干扰Sestrin2表达对ox-LDL诱导的人脐静脉内皮细胞凋亡的影响

目的观察干扰Sestrin2表达对氧化型低密度脂蛋白(ox-LDL)诱导的人脐静脉内皮细胞(HUVEC)凋亡的影响。方法用ox-LDL处理HUVEC复制内皮细胞损伤模型。Western blot检测不同浓度ox-LDL处理HUVEC不同时间Sestrin2的蛋白表达水平及转染Sestrin2 siRNA后Sestrin2的蛋白表达水平;流式细胞术检测干扰Sestrin2表达后对ox-LDL诱导的HUVEC凋亡率的影响;Western blot检测干扰Sestrin2表达后对ox-LDL诱导的HUVEC中Caspase-3表达的影响及ox-LDL对HUVEC中JNK通路的影响。结果不同浓度(20、50和100 mg/L)的ox-LDL处理HUVEC 24 h均能明显上调Sestrin2表达;50 mg/L ox-LDL处理HUVEC不同时间(24 h和48 h)均能明显上调Sestrin2表达,24 h达高峰;转染Sestrin2 siRNA能明显抑制Sestrin2的表达;ox-LDL能明显诱导HUVEC凋亡,转染Sestrin2 siRNA能明显促进由ox-LDL诱导的HUVEC凋亡;ox-LDL能明显诱导p-JNK和p-c-Jun的表达,且SP600125能明显抑制由ox-LDL诱导的Sestrin2表达。结论 ox-LDL通过JNK/c-Jun信号通路诱导Sestrin2表达,干扰Sestrin2表达能明显促进ox-LDL诱导的HUVEC凋亡。     

Irisin通过激活AMPK促进C2C12细胞葡萄糖摄取

目的研究鸢尾素(irisin)对C2C12细胞葡萄糖摄取的影响及机制。方法将C2C12细胞分为:Control组,不同浓度的irisin组(分为0.1μg/ml,0.3μg/ml和1μg/ml组),高糖/高脂(HG/HF)处理组,HG/HF+不同浓度的irisin组(分为0.1μg/ml,0.3μg/ml和1μg/ml组),Control+Scramble siRNA组,HG/HF+AMPKα2 siRNA组,HG/HF+scramble siRNA组,HG/HF+scramble siRNA+irisin组,HG/HF+AMPKα2 siRNA+irisin组。采用荧光葡萄糖(2-NBDG)检测细胞的葡萄糖摄取;Western blot检测细胞葡萄糖转运体4(GLUT4)转位情况和AMPK磷酸化水平。结果 (1)与Control组相比,HG/HF组C2C12细胞葡萄糖摄取降低(P0.05),不管是否经过高糖处理,Irisin均增加C2C12细胞葡萄糖摄取(P0.05或P0.01)。与Control组相比,HG/HF降低鼠骨骼肌细胞膜GLUT4表达(P0.01),Irisin显著增加HG/HF孵育骨骼肌细胞细胞膜GLUT4表达(P0.01);(2)与Control组相比,HG/HF降低C2C12细胞细胞膜AMPK磷酸化水平(P0.05),在Control组及HG/HF组,Irisin显著增加骨骼肌细胞AMPK的磷酸化水平(P0.05);(3)与HG/HF+scramble siRNA+irisin组相比,HG/HF+AMPKα2 siRNA+irisin转染组细胞葡萄糖摄取及细胞表面GLUT4表达均显著减少(P0.05或P0.01)。结论 Irisin通过激活AMPK促进小鼠骨骼肌细胞葡萄糖摄取。     

Pin1对oxLDL诱导内皮细胞炎症反应的作用及机制研究

目的探讨肽基脯氨酰顺反异构酶1(Pin1)对氧化低密度脂蛋白(oxLDL)诱导内皮细胞炎症反应的影响。方法人脐静脉内皮细胞系予以不同浓度oxLDL干预12、24、48h;并预先转染Pin1 siRNA或给予STAT3抑制剂后,采用50μg/ml oxLDL干预内皮细胞15min或24h,检测内皮细胞中血管细胞黏附分子1(VCAM-1)、Pin1蛋白表达水平,STAT3活化水平和单核-内皮细胞黏附情况。结果与对照组相比,oxLDL 50μg/ml组和100μg/ml组细胞内VCAM-1、Pin1蛋白表达明显增加(P0.05); oxLDL50μg/ml干预12、24、48h后,细胞内VCAM-1、Pin1蛋白表达亦明显增加(P0.05)。与对照组相比,oxLDL50μg/ml干预15min后,胞内p-STAT3明显增加(P0.05);oxLDL50μg/ml干预24h后,内皮细胞上黏附的单核细胞数量明显增多(P0.05)。与oxLDL组相比,Pin1 siRNA干扰后显著降低了oxLDL诱导的内皮细胞p-STAT3水平、VCAM-1蛋白表达水平(P0.05),也明显减少了oxLDL诱导的单核细胞黏附水平(P0.05)。此外,STAT3抑制剂显著降低了oxLDL诱导的内皮细胞VCAM-1蛋白表达与单核细胞黏附水平(P0.05)。结论 Pin1通过上调STAT3信号通路活化水平促进oxLDL诱导的内皮细胞炎症反应。     

HIF-2α基因沉默对人肝癌细胞株HepG2增殖能力的影响

目的探讨缺氧诱导因子-2α(hypoxia-inducible factors-2α,HIF-2α)表达水平对人肝癌细胞株HepG2增殖能力的影响。方法用氯化钴(CoCl_2)诱导细胞模拟缺氧微环境,并根据CoCl_2浓度不同分为50μmol/L、100μmol/L、200μmol/L、400μmol/L组,选择CoCl_2浓度为200μmol/L模拟肿瘤内缺氧微环境。采用siRNA干扰沉默HepG2细胞中HIF-2α的表达,分别应用RT-PCR、Western blotting方法检测HIF-2αmRNA和蛋白的表达,MTT方法检测HepG2细胞增殖,绘制细胞生长曲线,流式细胞技术观察细胞周期的改变。结果建立细胞缺氧模型,作为低氧组;采用浓度为50 nmol/L的HIF-2αsiRNA转染缺氧环境下的HepG2细胞,作为低氧+HIF-2αsiRNA组,结果显示:低氧组、低氧+Control siRNA组的HIF-2αmRNA和蛋白表达显著高于常氧组和低氧+HIF-2αsiRNA组(P0.05);细胞生长曲线结果显示:低氧组、低氧+Control siRNA组的光密度值显著高于常氧组和低氧+HIF-2αsiRNA组(P0.05);细胞周期结果显示:低氧组、低氧+Control siRNA组HepG2细胞在DNA合成前期(G0/G1期)细胞比率显著低于常氧组和低氧+HIF-2αsiRNA组,合成期(S)及合成后期(G2/M)细胞比率显著高于常氧组和低氧+HIF-2αsiRNA组(P0.05)。结论缺氧微环境通过促进人肝癌细胞株HepG2细胞中HIF-2α的表达进一步促进HepG2细胞增殖,而针对HIF-2α的靶向siRNA能有效抑制肝癌HepG2细胞中HIF-2α的表达,从而抑制HepG2细胞的增殖。     

高尿酸血症诱导NLRP3炎症小体活化GSDMD对血管内皮细胞的影响

目的分析高尿酸血症是否通过诱导亮氨酸富集的核苷酸结合寡聚结构域3蛋白(NLRP3)炎症小体活化蛋白质-gasdermin D(GSDMD)导致内皮细胞焦亡,观察尿酸对血管内皮细胞的作用机制。方法选择ATCC细胞库的120份人脐静脉内皮细胞(HUVEC),制备HUVEC培养基、内皮细胞(ECM)培养基,培养HUVEC细胞,按随机数表法将培养成功的120份HUVEC细胞分为3组,分别为HUVEC+ECM+500μmol/L浓度尿酸,HUVEC+ECM+1000μmol/L浓度尿酸,HUVEC+ECM+1500μmol/L浓度尿酸。分别将各浓度尿酸置于5%CO2 95%空气细胞培养箱内分别培养24 h、48 h、72 h。计算不同浓度内皮细胞活力下降率;采用Western blot与免疫荧光测定细胞caspase-3、caspase-4、NLRP3及GSDMD表达。结果在处理12 h的实验模型内,仅有1500μmol/L浓度的尿酸能够明显降低HUVECS的细胞活力,细胞活力下降率为50.00%;在处理24 h的实验模型内,各组细胞活力下降率分别为25.00%、65.00%、80.00%,随着尿酸浓度增加,HUVECS细胞的细胞活力随之下降,各浓度比较(P0.05);随着尿酸浓度的升高,内皮细胞caspase-3、caspase-4、NLRP3及GSDMD表达逐渐上升(P0.05)。结论高尿酸血症可引起内皮细胞NLRP3炎症小体激活,诱发细胞炎症反应,通过介导细胞中GSDMD活性,从而引起内皮细胞焦亡的发生。     

沙格列汀对过氧化氢诱导损伤的血管内皮细胞二甲基精氨酸二甲胺水解酶/非对称性二甲基精氨酸/内源性一氧化氮合酶通路影响的研究

目的观察二肽基肽酶-4(DPP-4)抑制剂沙格列汀对过氧化氢(H2O2)诱导损伤的血管内皮细胞二甲基精氨酸二甲胺水解酶/非对称性二甲基精氨酸/内源性一氧化氮合酶(DDAH/ADMA/eNOS)通路的影响。方法以培养人脐静脉内皮细胞株(HUVEC)作为靶细胞,在内皮细胞培养基中加入100μmol/L的H2O2制备细胞损伤模型,以20μmol/L沙格列汀进行干预,观察24~72h,检测细胞上清一氧化氮(NO)含量、ADMA浓度,检测细胞中NOS活性、DDAH活性及DDAH蛋白表达量。结果H2O2作用HUVEC后,细胞上清中NO含量降低,而ADMA浓度增加(P0.05)。细胞中NOS活性、DDAH活性、DDAH-Ⅱ蛋白表达量降低(P0.05);而加入沙格列汀,细胞上清中NO含量升高,ADMA浓度降低(P0.05)。细胞中NOS活性、DDAH活性、DDAH-Ⅱ蛋白表达量升高(P0.05)。结论沙格列汀通过对DDAH/ADMA/eNOS通路的调节作用改善H2O2诱导的内皮细胞NO生成减少。     

半枝莲黄酮类化合物对体外肿瘤血管生成的影响

目的:探讨中药半枝莲黄酮类化合物A06对体外肿瘤血管生成的影响.方法:分别采用小管形成实验、Transwell小室、酶联免疫吸附测定法(ELISA)、硝酸还原酶法检测半枝莲黄酮类化合物A06对人脐静脉内皮细胞(HUVEC)小管形成、迁移及人宫颈癌Hela细胞血管内皮细胞生长因子(VEGF)、一氧化氮(NO)表达的影响.结果:与对照组相比,半枝莲黄酮类化合物A06(50和100 mg/L)都能有效抑制HUVEC细胞的迁移(37.7%±10.7%,13.7%±6.0%vs 68.0%±8.2%,均P<0.01),减少小管样结构形成(9.7%±4.5%,1.8%±1.2%vs 30.2%±5.4%,均P<0.05),以及降低24 h后Hela细胞VEGF和NO的表达(VEGF:135.6±14.6 ng/L,108.3±7.7 ng/L vs 231.1±6.4 ng/L,均P<0.01;NO:42.3±2.3μmol/L,25.9±5.2μmol/L vs 70.1±8.1μmol/L,均P<0.01).结论:半枝莲黄酮类化合物A06有抑制体外肿瘤血管生成的作用,可能与抑制肿瘤细胞血管生成相关因子的表达,抑制内皮细胞迁移、形成管样结构有关.     

莱菔硫烷对过氧化氢诱导人脐静脉血内皮细胞损伤的保护作用及机制研究

目的探讨莱菔硫烷(Sulforaphane,SFN)对过氧化氢(H_2O_2)诱导的人脐静脉血内皮细胞(HUVEC)氧化损伤的保护作用,并进一步研究莱菔硫烷在保护内皮细胞损伤中的机制。方法复苏由中国科学院上海细胞库提供的人脐静脉内皮细胞株,37℃、5%CO2条件下培养细胞至80%以上融合度时,分组进行干预。实验分组:(1)空白对照组(对照组):细胞正常培养2.5 h;(2)过氧化氢组(H_2O_2组):分别给予100、200、400、600、800μmol/L浓度培养细胞2 h,选择400μmol/L为其最佳干预浓度(后续各组如未特殊注明,默认H_2O_2浓度为400μmol/L);(3)莱菔硫烷+过氧化氢组(SFN+H_2O_2组):不同浓度SFN(2.5、5、10μmol/L)预孵育30 min后,加入400μmol/L的H_2O_2继续培养2 h。收集干预后的各组细胞,用MTT法比较细胞活性,流式细胞技术测定细胞内ROS水平,逆转录聚合酶链反应法(RT-PCR法)测定细胞内转录因子核因子E2(Nrf2)m RNA和血红素加氧酶-1(HO-1) mRNA表达情况。结果与对照组相比,H_2O_2组MTT法测定的细胞吸光光度值(A值)降低(P0.05),并浓度依赖性的使A值下降,SFN+H_2O_2组细胞吸光光度值高于H_2O_2组(P0.05)。H_2O_2组ROS水平高于对照组(P0.05),SFN+H_2O_2组ROS水平低于H_2O_2组(P0.05)。H_2O_2组Nrf2 mRNA、HO-1 mRNA表达水平低于对照组(P0.05),SFN+H_2O_2组Nrf2 mRNA、HO-1 mRNA表达水平高于H_2O_2组(P0.05)。结论莱菔硫烷对过氧化氢致HUVEC的氧化损伤有保护作用,其机制可能与莱菔硫烷在转录水平激活Nrf2/HO-1表达有关。     

利拉鲁肽通过调节Sestrin2改善棕榈酸诱导的L6骨骼肌细胞胰岛素抵抗

目的探讨应激诱导蛋白Sestrin2(Sesn2)在利拉鲁肽改善大鼠L6骨骼肌细胞胰岛素抵抗中的作用。方法采用棕榈酸诱导法建立大鼠L6骨骼肌细胞的胰岛素抵抗, 将实验细胞分为对照组(Con组)、棕榈酸0.6 mmol/L处理组(PA组)、棕榈酸0.6 mmol/L+利拉鲁肽10 nmol/L处理组(PA+Lir10组)、棕榈酸0.6 mmol/L+利拉鲁肽100 nmol/L处理组(PA+Lir100组)和棕榈酸0.6 mmol/L+利拉鲁肽1 000 nmol/L处理组(PA+Lir1000组)。细胞计数试剂盒8(CCK8)法检测各组细胞活性, Western印迹法检测各组葡萄糖转运蛋白4(GLUT4)、蛋白激酶B(Akt)、磷酸化的蛋白激酶B(p-Akt)和Sesn2蛋白的表达。小干扰RNA(siRNA)转染L6细胞, 抑制Sesn2的表达后, 用棕榈酸0.6 mmol/L和利拉鲁肽100 nmol/L干预细胞, 采用Western印迹法检测细胞中Sesn2、Akt、p-Akt、GLUT4蛋白表达水平。结果与Con组相比, PA组细胞存活率明显下降(P<0.05), p-Ak...     

雌激素对肿瘤坏死因子诱导的内皮细胞凋亡的影响

目的 观察17β-雌二醇(E2)及肿瘤坏死因子(TNFα)对培养的人脐静脉内皮细胞(HUVEC)凋亡的影响.方法 将HUVEC分为4组,空白对照组、TNFα(40 μg/L)组、TNFα(40 μg/L)加E2(1 μmol/L)和E2(10 μmol/L)组,各组细胞经维持液培养48 h使细胞生长同步,加入相应药物培养24 h后收获细胞.采用免疫组化方法测定bcl-2蛋白表达及流式细胞仪测定细胞凋亡发生百分率.结果 TNFα诱导的细胞凋亡发生率较正常对照组明显增多(P＜0.001),bcl-2蛋白表达少,DNA直方图上可见高大的凋亡峰,而雌激素干预后凋亡发生率明显降低(P＜0.001),bcl-2蛋白表达增加.结论 E2可增加bcl-2蛋白的表达,抑制TNFα诱导的人内皮细胞细胞凋亡的发生率,维持内皮细胞结构和功能的完整,可能为其抗动脉粥样硬化的机制之一.     

The immunoneuroendocrine role of melatonin

Abstract: A tight, physiological link between the pineal gland and the immune system is emerging from a series of experimental studies. This link might reflect the evolutionary connection between self-recognition and reproduction. Pinealectomy or other experimental methods which inhibit melatonin synthesis and secretion induce a state of immunodepression which is counteracted by melatonin. In general, melatonin seems to have an immunoenhancing effect that is particularly apparent in immunodepressive states. The negative effect of acute stress or immunosuppressive pharmacological treatments on various immune parameters are counteracted by melatonin. It seems important to note that one of the main targets of melatonin is the thymus, i.e., the central organ of the immune system. The clinical use of melatonin as an immunotherapeutic agent seems promising in primary and secondary immunodeficiencies as well as in cancer immunotherapy. The immunoenhancing action of melatonin seems to be mediated by T-helper cell-derived opioid peptides as well as by lymphokines and, perhaps, by pituitary hormones. Melatonin-induced-immuno-opioids (MHO) and lymphokines imply the presence of specific binding sites or melatonin receptors on cells of the immune system. On the other hand, lymphokines such as -γ-interferon and interleukin-2 as well as thymic hormones can modulate the synthesis of melatonin in the pineal gland. The pineal gland might thus be viewed as the crux of a sophisticated immunoneuroendocrine network which functions as an unconscious, diffuse sensory organ.     

Changes in connexin43, the gap junction protein of astrocytes, during development of the rat pineal gland

Abstract: The abundance of gap junctions between rat pineal astrocytes formed by connexin43 (Cx43) was studied during development. Levels and distribution of Cx43 were measured by immunoblotting and indirect immunofluorescence, respectively. The amount of Cx43 in cells located within the gland was low until about the 7th postnatal day and increased to adult values between the 14th and 21st days postpartum. Although astrocytes, recognized by their vimentin immunoreactivity, were scarce before birth, they were abundant by the 7th postnatal day suggesting that the low levels of Cx43 found at this age corresponded to a low expression of this protein. Localization of the immunoreactivity to Cx43 and vimentin showed a close correlation, indicating that mature or immature pineal astrocytes form gap junctions made of Cx43. Since Cx43 levels attained their adult values at about the time the innervation and the functional state of the gland reached maturity (2–3 weeks after birth), it is proposed that astrocyte gap junctions are involved in the function of the adult rat pineal gland.     

Conservative management of perforated duodenal diverticulum： A case report and review of the literature

Duodenal diverticula are a relatively common condition. They are asymptomatic, unless they become complicated, with perforation being the rarest but most severe complication. Surgical treatment is the most frequently performed approach. We report the case of a patient with a perforated duodenal diverticulum, which was diagnosed early and treated conservatively with antibiotics and percutaneous drainage of secondary retroperitoneal abscesses. We suggest this method could be an acceptable option for the management of similar cases, provided that the patient is in good general condition and without septic signs.     

Response of rat pineal melatonin to calcium, magnesium, and lithium is circadian stage dependent

Abstract: Herein we documented the response of pineal melatonin production to electrolytes known to be effective on pineal function in view of a possible circadian stage dependence. We studied the release of melatonin by perifused rat pineal glands at 2 different circadian stages corresponding to the middle of the light and dark periods, i.e., respectively, 7 and 19 HALO (Hours After Light Onset, L:D = 12:12). The initial efflux rates were, as expected, much higher in the perifusates of glands removed from rats sacrificed during the dark phase than of those removed during the light phase. After 3 hr of perifusion, melatonin release reached similar levels which were found constant up to the 8th hr of perifusion, whatever the circadian stage. Perifusion of the glands with physiological concentrations for the rat of calcium (5.2 mmol/1) and magnesium (1.34 mmol/1) resulted in a stimulatory effect on the pineal glands removed from rats sacrificed in the middle of the dark period (19 HALO), whereas no effects were observed on the pineal glands removed from rats sacrificed during the light (7 HALO). Lithium (0.28 and 0.55 mmol/1) was ineffective on melatonin release in pineal glands removed 7 and 19 HALO. Our results show differences in the initial efflux rates of melatonin and in the response of perifused pineal glands to calcium and magnesium according to the circadian stage.     

Presence of immunoreactive growth hormone and prolactin in the ovine pineal gland

Abstract: The use of antisera raised against bovine growth hormone (GH) and ovine prolactin (PRL) enabled the detection of related immunoreactive (ir) sequences of proteins in ovine pineal tissue. The isolation of PRL-like ir-material was accomplished using a 0.25 M ammonium sulphate (pH 5.5) extraction followed by ethanol precipitation, whereas the resulting 2.0 M ammonium sulphate (pH 7.0) precipitate contained a GH-like immunoreactivity. Gel chromatography of the GH-like immunoreactivity (Sephadex G-100) indicated the presence of several GH-like fragments ranging in the M_r range of 7,000 to 55,000. Analyses of the PRL-like ir-material found in pineal tissue on HPLC using a TSK 545-DEAE column led to the resolution into a single peak of immunoreactivity. A single peak of activity was also observed following chromatofocusing and hydrophobic interaction chromatography of the ir-peak from the TSK 545-DEAE column. The PRL-like ir-material inhibited the binding of [¹²⁵I]ovine PRL-S14 to anti-ovine PRL antibodies without showing an affinity for binding to anti-rat PRL or anti-bovine GH antibodies. Scatchard analysis of the binding of pineal PRL-like ir-material and pituitary ovine PRL-S14 to liver membranes from day-20 pregnant rats revealed similar affinity constants (K_a of 4.7 ± 0.2 × 10⁹ M^-1). In addition, the replication of Nb 2 Node rat lymphoma cells was stimulated by pineal PRL-like ir-material, an effect known to be specific for lactogenic hormones. The pineal PRL-like immunoreactivity appeared on sodium dodecyl sulfate polyacrylamide gels as a single major band of M_r 24,000. The functional status of PRL-and GH-like ir-material in the ovine pineal remains to be determined, but evidence is presented that the overall protein synthesis rate of the rat pineal responded to circulating concentrations of PRL.     

Microbiology of human immunodeficiency virus anorectal disease

PURPOSE: Individuals who are seropositive for the human immunodeficiency virus are at high risk for opportunistic infection and anorectal disorders. Little prospective information is available regarding anorectal pathogens in these patients. METHODS: One hundred sixty-three HIV-seropositive patients presented to the colorectal clinic between 1989 and 1992. Forty-seven (29 percent) patients were thought to have an infectious process and were prospectively studied using a standardized multiculture protocol. RESULTS: Mean age was 33 (range, 19–59) years. All were male; high-risk behavior accounted for 87 percent of HIV transmissions. Presenting complaints included anorectal pain (79 percent), pus per anum (28 percent), and blood per anum (26 percent). Examination revealed perianal tenderness (60 percent), condyloma (38 percent), perianal ulcers (38 percent), and anal fissures (34 percent). Sixty-six sets of cultures were performed; 28 patients had one set, 15 had two sets, and 4 had three sets. Thirty-two of these 47 patients (68 percent) had positive cultures including herpes (50 percent), cytomegalovirus (25 percent),Neisseria gonorrhoeae (16 percent), chlamydia (16 percent), acidfast bacilli (2 percent), and others (9 percent). Six of 32 patients with positive cultures had more than one organism cultured. Sixteen (50 percent) patients with positive cultures were treated medically, 8 (25 percent) were treated surgically and 8 (25 percent) were treated with both modalities. Sixty-one procedures were performed on 17 patients for condylomata. Eighteen patients had 20 procedures for abscesses, 50 percent of whom had positive cultures for other than common bowel flora; all improved. Fourteen patients underwent 33 procedures for perianal fistulas.Mycobacterium fortuitum was cultured from one patient who required 13 procedures for abscesses and fistulas. Forty-five (96 percent) patients were followed for an average of 12.5 months ±2.9 SEM (range, 1–94 months). Symptoms were improved or resolved in 22 of 32 (69 percent) patients with positive cultures and in 11 of 13 (84 percent) with negative cultures. CONCLUSIONS: Specific pathogens may often be identified in human immunodeficiency virus-seropositive patients with anorectal disorders if aggressively sought. Although patients without specific pathogens identified may be expected to improve with planned empiric treatment, positive identification allows more directed therapy.