胰岛素和葡萄糖对缺氧血管内皮细胞分泌tPA和PAI-1抗原的影响

目的: 观察胰岛素和葡萄糖对缺氧血管内皮细胞分泌组织型纤溶酶原激活物(tPA)及其抑制物-1(PAI-1)的影响。 方法:培养人脐静脉内皮细胞株ECV-304。分组实验:(1)常氧组;(2)在缺氧条件下又分为Ⅰ:缺氧对照组;Ⅱ:低浓度组:胰岛素150 mU/L、葡萄糖5.5 mmol/L;Ⅲ:中浓度组:胰岛素450 mU/L、葡萄糖15 mmol/L;Ⅳ:高浓度组:胰岛素900 mU/L、葡萄糖30 mmol/L;Ⅴ:渗透压对照组:甘露醇24.5 mmol/L。取培养4、8、12 h 3个时点,用ELISA法测定细胞培养上清液tPA、PAI-1抗原。结果:缺氧明显增加tPA和PAI-1抗原分泌,tPA/PAI-1值明显增加(P＜0.01)。胰岛素和葡萄糖能够刺激缺氧内皮细胞分泌tPA和PAI-1抗原,在缺氧8 h以内,tPA/PAI-1比值明显增加(P＜0.05)。随缺氧时间的延长,tPA/PAI-1值逐渐下降。结论:胰岛素和葡萄糖能够刺激内皮细胞分泌tPA和PAI-1抗原,在缺氧8 h以内,tPA/PAI-1值升高,纤溶活性升高,有利于局部自发性纤溶的发生。此作用可能是IGK治疗急性心肌梗死的机制之一。     

The urokinase-type plasminogen activator and inhibitors in resectable lung adenocarcinoma

BackgroundThe urokinase-type plasminogen activator (uPA) system is closely related to the occurrence and progression of cancer in many aspects. Previous studies demonstrated that the conclusions about the prognosis value of uPA, plasminogen activator inhibitor 1 (PAI-1) and plasminogen activator inhibitor 2 (PAI-2) in lung cancer are controversial, so this study was performed for the exploration of the predictive effect of uPA, PAI-1 and PAI-2 on the overall survival (OS) of resectable pulmonary adenocarcinoma patients.MethodsUPA, PAI-1 and PAI-2 expression levels were assayed by immunohistochemical staining based on tissue microarray (TMA) that is composed of formalin-fixed paraffin-embedded specimens from 84 resectable lung adenocarcinoma patients from July 2004 to June 2009. The relationship of IHC, mRNA expression levels of three molecules were investigated respectively. The three molecules’ relationship with clinicopathologic parameters and OS was explored by Chi-square, Kaplan-Meier, and Cox regression analyses. The Cancer Genome Atlas (TCGA) database was used to analyze differential gene expressions of RNA-sequencing data of pulmonary adenocarcinoma and normal tissues, and Kaplan-Meier methods were adopted to confirm the prognostic value of uPA, PAI-1 and PAI-2 in resectable lung adenocarcinoma in TCGA database and the R package MethylMix was used to conduct an analysis integrating methylation data and gene expression of RNA-sequencing data based on TCGA.ResultsUPA, PAI-1 and PAI-2 had much higher IHC expression levels in tumor than those in the normal tissues (uPA, Z = -10.511; PAI-1, Z = -4.836; PAI-2, Z = -6.794; all P < 0.0001). High DNA methylation level of gene uPA resulted in the decrease of its expression. In addition, expression level of PAI-2 was positively associated with tumor size (χ² = 8.372, P = 0.004). Multivariate analyses showed TNM stage III was an independent adverse prognostic factor (hazard ratio = 3.736, 95 % confidence interval = 1.097–12.72, P = 0.035). Kaplan-Meier method revealed that uPA, PAI-1 and PAI-2 expression levels were not related to the OS for 84 resectable lung adenocarcinoma patients. According to TCGA data, PAI-1 expression level was identified as a potential adverse predictor for prognosis of resectable lung adenocarcinoma (Gehan-Breslow-Wilcoxon test, P = 0.025).ConclusionsOur data show that, the expression levels of uPA, PAI-1 and PAI-2 are significantly up-regulated in resectable lung adenocarcinoma. Besides, this study highlights PAI-1 as a latent adverse prognostic factor in resectable adenocarcinoma of lung.     

大黄素对人血管平滑肌细胞增殖的影响

目的 观察大黄素对人血管平滑肌细胞周期时相和cyclin D1表达的影响,探讨大黄素抑制平滑肌细胞增殖的作用机制。方法 取对数增长期的平滑肌细胞同步于G0期,药物组:加入含37.5mg·L-1大黄素10％胎牛血清的培养液,对照组:仅加入10％胎牛血清的培养液;作用12、24、36、48 h后分别用流式细胞仪和Westemblot法进行细胞周期时相和cyclin D1表达的测定。结果 与对照组比较,药物组在各相同时间点C0/G1期细胞百分比升高,S期细胞百分比下降,且随着时间的延长差值增大,cyclin D1表达高峰延迟,表达量下调,细胞周期受阻于G0/G1期。结论 大黄素在抑制平滑肌细胞增殖的过程中通过下调,cyclin D1表达,从而阻滞细胞周期进程。     

PAI-1反义RNA对人主动脉平滑肌细胞中血管内皮生长因子的影响

目的 探讨纤溶酶原激活物抑制剂－1（plasminogen activator inhibitor-1,PAI-1)反义RNA对离体培养的主动脉平滑肌细胞（smooth muscle cell,smc)PAI-1表达的作用及对血管内皮生长因子（vscular endothelial growth factor,VEGF)表达的影响。方法 PCR扩增PAI－1第2外显子,将PCR产物纯化克隆后连入真核细胞表达载体pcDNA3．1,构建PAI－1反应RNA重组质粒。将pcDNA3.1-反应PAI－1重组质粒转染SMC中。通过免疫组化、Western印迹、ELISA检测细胞中PAI－1表达的改变;通过免疫荧光技术观察细胞中PAI－1表达量的变化对VEGF的影响。 结果 转染后第3天、细胞中PAI－1含量最低,VEGF的表达也减少。第5天、PAI－1含量逐渐高,VEGF也相应增加。第7天、PAI－1含量接近于正常,VEGF也增至正常水平。结论 反义PAI－1RNA能有效阻断SMC中PAI－1的蛋白合成,同时抑制细胞中VEGF的表达。     

小檗碱对糖尿病大鼠血管平滑肌细胞增殖影响研究

目的:观察小檗碱(Berberine)对糖尿病大鼠血管平滑肌细胞(VSMC)增殖影响及对核因子-κB(NF-κB)的影响。方法:采用一次性腹腔注射链脲佐菌素55mg.kg-1诱发糖尿病大鼠模型,取建模成功大鼠胸主动脉中膜血管平滑肌细胞进行培养,倒置显微镜下观察细胞形态,SMA免疫组化染色鉴定细胞,计数法观察细胞的生长曲线,CCK-8法测定小檗碱IC50,共聚焦显微镜下观察小檗碱对VSMC中NF-κB核转移的影响。结果:与正常组对比,糖尿病组VSMC生长速度快,小檗碱能明显抑制糖尿病血管平滑肌细胞增殖,IC50为6.001±0.853μmol.L-1,而对正常大鼠VSMC增殖的IC50为18.229±0.434μmol.L-1(P<0.05)有剂量依赖性;小檗碱能抑制糖尿病VSMC中NF-κB从胞浆转移到胞核。结论:小檗碱呈剂量依赖性能抑制糖尿病VSMC增殖与NF-κB的核转移。揭示小檗碱可能有抑制糖尿病大血管病变作用,而此作用可能与抑制NF-κB激活有关。     

人脑星形细胞瘤纤溶酶原激活抑制因子1基因表达研究

目的研究人脑星形细胞瘤纤溶酶原激活抑制因子１（ＰＡＩ１）基因表达及其临床意义。方法采用Ｎｏｒｔｈｅｒｎ杂交和免疫组化ＡＢＣ方法检测３６例人脑星形细胞瘤ＰＡＩ１ｍＲＮＡ和蛋白表达，分析其与临床病理因素之间的关系。结果所有星形细胞瘤组织均可表达３．０ｋｂ和２．２ｋｂ的ＰＡＩ１ｍＲＮＡ转录物；高分级星形细胞瘤ＰＡＩ１ｍＲＮＡ表达水平显著高于低分级星形细胞瘤（Ｐ＜００１）；正常脑组织未检测出ＰＡＩ１ｍＲＮＡ表达。ＰＡＩ１ｍＲＮＡ表达水平与星形细胞瘤的坏死（ｒ＝０．５１，Ｐ＜０．０１）、微血管数（ｒ＝０．３３，Ｐ＜０．０１）及脑水肿（ｒ＝０．２７，Ｐ＜０．０１）呈显著正相关，与患者性别、年龄及瘤体大小无显著相关性。免疫组化染色显示，ＰＡＩ１蛋白主要分布在高分级星形细胞瘤的瘤细胞和内皮细胞，尤以血管增殖部位和坏死灶周围较为显著，低分级星形细胞瘤呈低水平表达。结论ＰＡＩ１基因表达与人脑星形细胞瘤的分级、坏死、血管生成及脑水肿密切相关，可作为星形细胞瘤恶性程度的分子标记。     

糖基化终末产物对大鼠肾系膜细胞纤溶酶原激活物抑制物-1表达韵影响

目的：观察糖基化终末产物（AGEs）对大鼠肾系膜细胞纤溶酶原激活物抑制物-1（PAI-1）表达的影响及其与细胞外基质（ECM）成分含量的关系。方法：体外培养正常大鼠肾系膜细胞，分别用糖化牛血清白蛋白（AGEs）及未经糖化的牛血清白蛋白（BSA）处理，以常规培养的肾系膜细胞作为对照，检测不同时间、不同浓度AGEs对纤维连接蛋白（FN）、Ⅳ型胶原、PAI-1表达的影响。MTT法检测AGEs对系膜细胞增殖的作用，ELISA测定条件培养基中FN、Ⅳ型胶原及PAI-1蛋白含量，逆转录聚合酶链式反应（RT—PCR）检测系膜细胞PAI-1 mRNA的表达。结果：与相应浓度的BSA比较，AGEs（0—200mg／L）对系膜细胞增殖无明显影响，但可不同程度地刺激系膜细胞FN、Ⅳ型胶原、PAI-1蛋白的产生。RT—PCR检测显示，给予AGEs（100mg／L）的系膜细胞PAI-1 mRNA的表达明显增加（P〈0．01）。结论：AGEs促进系膜细胞PAI—1的表达，提示AGEs通过上调PAI-1的表达而减少细胞外基质降解，可能是糖尿病肾病细胞外基质积聚的原因之一。     

雌激素对大鼠血管平滑肌细胞囊泡素-1基因表达的影响

目的:探讨雌激素对血管平滑肌细胞囊泡素-1基因表达的影响。方法:取Wistar雌性大鼠,分为3组:假手术组,卵巢切除后皮下埋植雌激素组 (OVX+E组)及卵巢切除后皮下埋植安慰剂组(OVX+V组)。用药2周后处死大鼠,剥离主动脉平滑肌组织,提取总RNA进行半定量RT-PCR分析,检测雌激素对囊泡素-1(caveolin-1)基因表达的影响。为进一步明确雌激素是否直接调节血管平滑肌细胞caveolin-1基因表达,又采用100 nmol/L 17β-雌二醇(17β-E₂)处理培养的大鼠血管平滑肌细胞24 h,通过Northern blot分析检测雌激素对细胞caveolin-1 mRNA表达的影响。结果:OVX+E组大鼠主动脉平滑肌组织caveolin-1基因表达量明显高于OVX+V组,17β-E₂处理的培养细胞中caveolin-1基因mRNA表达量高于未用药的培养细胞。 结论:雌激素可促进血管平滑肌细胞caveolin-1基因表达,反映了雌激素心血管作用机理的一个方面。     

Neuropeptide Y potentiates calcium-channel currents in single vascular smooth muscle cells

The effect of neuropeptide Y (NPY) on Ca²⁺- channel currents in isolated vascular smooth muscle cells was studied with the perforated-patch recording technique. Using Ba²⁺ (10 mM) as the charge carrier, inward currents sensitive to Cd²⁺ and nifedipine were potentiated by NPY in a concentration-dependent manner. The threshold concentration for the potentiating effect of NPY was 50 nM and reached a maximum at 150 nM. NPY shifted the steady-state activation curve to less positive membrane potentials by about 6 mV so that the potentiating effect was most prominent near the activation threshold of the current. It had no effect on steady-state inactivation of the current. These results suggest that NPY may potentiate vasoconstriction by promoting calcium entry through L-type voltage-dependent Ca²⁺-channels.     

人组织激肽释放酶基因重组腺病毒转染对血管平滑肌细胞增殖和迁移的影响

目的:探讨重组腺病毒介导的人组织激肽释放酶(hKLK1)基因转移对血小板源性生长因子-BB(PDGF-BB)诱导下的自发性高血压大鼠(SHR)血管平滑肌细胞(VSMCSHR)增殖和迁移的影响。方法:自行构建双顺反子重组腺病毒载体,携带强绿色荧光蛋白(EGFP)标志基因和目的基因hKLK1;用细胞计数法和四甲基偶氮唑盐(MTT)比色法检测细胞增殖,流式细胞仪检测细胞生长周期;蛋白免疫印迹法(Western blotting)测定细胞周期素依赖性激酶抑制蛋白p27Kip1、p21Cip1的表达。采用改良Boyden微孔膜双槽法测定VSMCSHR迁移。结果:(1)hKLK1基因转移呈感染复数依赖性(20-100MOI)抑制PDGF-BB诱导的VSMCSHR生长,100MOI时抑制率为39.3%;呈时间依赖性抑制VSMCSHR生长,第5d时达高峰,抑制率为35.2%。(2)hKLK1基因转移可显著抑制PDGF-BB诱导的VSMCSHR增殖,峰值抑制率为30.2%(P0.01);细胞周期阻滞于G0/G1期的VSMCSHR明显增多,最大阻滞率为36.4%(P0.01),而缓激肽B2受体特异性阻断剂Hoe140逆转了hKLK的抑制作用。(3)hKLK1基因转移明显上调PDGF-BB诱导VSMCSHR的p27Kip1、p21Cip1表达,Hoe140明显降低p27Kip1、p21Cip1表达。(4)hKLK1基因转移可明显抑制PDGF-BB诱导的VSMCSHR细胞迁移,抑制率为34.6%,且Hoe140不影响该抑制作用。结论:hKLK1基因转移可抑制PDGF-BB诱导的VSMCSHR增殖,主要由缓激肽B2受体介导的,通过上调细胞周期素依赖性激酶抑制蛋白p27Kip1、p21Cip1表达的途径。而hKLK1基因转移抑制VSMCSHR迁移效应可能不通过B2受体。     

Expression and localization of plasminogen activator inhibitor 1 mRNA in transplant kidneys

Fibrinolysis in renal transplant rejection has not been systematically investigated but is known to be impaired. By an in situ hybridization technique, we have studied gene expression of plasminogen activator inhibitor 1 (PAI-1) in human renal tissue showing severe acute vascular rejection (n=8), clinically irreversible (chronic) vascular rejection (n = 3), mild vascular rejection (n= 8), parenchymal rejection (n=4), ‘normal’ kidneys (n=6), and non-rejecting kidneys (n=6). The results showed that in 7/8 cases showing severe acute vascular rejction and in all three chronic vascular rejection cases, PAI-1 mRNA was expressed by endothelial cells of arterioles and arteries, and interstitial inflammatory cells but was not detectable in any other groups. The expression of PAI-1 was frequently associated with areas of haemorrhage. Expression of PAI-1 might be expected to promote thrombosis and ischaemia, the catastrophic consequence of severe vascular rejection. In irreversible chronic rejection, this seems to be the principal mode of action. However, our observation that there is expression of PAI-1 in areas of haemorrhage in severe acute vascular rejection may suggest an additional potentially protective role, if it were produced as a response to haemorrhage.     

RNA interference targeting ORC1 gene suppresses the proliferation of vascular smooth muscle cells in rats

Background

The proliferation of vascular smooth muscle cells (VSMCs) plays an important role in the pathogenesis of vascular diseases such as atherosclerosis and postangioplasty restenosis. The largest subunit of the origin recognition complex (ORC), ORC1, plays a critical role during the initiation of DNA replication in eukaryotes. However, the involvement of ORC1 in the initiation of DNA replication in VSMCs has not been studied yet.

Objective

The aim of this study was to silence ORC1 gene selectively by using RNA interference and analyze the effects of ORC1 gene on the proliferation and apoptosis of rat VSMCs.

Methods

Freshly isolated rat VSMCs were transfected with siRNA targeting ORC1 gene capsulated in liposome. ORC1 protein expression was determined by Western blotting and ORC1 mRNA level by RT-PCR. DNA synthesis was analyzed by ³H thymidine (³H-TdR) incorporation and cell proliferative activity and cell cycle distribution by flow cytometry. Two apoptosis-related proteins, Bax and Bcl-2, were examined immunohistochemically.

Results

Down-regulation of ORC1 mRNA and protein expression was observed in rat VSMCs at 24 h after transfection with the three pairs of siRNA targeting ORC1 gene and this reduction persisted at least 7 days post-transfection. Down-regulation of ORC1 mRNA (60%) and protein (80%) expression was observed at 72 h post-transfection in the cells transfected with B-ORC1 siRNA. A significant decrease in ³H thymidine incorporation was observed in rat VSMCs with ORC1 gene silencing after serum challenge, but not in the non-silenced control. A significant increase in the proliferation index and a significant decrease in the percentage of cells at G₀/G₁ phase after serum challenge were observed in the non-silenced control, but not in ORC1 gene silenced cells. A significant increase in the ratio of Bcl-2/Bax was observed after serum challenge in the non-silenced control, but only a slight increase was found in the ORC1 gene silenced cells. ORC1 gene silencing disappeared 7 days after transfection. Continuous serum challenge stimulated VSMCs to synchronously reenter the cell cycle as evidenced by increases in [³H] thymidine incorporation, the proliferation index, and the ratio of Bcl-2/Bax, as non-silenced cells were induced to resume cell cycle progression by the addition of 15% fetal bovine serum to the culture medium.

Conclusion

ORC1 gene silencing causes rat VSMCs to enter a reversible G₀ quiescent, growth arrested state; thus, ORC1 gene may be an important new target for suppressing VSMCs proliferation.     

mmLDL对人脐静脉内皮细胞PAI-1表达的影响及其转录调控机制

目的:探讨弱氧化修饰低密度脂蛋白(mmLDL)对人脐静脉内皮细胞(HUVEC)PAI-1活性和mRNA表达的影响及其转录调控机制。方法:人脐静脉内皮细胞的培养和鉴定。用发色底物法测定PAI-1活性。Northern印迹分析法检测PAI-1mRNA的水平。采用基因重组技术构建含不同长度PAI-1 5'上游序列的荧光素酶报告基因质粒, 瞬时转染进入内皮细胞, 并检测荧光素酶的表达情况。 利用PCR和测序技术, 对构建质粒上AP-1元件进行定点突变。Western blot印迹杂交检测内皮细胞核内激活蛋白-1(AP-1)蛋白水平。结果:50 mg/L mmLDL诱导HUVECs PAI-1活性和mRNA表达量明显增高, 同时提高核内AP-1蛋白水平。mmLDL显著诱导构建质粒pGL3-PAI-1-1509/+90和pGL3-PAI-1-823/+90的荧光素酶活性, 但对质粒pGL3-PAI-1-553/+90和pGL3-PAI-1-47/+90诱导作用不明显。当PAI-1 5'上游序列的3个AP-1元件突变后, mmLDL的诱导作用明显降低。结论:(1)mmLDL增强血管内皮细胞 PAI-1活性与mRNA表达;(2)PAI-1活性提高与其mRNA表达增加呈正相关;(3) PAI－1 5'上游序列中3个AP-1元件在mmLDL对PAI－1诱导中具有重要调控作用。     

不饱和脂肪酸影响HepG-2细胞PAI-1表达的机制初探

目的：探讨不饱和脂肪酸对HepG-2细胞纤溶酶原激活物抑制剂-1(PAI-1)表达的影响及其机制。方法： 以发色底物法检测PAI-1活性，RT-PCR法检测PAI-1 mRNA水平；构建两个含不同片段缺失的PAI-1启动子序列控制表达的氯霉素转移乙酰酶（CAT）报告基因质粒，转染HepG-2细胞，ELISA法检测CAT表达量。结果： 油酸、亚油酸诱导下HepG-2细胞PAI-1mRNA表达及蛋白活性显著高于对照组；共转染过氧化体增殖物激活型受体表达质粒（PPARα-pSG5）PAI-1转录活性显著增加；转染NF-κB样蛋白结合序列缺失的重组质粒，亚油酸诱导下PAI-1转录活性显著增加，而转染VLDL/脂肪酸反应元件缺失的重组质粒则无显著变化。结论： 不饱和脂肪酸增强HepG-2细胞PAI-1 mRNA表达及活性；PPARα可能是其上调PAI-1表达所涉及的转录因子之一，且VLDL/脂肪酸反应元件在该调控中具有重要作用，但可能并不涉及NF-κB信号转导途径。     

罗格列酮减轻糖基化终产物诱导的大鼠血管平滑肌细胞钙化

目的观察糖基化终产物(advanced glycation end products,AGEs)对血管平滑肌细胞钙化的影响以及罗格列酮对这种影响的干预作用,探讨罗格列酮在血管钙化产生中的作用。方法在含10mmol.L-1β-甘油磷酸培养液中加入200mg·mL-1的AGE-BSA及不同浓度(10-7mol.L-1～10-5mol.L-1)的罗格列酮体外培养大鼠血管平滑肌细胞,采用磷酸苯二钠法检测碱性磷酸酶活性,甲-酚酞络合酮方法测定细胞层钙含量,RT-PCR检测骨桥蛋白mRNA表达,Western blotting检测骨桥蛋白(osteopontin,OPN)蛋白表达。结果与对照组相比,AGE-BSA组细胞层钙含量增加、碱性磷酸酶活性升高,细胞OPNmRNA表达升高,OPN蛋白表达增加(P<0.01);10-7mol.L-1～10-5mol.L-1罗格列酮可抑制AGE-BSA对细胞的上述影响(P<0.01)。结论AGEs可促进血管平滑肌细胞钙化,罗格列酮可减轻AGEs对细胞的上述影响。     

Angiotensin-(1-7)对血管平滑肌细胞钙化的影响及信号转导通路

目的： 在钙化大鼠主动脉血管平滑肌细胞上观察血管紧张素-(1-7)［Angiotensin-(1-7)］对钙化的影响及其信号通道。方法: 用β-磷酸甘油制备钙化的大鼠血管平滑肌细胞，再以血管紧张素-(1-7)、血管紧张素Ⅱ、血管紧张素Ⅱ +血管紧张素-(1-7)、选择性蛋白激酶A（PKA）或蛋白激酶C（PKC）抑制剂等干预，通过Von Kossa染色及检测钙含量、碱性磷酸酶活性、骨钙素浓度和Cbfa1 mRNA表达来探讨血管紧张素Ⅱ对钙化的影响及其信号通道。结果: 血管紧张素-(1-7)抑制钙化大鼠血管平滑肌细胞的钙含量、碱性磷酸酶活性（P>0.05）、骨钙素浓度和Cbfa1 mRNA表达（P<0.05），也抑制血管紧张素Ⅱ对血管平滑肌细胞的钙含量、碱性磷酸酶活性、骨钙素浓度和Cbfa1 mRNA表达的促进作用（P<0.05）；血管紧张素-(1-7)提高血管平滑肌细胞内cAMP浓度（P<0.05），PKA抑制剂可阻断血管紧张素-(1-7)对钙化血管平滑肌细胞的钙含量、碱性磷酸酶活性、骨钙素浓度和Cbfa1 mRNA表达的抑制作用（P<0.05）。结论: 血管紧张素-(1-7)可抑制β-磷酸甘油诱导的血管平滑肌细胞钙化，并拮抗血管紧张素Ⅱ促进的血管平滑肌细胞钙化；这些效应与cAMP-PKA-Cbfa1信号途径有关。     

Effects of black cohosh on estrogen biosynthesis in normal breast tissue in vitro

OBJECTIVES: To investigate the effect of black cohosh on the estrogen biosynthesis in the breast in vitro. METHODS: Steroid sulfatase (STS) activity was studied in normal breast tissue obtained from pre- and postmenopausal women undergoing reduction mammoplasty. STS protein expression was studied by immunohistochemistry and western blotting. Breast tissue was incubated in vitro without or with black cohosh (iCR) at concentrations ranging from 0.1mg/ml to 1 ng/ml. STS activity was evaluated by incubating homogenized breast tissue with [3H]-estrone sulfate, separating the formed products, estrone (E1) and estradiol (E2), by thin layer chromatography and measuring the amounts of E1 and E2 by scintillation counting. RESULTS: STS protein expression and enzymatic activity were detected in all specimens investigated. In all groups, significantly more E1 than E2 was produced. Local estrogen formation was decreased in premenopausal breast tissue by treatment with iCR at 0.1mg/ml (p相似文献   

溶血磷脂酸与血管平滑肌细胞表型转化

2003年人类基因组计划的完成开辟了人类遗传学、人类病理学等研究领域的新时代,然而近年来解析人类基因组意义过程中遇到一系列的难题使人们认识到DNA序列本身并不能解释所有关于遗传信息传递、人类疾病生物学基础等问题.随着研究的深入,人们逐渐认识到不仅蛋白编码序列,而且非编码序列、表观遗传机制等也是影响遗传信息传递、疾病发生等过程中的重要因素.     

STAT3介导了血小板源性生长因子BB诱导的大鼠血管平滑肌细胞Pim-1表达

 目的： 观察血小板源性生长因子BB(PDGF-BB)是否可以诱导大鼠血管平滑肌细胞（VSMCs）表达Pim-1及Pim-1对VSMCs增殖的影响,探讨STAT3信号分子在这一过程中的作用,为血管重建性疾病（VRD）的研究提供实验依据。方法： 不同浓度PDGF-BB作用不同时间刺激体外培养的VSMCs,用细胞计数法检测增殖;用real-time RT-PCR 检测Pim-1 mRNA表达水平;Western blotting 检测STAT3 的活性变化;用放线菌素D（actinomycin D）、AG490（JAK特异性抑制剂）及siRNA沉默Pim-1和STAT3进行干预。结果： PDGF-BB（20 μg/L）作用VSMCs 24 h,可以诱导细胞增殖,Pim-1沉默抑制了这一过程;正常未经处理的VSMCs Pim-1 mRNA表达量较低,不同浓度PDGF-BB（10 μg/L~50 μg/L ）作用VSMCs 1 h,Pim-1 mRNA表达明显增加,其中以20 μg/L最显著;用PDGF-BB（20 μg/L）作用VSMCs 不同时间（0.5 h~4 h）,可显著上调Pim-1 mRNA表达,以0.5 h最显著。用actinomycin D及AG490预处理后Pim-1 mRNA表达随之降低。PDGF-BB可激活VSMCs中磷酸化STAT3水平,AG490和转染STAT3-siRNA可抑制 STAT3的磷酸化以及相应的Pim-1 mRNA表达。结论： PDGF-BB可通过Pim-1调节VSMCs增殖;STAT3可能参与了PDGF-BB诱导的VSMCs Pim-1表达。