ABSTRACT
PURPOSE: To investigate the effects of irradiation on transforming growth factor beta1 (TGF-beta1) mRNA expression and calcific nodule formation in MC3T3-E1 osteoblastic cell line. MATERIALS AND METHODS: Cells were cultured in alpha-minimum essential medium (alpha-MEM) supplemented with 10% fetal bovine serum and antibiotics. When the cells reached the level of 70-80% confluence, culture media were changed with alpha-MEM supplemented with 10% FBS, 5 mM beta-glycerol phosphate, and 50 microgram/mL ascorbic acid. Thereafter the cells were irradiated with a single dose of 2, 4, 6, 8 Gy at a dose rate of 1.5 Gy/min. The expression pattern of TGF-beta1 mRNA, calcium content and calcific nodule formation were examined on day 3, 7, 14, 21, 28, respectively, after the irradiation. RESULTS: The amount of TGF-beta1 mRNA expression decreased significantly on day 7 after irradiation of 4, 6, 8 Gy. It also decreased on day 14 after irradiation of 6, 8 Gy, and decreased on day 21 after irradiation of 8 Gy. The amount of calcium deposition decreased significantly on day 7 after irradiation of 4, 8 Gy (P.0.01) and showed a decreased tendency on day 14, 21 after irradiation of 4, 6, 8 Gy. The number of calcific nodules was decreased on day 7 after irradiation of 4, 8 Gy. CONCLUSION: Irradiation with a single dose of 4, 6, 8 Gy influences negatively the bone formation at the molecular level by affecting the TGF-beta1 mRNA expression that was associated with proliferation and the production of extracellular matrix in MC3T3-E1 osteoblastic cell line.
Subject(s)
Anti-Bacterial Agents , Ascorbic Acid , Calcium , Cell Line , Culture Media , Extracellular Matrix , Glycerophosphates , Organic Chemicals , Osteoblasts , Osteogenesis , RNA, Messenger , Transforming Growth Factor beta1ABSTRACT
The functional expression of potassium (K+) channels has electrophysiologically been studied in bone cells from several species, however, their identity and regulation of gene expressions in bone cells are not well known. In the present study, to investigate how K+ channel expressions are regulated by estrogen, we measured changes of transcript levels of various Ca2+-activated (K(Ca)) and ATP-sensitive K+ channels in rat osteoblastic ROS 17/2.8 cells after treatment with estrogen. Application of 17beta-estradiol (E2) for 24 h and 48 h increased mRNA and protein expressions of inwardly rectifying K+ channel (Kir) 6.2 and type 2 small conductance K(Ca) channel (SK2), respectively. Combined treatment of cells with 17beta-E2 and ICI 182,780, a pure antiestrogen, suppressed 17beta-E2-induced alterations of SK2 and Kir6.2 mRNA levels. In addition, treatment of cells with U0126, a specific inhibitor of extracellular receptor kinases (ERK)1/2, and SP600125, a specific inhibitor of c-jun N-terminal kinase (JNK) blocked the enhancing effects of 17beta-E2 on SK2 and Kir6.2 protein expressions. On the other hand, blocking of p38 mitogen-activated protein kinase had no effect. Taken together, these results indicate that 17beta-E2 modulates SK2 and Kir6.2 expressions through the estrogen receptor, involving ERK1/2 and JNK activations.
Subject(s)
Animals , Rats , Estrogen Receptor Modulators , Estrogens , Gene Expression , Hand , JNK Mitogen-Activated Protein Kinases , MAP Kinase Signaling System , Osteoblasts , Phosphotransferases , Potassium , Protein Kinases , RNA, Messenger , Up-RegulationABSTRACT
PURPOSE: To investigate the effects of irradiation on the calcium content and calcific nodule formation in the MC3T3-E1 osteoblastic cell line. MATERIALS AND METHODS: Cells were irradiated with a single dose of 2, 4 and 8 Gy at a dose rate of 5.38 Gy/min using a Cs-137 irradiator. After irradiation, the calcium content and calcific nodule formation were examined on the 1st, 2nd, 3rd and 4th week. RESULTS: A decreasing dose-dependent tendency of the cell proliferation rate was found in all irradiated groups of this experiment when compared with the unirradiated control group. In accordance with the duration of culture, there was no significant difference in the cell proliferation rate after irradiation of 2 Gy when compared with the unirradiated group, however a decreasing tendency was found in 4 Gy- and 8 Gy-irradiated groups. While an increase in total calcium content after irradiation of 2 Gy was found at week 1, week 2, and week 4, there was a decrease in calcium content at week 1 through 4 in the 8 Gy- irradiated group. Calcific nodule formation was increased in irradiated experimental groups when compared with the unirradiated control group in the 2 Gyirradiated group, but decreased in the 4Gy- and 8Gy-irradiated groups at the same stage. CONCLUSION: The results showed a mild increasing tendency of the calcific nodule formation after irradiation of 2 Gy. However, a decreased calcific nodule formation in 4Gy- and 8Gy-irradiated groups was found. Taken together, the irradiation of 2 Gy mildly activated bone formation, however 4 Gy or 8 Gy suppressed bone formation by decreasing cell numbers in the MC3T3-E1 osteoblastic cell line.
Subject(s)
Calcium , Cell Count , Cell Line , Cell Proliferation , Osteoblasts , OsteogenesisABSTRACT
PURPOSE: To investigate the effects of low dose irradiation on the calcium content and calcific nodule formation of the MC3T3-E1 osteoblastic cell line. MATERIALS AND METHODS: Cells were irradiated with a single dose of 0.2, 0.4 and 0.6 Gy at a dose rate of 5.38 Gy/min using Cs-137 irradiator. After irradiation, the calcium content and calcific nodule formation were examined on the 1st, 2nd, 3rd and 4th week. RESULTS: We did not find any significant difference of total calcium content after irradiation of 0.2, 0.4 and 0.6 Gy when compared with the unirradiated control group. There was no significant difference of total calcium content between 0.2, 0.4 and 0.6 Gy irradiated groups. We found an increased tendency of the calcific nodule formation after irradiation of 0.2, 0.4 and 0.6 Gy when compared with the unirradiated control group without significant difference of calcific nodule formation between 0.2, 0.4 and 0.6 Gy irradiated groups. CONCLUSION: The results showed an increased tendency of the calcific nodule formation after low dose irradiation. However, this tendency did not increase with the increase of irradiation dose.
Subject(s)
Calcium , Cell Line , OsteoblastsABSTRACT
PURPOSE: To investigate the effects of irradiation on the phenotypic expression of the MC3T3-E1 osteoblastic cell line, especially on the osteonectin and bone sialoprotein. MATERIALS AND METHODS: Cells were irradiated with a single dose of 0.5, 1, 4 and 8 Gy at a dose rate of 5.38 Gy/min using Cs-137 irradiator. After specimens were harvested, total RNA was extracted on the 3rd, 7th, 14th, 21st day after irradiation. The total RNA was reverse-transcribed and the resulting cDNAs were subjected to amplification by PCR with a pair of primers. RESULTS: The irradiated cells showed a dose-dependent increase in osteonectin mRNA expression when compared with the unirradiated control group. The irradiated cells showed no difference in bone sialoprotein mRNA expression when compared with the unirradiated control group. In accordance with the duration of culture period after irradiation, the level of osteonectin mRNA expression showed no difference, but it increased a little at the 21st day in the 4 and 8 Gy exposure groups. In the case of bone sialoprotein, however, the level of mRNA expression increased significantly at the 3rd and 7th day after irradiation, but it showed no difference at the 14th and 21st day when compared with the control group. CONCLUSION: These results showed that each single dose of 0.5, 1, 4 and 8 Gy influenced the mRNA expression of osteonectin and bone sialoprotein at the calcification stage of osteoblastic cells, suggesting that single dose of irradiation affected the osteoblastic bone formation at the cell level.
Subject(s)
Cell Line , DNA, Complementary , Integrin-Binding Sialoprotein , Osteoblasts , Osteogenesis , Osteonectin , Polymerase Chain Reaction , RNA , RNA, MessengerABSTRACT
PURPOSE: To investigate the effects of irradiation on the phenotypic expression of the MC3T3-E1 osteoblastic cell line, particularly on the expression of osteocalcin and osteopontin. MATERIALS AND METHODS: Cells were irradiated with a single dose of 0.5, 1, 4, and 8 Gy at a dose rate of 5.38 Gy/min using a cesium 137 irradiator. After the specimens were harvested, RNA was extracted on the 3rd, 7th, 14th, and 21st day after irradiation. The RNA strands were reverse-transcribed and the resulting cDNAs were subjected to amplification by PCR. RESULTS: The irradiated cells demonstrated a dose-dependent increase in osteocalcin and a dose-dependent decrease in osteopontin mRNA expression compared with the non-irradiated control group. The amount of osteocalcin mRNA expression decreased significantly at the 3rd day after irradiation of 0.5, 1, 4, and 8 Gy, and also decreased significantly at the 3rd, 14th, and 21st day after irradiation in the 8 Gy exposed group compared with the control group. The degree of osteopontin mRNA expression increased significantly at the 7th day after irradiation of 0.5, 1, 4, and 8 Gy. CONCLUSION: These results showed that each single dose of 0.5, 1, 4, and 8 Gy influenced the mRNA expression of osteocalcin and osteopontin associated with the calcification stage of osteoblastic cells, suggesting that each single dose affected bone formation at the cell level.
Subject(s)
Cell Line , Cesium , DNA, Complementary , Osteoblasts , Osteocalcin , Osteogenesis , Osteopontin , Polymerase Chain Reaction , RNA , RNA, MessengerABSTRACT
Objective To give a summary of the current researches on hematopoietic stem cell niche.Methods Through extensive reviewing the related domestic and abroad literatures,the present summary reviews the compo- nents of hematopoietic stem cell niche,related cell factors and related cell signaling pathway participating in regula- tion of hematopoietic stem cell.Results The activities of hematopoietic stem cells are regulated by the niche(micro- environment),which is composed of hematopoietic stem cell and its surrounding cells.The regulation cannot be completed through one signaling pathway.Also,the self-renewal and differentiation of hematopoietic stem cell can- not be completed only through osteoblastic cells.Conclusion The niche regulates hematopoietic stem cells by differ- ent ways.With study in-depth,we will comprehensively understand the nature of stem cells and the study will pro- vide a broader space to stem cell-based therapy.