2型糖尿病与血小板P2Y12受体

ADP-P2Y12受体途径在血小板激活、血栓形成中发挥重要作用。2型糖尿病血小板P2Y12受体途径异常可能是血小板功能亢进的重要原因之一。在正常人，胰岛素对P2Y12途径有抑制作用，而2型糖尿病由于血小板存在胰岛素抵抗，导致P2Y12途径的高敏感性。许多2型糖尿病对普通剂量的抗血小板药物反应性降低，但是加大P2Y12阻断剂的剂量后可能会克服这种耐药现象。     

经皮冠状动脉介入治疗术后支架内再狭窄的遗传学与表观遗传学研究进展

冠状动脉粥样硬化性心脏病(简称冠心病)是人类最主要的死亡原因之一,也是消耗巨大医疗资源的疾病.经皮冠状动脉介入治疗(percutaneous coronary intervention,PCI)是冠心病治疗的重大进展之一.然而,PCI治疗面临支架内再狭窄(in-stent restenosis,ISR)的重大挑战.尽管药物支架减少了ISR的发生,再狭窄发生率仍达5％～10％.支架后血管内皮细胞过度增生和平滑肌细胞移行是支架术后再狭窄的主要原因.再狭窄个体差异的确切机制还不完全明确,可能存在多种原因.除了高年龄、吸烟、糖尿病等可能增加再狭窄率外,越来越多的研究发现遗传因素和表观遗传学因素对疾病的进展有着重要影响.本文主要对冠心病患者PCI术后发生支架内再狭窄的遗传学和表观遗传学研究进行综述,以期初步确定冠心病患者PCI术后再狭窄发生的遗传危险因素.     

Molecular and cellular basis of restenosis after percutaneous coronary intervention: the intertwining roles of platelets, leukocytes, and the coagulation-fibrinolysis system

The major limitation of percutaneous coronary intervention (PCI) is restenosis. Restenosis is considered to be an overreaction of the natural healing process after traumatic balloon dilatation. An elaborate web of cellular and molecular responses, including the interaction of platelets, leukocytes, and the coagulation-fibrinolysis system, as well as the secretion of various growth factors and pro-inflammatory cytokines, contributes to neointimal hyperplasia and the development of restenosis. Moreover, platelet and neutrophil activation after stenting appears to be different from that after balloon angioplasty alone. Pharmacotherapy targeting the cell-to-cell interaction between platelets and neutrophils may potentially offer an effective treatment strategy against restenosis after PCI.     

Involvement of P2Y13 receptor in suppression of neuronal differentiation

We examined the receptor-mediated effects of extracellular ATP on neuronal differentiation of PC12 cells, Neuro2a cells and MEB5 cells by using a series of receptor antagonists. The P2Y13 receptor antagonist MRS2211 significantly accelerated neurite outgrowth in all cases. Treatment with nerve growth factor (NGF) alone activated ERK1/2 in PC12 cells, and the activation was further increased by MRS2211. These results suggest involvement of P2Y13 receptor in suppression of neuronal differentiation. Thus, P2Y13 receptor antagonists might be candidates for treatment of neurodegenerative diseases.     

天津地区汉族人群IL-10-592C/A基因多态性与冠状动脉支架术后再狭窄的关系

目的 探讨中国天津地区汉族人群白细胞介素-10(interleukin-10,IL-10)-592C/A基因多态性的功能性以及其对经皮冠状动脉介入治疗(percutaneous coronary intervention,PCI)术后再狭窄的发病,PCI术后血清IL-10水平的影响.方法 对437例接受PCI并进行冠状动脉造影随访的患者,按冠状动脉造影结果分为再狭窄组(166例)和非再狭窄组(271例),应用聚合酶链反应-限制性片段长度多态性方法检测IL-10-592位点基因型和等位基因频率的分布;用酶联免疫吸附试验法测定2组PCI术前及PCI术后24 h血清IL-10浓度,并比较两组间和各基因型间IL-10水平.结果 (1)IL-10-592C/A基因型和等位基因频率在再狭窄组和非再狭窄组之间差异无统计学意义(P均＞0.05);(2)PCI术后24 h血清IL-10水平再狭窄组显著低于非再狭窄组[(82.67±35.02)ng/Lvs.(95.08±32.26)ng/L,P＜0.05];(3)IL-10-592位点A等位基因携带者(AA+AC基因型)术后24 h血清IL-10水平明显低于非携带者(CC型)[(86.13±34.77)ng/L vs.(102.50±27.52)ng/L,P＜0.05];(4)再狭窄组A等位基因携带者术后24 h血清IL-10水平明显低于非携带者[(78.51±34.09)ng/L vs.(102.19±33.66)ng/L,P＜0.05];(5)再狭窄危险的多因素Logistie回归分析显示:急性冠状动脉综合征、术前狭窄程度、靶病变长度与冠状动脉内支架再狭窄呈正相关(()R值分别为5.90、1.86、2.83),术后24 h血清IL-10水平、参照血管直径、支架直径与冠状动脉内支架再狭窄呈负相关(OR值分别为0.99、0.70、0.46).结论 (1)IL-10基因-592 C/A多态性与中国天津地区汉族人群再狭窄发病无关;(2)IL-10是PCI术后早期的炎症细胞因子,术后24 h血清IL-10水平为再狭窄的独立预测因素,携带A等位基因的个体可能通过降低其表型血清IL-10水平而增加了冠状动脉内支架术后再狭窄的发病.

Abstract：

Objective To investigate the relationship of interleukin-10 gene (IL-10)polymorphism and the serum IL-10 level with restenosis after percutaneous coronary intervention (PCI) in Tianjin Chinese Han population and study the effect of IL-10 gene polymorphism on serum IL-10 level. Methods Four hundred and thirty-seven patients who successfully underwent PCI with a follow-up angiography were divided into a restenosis group (n= 166) and non-restenosis group (n= 271). The IL-10 gene promoter polymorphism at position -592 was determined by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. Meanwhile their serum IL-10 level before and 24 h after PCI was determined by enzyme-linked immunosorbent assay (ELISA). Results (1) There was no significant difference in frequencies of -592 genotypes and alleles between the two groups (P＞0. 05); (2) The 24 hpost-PCI IL-10 serum level of restenosis group was significantly lower than that of the non-restenosis group [(82. 67±35. 02) ng/L vs. (95.08±32.26) ng/L, P＜0.05]; (3) The serum level of the A allele carriers (AA+AC) was significant lower than that of the CC carriers [(86.13±34.77) ng/L vs. (102. 50±27.52)ng/L,P＜0.05]; (4) In the restenosis group, the 24 h post-PCI serum level of IL-10 in the A allele carriers was also significantly lower than that in those without the A allele [(78.51 ± 34.09) ng/L vs. (102.19 ±33.66) ng/L, P＜ 0. 05]; (5) Logistic regression analysis revealed positive correlations between acute coronary syndrome patients, pre-PCI degree of stenosis, length of target stenosis lesion and restenosis (OR=5.90, 1.86, 2.83 respectively); and there were negative correlations between 24 h post-PCI serum level of IL-10, the stent diameter, the diameter of reference vessel before stent implantation and restenosis (OR=0. 99, 0. 70, 0. 46 respectively). Conclusion (1) TheIL-10 gene -592 C/A polymorphism was not associated with restenosis in the Tianjin Chinese Han population; (2) IL-10 is an early post-PCI inflammatory cytokine, 24 h post-PCI serum IL-10 level was an independent predictive factor for restenosis,the IL-10 A allele carriers may have increased incidence of in-stent restenosis (ISR) by reducing the serum IL-10 levels.     

Angiotensin type 1 receptor A1166C gene polymorphism is associated with endothelial dysfunction and in-stent restenosis after percutaneous coronary intervention

Background and purpose: Percutaneous coronary intervention (PCI) has been commonly used in the treatment of ischemic cardiovascular diseases, but the postprocedural in-stent restenosis (ISR) associated with altered endothelial functions has limited the clinical application of it; preventive medication with aspirin and statins has underlying adverse effects despite lowered risk of ISR. The purpose of this study was to investigate the role of angiotensin type 1 receptor (AT1R) A1166C gene polymorphisms in the development of endothelial dysfunction and ISR after PCI. Methods: A total of 483 ST-segment elevation myocardial infarction (STEMI) patients undergoing PCI were prospectively genotyped using polymerase chain reaction (PCR) and restriction fragment length polymorphism assay. The demographic, clinical, laboratory and angiographic parameters were recorded peri-procedurally and the patients were followed within 3 years. The flow-mediated dilation (FMD) was used to reflect the short-term changes in endothelial functions among different genotypes. The significance of AT1R gene polymorphisms in the development of ISR was analyzed using univariable and multivariable models. Results: Amongst 483 patients, the distribution of the AT1R genotypes (AA, AC and CC) was associated with the levels of blood biomarkers of oxidative stress and deteriorated FMD after PCI (P<0.05). In univariable and multivariable logistic regression analysis, it was shown that AT1R CC genotype is strongly associated with the development of restenosis within 3 years after PCI (OR=3.736; P<0.001; calibrated OR=4.104; P<0.001). Conclusion: The CC AT1R genotype was associated with deteriorated endothelial functions in the target vessels of PCI and intermediate to long-term ISR. Our findings contribute to the foundation of genome-based prevention for high risk groups of cardiovascular diseases and pretreatment for the patients undergoing PCI.     

Role of P2X and P2Y receptors in rat myocardial contractility during ontogeny

Stable agonist of P2 receptors 2-methylthio-ATP and selective antagonists of P2X and P2Y receptors PPADS and reactive blue-2 were used for evaluation of the role of P2 receptors in positive contractile reaction of atrial and ventricular myocardium in rats. PPADS significantly moderated the effects of 2-methylthio-ATP in 14-, 21-, and 56-day-old rat pups, but potentiated them in 100-day-old rats. Under conditions of reactive blue-2 treatment, the positive effect of the agonist was preserved in the atria and ventricles in all age groups and was age-dependent. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 143, No. 6, pp. 637–640, June, 2007     

Signalling and pharmacological properties of the P2Y14 receptor

The P2Y₁₄ receptor is a relatively broadly expressed G protein-coupled receptor that is prominently associated with immune and inflammatory cells as well as with many epithelia. This receptor historically was thought to be activated selectively by UDP-glucose and other UDP-sugars. However, UDP is also a very potent agonist of this receptor, and may prove to be one of its most important cognate activators.     

ATP通过P2Y1受体引发脊髓背角星形胶质细胞[Ca2+]i升高和GFAP表达上调

目的 观察体外培养的背角星形胶质细胞P2Y1受体激活对其[Ca2 ]i的变化和GFAP表达的影响.方法 培养并纯化脊髓背角星形胶质细胞,采用免疫组织化学染色观察背角星形胶质细胞P2Y1受体及GFAP的表达,激光共聚焦技术观察星形胶质细胞[Ca2 ]i的变化.结果 体外培养的大鼠脊髓背角星形胶质细胞大多表达P2Y1受体;P2Y受体激动剂ATP、ADP、ADP-βs剂量依赖性促进星形胶质细胞[Ca2 ]i升高;10 μg/mL的ATP、ADP和ADP-βs显著增加胞内[Ca2 ]i,此作用可被特异性P2Y1受体拮抗剂MRS2179所阻断,并具量效关系.免疫组织化学染色结果显示,100 μg/mL的ATP、ADP和ADP-βs作用下,星形胶质细胞GFAP表达上升,此效应可被100 μg/mL的MRS2179所抑制.结论 体外培养的大鼠脊髓背角星形胶质细胞表达P2Y1受体;P2Y1受体介导了ATP、ADP及ADP-βs促进星形胶质细胞[Ca2]i升高和GFAP表达增强的过程.     

Promoting MΦ transepithelial migration by stimulating the epithelial cell P2Y2 receptor

In intestine, neutrophils are recruited in response to bacterial infiltration and their anti‐cellular activities contribute to inflammatory bowel diseases. In contrast, little is known regarding the recruitment of MΦ to the intestinal epithelium. Extracellular adenosine and uridine 5′‐triphosphate (ATP and UTP) can function as leukocyte chemoattractants. We investigated the effects of these nucleotides on the ability of intestinal epithelial cells (IEC) to promote MΦ transepithelial migration and adhesion. ATP and UTP promoted the migration of neutrophil‐like PLB‐985 cells and MΦ across a Caco‐2 monolayer. The MΦ‐like U‐937 cells adhered to nucleotide‐stimulated IEC monolayers. In mice with intestinal inflammation, there were infiltrating CD68⁺ MΦ in the colonic epithelium and CD68⁺ MΦ present at the apical surface of colonocytes. We determined that ATP and UTP activated the P2Y₂ receptor P (P2Y₂R) to increase ICAM‐1 expression, which mediated the adhesion of MΦ to the apical surface of IEC. Intriguingly, stimulation of IEC with nucleotides did not increase the adhesion of neutrophils. However, in the presence of adherent MΦ, there was adhesion of neutrophils, suggesting that MΦ may serve as anchors for neutrophil adhesion. These studies provide insight into the inflammatory mechanisms that contribute to inflammatory bowel diseases and identify potential therapeutic targets for the treatment of gastrointestinal disorders.     

Stimulation of Xenopus P2Y1 receptor activates CFTR in A6 cells

Nucleotide binding to purinergic P2Y receptors contributes to the regulation of a variety of physiological functions in renal epithelial cells. Here, we investigate the regulatory mechanism of the P2Y1 receptor agonist 2-methylthioadenosine diphosphate (2-MeSADP) on Cl^– transport in A6 cells, a commonly used model of the distal section of the Xenopus laevis nephron. Protein and mRNA expression analysis together with functional measurements demonstrated the basolateral location of the Xenopus P2Y1 receptor. 2-MeSADP increased intracellular [Ca²⁺] and cAMP and Cl^– efflux, responses that were all inhibited by the specific P2Y1 receptor antagonist MRS 2179. Cl^– efflux was also inhibited by the cystic fibrosis transmembrane conductance regulator (CFTR) blocker glibenclamide. Inhibition of either protein kinase A (PKA) or the binding between A-kinase-anchoring proteins (AKAPs) and the regulatory PKA RII subunit blocked the 2-MeSADP-induced activation of CFTR, suggesting that PKA mediates P2Y1 receptor regulation of CFTR through one or more AKAPs. Further, the truncation of the PDZ1 domain of the scaffolding protein Na⁺/H⁺ exchanger regulatory factor-2 (NHERF-2) inhibited 2-MeSADP-dependent stimulation of Cl^– efflux, suggesting the involvement of this scaffolding protein. Activation or inhibition of PKC had no effect per se on basal Cl^– efflux but potentiated or reduced the 2-MeSADP-dependent stimulation of Cl^– efflux, respectively. These data suggest that the X. laevis P2Y1 receptor in A6 cells can increase both cAMP/PKA and Ca²⁺/PKC intracellular levels and that the PKC pathway is involved in CFTR activation via potentiation of the PKA pathway.     

High nocturnal sleep fragmentation is associated with low T lymphocyte P2Y11 protein levels in narcolepsy type 1

Study ObjectivesNarcolepsy type 1 (NT1) is associated with hypocretin neuron loss. However, there are still unexplained phenotypic NT1 features. We investigated the associations between clinical and sleep phenotypic characteristics, the NT1-associated P2RY11 polymorphism rs2305795, and P2Y₁₁ protein levels in T lymphocytes in patients with NT1, their first-degree relatives and unrelated controls.MethodsThe P2RY11 SNP was genotyped in 100 patients (90/100 H1N1-(Pandemrix)-vaccinated), 119 related and 123 non-related controls. CD4 and CD8 T lymphocyte P2Y₁₁ protein levels were quantified using flow cytometry in 167 patients and relatives. Symptoms and sleep recording parameters were also collected.ResultsWe found an association between NT1 and the rs2305795 A allele (OR = 2, 95% CI (1.3, 3.0), p = 0.001). T lymphocyte P2Y₁₁ protein levels were significantly lower in patients and relatives homozygous for the rs2305795 risk A allele (CD4: p = 0.012; CD8: p = 0.007). The nocturnal sleep fragmentation index was significantly negatively correlated with patients’ P2Y₁₁ protein levels (CD4: p = 0.004; CD8: p = 0.006). Mean MSLT sleep latency, REM-sleep latency, and core clinical symptoms were not associated with P2Y₁₁ protein levels.ConclusionsWe confirmed that the P2RY11 polymorphism rs2305795 is associated with NT1 also in a mainly H1N1-(Pandemrix)-vaccinated cohort. We demonstrated that homozygosity for the A risk allele is associated with lower P2Y₁₁ protein levels. A high level of nocturnal sleep fragmentation was associated with low P2Y₁₁ levels in patients. This suggests that P2Y₁₁ has a previously unknown function in sleep-wake stabilization that affects the severity of NT1.     

P2X4受体在高糖介导PC12细胞损伤中的作用及其机制研究

目的 探讨P2X4受体在高糖介导PC12细胞损伤中的作用及其可能的作用机制.方法 通过高糖处理PC12细胞建立拟糖尿病细胞损伤模型,应用MTS比色法检测不同糖浓度对细胞的损伤作用并确定最佳建模浓度.对各组细胞进行不同干预后,通过ELISA方法检测各组细胞上清液中TNF-α和IL-1β的含量;通过qPCR和Western Blot方法检测细胞内P2X4 mRNA、受体蛋白和P65蛋白变化.结果 不同糖浓度处理细胞24h后,细胞存活率随糖浓度增大逐渐降低;通过高糖建模并经不同处理发现,与对照组相比,高糖组细胞存活率明显下降,高糖+5-BDBD组与之相比显著上升;通过qPCR和Western Blot检测发现,与对照组相比,高糖组细胞P2X4 mRNA、受体蛋白和NF-κB蛋白的表达均明显上升,高糖+5-BDBD组与之相比有所下降,且与对照组无明显差异.结论 P2X4受体参与高糖介导PC12细胞损伤,高糖能诱导PC12细胞NF-κB转录复合体形成,进而介导P2X4受体表达上调,使细胞过度激活并大量释放TNFα、IL-1β等炎性因子对细胞产生毒副作用.     

Expression of P2Y(6) receptors in the developing mouse skeletal muscle and after injury and repair

In this study, single and double-labeling immunofluorescence histochemistry, Western blot and real-time polymerase chain reaction were used to study the expression of P2Y(6) receptors in developing mouse skeletal muscle and during injury and repair. The results show that P2Y(6) receptor immunoreactive (ir) cells were first detected in the dermamyotome at embryonic (E) day 9. The number and immunostaining intensity of the P2Y(6) receptor-ir cells increased from E9 to E13, but decreased from E15 to postnatal day 60 in the developing skeletal muscle system. The expression levels of P2Y(6) receptor protein and mRNA increased rapidly from 1 to 5 days after skeletal muscle injury and then decreased almost to the control level from 7 to 10 days, at the beginning of regeneration. P2Y(6) receptor-immunoreactivity was mainly localized to the ends of single myoblasts and myotube processes in the developing and injury-repair skeletal muscle tissues. These data suggest that the P2Y(6) receptor may be involved in the development and regeneration of skeletal muscle, especially in the migration and extension of the myoblast and myotube in developing and regenerating skeletal muscle.     

Increase of intracellular Ca by P2X and P2Y receptor-subtypes in cultured cortical astroglia of the rat

Astrocytes express purinergic receptors that are involved in glial–neuronal cell communication. Experiments were conducted to characterize the expression of functional P2X/P2Y nucleotide receptors in glial cells of mixed cortical cell cultures of the rat. The vast majority of these cells was immunopositive for glial fibrillary acidic protein (GFAP) and was considered therefore astrocyte-like; for the sake of simplicity they were termed “astroglia” throughout. Astroglia expressed predominantly P2X_4,6,7 as well as P2Y_1,2 receptor-subtypes. Less intensive immunostaining was also found for P2X₅ and P2Y_4,6,13,14 receptors. Pressure application of ATP and a range of agonists selective for certain P2X or P2Y receptor-subtypes caused a concentration-dependent increase of intracellular Ca²⁺ ([Ca²⁺]_i). Of the agonists tested, only the P2X_1,3 receptor-selective α,β-methylene ATP was ineffective. Experiments with Ca²⁺-free solution and cyclopiazonic acid, an inhibitor of the endoplasmic Ca²⁺-ATPase, indicated that the [Ca²⁺]_i response to most nucleotides, except for ATP and 2′,3′-O-(benzoyl-4-benzoyl)-ATP, was due primarily to the release of Ca²⁺ from intracellular stores. A Gprotein–mediated release of Ca²⁺ is the typical signaling mechanism of various P2Y receptor-subtypes, whose presence was confirmed also by cross-desensitization experiments and by using selective antagonists. Thus, our results provide direct evidence that astroglia in mixed cortical cell cultures express functional P2Y (P2Y_1,2,6,14 and probably also P2Y₄) receptors. Several unidentified P2X receptors, including P2X₇, may also be present, although they appear to only moderately participate in the regulation of [Ca²⁺]_i. The rise of [Ca²⁺]_i is due in this case to the transmembrane flux of Ca²⁺ via the P2X receptor-channel. In conclusion, P2Y rather than P2X receptor-subtypes are involved in modulating [Ca²⁺]_i of cultured astroglia and thereby may play an important role in cell-to-cell signaling.     

P2Y2受体激动剂2-硫代-UTP及拮抗剂苏拉明对Hela细胞活性影响

目的 研究P2Y2受体激动剂2-硫代-UTP及其拮抗剂苏拉明对Hela细胞活性的影响,从而在细胞水平探究P2Y2受体在人子宫颈癌病理生理过程中的作用.方法 应用CCK-8法检测不同时间点各处理组Hela细胞的活性;ELISA法分别检测细胞上清液中TNF-α和IL-6的水平;qPCR和Western blot法分别检测细胞P2Y2mRNA和蛋白的表达变化.结果 2-硫代-UTP可明显提高Hela细胞的增殖能力.且经2-硫代-UTP处理后的细胞IL-6的水平明显增加,P2Y2mRNA和蛋白表达水平均有所提高;苏拉明可明显抑制Hela细胞增殖,经其处理后的细胞TNF-α水平有所提高,P2Y2mRNA和蛋白表达水平无明显变化.结论2-硫代-UTP可通过激活P2Y2受体的活化细胞,引起细胞过表达P2Y2受体,从而促进Hela细胞增殖.苏拉明可通过阻断P2Y2受体及其下游活动,抑制Hela细胞增殖.以上结果表明,P2Y2受体可能成为治疗子宫颈癌等疾病的新靶点,这将为探索子宫颈癌新型治疗手段等方面提供新思路.