Notch信号通路介导血小板源生长因子AA诱导的血管平滑肌细胞增殖和迁移

目的探讨Notch信号通路在血小板源生长因子AA(PDGF-AA)诱导血管平滑肌细胞(VSMC)增殖和迁移中的作用及机制。方法脱臼处死1~2月龄雄性SD大鼠,无菌取腹主动脉,剥弃外膜及去除内皮后,贴块法培养VSMC,α-SM-actin染色鉴定。实验分为四组:对照组、γ-secretase抑制剂组(DAPT组)、PDGF-AA组和PDGFAA+γ-secretase抑制剂组(PDGF-AA+DAPT组)。实时荧光定量PCR(Real-time q PCR)检测Notch信号分子mRNA表达;Cell Titer96 Aqueous One Solution试剂盒检测细胞增殖活性;采用细胞划痕实验检测VSMC的迁移能力。结果腹主动脉VSMC表达Notch信号通路Jagged1、Jagged2、Notch1~3等几种主要的配体及受体;PDGF-AA刺激24h和48 h后分别导致Jagged2和Jagged1 mRNA表达显著增强(P0.01,n=4);与0 h相比,PDGF-AA刺激72 h后,Notch1、Notch3 mRNA表达显著增高(P0.01,n=4),而刺激24 h后Notch2 mRNA表达显著降低。PDGF-AA显著促进HES1(P0.05,n=4)、HEY2(P0.05,n=4)和转录因子Rbp-jКmRNA(P0.05,n=4)表达,DAPT抑制PDGF-AA介导的HES1、HEY2 mRNA表达增强(P0.01,n=4),但是进一步促进PDGF-AA诱导的Rbp-jКmRNA表达(P0.05,n=4);PDGF-AA刺激促进VSMC增殖(P0.01,n=5)和减少划痕余留面积(P0.01,n=4),两种效应均可被DAPT显著阻抑(P0.01,n=4)。结论 PDGF-AA调节了VSMC Notch信号分子表达,PDGF-AA部分通过激活Notch信号通路调节VSMC的增殖和迁移。     

NO-dependent Smooth Muscle Vasodilatation is Reduced in NIDDM Patients with Peripheral Sensory Neuropathy

In order to determine the involvement of denervation in endothelium-independent, nitric oxide (NO)-dependent smooth muscle vasodilatation, we have measured vascular endothelial and smooth muscle function in three groups of age- and sex-matched patients: 8 patients with non-insulin-dependent (Type 2) diabetes mellitus (NIDDM) with neuropathy; 7 NIDDM patients without neuropathy; and 10 non-diabetic control subjects. Laser Doppler probes were used to measure blood flow in the dorsum of the left foot. Vascular endothelial response was assessed by measuring vasodilatory responses to iontophoretic application of acetylcholine to the dorsum of the foot. Vascular smooth muscle activity was assessed by the response to iontophoresis of sodium nitroprusside (SNP)—a NO donor and direct vasodilator. The vasodilator response to acetylcholine, expressed as the ratio of peak to basal blood flow, was significantly reduced in both diabetic groups when compared to non-diabetic controls (geometric mean ×/÷ anti-logged SD 9.81 ×/÷ 1.65 versus patients with neuropathy 3.50 ×/÷ 2.03, p < 0.005 and diabetic non-neuropathic subjects 3.49 ×/÷ 1.67, p < 0.005). The difference between the two groups of diabetic patients was not significant. In contrast, the vasodilatation to nitroprusside was significantly reduced only in the diabetic neuropathic patients, significantly lower than in either the non-neuropathic diabetic controls or the non-diabetic controls (2.1 ×/÷ 2.0 versus 6.42 ×/÷ 1.56 and 7.02 ×/÷ 2.05, p < 0.005). This indicates that neuropathy is important in abnormalities of endothelium-independent vasodilatation. © 1997 by John Wiley & Sons, Ltd.     

家兔主动脉平滑肌细胞条件培养基抑制血管平滑肌细胞增殖

为探讨血管平滑肌细胞自分泌和旁分泌对血管平滑肌细胞增殖的抑制作用 ,采用家兔主动脉贴块培养法培养血管平滑肌细胞 ,并制备平滑肌细胞条件培养基。采用孔径 10 0kDa和 10kDa的Millipore滤膜 ,将平滑肌细胞条件培养基分部。平滑肌细胞增殖测定采用宝灵曼试剂盒XTT法。结果提示 ,家兔血管平滑肌细胞条件培养基抑制血管平滑肌细胞的增殖 ,浓度 5 0 %的平滑肌细胞条件培养基抑制血管平滑肌细胞的增殖率高达 45 % ,且该生长抑制活性物质具有剂量依赖、加热 5 6℃ 30min稳定和分子质量小于 10kDa的特点。     

The PML/RAR alpha oncoprotein is a direct molecular target of retinoic acid in acute promyelocytic leukemia cells

Acute promyelocytic leukemia (APL) is characterized by the translocation, t(15;17) and the expression of a PML/RAR alpha fusion protein that is diagnostic of the disease. There is evidence that PML/RAR alpha protein acts as a dominant negative inhibitor of normal retinoid receptor function and myeloid differentiation. We now show that the PML/RAR alpha fusion product is directly downregulated in response to retinoic acid (tRA) treatment in the human APL cell line, NB4. tRA treatment induces loss of PML/RAR alpha at the protein level but not at the level of mRNA, as determined by Northern blots, by Western blots, and by ligand binding assays and in binding to RA- responsive DNA elements. We present evidence that this regulation is posttranslational. This evidence suggests that tRA induces synthesis of a protein that selectively degrades PML/RAR alpha. We further show that this loss of PML/ RAR-alpha is not limited to the unique APL cell line. NB4, because PML/RAR alpha protein is selectively downregulated by tRA when expressed in the transfected myeloid cell line U937. The loss of PML/RAR alpha may be directly linked to tRA-induced differentiation, because in a retinoid-resistant subclone of NB4, tRA does not decrease PML/RAR alpha protein expression. In NB4 cells, the specific downregulation of the fusion protein decreases the ratio of PML/RAR alpha to wild-type RAR alpha. Because the ratio of expression of PML/RAR alpha to wild-type RAR alpha and PML may be important in maintaining the dominant negative block of myelocytic differentiation, these data suggest a molecular mechanism for restoration by tRA normal myeloid differentiation in APL cells.     

The Interleukin-1 receptor antagonist is a direct target gene of PPARalpha in liver

BACKGROUND/AIMS: The Peroxisome Proliferator-Activated Receptor (PPAR) alpha belongs to the superfamily of Nuclear Receptors and plays an important role in numerous cellular processes, including lipid metabolism. It is known that PPARalpha also has an anti-inflammatory effect, which is mainly achieved by down-regulating pro-inflammatory genes. The objective of this study was to further characterize the role of PPARalpha in inflammatory gene regulation in liver. RESULTS: According to Affymetrix micro-array analysis, the expression of various inflammatory genes in liver was decreased by treatment of mice with the synthetic PPARalpha agonist Wy14643 in a PPARalpha-dependent manner. In contrast, expression of Interleukin-1 receptor antagonist (IL-1ra), which was acutely stimulated by LPS treatment, was induced by PPARalpha. Up-regulation of IL-1ra by LPS was lower in PPARalpha -/- mice compared to Wt mice. Transactivation and chromatin immunoprecipitation studies identified IL-1ra as a direct positive target gene of PPARalpha with a functional PPRE present in the promoter. Up-regulation of IL-1ra by PPARalpha was conserved in human HepG2 hepatoma cells and the human monocyte/macrophage THP-1 cell line. CONCLUSIONS: In addition to down-regulating expression of pro-inflammatory genes, PPARalpha suppresses the inflammatory response by direct up-regulation of genes with anti-inflammatory properties.     

血管平滑肌细胞凋亡机制的研究进展

血管平滑肌细胞凋亡与很多心血管疾病的发生、发展密切相关,目前已经成为心血管疾病防治研究的热点。现对血管平滑肌细胞凋亡机制的研究现状进行了较为深入的探讨,从其诱导因素以及基因调控机制等角度综述了近年来国内外在这方面的研究进展。     

The Collagenous Exocytoskeleton of Smooth Muscle Cells

Using specific antibodies in immunoelectron microscope studies with vascular tissues, type V collagen has been localized in basement membrane structures associated with discrete regions of the smooth muscle cell surface as well as membranous structures adjacent to endothelial cell membranes. These results confirm and extend earlier light microscope observations suggesting that type V collagen is largely associated with the pericellular environment in such tissues.     

川芎嗪对血管平滑肌细胞增殖及表达c-myc基因的影响

为观察川芎嗪对血管平滑肌细胞增殖的影响,在建立凝血酶诱导的体外培养的兔主动脉血管平滑肌细胞增殖模型后,应用免疫细胞化学方法观察川芎嗪对血管平滑肌细胞表达ｃ－ｍｙｃ基因蛋白的影响;并用流式细胞术观察了血管平滑肌细胞增殖周期的变化。结果发现,川芎嗪能够显著抑制凝血酶诱导的血管平滑肌细胞ｃ－ｍｙｃ基因蛋白表达增加,使血管平滑肌处于ＧＩ期的细胞数显著增多,Ｓ期和Ｇ２＋Ｍ期的细胞数显著减少。结果提示,川芎嗪对凝血酶诱导的血管平滑肌细胞增殖有显著抑制作用,其机制与抑制ｃ－ｍｙｃ基因表达有关。     

Ethanol Inhibits Contractility of Esophageal Smooth Muscle Strips

Acute ethanol (EtOH) in vivo decreases both the pressure of the lower esophageal sphincter (LES) and the amplitude of contractions of the smooth muscle of the lower esophageal body (LEB) in both man and cat. However, the mechanism of this inhibitory effect of EtOH is unclear. This inhibitory effect could be caused by a direct effect of EtOH on the esophagus or be secondary to known inhibitory effects of EtOH on the central nervous system. To this end, we evaluated the in vitro effect of EtOH on contractility of smooth muscle strips from both LES and LEB. Circular muscle strips from LES and LEB were isolated from cats. Changes in resting tension of LES strips and changes in stimulant-induced tension of LES or LEB strips were measured in the presence of up to five concentrations of EtOH (12.5–100 mM). Stimulants included electric field stimulation (EFS) and carbachol. EtOH at 75 mM significantly decreased resting LES tension. EtOH also decreased maximal contractile responses to carbachol in both LES and LEB and increased the EC₅₀ of carbachol for LES, but not LEB. EtOH also modulated EFS-induced esophageal contractility; EtOH potentiated EFS-induced "on-response relaxation" in LES and decreased EFS-induced "off-response contractions" in LEB. EtOH-induced inhibition of esophageal contractility seemed to be reversible. EtOH did not result in muscle fatigue. Thus, EtOH can directly inhibit contractility of the esophagus, and does so reversibly and at pharmacologically relevant concentrations.     

平滑肌细胞源性生长因子的部分纯化

本文用超滤和肝素亲和层析法对培养的兔主动脉平滑肌细胞的无血清条件培养基进行纯化。用３Ｈ－ＴｄＲ掺入细胞ＤＮＡ法检测层析所得各组分对ＮＩＨ３Ｔ３纤维母细胞的致有丝分裂活性。用ＮａＤｏｄＳＯ４－聚丙烯酰胺凝胶电泳及等电聚焦电泳检测其分子量及等电点。结果表明，该条件培养基经肝素亲和层析，被１．０～１．６ｍｏｌ·Ｌ＇ＮａＣｌ洗脱下来的蛋白质成分对３Ｔ３细胞有致有丝分裂活性，其分子量范围为２３．０～２６．９ｋＤａ，等电点约为４．６。这些生物化学特性不同于ＰＤＧＦ、ＩＧＦ及ＦＧＦ，提示这种致有丝分裂蛋白质可能是一种独立的生长因子。