The influence of insulin on secretion of IGF-Ⅰ and IGFBP-Ⅰ in cultures of human endometrial stromal cells

Objectives To study the influence of insulin on IGF-Ⅰ and IGFBP-Ⅰ secretion of the human endometrial stromal cells. Methods Late proliferative phase endometrial stromal cells were isolated from endometrium tissues and then cultured for 24 h in Hams F-12 only as a control and in Hams F-12 with different concentrations of estradiol (E2) and insulin (INS) as treated groups. Simultaneously, the endometrial stromal cells from late secretory phase endometrium were cultured for 24 h in Hams F-12 only as a control and in Hams F-12 supplemented with different concentrations of progesterone (P) and insulin as treated groups. After 24 h of culturing, the mediums were collected for either IGF-Ⅰ or IGFBP-Ⅰ assays. Result The concentrations of IGF-Ⅰ in medium from cultured endometrial stromal cells in the proliferative phase were 0.78±0.47 ng/ml in the hormone-free control group; 1.44±0.59 ng/ml and 1.39± 0.33 ng/ml in 100 pg/ml E2 group and 20 μU/ml INS group, which was higher than that of the control group (P<0.05 and P<0.01, respectively). The IGF-Ⅰ concentration in the 100 μU/ml INS group was 2.03±0.53 ng/ml, which was higher than that of the 20 μU/ml INS group (P<0.01). Levels of IGF-Ⅰ in the 100 pg/ml E2 plus 20 μU/ml INS group was 2.18±0.36 ng/ml, which was significantly higher than that of the 20 μU/ml INS and 100 pg/ml E2 group (P<0.01), but lower than that of the 100 pg/ml E2 plus 100 μU/ml INS group (3.42±0.75 ng/ml), P<0.01. The concentration of IGFBP-Ⅰ in medium from cultured endometrial stromal cells in the secretory phase was 2.50±1.39 ng/ml in the hormone-free control group and 5.44±2.09 ng/ml in the 10 pg/ml P group, which was significantly higher than that of the control (P<0.01). IGFBP-Ⅰ concentration in 20 μU/ml INS group was 0.16±0.58 ng/ml, which was lower compared with control, but higher compared with the 100 μU/ml INS group (P<0.01). The level of IGFBP-Ⅰ in the 10 ng/ml P plus 20 μU/ml INS group was 2.10±1.17 ng/ml, lower compared with the 10 ng/ml P group, but higher compared with the 10 pg/ml P plus 100 μU/ml INS group, P<0.01. Conclusions Insulin can stimulate basal (without hormone) and E2-stimulated IGF-Ⅰ secretion in cultured stromal cells from human late proliferative endometrium in a dose-dependent manner. Insulin can suppress basal (without hormone) and P-stimulated IGFBP-Ⅰ secretions in cultured stromal cells from human secretory endometrium in a dose-dependent manner.     

Inhibitory Effects of Parthenolide on the Angiogenesis Induced by Human Multiple Myeloma Cells and the Mechanism

The inhibitory effects of parthenolide （PTL） on angiogenesis induced by multiple myeloma （MM） cells in vitro and the mechanism were investigated. Human MM line RPMI8226 cells were cultured in vitro. The effects of MM culture supernatant on the migration and tubule formation ability of human umbilical vein endothelial cells （HUVECs） treated with PTL were observed. By using Western blot, the expression of p65 and IкB-α in MM cells was detected. RT-PCR was used to assay the expression of VEGF, IL-6, MMP2 and MMP9 mRNA in MM cells. ELISA was used to measure the levels of VEGF and IL-6 in MM cell culture supernatant. The expression of MMP2 and MMP9 in MM cells was examined by immunohistochemistry. （1） In 3.5, 5.0, 7.5 and 10 μmol/L PTL groups the number of migrated cells was 310±56, 207±28, 127±21 and 49±10 respectively, which was significantly different from that in positive control group （598±47） （P〈0.01）. In 3.5 and 5.0 μmol/L PTL groups the areas of capillary-like structures were 0.092±0.003 and 0.063±0.002 mm2, significantly less than in positive control group （0.262±0.012 mm2） （P〈0.01）, but in 7.5 and 10 μmol/L PTL groups no capillary-like structures were found; （2） After treatment with different concentrations of PTL for 48 h, the expression of p65 protein was gradually decreased, while that of IкB-α was gradually enhanced with the increased concentration of PTL; （3） After treatment with 3.5, 5.0, 7.5 and 10 μmol/L PTL for 48 h, the VEGF levels in the supernatant were 2373.4±392.2, 1982.3±293.3, 1247.0±338.4 and 936.5±168.5 pg/mL respectively, significantly different from those in positive control group （2729±440.0 pg/mL） （P〈0.05）. After treatment with 7.5 and 10 μmol/L PTL, the IL-6 levels in the culture supernatant were 59.6±2.8 and 41.4±9.8 pg/mL respectively, signifi- cantly lower than in positive control group （1287.3±43.5 pg/mL） （P〈0.05）; （4） RT-PCR revealed that PTL could significantly inhibit the expression of V     

Influence of Isoflavones on Cadmium-induced Adverse Effects in Vascular Endothelial Cells （ECV 304）

To study the possible intervention of isoflavones in cytotoxicity induced by cadmium in vascular endothelial cells. Methods An ECV 304 cell line derived from human umbilical vein endothelial cells was adopted. Genistein / daidzein was added prior to or simultaneously with CdCl2, cell viability was determined by MTT assay, and metallothionein mRNA expression was monitored by RT-PCR method. Results Cell viability was higher in isoflavone and CdCl2. co-treated groups than that in CdCl2 treated group, with CdCl2 concentration at 10, 20, 40, and 80 μmol/L, respectively. However this increase was not observed in the group treated with CdCl2 at a concentration of 60 μmol/L, lsoflavones (10^-1mol/L to 10^-5 mol/L) were added 24 h before cells were challenged with 80 μmol/L CdCl2 for 24 h or simultaneously with 80 ^-10mol/L CdCl2. Genisteinincreased cell viability only at 10^-5 mol/L, while daidzein caused a dose-dependent increase from 10^-10 mol/L to 10^-5 mol/L in co-treatment with CdCl2. In pre-treatment, genistein (10^-7 to 10^-5 mol/L) increased cell viability whereas only 10^-5 mol/L of daidzein exerted protection. Apparent protection could be found when the cells were pre-treated with 10^-5 mol/L isoflavones for over 12 h, whereas 24 h incubation was required in such a co-treatment, with the exception of daidzein that had a significant protection in only 3 h. Isoflavones (10^-6 mol/L) incubated for 3 h to 24 h, increased MT ⅡA and MT IF mRNA expression, but the induction could not last for more than 24 h. Co-treatment with isoflavones could induce an additional induction of MT ⅡA mRNA expression in cells exposed to cadmium. However, the additional induction of MT ⅡA and MT IF mRNA was not seenwhen pre-treatment was carried out with isoflavones, with the exception of an increase in MT ⅡA mRNA expression in the daidzein pre-treated group. Conclusion Genistein/daidzein could reverse the cytotoxicity of cadmium either in pre-treatment or in co-treatment. The protection is the strongest in 10^-5 mol/L of isoflavones with a dose-dependent pattern. There are differences between genistein and daidzein in their protective effects. Whether the protection of isoflavones is related to their capacity of inducing MT mRNA expression remains to be elucidated.     

Effect of puerarin on the release of interleukin-8 in co-culture of human bronchial epithelial cells and neutrophils

Objective:To investigate the effect of puerarin on interleukin(IL)-8 mRNA expression and the protein release in the co-culture of human bronchial epithelial(BEAS-2B) cells and human neutrophils.Methods:BEAS2B cells and neutrophills were cultured separately and co-cultured with puerarin(50,100,and 200μg/mL) for a predetermined time.Cytokines in culture supematant were evaluated by protein array and IL-8 quantified by enzymelinked immunosorbent assay(ELISA).IL-8 mRNA expression was evaluated by real-time quantitative polymerase chain reaction(real-time qPCR).Results:The co-culture of BEAS-2B cells and neutrophils exhibited synergistic effects on IL-8 mRNA expression in BEAS-2B cells,but not in neutrophils after 12 h incubation(P<0.01),as compared with that in BEAS-2B cells or neutrophils alone.IL-8 protein release in the culture supematant was obviously elevated when BEAS-2B cells were co-cultured with human neutrophils as compared with that in the supematant of BEAS-2B cells or neutrophils alone after incubated for 2,6,12,and 18 h(P<0.01).Treatment with puerarin could significantly down-regulate the expression of IL-8 mRNA in BEAS-2B cells and IL-8 release in the supernatant of the co-culture of BEAS-2B cells and neutrophils(P<0.01).Conclusion:Puerarin could exhibit anti-inflammatory activity by suppressing IL-8 production from the co-culture of human bronchial epithelial cells and neutrophils.     

Effects of estradiol on proliferation and metabolism of rabbit mandibular condylar cartilage cells in vitro

Objective To investigate the effects in vitro of 17β-estradiol ( E2 ) on the proliferation and metabolism of rabbit mandibular condylar cartilage cells.Methods Chondrocytes were derived from neonatal rabbit mandibular condylar cartilage using a modified enzyme method. 17β-estradiol was added to the culture medium in a variety of concentrations. Cell growth and DNA, collagen, and proteoglycan synthesis were used as indicators of proliferation and differentiation of condylar chondrocytes. These were measured by cell number, ^3 H-proline and ^35S-incorporation, respectively.Results E2 increased cell number and ^3H-thymidine incorporation at 10^-8 to 10^-10mol/L, and 10^-8 to 10^-11 mol/L in a dose-dependent manner, peaking at 10^-8 mol/L and 10^-9mol/L, respectively.However, further increase in the concentration of estradiol caused inhibition of both cell number and ^3H-thymidine incorporation, and this was significant at 10^-6mol/L. The effect of E2 on proteglycan synthesis was similar; the maximum stimulating effect was at 10^-8mol/L, and inhibition was significant at 10^-6mol/L. There was no obvious stimulatory effect of E2 on ^3H-thymidine incorporation observed.Conclusions Estradiol affects condylar chondrocyte cell growth, DNA, and proteoglycan synthesis in a biphasic manner depending on its concentration. This indicates that estrogen may be important in the proliferation and differentiation of mandibular condylar chondrocytes, and could be relevant to some aspects of certain tempormandibular joint diseases by modulating the function of the chondrocytes.     

Effects of dexamethasone on proliferation, differentiation and apoptosis of adult human osteoblasts in vitro

Objective To observe the effects of dexamethasone on proliferation, differentiation and apoptosis of adult human osteoblasts in vitro.Methods Iliac trabecular bone specimens were obtained from adult patients undergoing necessary surgery. After the bone pieces were digested with collagenase-trypsin, osteoblasts were released and incubated at 37℃ in a relative humidity of 95% and 5% CO2. Then, the ceils were purified, and their passages were given DMEM-F12 and fetal bovine serum medium. Subsequently, 10^8 mol/L dexamethasone was added into the culture medium to incubate the osteoblasts for three days, and the cells from control groups were incubated without any drugs. All cells were observed continually with phase contrast microscope and transmission electron microscope. Finally, apoptosis was detected by the use of terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL)and biochemical indices, alkaline phosphatase (ALP) and osteocalcin (OCN) were used to determine the effects of dexamethasone on proliferation, differentiation and apoptosis of adult osteoblasts in vitro.Results In the adult osteoblasts obtained by collagenase-trypsin digestion, it achieved high survial,stable biochemical indices and excellent purification. Under the condition of dexamethasone 10^8mol/L and osteoblasts 10 000/ml, there was significant promotion of ALP and OCN secretion without cell apoptosis.Conclusions Dexamethasone has a significant effect on the proliferation and differentiation of adult osteoblasts in vitro without apoptosis, and dexamethasone at the suggested concentration can be used as positive control in drug studies for osteoporosis treatment.     

Effects of prolactin on HLA- DR and CD40 expressions by human thyrocytes

Objective To study the effects of prolactin (PRL) on HLA- DR and CD40 expressions by human thyrocytes, and to investigate the possible mechanisms for PRL to affect the de velopment of Graves’ disease (GD). Methods Thyrocytes in secondary culture, which were from GD thyroid glands and the tissu es adjacent to the lesions of multinodular goiter and adenoma (control group), w ere treated respectively with ovine PRL (oPRL), interferon- γ (IFN- γ), interl ukin- 4 (IL- 4) and oPRL plus IFN- γ (10 U/ml) or IL- 4 (5 ng/ml) for 7 days . HLA- DR and CD40 expressions on the thyrocytes were determined by immunofluor escent staining and flow cytometry. Results oPRL (12. 5 ng/ml-1000 ng/ml) had no significant direct effect on HLA- DR or C D40 expression. It did not significantly affect the stimulation of HLA- DR exp ressions on the two groups of thyrocyte treated with IFN- γ or on GD thyrocytes treated with IL- 4. oPRL could antagonize the stimulation of CD40 expressions by IFN- γ and the inhibition by IL- 4 on both groups of thyrocytes. The antago nizing effects were related to the concentrations of PRL. IFN- γ- stimulated p ercentages of CD40［+］ thyrocytes and delta mean fluorescence intensity (dMF) in both thyrocyte sources were significantly reduced in the presence of 200 ng/ml oPRL (both GD and Control: P&lt;0. 01 and P&lt;0. 05, respectively; Contro l: P&lt;0. 05) and 1000 ng/ml oPRL (GD: P&lt;0. 01; Control: P&lt;0. 05). oPRL caused significant increasing in IL- 4- inhibited percentages of CD40［+］ ce lls from the two groups of thyrocytes at 12. 5 ng/ml and 1000 ng/ml and dMF fr om GD thyrocytes at 1000 ng/ml (P&lt;0. 05). Conclusions PRL can exert indirect effects on CD40 expressions on thyrocytes by antagonizing the modulatory actions of IFN- γ and IL- 4 with dose- related effects. This m ay be one important mechanism for PRL to affect the development of GD.     

Recombinant human Flt3 ligand exerts both direct and indirect effects on hematopoiesis

Objective To investigate the direct effects of the Flt3 ligand (FL) on hematopoiesis, such as the stimulation of the formation of hematopoietic colonies and the proliferation of dendritic cells, as well as the indirect stimulation of hematopoiesis, especially via the proliferation of endothelial cells.Methods Mononuclear cells from human cord blood were plated in methylcellulose medium containing different cytokines to induce hematopoietic colony formation. Dendritic cells (DCs) were induced from the mononuclear cells with a cytokine cocktail with or without recombinant human soluble FL (rhFL; 100ng/ml).The Flt3 receptors on the surface of a human microvascular endothelial cell line (ECV) were analyzed by flow cytometry. The proliferation of ECV stimulated by rhFL was measured with the microculture tetrazolium assay. The levels of FL, IL-6, IL-8, G-CSF and GM-CSF in the supernatant of ECV cultures were measured by enzyme linked immunoabsorbent assay (ELISA).Results rhFL stimulates colony formation from cord blood when used as a sole stimulant. FL in combination with other cytokines increased colony formation significantly. The number of DCs was approximately 2.5 times higher when rhFL was used. rhFL stimulates the proliferation of ECV on which Flt3 receptors are expressed. Furthermore, ECV secretes FL, IL-6, IL-8, G-CSF and GM-CSF, which were augmented by tumor necrosis factor-α and rhFL.Conclusions rhFL enhances hematopoietic colony formation and DC proliferation from human cord blood cells. FL not only     

Relationship between Carbachol Hyperstimulation-Induced Pancreatic Acinar Cellular Injury and Trypsinogen or NF-KB Activation in Rats in vitro

The relationship between M3 cholinergic receptor agonist （carbachol） hyperstimulationinduced pancreatic acinar cellular injury and trypsinogen activation or NF-κB activation in rats was studied in vitro. Rat pancreatic acinar ceils were isolated, cultured and treated with carbachol, the active protease inhibitor （pefabloc）, and NF-κB inhibitor （PDTC） in vitro. Intracellular trypsin activity was measured by using a fluorogenic substrate. The cellular injury was evaluated by measuring the leakage of LDH from pancreatic acinar ceils. The results showed that as compared with control group, 10-3 mol/L carbachol induced a significant increase of the intracellular trypsin activity and the leakage of LDH from pancreatic acinar cells. Pretreatment with 2 mmol/L pefabloc could significantly decrease the activity of trypsin and the leakage of LDH from pancreatic acinar cells （P〈0. 01） following the treatment with a high concentration of carbachol （10^-3 mol/L） in vitro. The addition of 10^-2mol/L PDTC didn＇t result in a significant decrease in the activity of trypsin and the leakage of LDH from pancreatic acinar cells treated with a high concentration of carbachol （10^-3 mol/L） in vitro （P〉0. 05）. It was concluded that intracellular trypsinogen activation is likely involved in pancreatic acinar cellular injury induced by carbachol hyperstimulation in vitro. NF-κB activation may not be involved in pancreatic acinar cellular injury induced by carbachol hyperstimulation in vitro.     

地塞米松抑制聚肌胞苷酸诱导人气道上皮细胞趋化因子表达及机制的初步研究

目的 初步探讨地塞米松对聚肌胞苷酸刺激气道上皮细胞后趋化因子表达的影响及其机制.方法 将人气道上皮细胞16hBE给予不同浓度的聚肌胞苷酸(0.001、0.01、0.1、1 μg/ml)及地塞米松(0.1、1、10 μmol/L)处理,用RT-PCR检测刺激6h后IL-8、IP-10 mRNA的表达水平,用ELISA法检测刺激24 h后培养上清液中IL-8和IP-10蛋白含量,用免疫组化方法检测细胞NF-κB p65亚单位的表达强度.结果 0.001、0.01、0.1 μg/ml聚肌胞苷酸处理16hBE细胞后IL-8和IP-10mRNA表达水平和蛋白分泌量呈浓度依赖性升高,在0.01 μg/ml及0.1 μg/ml浓度时,与对照组比较差异有统计学意义(P＜0.05,P＜0.01);但在聚肌胞苷酸浓度为1μg/ml时,IL-8和IP-10 mRNA表达水平和蛋白分泌量都出现了下降.地塞米松(1、10 μmol/L)预处理0.5h明显抑制聚肌胞苷酸诱导的IL-8和IP-10 mRNA表达及蛋白分泌(P＜0.05,P＜0.01).1 μmol/L地塞米松预处理明显抑制了聚肌胞苷酸诱导的NF-κB p65表达强度(P＜0.01).结论 糖皮质激素可抑制聚肌胞苷酸诱导的气道上皮细胞趋化因子的表达,其机制可能与NF-κB的活化有关.     

异丙酚对缺氧复氧大鼠海马星形胶质细胞的影响

目的:观察异丙酚对缺氧复氧大鼠海马星形胶质细胞及其分泌促炎性细胞因子(TNF-α、IL-1β、IL-6)和抗炎性细胞因子IL-10变化的影响。方法:取新生2～3dWistar大鼠海马星形胶质细胞,原代纯化培养3周。将细胞分为正常对照组(Nor组),乳剂对照组(Nor-L组),缺氧6h后复氧24h组(Ano组),加异丙酚50μmol/L缺氧6h后复氧24h组(Pro1组)、加异丙酚500μmol/L缺氧6h后复氧24h组(Pro2组)。取各组培养孔上清液检测IL-1β、TNF-α、IL-6和IL-10含量,用神经胶质纤维酸性蛋白(GFAP)免疫细胞化学法标记星形胶质细胞,用多媒体彩色病理图像分析系统检测阳性细胞平均光密度(AOD)。结果:与Nor组比较,Ano组细胞被激活增生肥大,AOD升高(P<0.01),其上清液中IL-1β、IL-6和TNF-α含量上升(P<0.01),IL-10无明显变化(P>0.05)。与Ano组比较,Pro1组和Pro2组细胞激活被抑制,AOD、IL-1β、IL-6和TNF-α含量均降低(其中Pro1组P<0.05,Pro2组P<0.01),而IL-10含量均上调(P<0.01)。Nor组和Nor-L组比较无明显差异。结论:异丙酚可抑制缺氧复氧大鼠海马星形胶质细胞的激活作用,同时抑制星形胶质细胞分泌IL-1β、IL-6和TNF-α,增强IL-10的合成释放,其作用程度与剂量有关。     

桥本病中白细胞介素18表达可能参与甲状腺细胞的自我毁损过程

目的 观察白细胞介素18(IL-18)在桥本病甲状腺组织中的表达及其细胞定位,并探讨干扰素γ(IFN-γ)对甲状腺细胞中IL-18表达的影响.方法 采用逆转录-聚合酶链反应(RT-PCR)和免疫组织化学染色,检测IL-18在正常和桥本病甲状腺组织中的表达及其细胞定位.原代培养的人甲状腺细胞与IL-1B、肿瘤坏死因子α(TNF-α)或IFN-γ孵育后,应用RT-PCR和Western印迹检测IL-18表达的变化.结果 新鲜甲状腺组织的RT-PCR分析显示,IL-18 mRNA在正常甲状腺组织中表达水平极低,但在桥本病的甲状腺组织中表达显著增高,IL-18/β-肌动蛋白的灰度比分别为0.15±0.02和0.90±0.13(P<0.05).免疫组织化学染色显示,IL-18表达在桥本病的甲状腺组织中明显增加,且主要定位于甲状腺滤泡上皮细胞.在离体培养的甲状腺上皮细胞中,IFN-γ可剂量依赖性增加IL-18 mRNA的表达,而TNF-α和IL-1β则无类似的效应.Western印迹显示,IL-18原在离体甲状腺细胞中可见表达,IFN-γ使IL-18原表达增加.结论 IL-18在桥本病的甲状腺滤泡上皮细胞中表达明显增加,IFN-γ可上调离体甲状腺细胞IL-18表达.IL-18与IFN-γ之间的相互作用在桥本病的甲状腺细胞破坏中可能具有重要的意义.     

碘化钠和IL-1β对人甲状腺上皮细胞TRAIL表达的影响

目的 研究不同浓度碘化钠(NaI)和白细胞介素-1β(IL-1β)对体外培养的原代人甲状腺上皮细胞(TEC)肿瘤坏死因子相关凋亡诱导配体(TRAIL)的表达及凋亡的影响,探讨自身免疫性甲状腺疾病(AITD)的可能发病机制.方法 收集正常、Graves病(GD)和桥本甲状腺炎(HT)患者甲状腺组织进行原代细胞培养,以不同...     

Cytokine-Induced Cell Surface Expression of Adhesion Molecules in Vascular Endothelial Cells In vitro

Brain ischemia is commonly accompanied by anacute inflammatory response characterized by cy-tokines release and leukocytes infiltration and the mi-gration of leukocytes into injured cerebral tissue,which is regulated,in part,by immunoglobulin su-perfamilies of adhesion molecules,intercelluar adhe-sion molecule- 1 ( ICAM- 1 ) and vascular celladhesionmolecule- 1 ( VCAM- 1 ) [1] .The expression climax ofcytokines( such as TNF- α and IL- 1 β) precede thatofadhesion molecules by 1 2 - 2 4 h …     

白藜芦醇对小鼠肺泡巨噬细胞释放IL-6、TNF-α的影响

目的:探讨白黎芦醇(Res)对小鼠肺泡巨噬细胞释放细胞因子IL-6,TNF-α的影响。方法:经从小鼠支气管肺泡灌洗中获得肺泡巨噬细胞,分5组进行培养,A组(LPS 10mg/L),B组(LPS 10mg/L+Res 50μg/ml),C组(LPS 10mg/L+Res 100μg/ml),D组(LPS 10mg/L+Res 150μg/ml),E组(LPS 10mg/L+Res 200μg/ml).各组在培养4,6,12,24h提取上清液,应用ELISA法测定上清液中IL-6,TNF-α含量。结果:在LPS刺激下IL-6于12h达释放高峰,在高峰期随着白黎芦醇浓度的逐步加大,IL-6的含量相应逐渐降低,E组IL-6含量明显低于只有内毒素刺激的A组(P<0.05)。同样在LPS刺激下TNF-α于6h达释放高峰,在高峰期随着白黎芦醇浓度的逐步加大,TNF-α含量也相应逐渐降低,而C组、D组、E组TNF-α含量明显低于A组(P<0.05)。结论:白黎芦醇对小鼠肺肺泡巨噬细胞过度释放炎症性细胞因子IL-6,TNF-α具有明显抑制作用。     

慢性炎症和能量代谢因子对乳腺癌细胞系KISS-1表达的影响

目的下丘脑弓状核KISS-1神经元,是调控下丘脑-垂体-性腺轴功能的神经中枢。各种因子可能通过影响下丘脑细胞KISS-1表达而调控性腺轴功能。因此,本研究旨在评价慢性炎症因子脂多糖（lipopolysaccharide,LPS）,肿瘤坏死因子α（tumor necrosis factor alpha,TNF-α）,白介素-6（interleukin6,IL-6）、地塞米松和能量代谢相关因子胰岛素、胰高糖素样肽（glucagon-like peptide,GLP-1）对乳腺癌MCF-7细胞系KISS-1表达的影响。方法对MCF-7细胞系进行培养,测定KISS-1 mRNA和蛋白表达水平。在培养液中添加不同浓度LPS、TNF-α、IL-6、地塞米松、胰岛素和GLP-1刺激24 h、48 h和72 h,观察不同浓度的因子对KISS-1表达的影响。用实时聚合酶链反应（real-time polymerase chain reaction,RT-PCR）方法定量测定KISS-1 mRNA水平;用Western blotting方法半定量评价KISS-1蛋白表达变化。结果 1LPS（10μg/ml）和地塞米松（10-6 M和10-7 M）可抑制KISS-1 mRNA和蛋白表达;2TNF-α（100ng/ml）、IL-6（10 ng/ml,20 ng/ml）和胰岛素（0.1 mIU/ml,0.01mIU/ml）可增加KISS-1 mRNA和蛋白的表达;3GLP-1类似物（艾塞那肽,1 ng/ml和10 ng/ml）,在6 h一过性增加KISS-1mRNA和蛋白表达。结论慢性炎症和能量代谢相关因子,如LPS、TNF-α、IL-6、地塞米松、胰岛素和GLP-1影响MCF-7细胞系KISS-1表达,可能通过影响下丘脑细胞KISS-1表达,从而调控下丘脑-垂体-性腺轴的功能。     

睾酮对小鼠RAW264.7巨噬细胞肿瘤坏死因子-α和单核细胞趋化蛋白-1表达的影响及其分子机制

目的 探讨雄激素对RAW264.7巨噬细胞肿瘤坏死因子(TNF)-α和单核细胞趋化蛋白(MCP)-1表达的影响及其分子机制.方法 (1)用不同浓度睾酮处理小鼠RAW264.7巨噬细胞不同时间,分别用RT-PCR和ELISA法检测细胞TNF-α和MCP-1 mRNA和培养上清液中的表达;(2)10-7 mol/L睾酮处理细胞0、10、30、60、180、360 min后,Western印迹检测磷酸化NF-κB(p-NF-κB)的表达.(3)NF-κB的抑制剂PDTC处理细胞1 h后再用不同浓度睾酮处理,用RT-PCR和ELISA法检测TNF-α和MCP-1的表达.结果 (1)①10-9和10-7 mol/L睾酮处理细胞6 h后,TNF-α mRNA表达分别增加1.78倍和1.87倍(均P<0.05),MCP-1 mRNA的表达也显著增加(均P<0.01);②睾酮处理6 h后TNF-α和MCP-1的分泌量的变化差异均无统计学意义;睾酮处理24 h后,TNF-α和MCP-1的分泌量显著增加(均P<0.05);10-7 mol/L睾酮处理组的分泌量显著高于10-9 mol/L睾酮处理组(均P<0.05).(2)随着睾酮处理时间的增加,p-NF-κB的表达增加,60 min达峰值(2.49±0.40)倍,P<0.05,以后逐渐降低.(3)PDTC预处理后再用睾酮处理6 h,TNF-α和MCP-1 mRNA的表达均低于同浓度单纯睾酮处理组(均P<0.05),但仍高于空白对照组(均P<0.05).PDTC预处理后再用睾酮处理24 h,TNF-α和MCP-1的分泌量均低于同浓度单纯睾酮处理组(除10-9mol/L睾酮组外,均P<0.05),但仍高于空白对照组(均P<0.05).结论 睾酮能促进离体的小鼠RAW264.7巨噬细胞TNF-α和MCP-1表达.NF-κB的活化是介导睾酮这一效应的分子机制之一.     

大豆异黄酮类药genistein抑制IL-1β的促破骨细胞骨吸收功能

目的:观察大豆异黄酮类药genistein抑制IL-1 β的促破骨细胞骨吸收功能.方法:新生兔长干骨机械分离法获得破骨细胞(osteoclast,OC),鼠颅盖骨机械法体外器官培养.按照加入药物的不同分为4组:对照组(不加genistein和IL-1β)、10-6mol/L genistein、10 μg/L IL-1β、10-6 mol/L genistein 10 μg/L IL-1β组.图像分析体外骨吸收陷窝面积,原子分光光度计法测定OC培养上清液和器官培养上清液中的Ca2 含量,用生化法检测器官培养上清液中的酸性磷酸酶(acid phosphatase,ACP)含量.计算实验组与对照组平均值比值,作为骨吸收指数,其值大于1.0表示骨吸收增强,小于1.0表示骨吸收受到抑制.结果:10-6mol/L genistein 10μg/L IL-1β组骨吸收陷窝面积显著高于10-6mol/L genistein组,Ca2 含量显著低于10μg/L IL-1β组,骨吸收指数小于10μg/L IL-1β组,大于10-6mol/L genistein组.器官培养上清液中,各组ACP含量差异无显著性;而10-6 mol/L genistein 10μg/LIL-1β组的骨吸收指数低于10μg/L IL-1β组,且二者都大于1.0;10-6mol/L genistein组的骨吸收指数低于1.0.结论:10-6mol/L genistein和10μg/LIL-1β联合使用显著降低了单独使用IL-1β导致的骨吸收陷窝面积和Ca2 浓度的上升,与对照组数值接近,说明genistein抑制了IL-1β诱导的骨吸收亢进.     

地塞米松对甲状腺相关眼病眼眶成纤维细胞ICAM-1表达的影响

目的 探讨地塞米松对甲状腺相关眼病(TAO)眼眶成纤维细胞(OF)细胞间粘附分子(ICAM-1)表达的影响.方法 取10例TAO患者和5例正常对照的眼眶脂肪组织与眼外肌,体外培养OF,在培养基中分别加入10-6 mol/L地塞米松、300 U/mL 干扰素-γ(IFN-γ)或10-6 mol/L地塞米松与300 U/mL IFN-γ的混合液,作用72 h,用流式细胞仪检测OF上ICAM-1的表达水平.结论 体外培养的OF均表达ICAM-1,TAO患者的表达量高于正常对照者(P＜0.05).IFN-γ促进OF表达ICAM-1,地塞米松不仅抑制OF表达ICAM-1,而且能抑制IFN-γ诱导的ICAM-1表达.结论 TAO患者OF表达ICAM-1增强可能是促进眶内炎症发生的原因之一,糖皮质激素发挥治疗作用的可能机制之一是其抑制OF表达ICAM-1以及IFN-γ诱导的ICAM-1表达.