松花粉抗成纤维细胞复制性衰老的机制

目的 研究松花粉对衰老成纤维单细胞面积、β-半乳糖苷酶染色(SA-β-gal)阳性率、p16INK4A和p21CIP-1表达以及细胞周期的影响.方法 以二倍体成纤维(2BS)细胞建立衰老细胞模型.生物体视学法分析单细胞面积变化;免疫组化法测SA-β-gal阳性率;流式细胞术分析细胞周期;RT-PCR法测定p16INK4A、p21CIP-1基因mRNA表达量.结果 56代细胞出现典型的衰老细胞形态改变,同时SA-β-gal染色阳性率与G1期细胞比例均增高.经松花粉处理后,衰老细胞的单细胞面积、SA-β-gal染色阳性率与G1期细胞比例均显著减少(P＜0.05);p16INK4A与p21CIP-1mRNA的表达量均较衰老模型组明显下调(P＜0.05). 结论松花粉具有改善细胞复制性衰老的作用,其分子机制可能与下调p16INK4A及p21CIP-1基因mRNA的表达从而改善衰老细胞G1期阻滞有关.     

偏硅酸钠对氧化低密度脂蛋白刺激iNOS表达的抑制作用

目的 观察偏硅酸钠对氧化低密度脂蛋白(ox-LDL)刺激的ECV-304细胞诱导型一氧化氮合酶(iNOS)表达的抑制作用及机制.方法 ECV-304细胞分成5组,对照组以及oxLDL组和加偏硅酸钠干预3个组(68.5、34.2、17.1 mg/L组),给予ox-LDL刺激后比较各组细胞培养液NO浓度、细胞内NOS、iNOS活性和蛋白以及mRNA,同时检测LOX-1和NOX4的mRNA.结果 与oxLDL组比较,加偏硅酸钠干预的3个组NO浓度上升、NOS活性提高(P ＜0.01);iNOS活性、mRNA水平、蛋白量均增加(P＜0.01).加偏硅酸钠的各组LOX-1和NOX4的mRNA也比oxLDL组显著下降.结论 偏硅酸钠能抑制ox-LDL刺激的ECV-304细胞iNOS基因表达,其作用机制与下调LOX-1和NOX4表达有关.     

抑制细胞间通讯功能可加速人成纤维细胞的衰老

目的研究间隙连接蛋白connexin43在人成纤维细胞衰老过程中的调节作用。方法设计并合成针对人connexin43的双链RNA(siRNA),抑制人二倍体成纤维细胞WI-38细胞的connexin43表达和细胞间通讯功能,观察对其衰老相关β-半乳糖苷酶(SA-β-gal)染色阳性率、细胞增殖能力、细胞周期调节蛋白P27、P21表达水平等衰老表型的影响。结果我们合成的siRNA-cx43可高效、特异的抑制connexin43 mRNA和蛋白质表达及细胞间通讯功能。转染WI-38细胞后,细胞增殖能力降低、细胞SA-β-gal染色阳性率(75.32±5.17)较对照组(32.48±3.94)和转染siRNA-con组(37.81±4.1 2)细胞升高(P<0.05),P27、P21表达增加,转染siRNA-cx43的WI-38细胞增殖能力显著降低,细胞变扁平、肥大,细胞SA-β-gal染色阳性率达100%,细胞提前出现衰老。结论间隙连接蛋白connexin43对WI-38细胞的衰老进程有重要的调节作用,其表达减少和细胞间通讯功能降低,可以促进WI-38细胞衰老进程。     

急性心肌梗死病人外周血单个核细胞中miR-146a表达及与短期预后的关系探讨

目的探讨急性心肌梗死病人外周血单个核细胞中miR-146a表达以及与短期预后的关系。方法选取2015年2月—2016年8月在河南焦作市人民医院心内科治疗的急性心肌梗死病人155例作为病例组,选取同期健康者60名作为对照组,收集研究对象的临床资料,利用实时荧光定量PCR技术检测单个核细胞中miR-146a表达,所有病人顺利出院,且均进行门诊或电话随访12个月,根据主要不良心血管事件(MACE)发生情况分为MACE组(46例)和非MACE组(109例)。结果病例组外周血单个核细胞中miR-146a相对表达量高于对照组(P 0.05);外周血单个核细胞中miR-146a相对表达量在诊断急性心肌梗死时,曲线下面积0.964(95%CI 0.942～0.986),当miR-146a相对表达量取界值1.65时,灵敏度91.6%,特异度91.7%。MACE组病人miR-146a表达量、心肌肌钙蛋白I(cTnI)和B型钠尿肽(BNP)高于非MACE组(P 0.05);多因素Logistic回归分析显示,cTnI、BNP和miR-146a表达量是影响急性心肌梗死病人预后的独立危险因素(P 0.05);外周血单个核细胞中miR-146a相对表达量在评估病人预后时,曲线下面积0.873(95%CI 0.800～0.946),当miR-146a相对表达量取界值1.83时,灵敏度76.1%,特异度90.8%。结论急性心肌梗死病人外周血单个核细胞中miR-146a呈高表达,且与病人不良预后有关,有望成为急性心肌梗死病人诊断及预后评估的潜在标志物。     

类风湿关节炎患者外周血单个核细胞和血浆中miR-155和miR-146a的表达

目的 通过检测miR- 155和miR-146a在类风湿关节炎(RA)患者外周血单个核细胞( PBMC)和血浆中的表达,探讨miR-155和miR-146a表达在RA发病中的作用.方法 RA患者34例,其中高活动组23例,低活动组11例;健康对照组15名.取外周血分离PBMC和血浆,提取和纯化miRNA,实时定量聚合酶链反应(PCR)检测miR-155和miR-146a的表达,以小分子RNA U6作为内对照,计算标准化后的2-ΔCt值表示miRNA的相对表达含量.采用SPSS 13.0软件包进行方差齐性分析和t检验.结果 RA患者PBMC和血浆miR-155的表达分别是对照组的1.74倍(0.08±0.08与0.05±0.03,t=-2.225,P＜0.05)和4.65倍(5.9±6.7与1.3±2.0,t=-3.677,P＜0.05);miR-146a的表达分别是对照组的1.68倍(1.3±1.2与0.8±0.6,t=-2.154,P＜0.05)和2.47倍(741±1001与300±295,t=-1.669,P＞0.05);RA患者中高活动组和低活动组PBMC中miR-155和miR-146a的表达差异无统计学意义;而血浆miR-155和miR-146a的表达高活动组明显高于低活动组,差异有统计学意义(P＜0.05).结论 miR-155和miR-146a在RA患者PBMC和血浆中异常高表达,且血浆miR-155和miR-146a的表达与RA活动度相关,提示血浆miR-155和miR-146a可以作为RA及RA活动度的预测指标.     

新诊断1型糖尿病患者外周血miR-146a与辅助性T细胞17的相关性研究

目的探讨新诊断TIDM患者外周血单核细胞(PBMCs)中miR-146a的表达水平与辅助性T细胞17 (Th17)、IL-17、IL-6的相关性。方法选取2013年1月至2016年1月于南京医科大学康达学院附属医院内分泌科住院治疗的新诊断TIDM患者68例(TIDM组),另选取同期健康体检者30名为正常对照(NC)组。应用实时荧光定量聚合酶反应测定PBMCs中miR-146a的表达,流式细胞术检测PBMCs中Th17细胞比例,ELISA法测定血浆IL-17、IL-6、TNF-α水平,分析Th17细胞比例与miR-146a、炎性因子等的相关性。结果与NC组比较,TIDM组PBMCs中miR-146a表达水平下降[(2.04±0.72)vs(-1.39±0.499),P0.05],Th17细胞比例、IL-17、IL-6及TNF-α升高[(1.88±0.09)%vs (4.23±0.86)%,(2.51±0.34) vs (4.77±0.65)ng/L,(3.87±1.78) vs (5.98±2.43)ng/L,(8.28±2.76) vs (13.93±3.53)pg/ml,P0.05]。Pearson相关分析显示,新诊断T1DM患者Th17细胞比例与IL-17、IL6、TNF-α呈正相关,与miR-146a表达呈负相关(P0.05)。多元逐步回归分析结果显示,IL-6、miR-146a是新诊断T1DM患者Th17细胞比例的影响因素(P0.05)。结论T1DM患者外周血miR-146α与Th17细胞比例存在相关性。     

miR-146a在慢性ITP患者外周血单个核细胞的表达及其临床意义

目的:探讨miR-146a在慢性免疫性血小板减少症(ITP)患者外周血单个核细胞(PBMC)的表达及其临床意义。方法:选取本院血液科收治的ITP患者35例,选取同期健康者38例作为对照组,比较2组研究对象miR-146a在PBMC和血清中表达量的差异;比较2组血小板计数(PLT)、细胞因子、T细胞亚群的差异;并对ITP患者PBMC中miR-146a的表达量与血清中miR-146a的表达量、PLT、细胞因子、T细胞亚群进行相关性分析。结果:ITP组PBMC中miR-146a的表达量和血清中miR-146a的表达量均显著低于对照组[(0.071±0.004)%∶(0.089±0.008)%,(1.549±0.412)%∶(4.485±0.396)%,均P0.01]。ITP组外周血中的PLT和Treg显著低于对照组,而IFN-γ、TNF-α、IL-2、Th22显著高于对照组(均P0.05)。ITP患者PBMC中miR-146a的表达量与血清中miR-146a的表达量、PLT、Treg呈正相关,与IFN-γ、TNF-α、IL-2呈负相关(均P0.05)。结论:ITP患者PBMC中miR-146a的表达量降低,IFN-γ、TNF-α、IL-2、Th22升高。ITP患者PBMC中miR-146a表达与血清中miR-146a的表达量、PLT、Treg呈正相关,与IFN-γ、TNF-α、IL-2呈负相关。     

MicroRNA-196a抑制序列对人胰腺癌PANC1细胞株HOXB8基因表达的影响

目的 观察microRNA-196a(miR-196a)抑制序列转染胰腺癌细胞株PANC1后对其HOXB8基因表达的影响.方法 将PANC1细胞分为对照组、miR-196a抑制序列组和siRNA对照组.采用脂质体法将miR-196a抑制序列及对照siRNA分别转染PANCI细胞.应用RT-PCR和蛋白质印迹法检测转染细胞miR-196a及其下游靶基因HOXB8 mRNA和蛋白的表达.结果 转染miR-196a抑制序列后,PANC1细胞miR-196a表达量较siRNA对照组显著减少(0.050±0.054比0.839±0.025,t=3.12,P＜0.05);HOXB8 mRNA表达量较siRNA对照组增高1.57倍(2.20 ±0.07比1.29±0.10,t=3.86,P＜0.05);HOXB8蛋白表达量也显著增强(0.90±0.03比0.40±0.10,t=3.11,P＜0.05).结论 miR-196a可以下调HOXB8基因的表达.     

微小RNA-146a通过靶向信号转导与转录激活子1表达调节颈动脉粥样硬化中血管内皮细胞的生物学特性

目的探讨微小RNA-146a(miR-146a)靶向调控信号转导与转录激活子1(STAT1)的表达,以及其在颈动脉粥样硬化(CAS)血管内皮细胞中的功能。方法将4月龄雄性新西兰白兔按随机数字表法分成4组,每组10只:假手术组(sham组)、模型组、miR-146a-mimics-NC组(对照组)和miR-146a-mimics组(实验组)。分别尾静脉注射Lipofectamine~(TM )RNAiMAX和miR-146a-mimics-NC、miR-146a-mimics的混合液到对照组和实验组兔体内,sham组和模型组注射等剂量的生理盐水;采用L-蛋氨酸饲料喂养配合颈总动脉球囊损伤法构建CAS模型,sham组只剥离颈内动脉,不进行球囊扩张和结扎。4周后处死动物留取标本,HE染色、qRT-PCR、Western blot检测各组兔CAS病理改变、miR-146a和STAT1蛋白的mRNA表达、Janus激酶2(JAK2)和磷酸化STAT1(p-STAT1)的表达水平。分别提取标本中颈动脉的血管内皮细胞进行培养,EdU标记、Transwell小室、小管形成、TUNEL染色检测各组细胞的增殖能力、迁移能力、小管生成能力和凋亡。荧光素酶报告基因分析miR-146a和STAT1的调控关系。结果与sham组相比,模型组兔颈动脉标本中miR-146a的表达水平明显降低,而STAT1的mRNA、JAK2和p-STAT1的表达水平明显升高,内皮细胞的增殖能力、迁移能力、小管生成能力降低,细胞凋亡率升高,CAS病理明显;与模型组和对照组相比,实验组兔颈动脉标本中miR-146a的表达水平明显升高,而STAT1的mRNA、JAK2和p-STAT1的表达水平明显下降,内皮细胞的增殖能力、迁移能力、小管生成能力增强,细胞凋亡率下降,CAS病理明显好转,差异均有统计学意义(均为P0.05)。荧光素酶报告基因分析证实miR-146a与STAT1靶向结合。结论 MiR-146a能靶向调控STAT1的表达,过表达miR-146a能够抑制STAT1的表达,发挥其保护颈动脉内皮细胞的功能。     

虫草提取物对高糖诱导的脐静脉内皮细胞损伤模型的保护作用

目的研究虫草提取物对人脐静脉内皮细胞损伤模型的影响,探讨其保护内皮功能的机制。方法原代培养人脐静脉内皮细胞,高糖(40mmol/L)孵育,建立细胞损伤模型。实验分组:空白组、高糖组、冬虫夏草组、蛹虫草组。MTT法检测细胞增殖;SA-β-半乳糖苷酶(SA-β-gal)染色法检测SA-β-gal染色阳性率;流式细胞术测定细胞周期及细胞内活性氧;实时荧光定量PCR检测衰老相关的p16、p21、沉默信息调节因子1(SIRT1)、Bcl-2mRNA表达水平。结果与空白组比较,高糖组细胞增殖明显下降,细胞内活性氧增加(P<0.05)。与高糖组比较,蛹虫草组及冬虫夏草组细胞增殖增加,S期细胞明显增多,SIRT1mRNA水平显著升高,细胞内活性氧和p21mRNA水平均显著降低(P<0.05)。结论虫草提取物对损伤内皮具有保护作用,其机制可能与促进SIRT1mRNA表达,减轻细胞氧化应激损伤,抑制p21mRNA表达,促进内皮细胞生长增殖有关。     

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.     

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.     

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.