investigated, and changes of stem cell properties were examined using RT-PCR and immunocytochemistry during serial subcultures. Results: Morphology and PDT of HUC began to change slightly from the 7th passage (p7). Expression level of nestin and vimentin mRNAs increased along with the culture period from p4 until p10. In contrast, expression level of SCF mRNA decreased during the same culture period. Expression level of Oct-4 and HNF-4${\alpha}$ mRNAs was not significantly changed throughout the culture period until p10. Expression level of BMP-4, FGF-5, NCAM and HLA-ABC mRNAs appeared to increase as the culture continued, however, the difference was not significant. Immunocytochemical studies showed that HUC at p3, p6 and p9 positively were stained with antibodies against SSEA-3 and SSEA-4 proteins. Interestingly, staining intensity of HUC for ICAM-1 and HLA-ABC gradually increased throughout the culture period. Intensity against thy-1 and fibronectin antibodies increased at p9 while that against TRA-1-60 and VCAM-1 antibodies began to decrease at p6 until p9. Conclusions: These results suggest that HUC change some of their stem cell characteristics during in vitro culture. Development of culture system might be needed for the maintenance of characteristics.

• Clinical and Experimental Reproductive Medicine
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• v.35 no.1
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• pp.49-60
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• 2008

Objective: This study was carried out to investigate the effects of 3-dimensional co-culture of human endometrial cells decidualized with progesterone and TGF-${\beta}1$ on the development of 2-cell mouse embryos. Methods: Stromal and epithelial cells isolated from human endometrial tissue were immunostained for cytokeratin and vimentin. Expression of TGF-${\beta}1$, its receptor-1, -2, integrin-${\beta}3$ and prolactin in mono or co-culture according to three different hormone conditions was investigated by RT-PCR. Differential staining was used to investigate the number of ICM and trophectoderm of hatched mouse blastocysts in different three conditions. Results: The immunohistochemical study was positive for cytokeratin or vimentin and confirmed that epithelial and stromal cells were isolated from endometrial tissue successfully. In co-culture, TGF-${\beta}1$, its receptor-1, integrin-${\beta}3$ and prolactin except TGF-${\beta}1$-r2 were expressed in progesterone dominant condition. The hatching and attaching rate were higher in the co-culture with decidualized cells (p<0.05). However, we observed that lots of the incomplete hatched blactocysts attached on non-decidualized cells. The ICM number of hatched mouse blastocysts was higher in co-culture with decidualized and non decidualized cells than media only culture (p<0.05). The trophectoderm number of hatched blastocyst was higher in the co-culture with decidualized cells than non-decidualized cells or media only culture (p<0.05). Conclusion: The administration of progesterone, estrogen and TGF-$\beta$ could induce decidualization of stromal and epithelial cells isolated from human endometrial tissue using 3-dimensional co-culture, and the decidualization of human endometrial cells could increase the hatching and attaching rate of 2-cell mouse embryos.

• The korean journal of orthodontics
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• v.35 no.4 s.111
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• pp.275-285
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• 2005

Ipriflavone (isoprofoxyisoflavone), a synthetic derivative from soy isoflavone diazein, has been shown to inhibit bone resorption and perhaps stimulate bone formation This study was performed to examine the effects of ipriflavone on the proliferation and bone remodeling in rat calvarial cells in vitro The rat calvarial cells were isolated from fetus aged 20 to 21 days and cultured In BGJb media The graded concentration of ipriflavone $(10^{-9}\;10^{-5}M)$ was administered into cultured cells. When the cell proliferation was estimated through the measurement of MTT assay, there was no increase in cellular proliferation of the rat calvarial cell at any ipriflavone concentration. The cellular activity was evaluated through the formation of mineralized nodules stained by alizarin red. The formation of mineralized nodules significantly increased at concentrations of $10^{-8}M,\;10^{-7}M\;and\;10^{-6}M$ ipriflavone. Reverse transcription-polymerase chain reaction analyses (RT-PCR) were done at 7 and 14 days after culture to detect the expression of Bone Sialoprotein (BSP), Type I Collagen (COL I) and Osteocalcin(OCN) As a result, the expressions of BSP and COL I increased on the 7th day of culture and the expression of OCN increased on the 14th day of culture. These results indicate that ipriflavone facilitates the bone remodeling process bvy promoting rat calvarial cell differentiation aid stimulating mineralization through increased expression of extracellular matrix genes. such as BSP. COL I and OCN.

• The Korean Journal of Zoology
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• v.39 no.3
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• pp.239-247
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• 1996

An embryonic stem (ES) cell-based in vitro model system was examined to determine whether a simple differentiation of embryoid bodies (EB) in the suspension medium is useful to dissect early erythropoiesis. Characteristics of the differentiating EBs were monitored for their differentiation potential to generate hematopoietic cell types by general morphology, benzidine staining and two-step colony assays, and expressivity of several erythroid marker genes by the RT-PCR analysis for total cellular RNA prepared from the differentiating EBs. Every ematopoietic lineage cells were generated from the differentiating EBs with reproducible frequencies, similar to the other sophisticated differentiation protocols. Furthermore, the globin gene switching in differentiating ES cells paralleled the sequence of events found in the mouse embryo, and such that their expression was activated by at least 12 hrs later than those of erythroid-specific transcription factors, GATA-1 and Tal-1 The erythropoietic differentiation program initiated reproducibly and efficiently in this simple differentiation system in a suspension culture, such that this system may be useful for dissection of the molecular events of early erythropoiesis.

• Macromolecular Research
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• v.13 no.4
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• pp.285-292
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• 2005

We developed alginate beads loaded with transforming growth $factor-{\beta}_{1}(TGF-{\beta}_{1})$ to examine the possible application of the scaffold and cytokine carrier in tissue engineering. In this study, bone marrow stromal cells (BMSCs) and $TGF{\beta}_{1}$ were uniformly encapsulated in the alginate beads and then cultured in vitro. The cell morphology and shape of the alginate beads were observed using inverted microscope, scanning electron microscope (SEM), histological staining and RT-PCR to confirm chondrogenic differentiation. The amount of the $TGF{\beta}_{1}$ released from the $TGF-{\beta}_{1}$ loaded alginate beads was analyzed for 28 days in vitro in a phosphate buffered saline (pH 7.4) at $37^{\circ}C$. We observed the release profile of $TGF-{\beta}_{1}$ from $TGF-{\beta}_{1}$ loaded alginate beads with a sustained release pattern for 35 days. Microscopic observation showed the open cell pore structure and abundant cells with a round morphology in the alginate beads. In addition, histology and RT-PCR results revealed the evidence of chondrogenic differentiation in the beads. In conclusion, these results confirmed that $TGF-{\beta}_{1}$ loaded alginate beads provide excellent conditions for chondrogenic differentiation.

• Archives of Pharmacal Research
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• v.28 no.2
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• pp.232-237
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• 2005

Mast cells play an important role in allergic inflammation by releasing their bioactive mediators. The function of mast cells is enhanced by stimulation because of the induction of specific genes and their products. While many inducible genes have been elucidated, we speculated that a significant number of genes remain to be identified. Thus, we applied differential display (dd) PCR to establish a profile of the induced genes in bone marrow-derived mast cells (BMMCs) after they were co-cultured with 3T3 fibroblasts. To date, 150 cDNA fragments from the connective-type mast cells (CTMCs) were amplified. Among them, thirty cDNA fragments were reamplified for cloning and sequencing. The ddPCR strategy revealed that serine proteases were the most abundant genes among the sequenced clones induced during the maturation. Additionally, unknown genes from the co-culture of BMMCs with 3T3 fibroblasts were identified. We confirmed their induction in the CTMCs by Northern blot analysis and RT-PCR. Characterization of these induced genes during the maturation processes will provide insight into the functions of mast cells.

• BMB Reports
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• v.45 no.3
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• pp.189-193
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• 2012

Cyst formation is a major characteristic of ADPKD and is caused by the abnormal proliferation of epithelial cells. Renal cyst formation disrupts renal function and induces diverse complications. The mechanism of cyst formation is unclear. mIMCD-3 cells were established to develop simple epithelial cell cysts in 3-D culture. We confirmed previously that Mxi1 plays a role in cyst formation in Mxi1-deficient mice. Cysts in Mxi1 transfectanted cells were showed by collagen or mebiol gels in 3-D cell culture system. Causative genes of ADPKD were measured by q RT-PCR. Herein, Mxi1 transfectants rarely formed a simple epithelial cyst and induced cell death. Overexpression of Mxi1 resulted in a decrease in the PKD1, PKD2 and c-myc mRNA relating to the pathway of cyst formation. These data indicate that Mxi1 influences cyst formation of mIMCD-3 cells in 3-D culture and that Mxi1 may control the mechanism of renal cyst formation.

• Journal of Embryo Transfer
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• v.25 no.2
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• pp.117-125
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• 2010

Several cloned animals have been produced using somatic cell nuclear transfer (SCNT) and have interested in producing the transgenic cloned animals to date. But still its efficiency was low due to a number of reasons, such as sub-optimal culture condition, aberrant gene expression and nuclear reprogramming. The purpose of this study was to analyze gene expression pattern in in vitro fertilized (IVF) or SCNT pre-implantation embryos. IVF- or SCNT-embryos were cultured in media supplemented with different proteins (FBS and BSA) or energy sources (glucose or fructose). Blastocysts from IVF or SCNT were analyzed using semi-quantitative RT-PCR in terms of developmentor metabolic-related genes. Culture medium supplemented different proteins or energy sources had affected on the expression of developmental or metabolic genes in the SCNT blastocysts.

• Biotechnology and Bioprocess Engineering:BBE
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• v.5 no.2
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• pp.110-117
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• 2000

Relationship between monoclonal antibody (MAb) productivity and growth rate, and effects of high cell density on MAb production rate increased with increasing specifis growth rate in both suspended and immobilized continuous cultures indicate a positively growth-associated relationship between MAb productivity and growth rate. moreover, the specific production rate was higher in the immobilized cell culture than that in suspended one at all dilution rates. In order to clarify these phenomana, MAb mPNA experession and cell cycle distribution were investigated in bacth cultures with immobilized cells and suspended cells. RT-PCR was used for observation of MAb mRNA expression and a two-color bromodeoxyuridine (BrdU)/propidium iodide (PI) flow cytometry method for determination of cell cycle distribution. The results revealed that MAb nRNA expression until dead phase, which was longer than in suspended cell. The cell cycle distribution patterns were observed almost the same for both immobilized and suspended cells. Such results may imply that a high cell density state has positive influence on the mRNA expression and on growth-associated Mab productivity of T0405 cells.