ox-Lp(a)通过上调miR-125a-5p靶向抑制10,11-转位酶2增加单层血管内皮细胞通透性

目的探讨ox-Lp(a)损伤血管内皮细胞的表观遗传调控机制。方法生物信息学分析和筛选与10,11-转位酶2(TET2)mRNA 3’-UTR靶向结合的候选miRNA,荧光素酶报告基因系统验证其结合的靶向性;以0 mg/L、25 mg/L、50 mg/L、100 mg/L及200 mg/L的ox-Lp(a)与HUVEC-12内皮细胞孵育24 h,或用100 mg/L ox-Lp(a)与HUVEC-12内皮细胞孵育0 h、6 h、12 h、24 h及48 h,qRT-PCR和Western blot分别检测TET2 mRNA和蛋白的表达水平。qRT-PCR检测hsa-miR-125a-5p表达水平,以5hmc水平分析TET2活性的变化,Transwell检测ox-Lp(a)对单层血管内皮细胞通透性的影响。结果生物信息学分析和荧光素酶报告基因验证结果表明TET2为hsa-miR-125a-5p的靶基因,且hsa-miR-125a-5p与TET2 mRNA的3’-UTR结合的自由能值低(-30.1 kcal/mol)。ox-Lp(a)呈剂量和时间依赖性抑制TET2蛋白和mRNA的表达水平,以100 mg/L ox-Lp(a)作用HUVEC-12内皮细胞24 h的效果最佳;100 mg/L ox-Lp(a)作用HUVEC-12内皮细胞24 h后,TET2活性显著下降,且显著上调hsamiR-125a-5p的表达。anti-hsa-miR-125a-5p能逆转ox-Lp(a)对HUVEC-12内皮细胞TET2蛋白和mRNA表达水平的抑制作用和活性下降。ox-Lp(a)显著增加单层血管内皮细胞通透性,但可被anti-hsa-miR-125a-5p部分逆转。结论ox-Lp(a)通过上调hsa-miR-125a-5p并与TET2 mRNA 3’-UTR靶向性结合,抑制TET2蛋白和mRNA的表达水平及活性,从而增加单层血管内皮细胞通透性。     

母体表达基因3通过miR-125a-5p/TET2途径抑制HepG2细胞载脂蛋白(a)的表达

目的研究发现母体表达基因3(MEG3)对HepG2细胞载脂蛋白(a)[Apo(a)]表达的调控作用及其机制。方法用荧光素酶报告系统分析MEG3与miR-125a-5p的靶向性结合。采用实时定量PCR(qRT-PCR)检测高表达Apo(a)的HepG2细胞和低表达Apo(a)的SMMC7721细胞中MEG3的表达情况;向HepG2细胞转染MEG3,Western blot和qRT-PCR检测Apo(a)、TET2表达情况;采用小干扰RNA技术沉默TET2的表达。结果 (1)MEG3与hsa-miR-125a-5p能互补性结合,荧光素酶报告基因系统分析结果证实了MEG3与hsa-miR-125a-5p结合的存在。(2)miR芯片结果表明,在HepG2细胞中,hsa-miR-125a-5p表达水平升高,是对照组的近1.5倍,MEG3在HepG2细胞和SMMC7721细胞中均有表达,但前者MEG3的表达水平显著低于后者。(3)MEG3抑制Apo(a)表达。(4)MEG3下调miR-125a-5p的表达,上调TET2的表达; miR-125a-5p的mimics可逆转MEG3对Apo(a)的下调作用及TET2的表达,但可被miR-125a-5p的抑制剂逆转; TET2沉默可逆转MEG3对Apo(a)的下调作用。结论 MEG3通过miR-125a-5p/TET2途径下调HepG2细胞Apo(a)的表达。     

氨氯地平对氧化型低密度脂蛋白诱导的人脐静脉内皮细胞血小板源性生长因子B mRNA表达的影响

目的 观察氨氯地平对氧化型低密度脂蛋白诱导的HUVEC-12内皮细胞中血小板源性生长因子B mRNA表达的影响,从一个新的角度探讨氨氯地平的抗动脉粥样硬化作用机制.方法 预实验筛选出氧化型低密度脂蛋白对血小板源性生长因子B表达适宜的处理浓度(50 mg/L)与时间(24 h).实验分为5组:对照组、氧化型低密度脂蛋白组和三个不同剂量(0.1、1.0和10.0 μmol/L)氨氯地平组,用RT-PCR检测血小板源性生长因子B mRNA的表达.结果 以50 mg/L氧化型低密度脂蛋白刺激HUVEC-12内皮细胞24 h.随着预处理氨氯地平浓度增加,细胞血小板源性生长因子B mRNA的表达逐渐降低,并呈现浓度依赖性,当浓度为10.0 μmol/L时作用更明显.结论 氧化型低密度脂蛋白可影响HUVEC-12内皮细胞中血小板源性生长因子B mRNA的表达,氨氯地平可下调氧化型低密度脂蛋白诱导的HUVEC-12内皮细胞中血小板源性生长因子B mRNA的表达.     

氧化脂蛋白(a)通过抑制桥粒芯糖蛋白1和桥粒芯胶蛋白2的表达增加单层内皮细胞的通透性

目的探讨氧化脂蛋白(a)[oxLp(a)]对人脐静脉内皮细胞(HUVEC)上跨膜蛋白桥粒芯糖蛋白1(DSG1)以及桥粒芯胶蛋白(DSC2)的表达及单层内皮细胞通透性的影响。方法每次实验前6 h给HUVEC换上新鲜的无血清培养基,使细胞饥饿,6 h后再换上新鲜的培养基并加入不同浓度的oxLp(a)(0、25、50、100 mg/L)孵育HUVEC 24 h;100 mg/L oxLp(a)分别处理HU-VEC不同时间(0、6、12、24 h),RT-PCR和Western blot分别检测DSG1、DSC2 mRNA以及蛋白的表达。Transwell上种植单层HUVEC,100 mg/L oxLp(a)处理HUVEC 24 h后,于Transwell上室加入100μL1 g/L的FITC-dextran,分别在不同的时间点(0min,15 min,30 min,1 h,2 h,4 h)收集下室中的液体,用荧光分光光度计测量液体的荧光强度,确定测量通透性的最佳时间;用不同浓度的oxLp(a)(0、25、50、100 mg/L)分别或者与SOD(100 mg/L)共同孵育HUVEC 24 h后,在上室中加入FITC...     

氨氯地平对氧化型低密度脂蛋白诱导的人脐静脉内皮细胞CD40表达的影响

目的观察氨氯地平对氧化型低密度脂蛋白(ox-LDL)诱导的人脐静脉内皮细胞(HUVEC-12)中白细胞分化抗原CD40表达的影响,从一个新的角度探讨氨氯地平的抗动脉粥样硬化作用机制。方法实验分为6组:对照组、10.0μmol/L氨氯地平单独处理组、ox-LDL组、0.1、1.0及10.0μmol/L氨氯地平预先处理细胞1 h后加入50 mg/L ox-LDL共同孵育24 h组。用逆转录聚合酶链反应(RT-PCR)检测CD40 mRNA的表达,W estern b lot检测CD40蛋白的表达。结果随着氨氯地平浓度增加,细胞CD40的mRNA和蛋白表达逐渐降低,当浓度为10.0μmol/L时作用更明显。结论氨氯地平对ox-LDL诱导的HUVEC-12细胞CD40的表达具有抑制作用。     

氧化型低密度脂蛋白对人脐静脉内皮细胞TET2表达及自噬的影响

目的 观察氧化型低密度脂蛋白对人脐静脉内皮细胞自噬的影响及其调节机制.方法 氧化型低密度脂蛋白孵育人脐静脉内皮细胞24 h,RT-PCR检测TET2 mRNA表达,Western blot检测TET2以及自噬标记物Beclin 1、LC3的表达.TET2 siRNA转染人脐静脉内皮细胞,Western blot检测Beclin 1、LC3的表达.结果 人脐静脉内皮细胞经不同浓度氧化型低密度脂蛋白孵育24h后,浓度依赖性地下调TET2 mRNA以及蛋白的表达(P＜0.05),并且下调Beclin 1表达以及LC3Ⅱ/LC3 Ⅰ的比值.TET2 siRNA转染人脐静脉内皮细胞后,Beclin 1表达以及LC3Ⅱ/LC3 Ⅰ的比值明显降低.结论 氧化型低密度脂蛋白促进人脐静脉内皮细胞自噬,可能与下调TET2表达相关.     

氧化型低密度脂蛋白对人脐静脉内皮细胞Fractalkine表达的影响及机制分析

目的 观察氧化型低密度脂蛋白对人脐静脉内皮细胞Fractalkine表达的影响并探讨其可能作用机制.方法 采用胰蛋白酶消化法原代培养人脐静脉内皮细胞,取2～5代用于实验,随机分为:①对照组用RPMI1640培养基;②氧化型低密度脂蛋白不同浓度(5、25、50和75 mg/L)组;③氧化型低密度脂蛋白作用不同时间(6、12、24、48和72 h)组;④氧化型低密度脂蛋白+p38MAPK特异性阻断剂SB203580组;⑤p38MAPK特异性阻断剂SB203580组.用RT-PCR及EusA法检测Fractalkine mRNA及蛋白表达水平,用Western blot法检测p38MAPK磷酸化表达水平.结果 ①氧化型低密度脂蛋白在一定浓度范围及作用时间呈时间-剂量依赖方式诱导Fractalkine mRNA及蛋白表达增加,最佳作用时间为48 h,最佳作用浓度为50 mg/L;②与对照组比较,氧化型低密度脂蛋白诱导组人脐静脉内皮细胞p38MAPK磷酸化表达水平显著增加(P<0.05);使用SB203580干预后p38MAPK磷酸化水平明显下降,与氧化型低密度脂蛋白诱导组相比差异有统计学意义(P<0.05).③预先用p38MAPK特异性阻断剂SB203580(20 μmol/L)与内皮细胞共同孵育60 min后,再加入氧化型低密度脂蛋白作用48 h后,Fractalkine表达水平明显降低,与氧化型低密度脂蛋白组比较有统计学差异(P<0.05).结论 氧化型低密度脂蛋白可诱导内皮细胞Fractalkine表达增加,其作用机制可能是通过p38MAPK信号转导通路.     

miR-125a-5p抑制剂对大鼠心肌缺血再灌注损伤的影响

目的探讨miR-125a-5p抑制剂对大鼠心肌缺血再灌注(I/R)损伤的影响。方法将30只大鼠随机分为假手术组(sham组)、I/R损伤组(I/R组)和I/R+miR-125a-5p抑制组。采用生物信息学软件及荧光素酶报告体系预测和验证信号传导与活化转录因子3(STAT3)和miR-125a-5p之间靶向性。分析定量即时聚合酶连锁反应(qRT-PCR)鉴定体系中STAT3和miR-125a-5p表达;苏木素-伊红(HE)检测心肌组织细胞损伤情况;免疫组织化学法检测组织白介素-6(IL-6)表达情况;酶联免疫吸附测定(ELISA)法检测组织细胞氧化应激产物;免疫印迹试验(Western Blot)测定体系中凋亡相关分子的表达。结果经生物信息学和荧光素酶报告系统验证STAT3和miR-125a-5p具有较强的靶向性。I/R组miR-125a-5p表达量上调,STAT3表达受到抑制,细胞内氧化应激及炎症因子增加,细胞发生凋亡(P0.01);I/R+miR-125a-5p抑制组miR-125a-5p表达量下调,STAT3表达上升,细胞内氧化应激及炎症因子下调,凋亡现象有所改善(P0.01)。结论应用miR-125a-5p抑制剂可有效降低miR-125a-5p对STAT3的抑制效果,减少细胞凋亡和氧化应激反应,进而减缓大鼠心肌I/R损伤。     

1型糖尿病大鼠心肌纤维化microRNA表达变化筛选

目的应用链脲佐菌素(STZ)诱导1型糖尿病大鼠心肌纤维化模型,检测心肌组织miRNA表达谱,对差异miRNA调控的靶基因进行初步预测。方法 STZ诱导大鼠心肌纤维化,微距阵基因芯片技术筛选大鼠心肌组织差异表达的miRNA,对差异表达的miRNA调控的靶基因生物信息学分析。结果与对照组比较,模型组大鼠心肌组织中差异倍数改变大于2倍的miRNA共有25个,其中hsa-miR-29a-3p、hsa-miR-551a、hsa-miR-34a-5p、hsa-miR-885-5p等表达上调,hsa-miR-208a、hsa-miR-150-5p等表达下调。生物信息学分析,差异miRNA调控的靶基因多与细胞增殖、凋亡、糖代谢及血管生成等生物学功能相关。结论 STZ诱导的1型糖尿病大鼠心肌纤维化模型心肌中,miRNA表达谱有明显差异,其中有些miRNA可能靶向与1型糖尿病心肌纤维化发生发展密切相关。     

氨氯地平经核因子κB下调氧化型低密度脂蛋白诱导的人脐静脉内皮细胞血小板源生长因子B的表达

目的 观察氨氯地平对氧化型低密度脂蛋白(ox-LDL)诱导的人脐静脉内皮细胞(HUVEC-12)中血小板源生长因子B(PDGF-B)和核因子κB (NF-κB)表达的影响,通过氨氯地平对ox-LDL/NF-κB/PDGF-B的影响,进一步探讨氨氯地平相关的作用机制.方法 不同浓度氨氯地平(0、0.1、1.0、10.0μmol/L)预先处理细胞0.5h后加入50mg/L ox-LDL共同孵育24 h,RT-PCR和Western Blot检测细胞内PDGF-B的表达及NF-κB的蛋白表达；进一步通过NF-κB抑制剂吡咯烷二硫代氨基甲酸盐(PDTC)的加入,RT-PCR和Western blot观察其对PDGF-B及NF-κB表达的影响.结果 随着氨氯地平处理浓度的增加,HUVEC-12中PDGF-B的mRNA和蛋白表达,以及NF-κB的蛋白表达均明显下调,NF-κB抑制剂PDTC既可降低NF-κB的蛋白表达,也可下调PDGF-B的表达,作用与10.0 μmol/L氨氯地平组的结果相近.结论 氨氯地平可通过NF-κB下调ox-LDL诱导的HUVEC-12中PDGF-B的表达.     

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.     

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.     

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.     

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.