• Archives of Plastic Surgery
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• v.39 no.2
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• pp.106-112
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• 2012

Background : Complicated diabetic patients show impaired, delayed wound healing caused by multiple factors. A study on wound healing showed that platelet-rich plasma (PRP) was effective in normal tissue regeneration. Nonetheless, there is no evidence that when platelet-rich plasma is applied to diabetic wounds, it normalizes the diabetic wound healing process. In this study, we have analyzed matrix metalloproteinase (MMP)-2, MMP-9 expression to investigate the effect of PRP on diabetic wounds. Methods : Twenty-four-week-old male Otsuka Long-Evans Tokushima Fatty rats were provided by the Tokushima Research Institute. At 50 weeks, wounds were arranged in two sites on the lateral paraspinal areas. Each wound was treated with PRP gel and physiologic saline gauze. To determine the expression of MMP-2, MMP-9, which was chosen as a marker of wound healing, reverse transcription polymerase chain reaction (RT-PCR) was performed and local distribution and expression of MMP-2, MMP-9 was also observed throughout the immunohistochemical staining. Results : RT-PCR and the immunohistochemical study showed that the levels of MMP-2, MMP-9 mRNA expression in PRP applied tissues were higher than MMP-2, MMP-9 mRNA expression in saline-applied tissues. MMP-9 mRNA expression in wounds of diabetic rats decreased after healing began to occur. But no statistical differences were detected on the basis of body weight or fasting blood glucose levels. Conclusions : This study could indicate the extracellular matrix-regulating effect observed with PRP. Our results of the acceleration of wound healing events by PRP under hyperglycemic conditions might be a useful clue for future clinical treatment for diabetic wounds.

• Development and Reproduction
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• v.9 no.1
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• pp.33-41
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• 2005

Identification of differentially expressed genes at blastocyst stage embryos would provide insights into early development and differentiation. Here, we applied a new differential display reverse transcription polymerase chain reaction(DD RT-PCR) technology, called annealing control primers(ACP) system to identify the genes that are specifically or prominently expressed in mouse blastocysts compared to embryonic stem(ES) cells. Using 100 ACPs, 26 clones were perceived as differentially expressed genes in mouse blastocysts. A BLAST search revealed that cloned genes had significant sequence similarities with known genes in the GenBank/EMBL data base. Among them, 15 genes were selected and conformed by RT-PCR. This analysis suggests that the ACP system is a practical method for the identification of stage-specific genes using small numbers of mouse embryos.

• The Korean Journal of Physiology and Pharmacology
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• v.1 no.1
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• pp.45-53
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• 1997

In humans and many animal models with chronic progressive renal diseases, angiotensin-converting enzyme (ACE) inhibitor markedly attenuates the progression of nephropathy. Several studies have reported augmented gene expression and redistribution of renal renin in partial nephrectomized rats. Although precise mechanism(s) is not known, the renin-angiotensin system (RAS) may play an important role in the progression of renal diseases. Thus, this study was undertaken to examine the gene expression of renal renin, angiotensinogen, and $AT_1$ subtypes ($AT_{1A}$ and $AT_{1B}$) in rats with diabetic nephropathy, and the influences of lipopolysaccharide (LPS)-induced septicemia on the gene expression. Four weeks after streptozotocin (STZ) treatment (55 mg/kg, i.p.), rats were randomly divided into LPS-treated (1.6 mg/kg, i.p.) and control rats. At 6 hours after LPS treatment, the rats were killed and the kidney was removed from each rat. Northern blot and reverse transcription-polymerase chain reaction (RT-PCR)techniques were used to detect mRNA expression. STZ treatment markedly attenuated body weight gain and significantly increased blood glucose level. Renal renin content (RRC) was significantly decreased in the STZ-treated rats compared to that in control rats. The renal ACE activity between STZ-treated and control rats was not significantly different. Renal renin mRNA level was prominently increased, while angiotensinogen and $AT_{1A}$ mRNA levels were slightly decreased in STZ-treated rats compared to those in controls. $AT_1$B mRNA level did not differ in both groups. Acute LPS treatment did not show any significant changes of mRNA levels of intrarenal RAS components in both groups. These results suggest that intrarenal RAS components were differentially regulated in STZ-treated diabetic rats. Further studies are required to evaluate the relationship between intrarenal RAS and other vasomodulatory systems.

• The Korean Journal of Physiology and Pharmacology
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• v.1 no.1
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• pp.55-63
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• 1997

The present study was designed to quantify the alterations of renal renin, angiotensin type I receptor ($AT_1$), $TGF-{\beta}1$, and fibronectin gene expression in rats with unilateral ureteral obstruction (UUO). We also investigated the change of $AT_1$ density during UUO. Reverse transcription-polymerase chain reaction (RT-PCR) technique and receptor binding assay were used to detect mRNA expression and receptor density, respectively. At one day after UUO, renin mRNA level of the obstructed kidneys was decreased transiently and then subsequently increased to the level of sham kidneys. In the contralateral kidneys of the same rats, on the contrary, renin mRNA level was gradually decreased. Then, at 9 days after UUO, it was significantly lower than that of sham kidneys. The expressions of both $AT_1$ subtypes, called $AT_{1A}$ and $AT_{1B}$, mRNAs did not change at any time. UUO led to a significant decrease in $AT_1$ density in the obstructed kidneys compared with the sham kidneys at 1 and 3 days $(66\;{\pm}\;11.6%\;(p<0.005)\;and\;73\;{\pm}\;4.0%$ (p<0.01), respectively). Thereafter, $AT_1$ density was gradually increased and at 9 days it showed a marked elevation in the obstructed kidneys compared to the sham kidneys. In contrast, in the contralateral kidneys $AT_1$ density was significantly reduced from 3 to 9 days after UUO. The $TGF-{\beta}$1 mRNA level of the obstructed kidneys was unexpectedly decreased at 6 days after UUO. Then, at 9 days it was followed by a significant increase in the obstructed kidneys, whereas it showed an obvious decrease in the contralateral kidneys. In addition, fibronectin mRNA level was also significantly increased in the obstructed kidneys after UUO compared to the sham or the contralateral kidneys of the same rats. These results suggest a differential regulation of renal renin, $AT_1$ receptor, $TGF-{\beta}$1 and fibronectin mRNA levels at different stages of UUO.

• Journal of Yeungnam Medical Science
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• v.28 no.2
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• pp.153-164
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• 2011

Background: It has been reported that estrogen receptor beta ($ER{\beta}$) mRNA expression was down-regulated during carcinogenesis and was inversely related to estrogen receptor alpha ($ER{\alpha}$) expression in breast cancer. The association of $ER{\beta}$ mRNA expression to tamoxifen resistance has also been reported. In this study, the expression of $ER{\alpha}$ and $ER{\beta}$ via immunohistochemistry (IHC) and reverse transcription-polymerase chain reaction (RT-PCR) was prompted, and an attempt was made to find out the relationship between $ER{\beta}$ expression and recurrence in the hormonal therapy group, and between $ER{\beta}$ expression and known prognostic factors. Methods: Tumor specimens were obtained at surgery from 67 female breast cancer patients during the period of September 1995 to December 2000. All the specimens were frozen in liquid nitrogen and kept at $-70^{\circ}C$ until they were used. The medical records were analyzed retrospectively. The expressions of ER were analyzed using IHC and RT-PCR methods. Results: The median follow-up was at 93.0 months (range: 14-157 months). The percentage of $ER{\alpha}+/ER{\beta}+$, $ER{\alpha}+/ER{\beta}-$, $ER{\alpha}-/ER{\beta}+$, and $ER{\alpha}-/ER{\beta}$ group were 35.9% 9.4%, 47.2%, and 7.5%, respectively, in 53 patients with hormonal therapy. $ER{\beta}$ was positive in 42 (82.3%) of 51 ER-positive patients. In the hormonal therapy group, the recurrence rates of each group was 15.8%, 0%, 40.0%, and 0%, respectively. In this group, the $ER{\beta}$ expression tended to recur, but there was no clinical significance (p=0.084). Conclusion: The $ER{\beta}$ expression may be a predictive marker of a poor response to endocrine therapy in breast cancer patients, although this needs to be confirmed in additional studies.

• The Korean Journal of Physiology and Pharmacology
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• v.15 no.1
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• pp.23-29
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• 2011

Melatonin, which is the main product of the pineal gland, has well documented antioxidant and immune-modulatory effects. Macrophages produce molecules that are known to play roles in inflammatory responses. We conducted microarray analysis to evaluate the global gene expression profiles in response to treatment with melatonin in lipopolysaccharide (LPS) activated RAW 264.7 macrophage cells. In addition, eight genes were subjected to real-time reverse transcription polymerase chain reaction (RT-PCR) to confirm the results of the microarray. The cells were treated with LPS or melatonin plus LPS for 24 hr. LPS induced the up-regulation of 1073 genes and the down-regulation of 1144 genes when compared to the control group. Melatonin pretreatment of LPS-stimulated RAW 264.7 cells resulted in the down regulation of 241 genes and up regulation of 164 genes. Interestingly, among genes related to macrophage-mediated immunity, LPS increased the expression of seven genes (Adora2b, Fcgr2b, Cish, Cxcl10, Clec4n, Il1a, and Il1b) and decreased the expression of one gene (Clec4a3). These changes in expression were attenuated by melatonin. Furthermore, the results of real-time PCR were similar to those of the microarray. Taken together, these results suggest that melatonin may have a suppressive effect on LPS-induced expression of genes involved in the regulation of immunity and defense in RAW 264.7 macrophage cells. Moreover, these results may explain beneficial effects of melatonin in the treatment of various inflammatory conditions.

• Clinical and Experimental Reproductive Medicine
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• v.39 no.2
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• pp.87-93
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• 2012

Objective: Lin28 has been known to control the proliferation and pluripotency of embryonic stem cells. The purpose of this study was to determine the downstream effectors of Lin28 in mouse embryonic stem cells (mESCs) by RNA interference and microarray analysis. Methods: The control siRNA and Lin28 siRNA (Dharmacon) were transfected into mESCs. Total RNA was prepared from each type of transfected mESC and subjected to reverse transcription-polymerase chain reaction (RT-PCR) analysis to confirm the downregulation of Lin28. The RNAs were labeled and hybridized with an Affymetrix Gene-Chip Mouse Genome 430 2.0 array. The data analysis was accomplished by GenPlex 3.0 software. The expression levels of selected genes were confirmed by quantitative real-time RT-PCR. Results: According to the statistical analysis of the cDNA microarray, a total of 500 genes were altered in Lin28-downregulated mESCs (up-regulated, 384; down-regulated, 116). After differentially expressed gene filtering, 31 genes were selected as candidate genes regulated by Lin28 downregulation. Among them, neuropeptide Y5 receptor and oocyte-specific homeobox 5 genes were significantly upregulated in Lin28-downregulated mESCs. We also showed that the families of neuropeptide Y receptor (Npyr) and oocyte-specific homeobox (Obox) genes were upregulated by downregulation of Lin28. Conclusion: Based on the results of this study, we suggest that Lin28 controls the characteristics of mESCs through the regulation of effectors such as the Npyr and Obox families.

• Asian-Australasian Journal of Animal Sciences
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• v.25 no.5
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• pp.606-612
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• 2012

Insulin-like growth factor-binding protein-5 (IGFBP-5) is one of the six members of IGFBP family, important for cell growth, apoptosis and other IGF-stimulated signaling pathways. In order to explore the significance of IGFBP-5 in cells of the Inner Mongolian Cashmere goat (Capra hircus), IGFBP-5 gene complementary DNA (cDNA) was amplified by reverse transcription polymerase chain reaction (RT-PCR) from the animal's fetal fibroblasts and tissue-specific expression analysis was performed by semi-quantitative RT-PCR. The gene is 816 base pairs (bp) in length and includes the complete open reading frame, encoding 271 amino acids (GenBank accession number JF720883). The full cDNA nucleotide sequence has a 99% identity with sheep, 98% with cattle and 95% with human. The amino acids sequence shares identity with 99%, 99% and 99%, respectively. The bioinformatics analysis showed that IGFBP-5 has an insulin growth factor-binding protein homologues (IB) domain and a thyroglobulin type-1 (TY) domain, four protein kinase C phosphorylation sites, five casein kinase II phosphorylation sites, three prenyl group binding sites (CaaX box). The IGFBP-5 gene was expressed in all the tested tissues including testis, brain, liver, lung, mammary gland, spleen, and kidney, suggesting that IGFBP-5 plays an important role in goat cells.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.46 no.1
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• pp.70-76
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• 2013

Some fish live in aquatic environments with low or temporally changing $O_2$ availability. Variation in dissolved oxygen (DO) levels requires behavioral, physiological, and biochemical adaptations to ensure the uptake of sufficient $O_2$. Several species are relatively well adapted to tolerate low $O_2$ partial pressures (hypoxia). The medaka (Oryzias dancena ) is an important model organism for biomedical research that shows remarkable tolerance to hypoxia. We investigated the regulation and role of hypoxia-inducible factor-1 (HIF-$1{\alpha}$) as a general hypoxia-response gene and stanniocalcin-2 (STC2), which is one of the genes regulated by HIF-$1{\alpha}$ in mammals under hypoxia. We subjected adult male medaka to the following three acute hypoxia regimes: 1, 24, and 72 h at DO = $1.8{\pm}0.5$ ppm. The changes in STC2 and HIF-$1{\alpha}$ mRNA were monitored using quantitative real-time reverse-transcription PCR. We found strong upregulation of HIF-$1{\alpha}$ mRNA in the livers of fish exposed to hypoxia. Hypoxia rapidly upregulated STC-2 mRNA expression in muscle, but not in the brain, gills, liver, or intestine. Therefore, unlike in mammals, hypoxia might regulate O. dancena STC-2 expression in an HIF-$1{\alpha}$-independent manner.

• Journal of Korean Neurosurgical Society
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• v.57 no.5
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• pp.329-334
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• 2015

Objective : To comparatively investigate the expression of several integrins in specimens of human bone metastases and degenerative bone tissue. Methods : Degenerative cancellous tissue was obtained from a sample of human degenerative spine. Thirteen human specimens were obtained from metastatic spine tumors, whose primary cancer was colon cancer (n=3), hepatocellular cancer (n=3), lung cancer (n=4), and breast cancer (n=3). The expression of vimentin and integrins ${\alpha}v$, ${\beta}1$, and ${\beta}3$ was assessed in metastatic and degenerative specimens by immunohistochemistry and real-time reverse transcription-polymerase chain reaction (qRT-PCR). Results : Immunohistochemical staining showed that vimentin and integrin ${\alpha}v$ was broadly expressed in all tissues examined. By contrast, integrin ${\beta}1$ was weakly expressed only in 38.4% (5/13) of tissues. Integrin ${\beta}3$ was consistently negative in all cases examined. qRT-PCR analysis showed that vimentin gene expression was higher in all metastatic specimens, as compared to degenerative bone. The gene expression of integrin ${\alpha}v$ in breast specimen was significantly higher than others (p=0.045). The gene expression of integrin ${\beta}1$ was also higher in all metastatic specimens than in degenerative bone tissue. The gene expression of integrin ${\beta}3$ was variable. Conclusion : Spinal metastatic tumors have mesenchymal characteristics such as increased expression of vimentin. The increased expression of integrin ${\alpha}v$ and ${\beta}1$ in spine metastatic tumors suggests that adhesive molecules such as integrin may have implications for the prevention of spine metastasis.