152 PROLIFERATION- AND SENESCENCE-RELATED GENE EXPRESSION IN CANINE FIBROBLASTS DERIVED FROM DOGS OF DIFFERENT AGE

2016 ◽  
Vol 28 (2) ◽  
pp. 206
Author(s):  
Y. B. Choi ◽  
G. A. Kim ◽  
H. J. Oh ◽  
M. J. Kim ◽  
Y. K. Jo ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Proliferation ◽  
Somatic Cell ◽  
Cell Size ◽  
Growth Pattern ◽  
Doubling Time ◽  
Related Gene ◽  
Cell Donor ◽  
Significant Difference ◽  
Old Donor

In pig, cattle and mouse cloning, the age of the cell donor can influence cloning efficiency. However, little is known regarding the effects of the cell donor’s age related to cell proliferation and senescence and its importance for canine cloning. Therefore, the aim of this study was to investigate the effect of cell donor age on growth pattern and doubling time of cell populations, cell size, viability, and senescence-related gene expression (p53, p16, p21, Rb1, and E2F1) in canine fibroblasts. Cells were derived from 1-year-old and 7-year-old dogs. To obtain the fibroblasts, primary culture was performed using ear biopsies from three 1-year-old dogs (1yr) and three 7-year-old dogs (7yr), and cells at passage 2 to 6 were used for somatic cell cloning. Cells were plated at 1 × 105 cells well–1 and harvested every 24 h for 6 days. Cell number was determined for measurement of growth pattern and doubling time. The harvested cells were stained with trypan blue and their size and viability were analysed using a Countess automated cell counter (ThermoFisher Scientific, Waltham, MA, USA). Gene expression was measured by RT-qPCR. Cell proliferation and gene expression experiments were technically replicated 3 and 4 times, respectively. Statistical analysis was performed using GraphPad Prism (GraphPad Software, San Diego, CA, USA) and t-test (P < 0.05) was used. Growth curves in both groups showed typical S-shape and at Day 6, 1yr samples had significantly higher cell numbers compared with 7yr samples. For doubling times, passages 2 to 5 of 1yr (29.8 ± 4.4 h, 41.0 ± 1.6 h, 49.5 ± 4.8 h, and 71.3 ± 8.4 h) were significantly shorter than those of 7yr (47.0 ± 4.5 h, 57.1 ± 2.3 h, 65.8 ± 1.8 h, and 126.4 ± 12.4 h). Cells from passage 6 of 7yr and passage 7 of 1yr did not reach confluence at Day 6. Cell size of 1yr (14.1 ± 0.2 µm) was significantly smaller than that of 7yr (15.3 ± 0.2 µm). Additionally, there was no significant difference in cell viability between 1yr (90.7 ± 0.8%) and 7yr (88.7 ± 0.7%). However, mRNA expression of p53, p21, and Rb1 was significantly lower in 1yr cells compared with 7yr cells. The expression level of E2F1 was significantly higher in 1yr cells. There was no significant difference between 1yr and 7yr cells for p16 expression. In conclusion, proliferation analysis indicated that somatic cells derived from old donor dogs reach a state of senescence more rapidly compared with those of young donor dogs. In addition, analysis of gene expression demonstrated that cells from old donor dogs have significantly increased p53, p21, and Rb1 expression levels, which indicate senescence. Further studies are needed to produce dogs using 1yr and 7yr cells to evaluate the effect of donor aging in canine somatic cell nuclear transfer efficiency. This study was supported by RAD (#PJ0109282015), IPET (#311062-04-3-SB010), Nature Cell, Research Institute for Veterinary Science, (Natural Balance Korea or Nestle Purina Korea) and the BK21 plus program. We thank Barry D. Bavister for his valuable editing of the manuscript.

34 COMPARISON OF PROLIFERATION AND TELOMERASE ACTIVITY IN FIBROBLASTS DERIVED FROM GYEONG-JU DONGGYEONG DOGS ACCORDING TO THEIR AGE

2015 ◽  
Vol 27 (1) ◽  
pp. 110
Author(s):  
Y. B. Choi ◽  
G. A. Kim ◽  
H. J. Oh ◽  
M. J. Kim ◽  
B. C. Lee
Keyword(s):  
Cell Size ◽  
Growth Pattern ◽  
Doubling Time ◽  
Assisted Reproductive Technologies ◽  
Fibroblast Cell ◽  
Reproductive Technologies ◽  
Telomerase Activity ◽  
Cell Number ◽  
Primary Fibroblast ◽  
Significant Difference

Donggyeong dog is a breed considered a natural monument in Korea since 2012. Nevertheless, this breed is only found in Gyeong-ju and is classified as endangered. The use of assisted reproductive technologies (ART) could be applied to preserve these endangered dogs. Among various ART, somatic cell nuclear transfer (SCNT) may be a good technique for achieving propagation of genetically identical individuals. For this reason, we investigated the effect of age of the cell donor on several characteristics, including growth pattern, doubling time of cell populations, cell size, viability, and telomerase activity in Gyeong-ju Donggyeong dog fibroblast cultures before SCNT. Primary fibroblast cell cultures were performed using ear biopsies from 1-year-old (D-1yr) and 7-year-old (D-7yr) Donggyeong dogs and cells at passage 2 to 6 from in this study. Cells were plated at 1 × 105 cells well–1 in a 6-well plate. Cells were harvested every 24 h for 6 days, and cell number was determined to measure growth pattern and doubling time. The harvested cells were stained with trypan blue, their size and viability were analysed using a Countess Automated Cell Counter (Life Technologies, Carlsbad, CA, USA). Telomerase activity was measured by telomeric repeat amplification protocol using TeloTAGGG Telomerase PCR ELISAPLUS. All experiments were replicated at least 3 times. Statistical analysis was performed using Graphpad Prism (GraphPad Software Inc., San Diego, CA, USA), and t-test (P < 0.05) was used to compare the growth pattern, doubling time, cell size, viability, and telomerase activity between D-1yr and D-7yr groups. Growth curves in both groups showed the typical “S” shape and no significant differences between D-1yr and D-7yr groups. However, doubling times from 2nd to 5th passages of D-1yr (20.0 ± 0.3 h, 38.0 ± 0.5 h, 38.7 ± 0.5 h, and 53.4 ± 1.4 h) were significantly shorter than those of D-7yr (37.0 ± 1.2 h, 54.0 ± 3.6 h, 63.3 ± 1.8 h, and 100.9 ± 4.4 h). Cells from 6th passage of D-7yr and 7th passage of D-1yr did not reach confluence. Cell size of D-1yr (13.6 ± 0.2 μm) was significantly smaller than that of D-7yr (14.9 ± 0.3 μm; P < 0.001). There was no significant difference between D-1yr (89.4 ± 1.4%) and D-7yr (88.9 ± 1.2%) in cell viability but relative telomerase activity was significantly higher in D-1yr (37.8 ± 7.5) compared with D-7yr (19.0 ± 6.2, P < 0.001). In conclusion, these results suggest that fibroblasts derived from young Donggyeong dogs are more suitable for SCNT than those from old donors. For further study, dog cloning using D-1yr and D-7yr should be performed to evaluate the effect of donor age in canine SCNT efficiency.

scholarly journals Melatonin Receptor 2 Promotes Lipid Metabolism of Cumulus-oocyte Complexes via the cAMP/PKA Pathway

2021 ◽  
Author(s):  
Jun-Xue Jin ◽  
Hong-Di Cui ◽  
Chao-Qian Jiang ◽  
Zi-Cheng Qi ◽  
Ya Bian ◽  
...  
Keyword(s):  
Gene Expression ◽  
Energy Source ◽  
Lipid Metabolism ◽  
Signaling Pathway ◽  
Oocyte Maturation ◽  
Expression Patterns ◽  
Cumulus Cells ◽  
Related Gene ◽  
Melatonin Receptor ◽  
Significant Difference

Abstract Background: The importance of the processes of lipogenesis and lipolysis in providing an essential energy source during oocyte maturation is increasingly being recognized. Recent our studies have demonstrated that melatonin up-regulated lipid metabolism during oocyte maturation. Nevertheless, there is still limited information regarding the underlying molecular mechanisms of action of melatonin on lipid metabolism in porcine cumulus-oocyte complexes (COCs). Here, our aim was to investigate the effect of melatonin on COCs, and the melatonin receptor-mediated lipid metabolism signaling pathway.Materials/methods: To determine the melatonin-mediated lipolysis pathway in cumulus cells, COCs were treated with melatonin and the correlated metabolic responses were assessed using melatonin receptor-mediated signaling.Results: The results showed that exposure of COCs to melatonin during in vitro maturation significantly increased cumulus expansion index, blastocyst formation rate and total cell numbers/blastocyst, although nuclear maturation was no significant difference. The levels of proteins MT1, MT2, Gsα, PKA, and lipolysis-related factors (AGTL, HSL, PLIN A+B) were significantly increased by melatonin supplementation, and this effect was inhibited by simultaneous treatment with melatonin antagonists (luzindole or 4P-PDOT), although 4P-PDOT treatment did not completely block the effect of melatonin on MT1. Further, the gene expression patterns reflected their relevant protein levels in cumulus cells. Melatonin-mediated lipolysis could significantly reduce lipid droplets (LDs) numbers and increase fatty acid (FA) production and ATP levels by increasing the β-oxidation-related gene expression in cumulus cells. Simultaneously, melatonin significantly increased the amount of LDs, FAs, ATP, and enhanced the lipid metabolism-related gene expression in oocytes. Finally, the oocyte quality was improved by increasing GDF9, BMP15 and GSH and decreasing ROS levels.Conclusion: These findings revealed that the MT2-mediated cAMP/PKA signaling pathway promotes intracellular lipolysis and FA production in cumulus cells, which provided an essential energy source for COCs development.

scholarly journals Effect of Retinoic Acid on Cell Proliferation-Related Gene Expression in Raji Cells Induced by 12-O-tetra-Decanoylphorbol-13-Acetate.

2001 ◽  
Vol 30 ◽  
pp. 21-31
Author(s):  
Eiichiro ICHIISHI ◽  
Toshikazu YOSHIKAWA ◽  
Takako TAKAI ◽  
Harukuni TOKUDA ◽  
Yasuhiko YOSHIDA ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Proliferation ◽  
Retinoic Acid ◽  
Related Gene ◽  
Raji Cells

scholarly journals Effects of Developmental Arsenic Exposure on the Social Behavior and Related Gene Expression in C3H Adult Male Mice

2019 ◽  
Vol 16 (2) ◽  
pp. 174 ◽  
Author(s):  
Soe-Minn Htway ◽  
Mya-Thanda Sein ◽  
Keiko Nohara ◽  
Tin-Tin Win-Shwe
Keyword(s):  
Gene Expression ◽  
Social Behavior ◽  
Adult Male ◽  
Arsenic Exposure ◽  
Receptor Expression ◽  
Heme Oxygenase 1 ◽  
Male Mice ◽  
Related Gene ◽  
Gene Expressions ◽  
Significant Difference

Arsenic is carcinogenic and teratogenic. In addition, it is also a developmental neurotoxicant. Little is known however about the effect of arsenic exposure during brain development on social behavior. This study aimed to detect the effect of developmental arsenic exposure on social behavior and related gene expression in C3H adult male mice. Pregnant C3H mice were exposed to sodium arsenite (NaAsO2, 85 ppm in the drinking water) from gestational day (GD) 8 to 18. The F1 generation male pups from different mothers were taken and social behavior tasks were examined. Social behavioral-related gene expression in the prefrontal cortex was determined by the real-time RT-PCR method. The mice with developmental arsenic exposure showed poor sociability and poor social novelty preference. Glutamate receptor expression (NMDA and AMPA receptor subunits) showed no significant difference, but gene expressions of serotonin receptor 5B (5-HT 5B) and brain-derived neurotrophic factor (BDNF) were significantly decreased (p < 0.05) in the arsenic-exposed group compared to control group. The heme oxygenase-1 (HO-1) and cyclooxygenase-2 (COX-2) gene expressions were not significantly different. Our findings indicate that developmental arsenic exposure might affect social behavior by modulating serotonin receptors and reducing BDNF. Some oxidative stress markers and inflammatory markers were not affected.

scholarly journals Adipose-Derived Stem Cells Conditioned Media Promote In Vitro Osteogenic Differentiation of Hypothyroid Mesenchymal Stem Cells

2020 ◽  
Vol 7 (3) ◽  
Author(s):  
Tayebeh Sanchooli ◽  
Mohsen Norouzian ◽  
Mahtab Teimouri ◽  
Abdolreza Ardeshirylajimi ◽  
Abbas Piryaei
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Bone Marrow ◽  
Cell Proliferation ◽  
Mesenchymal Stem Cells ◽  
Osteogenic Differentiation ◽  
Calcium Content ◽  
Male Rats ◽  
Osteogenic Potential ◽  
Significant Difference

Background: Thyroid hormones have many effects on the physiological functions of cells, including growth, differentiation, and metabolism. Objectives: Recently, studies have shown that the adipose-derived mesenchymal stem cells conditioned medium (ADMSCs-CM) has many osteogenic factors, such as IGF-1, IL-6, and FGFs. Methods: In the current study, mesenchymal stem cells (MSCs) were isolated from two sources; the adipose tissue of the testicular fat pad and the bone marrow of rat, and then characterized by flow cytometry. ADMSCs-CM was collected from the ADMSC in the healthy adult male rats. Hypothyroidism was induced by the administration of the Methimazole during 60 days and confirmed by the analysis of the serum level of T4 and TSH hormones. Cell proliferation and osteogenic differentiation potential of bone marrow stem cells (BMSCs) derived from hypothyroid rats were investigated in the presence and absence of the CM by MTT assay, alkaline phosphatase (ALP) activity, calcium content assay, and bone-related gene expression. Healthy BMSCs were assigned to the control group. Results: Although Cell proliferation was decreased in the hypothyroid BMSCs, there was no significant difference between the control and the hypothyroid-CM groups. Similarly, osteogenic potential was significantly reduced in the hypothyroid group compared to the control and hypothyroid-CM groups according to the ALP, calcium content assays, and gene expression results. There was no significant difference between the hypothyroid-CM group and control. Conclusions: Our results indicated that hypothyroidism can decrease cell proliferation and osteogenic differentiation of BMSCs. Although ADMSCs-CM improved these parameters, it may be a promising candidate for the bone regeneration of the hypothyroidism cases.

Biological Analysis and Prognostic Significance of Proliferative Cellular Compartment in Chronic Lymphocytic Leukemia (CLL).

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 667-667 ◽  
Author(s):  
Pau Abrisqueta ◽  
Neus Villamor ◽  
Ana Muntañola ◽  
Carles Codony ◽  
Mireia Camós ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Cell Proliferation ◽  
Doubling Time ◽  
Conflicts Of Interest ◽  
Prognostic Significance ◽  
Time To Progression ◽  
Ki67 Expression ◽  
Paired Samples ◽  
Expression Of Genes

Abstract Abstract 667 Historically CLL has been considered a non-proliferative disease characterized by accumulation of leukemic cells. However, recent clinical and biological observations are questioning this concept. From the clinical standpoint, although some patients have lymphocyte counts stable during the course of the disease, others exhibit a short lymphocyte doubling time, suggesting the existence of a significant cell proliferation. Some specific anatomic locations (bone marrow (BM) and lymph nodes) seem to be more prone to proliferation than peripheral blood (PB). The amount of cell proliferation and its prognostic significance has not been properly analyzed. Against this background, gene expression profiling of proliferation genes and the amount of cell proliferation in different tissue compartments (BM and PB) were examined in patients with CLL. In isolated CD19/CD5+ tumoral cells from 20 paired PB and BM samples, expression of genes (n=93) involved in the initiation and development of the cell cycle was analyzed by low-density TaqMan® arrays. The amount of proliferative (Ki67 positive) CLL cells was measured by flow cytometry in 50 paired samples. In addition, coexpression of molecules associated with cellular activation (CD38, CD71, CD69), adhesion (CD49d), chemokine receptors (CXCR4, CXCR3, CCR7), interaction between T and B cells (CD86), signaling (ZAP-70), and Toll-like receptors (TLR9) was compared between Ki67+ and Ki67- CLL subpopulations. Finally, the degree of proliferation was correlated with the main clinical and biological characteristics. As assessed by gene expression profile, the great majority of genes involved in the initiation and development of cell cycle were more expressed in BM than in PB. Of note, Ki67+ CLL cells were significantly higher in BM than in PB (mean: 1.13% vs 0.88%; p= 0.004). This difference on Ki67+ expression between BM and PB was particularly significant (mean: 1.6% vs 1.1%; p=0.01) in patients who progressed of their disease at any particularly time (n=20), whereas it was not observed in patients with stable disease. Proliferating (Ki67+) CLL cells had significantly increased expression of ZAP-70 (mean fluorescence intensity (MFI): 162 vs 94, p<0.001), CD38 (MFI: 75 vs 27, p<0.001), CD86 (MFI: 31 vs 11, p=0.002), CD71 (MFI: 73 vs 24, p<0.001), and TLR9 (MFI: 49 vs 25, p<0.001) in comparison to non-proliferating Ki67- cells; CXCR4 was significantly decreased in proliferating cells (MFI: 212 vs 340, p=0.006). No differences were observed in CD49d, CD69, CCR7, and CXCR3 expression between Ki67+ and Ki67- CLL cells. When Ki67 expression was analyzed at diagnosis (n=41 paired samples, median follow-up of 4.2 years), patients with Ki67+ CLL cells ≥ 1% in BM had a shorter time to progression than those with Ki67 <1% (progression at 4 years: 47% vs 12%, respectively; p=0.008) (figure). In addition, patients with lymphocyte doubling time < 12 months, ZAP-70 expression ≥ 20%, or CD38 expression ≥ 30%, but not with increased CD49d expression, exhibit a higher percentage of Ki67+ CLL cells in both BM and PB (Table). In conclusion, in CLL expression of genes related to proliferation was significantly increased in BM compared to PB. Moreover, the number of proliferating CLL cells was also increased in BM, particularly in those patients with an aggressive disease, and presented different immunophenotype characteristics in comparison to non-proliferating CLL cells. Finally, the amount of Ki67+ CLL cells correlated with a shorter time to progression. These results challenge the concept of CLL as disease more accumulative than proliferative. These new insights on the proliferation pathways in CLL not only may provide a better understanding of the pathogenesis of this disease, but also would be of prognostic relevance and can support the use of new treatments aimed at inhibiting proliferation in CLL. Lymphocyte doubling timeZAP-70CD38CD49d<12 months (n=10)>12 months (n=37)≥20% (n=15)<20% (n=35)≥30% (n=19)<30% (n=31)≥30% (n=17)<30% (n=32)Mean% Ki67+ CLL cells in PB1.20.7P=0.021.40.6P<0.0011.10.7P=0.0151.10.8P=0.08Mean% Ki67+ CLL cells in BM1.60.8P=0.03220.8P=0.0011.31P=0.191.50.9P=0.053 Disclosures: No relevant conflicts of interest to declare.

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