Oestrogen is essential for maintaining bone mass, and it has been demonstrated to induce osteoblast proliferation and bone formation.In this study, complementary DNA（cDNA） microarrays were used to identify and study...Oestrogen is essential for maintaining bone mass, and it has been demonstrated to induce osteoblast proliferation and bone formation.In this study, complementary DNA（cDNA） microarrays were used to identify and study the expression of novel genes that may be involved in MC3T3-E1 cells’ response to 17-b estradiol. MC3T3-E1 cells were inoculated in minimum essential media alpha（a-MEM）cell culture supplemented with 17-b estradiol at different concentrations and for different time periods. MC3T3-E1 cells treated with1028mol？L2117-b estradiol for 5 days exhibited the highest proliferation and alkaline phosphatase（ALP） activity; thus, this group was chosen for microarray analysis. The harvested RNA was used for microarray hybridisation and subsequent real-time reverse transcription polymerase chain reaction（RT-PCR） to validate the expression levels for selected genes. The microarray results were analysed using both functional and pathway analysis. In this study, microarray analysis detected 5 403 differentially expressed genes,of which 1 996 genes were upregulated and 3 407 genes were downregulated, 1 553 different functional classifications were identified by gene ontology（GO） analysis and 53 different pathways were involved based on pathway analysis. Among the differentially expressed genes, a portion not previously reported to be associated with the osteoblast response to oestrogen was identified. These findings clearly demonstrate that the expression of genes related to osteoblast proliferation, cell differentiation, collagens and transforming growth factor beta（TGF-b）-related cytokines increases, while the expression of genes related to apoptosis and osteoclast differentiation decreases, following the exposure of MC3T3-E1 cells to a-MEM supplemented with 17-b estradiol. Microarray analysis with functional gene classification is critical for a complete understanding of complementary intracellular processes. This microarray analysis provides large-scale gene expression data that require further confirmatory studies.展开更多
Emerging evidence suggests that plasma membrane calcium ATPases (PMCAs) play a key role as regulators of calcium-triggered signal transduction pathways via interaction with partner proteins. PMCAs regulate these pathw...Emerging evidence suggests that plasma membrane calcium ATPases (PMCAs) play a key role as regulators of calcium-triggered signal transduction pathways via interaction with partner proteins. PMCAs regulate these pathways by targeting specific proteins to cellular sub-domains where the levels of intracellular freecalcium are kept low by the calcium ejection properties of PMCAs. According to this model, PMCAs have been shown to interact functionally with the calcium-sensitive proteins neuronal nitric oxide synthase, calmodulindependent serine protein kinase, calcineurin and endothelial nitric oxidase synthase. Transgenic animals with altered expression of PMCAs are being used to evaluate the physiological significance of these interactions. To date, PMCA interactions with calcium-dependent partner proteins have been demonstrated to play a crucial role in the pathophysiology of the cardiovascular system via regulation of the nitric oxide and calcineurin/nuclear factor of activated T cells pathways. This new evidence suggests that PMCAs play a more sophisticated role than the mere ejection of calcium from the cells, by acting as modulators of signaling transduction pathways.展开更多
AIM: The existence and properties of alpha-fetoprotein (AFP) receptor on the surface of NIH 3T3 cells and the effects of AFP on cellular signal transduction pathway were investigated. METHODS: The effect of AFP on the...AIM: The existence and properties of alpha-fetoprotein (AFP) receptor on the surface of NIH 3T3 cells and the effects of AFP on cellular signal transduction pathway were investigated. METHODS: The effect of AFP on the proliferation of NIH 3T3 cells was measured by incorporation of 3H-TdR. Receptor-binding assay of 125I-AFP was performed to detect the properties of AFP receptor in NIH 3T3 cells. The influences of AFP on the [cAMP]i and the activities of protein kinase A (PKA) were determined. Western blot was used to detect the change of K-ras P21 protein expression. RESULTS: The proliferation of NIH 3T3 cells treated with 0-80 mg/L of AFP was significantly enhanced. The Scatchard analysis indicated that there were two classes of binding sites with KD of 2.722 x 10(-9)M (Bmax=12810 sites per cell) and 8.931 x 10(-8)M (Bmax=119700 sites per cell) respectively. In the presence of AFP (20 mg/L), the content of cAMP and activities of PKA were significantly elevated . The level of K-ras P21 protein was upregulated by AFP at the concentration of 20 mg/L. The monoclonal antibody against AFP could reverse the effects of AFP on the cAMP content, PKA activity and the expression of K-ras p21 gene. CONCLUSION: The effect of AFP on the cell proliferation was achieved by binding its receptor to trigger the signal transduction pathway of cAMP-PKA and alter the expression of K- ras p21 gene.展开更多
目的探索桂皮醛诱导记忆性心脏移植耐受的作用及潜在分子机制。方法将60只小鼠随机分为正常对照组(假手术,n=10)、手术组(心脏移植,n=10)、模型组(注射T细胞+心脏移植,n=10)、桂皮醛低浓度组(注射T细胞+心脏移植+10 mg/kg桂皮醛,n=10)...目的探索桂皮醛诱导记忆性心脏移植耐受的作用及潜在分子机制。方法将60只小鼠随机分为正常对照组(假手术,n=10)、手术组(心脏移植,n=10)、模型组(注射T细胞+心脏移植,n=10)、桂皮醛低浓度组(注射T细胞+心脏移植+10 mg/kg桂皮醛,n=10)、桂皮醛中浓度组(注射T细胞+心脏移植+20 mg/kg桂皮醛,n=10)和桂皮醛高浓度组(注射T细胞+心脏移植+40 mg/kg桂皮醛,n=10)。观察各组移植物平均存活时间、移植物排斥程度,检测脾细胞增殖情况、移植物中相关基因白细胞介素-2(interleukin-2,IL-2)、白细胞介素-10(interleukin-10,IL-10)、γ-干扰素(interferon-γ,IFN-γ)和转化生长因子-β(transforming growth factor-β,TGF-β)的相对表达量,以及哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycin,mTOR)与信号转导及转录激活因子3(signal transduction and activator of transcription3,STAT3)蛋白磷酸化的表达情况。结果(1)与正常对照组相比,手术组存活时间明显缩短(P<0.05);与手术组相比,模型组存活时间明显缩短(P<0.05);与模型组相比,桂皮醛高浓度组的平均存活时间显著延长(P<0.05)。(2)对各组移植物排斥程度进行评估,模型组为Ⅳ级,桂皮醛治疗组的移植物排斥程度显著降低,且具有剂量依赖性。(3)与正常对照组相比,手术组及模型组脾细胞OD值升高(P<0.05);与模型组及桂皮醛低浓度组相比,桂皮醛中、高浓度组脾细胞OD值降低(P<0.05)。(4)与正常对照组相比,手术组IL-2、IFN-γmRNA表达显著上调(P<0.05),IL-10、TGF-βmRNA表达显著下调(P<0.05);与手术组相比,模型组IL-2、IFN-γmRNA表达显著上调(P<0.05),IL-10、TGF-βmRNA表达显著下调(P<0.05);与模型组比较,桂皮醛中、高浓度组移植物中IL-2、IFN-γmRNA表达明显下调(P<0.05),桂皮醛中、高浓度组IL-10、TGF-βmRNA表达明显上调(P<0.05)。(5)与正常对照组相比,手术组p-mTOR、p-STAT3蛋白表达显著上调(P<0.05);与手术组相比,模型组p-mTOR、p-STAT3蛋白表达显著上调(P<0.05);与模型组比较,桂皮醛中、高浓度组移植物中p-mTOR、p-STAT3蛋白表达明显下调(P<0.05)。结论对于记忆性心脏移植模型,高浓度桂皮醛可以获得移植物长期耐受,其机制可能是通过抑制mTOR和STAT3的表达,降低移植物记忆性T细胞的免疫应答水平。展开更多
基金supported by grants from the Natural Science Fund (ZR2010HM035) of Shandong Provincethe Shandong Provincial Health Development Project Fund (2011WSB19002) in China
文摘Oestrogen is essential for maintaining bone mass, and it has been demonstrated to induce osteoblast proliferation and bone formation.In this study, complementary DNA（cDNA） microarrays were used to identify and study the expression of novel genes that may be involved in MC3T3-E1 cells’ response to 17-b estradiol. MC3T3-E1 cells were inoculated in minimum essential media alpha（a-MEM）cell culture supplemented with 17-b estradiol at different concentrations and for different time periods. MC3T3-E1 cells treated with1028mol？L2117-b estradiol for 5 days exhibited the highest proliferation and alkaline phosphatase（ALP） activity; thus, this group was chosen for microarray analysis. The harvested RNA was used for microarray hybridisation and subsequent real-time reverse transcription polymerase chain reaction（RT-PCR） to validate the expression levels for selected genes. The microarray results were analysed using both functional and pathway analysis. In this study, microarray analysis detected 5 403 differentially expressed genes,of which 1 996 genes were upregulated and 3 407 genes were downregulated, 1 553 different functional classifications were identified by gene ontology（GO） analysis and 53 different pathways were involved based on pathway analysis. Among the differentially expressed genes, a portion not previously reported to be associated with the osteoblast response to oestrogen was identified. These findings clearly demonstrate that the expression of genes related to osteoblast proliferation, cell differentiation, collagens and transforming growth factor beta（TGF-b）-related cytokines increases, while the expression of genes related to apoptosis and osteoclast differentiation decreases, following the exposure of MC3T3-E1 cells to a-MEM supplemented with 17-b estradiol. Microarray analysis with functional gene classification is critical for a complete understanding of complementary intracellular processes. This microarray analysis provides large-scale gene expression data that require further confirmatory studies.
基金Supported by The Breast Cancer Campaign and the Research Institute in Healthcare Sciences (Armesilla AL)The Wellcome Trust (Emerson M)
文摘Emerging evidence suggests that plasma membrane calcium ATPases (PMCAs) play a key role as regulators of calcium-triggered signal transduction pathways via interaction with partner proteins. PMCAs regulate these pathways by targeting specific proteins to cellular sub-domains where the levels of intracellular freecalcium are kept low by the calcium ejection properties of PMCAs. According to this model, PMCAs have been shown to interact functionally with the calcium-sensitive proteins neuronal nitric oxide synthase, calmodulindependent serine protein kinase, calcineurin and endothelial nitric oxidase synthase. Transgenic animals with altered expression of PMCAs are being used to evaluate the physiological significance of these interactions. To date, PMCA interactions with calcium-dependent partner proteins have been demonstrated to play a crucial role in the pathophysiology of the cardiovascular system via regulation of the nitric oxide and calcineurin/nuclear factor of activated T cells pathways. This new evidence suggests that PMCAs play a more sophisticated role than the mere ejection of calcium from the cells, by acting as modulators of signaling transduction pathways.
基金This work was supported by National NaturalScience Fundation of China(No.39760077).
文摘AIM: The existence and properties of alpha-fetoprotein (AFP) receptor on the surface of NIH 3T3 cells and the effects of AFP on cellular signal transduction pathway were investigated. METHODS: The effect of AFP on the proliferation of NIH 3T3 cells was measured by incorporation of 3H-TdR. Receptor-binding assay of 125I-AFP was performed to detect the properties of AFP receptor in NIH 3T3 cells. The influences of AFP on the [cAMP]i and the activities of protein kinase A (PKA) were determined. Western blot was used to detect the change of K-ras P21 protein expression. RESULTS: The proliferation of NIH 3T3 cells treated with 0-80 mg/L of AFP was significantly enhanced. The Scatchard analysis indicated that there were two classes of binding sites with KD of 2.722 x 10(-9)M (Bmax=12810 sites per cell) and 8.931 x 10(-8)M (Bmax=119700 sites per cell) respectively. In the presence of AFP (20 mg/L), the content of cAMP and activities of PKA were significantly elevated . The level of K-ras P21 protein was upregulated by AFP at the concentration of 20 mg/L. The monoclonal antibody against AFP could reverse the effects of AFP on the cAMP content, PKA activity and the expression of K-ras p21 gene. CONCLUSION: The effect of AFP on the cell proliferation was achieved by binding its receptor to trigger the signal transduction pathway of cAMP-PKA and alter the expression of K- ras p21 gene.
文摘目的探索桂皮醛诱导记忆性心脏移植耐受的作用及潜在分子机制。方法将60只小鼠随机分为正常对照组(假手术,n=10)、手术组(心脏移植,n=10)、模型组(注射T细胞+心脏移植,n=10)、桂皮醛低浓度组(注射T细胞+心脏移植+10 mg/kg桂皮醛,n=10)、桂皮醛中浓度组(注射T细胞+心脏移植+20 mg/kg桂皮醛,n=10)和桂皮醛高浓度组(注射T细胞+心脏移植+40 mg/kg桂皮醛,n=10)。观察各组移植物平均存活时间、移植物排斥程度,检测脾细胞增殖情况、移植物中相关基因白细胞介素-2(interleukin-2,IL-2)、白细胞介素-10(interleukin-10,IL-10)、γ-干扰素(interferon-γ,IFN-γ)和转化生长因子-β(transforming growth factor-β,TGF-β)的相对表达量,以及哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycin,mTOR)与信号转导及转录激活因子3(signal transduction and activator of transcription3,STAT3)蛋白磷酸化的表达情况。结果(1)与正常对照组相比,手术组存活时间明显缩短(P<0.05);与手术组相比,模型组存活时间明显缩短(P<0.05);与模型组相比,桂皮醛高浓度组的平均存活时间显著延长(P<0.05)。(2)对各组移植物排斥程度进行评估,模型组为Ⅳ级,桂皮醛治疗组的移植物排斥程度显著降低,且具有剂量依赖性。(3)与正常对照组相比,手术组及模型组脾细胞OD值升高(P<0.05);与模型组及桂皮醛低浓度组相比,桂皮醛中、高浓度组脾细胞OD值降低(P<0.05)。(4)与正常对照组相比,手术组IL-2、IFN-γmRNA表达显著上调(P<0.05),IL-10、TGF-βmRNA表达显著下调(P<0.05);与手术组相比,模型组IL-2、IFN-γmRNA表达显著上调(P<0.05),IL-10、TGF-βmRNA表达显著下调(P<0.05);与模型组比较,桂皮醛中、高浓度组移植物中IL-2、IFN-γmRNA表达明显下调(P<0.05),桂皮醛中、高浓度组IL-10、TGF-βmRNA表达明显上调(P<0.05)。(5)与正常对照组相比,手术组p-mTOR、p-STAT3蛋白表达显著上调(P<0.05);与手术组相比,模型组p-mTOR、p-STAT3蛋白表达显著上调(P<0.05);与模型组比较,桂皮醛中、高浓度组移植物中p-mTOR、p-STAT3蛋白表达明显下调(P<0.05)。结论对于记忆性心脏移植模型,高浓度桂皮醛可以获得移植物长期耐受,其机制可能是通过抑制mTOR和STAT3的表达,降低移植物记忆性T细胞的免疫应答水平。
