苦瓜蛋白通过FXR/miR-23b-3p/HNF4α途径抑制载脂蛋白(a)表达

目的研究苦瓜蛋白(MD28)对Hep G2细胞载脂蛋白(a)[Apo(a)]表达的影响,探索其作用机制。方法离心、超滤、透析、阳离子交换色谱、反相高效液相色谱等方法提取苦瓜蛋白;MTT法检测苦瓜蛋白处理Hep G2细胞后细胞存活率;实时荧光定量PCR(qRT-PCR)和Western blot分别检测Apo(a)、法尼酯X受体(FXR)、肝细胞核因子4α(HNF4α)的mRNA和蛋白表达水平;qRT-PCR检测hsa-miR-23b-3p的表达水平,采用小干扰RNA转染沉默FXR。结果与对照组相比,0.1、0.2、0.4、0.8及1.6 g/L苦瓜蛋白处理Hep G2细胞24 h,细胞存活率无统计学差异;与对照组相比,苦瓜蛋白呈剂量和时间依赖性抑制Apo(a)表达,以1.6 g/L苦瓜蛋白作用48 h的效果最为显著;苦瓜蛋白上调FXR和hsa-miR-23b-3p表达水平,下调HNF4α表达水平,沉默FXR则逆转苦瓜蛋白的上述作用,沉默hsa-miR-23b-3p能逆转苦瓜蛋白对HNF4α的下调作用,但对FXR的表达无影响。结论苦瓜蛋白通过FXR/miR-23b-3p/HNF4α途径抑制Hep G2细胞Apo(a)的表达,有望成为临床降低脂蛋白(a)的候选药物。     

miR-135b-5p靶向KLF4基因调控胃癌SGC-7901细胞生物学行为的研究

目的 研究miR-135b-5p通过靶向KLF4基因对人胃癌SGC-7901细胞生物学行为的影响。方法 通过RT-PCR检测人正常胃黏膜上皮细胞株GES-1及胃癌细胞株SGC-7901、MKN-45、BGC-823、AGS中miR-135b-5p表达水平,将miR-135b-5p inhibitor转染至胃癌SGC-7901细胞中,RT-PCR和Western blotting分别检测转染后细胞中miR-135b-5p和KLF4蛋白表达水平,MTT法、流式细胞术、Transwell实验分别检测转染对细胞增殖、凋亡、侵袭和迁移能力的影响。生物信息学软件预测及双荧光素酶报告基因实验验证KLF4是否为miR-135b-5p的靶基因。结果 胃癌细胞SGC-7901、MKN-45、BGC-823、AGS中miR-135b-5p表达水平显著高于人正常胃黏膜上皮细胞GES-1,且胃癌SGC-7901细胞中miR-135b-5p表达水平最高。在SGC-7901细胞中转染miR-135b-5p inhibitor 48 h后,miR-135b-5p的表达水平显著降低,KLF4 mRNA和蛋白表达水平明显升高。下调miR-135b-5p后SGC-7901细胞增殖能力下降,凋亡率升高,侵袭和迁移能力减弱。双荧光素酶报告基因实验结果显示,KLF4是miR-135b-5p靶基因。结论 miR-135b-5p能够通过靶向KLF4抑制胃癌SGC-7901细胞增殖、侵袭和迁移,诱导细胞凋亡。     

苦瓜蛋白通过下调miR-23b-3p促进三磷酸腺苷结合盒转运体A1表达

目的前期工作发现苦瓜蛋白(MD28)可上调THP-1源性泡沫细胞三磷酸腺苷结合盒转运体A1(ABCA1)的表达而减少胞内的脂质蓄积,但其机制不清楚。文章拟从转录后水平分析其作用机制。方法首先用miRNA芯片技术分析经MD28干预后,50 mg/L ox-LDL处理48 h的THP-1源性泡沫细胞miRNA谱中下调1.5倍的micro RNA(miRNA),并与Genecards调控ABCA1基因表达的miRNA比较,找到二者共同miRNA,然后以荧光素酶报告基因系统验证其靶基因关系。qRT-PCR检测ABCA1 m RNA和miR-23b-3p的表达水平,Western blot检测蛋白表达水平,油红O染色测定胞内脂质蓄积。结果 miR-23b-3p是miRNA芯片和Genecards的交集,靶基因验证实验证实ABCA1是miR-23b-3p的靶基因。MD28剂量(0 g/L、0.4 g/L、1.2 g/L、3.6 g/L、5 g/L)和时间(0 h、6 h、12h、24 h、48 h)依赖性地上调ABCA1 m RNA及蛋白的表达水平,以1.2 g/L作用12 h最为显著。ox-LDL上调miR-23b-3p表达且被MD28抑制,MD28能减少胞内脂质蓄积,miR-23b-3p的抑制剂可拮抗MD28对ABCA1表达和胞内脂质蓄积的作用。结论 MD28通过下调miR-23b-3p介导其促进THP-1源性泡沫细胞ABCA1表达、减少胞内脂质蓄积作用。     

miR-302 b-3 p靶向AKT1调节皮肤成纤维细胞衰老的作用机制

目的 探讨miR-302b-3p靶向丝/苏氨酸蛋白激酶(AKT)1调节皮肤成纤维细胞衰老的作用及其分子机制.方法 建立复制性细胞衰老模型后,采用real-time PCR法检测细胞miR-302b-3p的表达情况;采用生物信息学软件分析miR-302b-3p的靶基因;分别将miR-302b-3p模拟物(mimic)及抑制剂(inhibitor)转染皮肤成纤维细胞后,β-半乳糖苷酶染色分析细胞衰老情况,qRT-PCR法检测AKT1 mRNA表达水平,Western印迹法检测AKT1蛋白表达情况.结果 与对照组比较,复制性衰老皮肤成纤维细胞中miR-302b-3p显著升高.转染miR-302b-3p mimic后,细胞衰老阳性染色细胞数目显著增加(P<0.01).AKT1为miR-302b-3p的预测靶基因.当细胞上调miR-302b-3p表达时,靶基因AKT1 mRNA及蛋白水平显著降低(P<0.05).反之,当细胞下调miR-302b-3p表达时,靶基因AKT1 mRNA及蛋白水平显著升高(P<0.05).结论 miR-302b-3p可能通过靶向抑制AKT1表达调节皮肤成纤维细胞的衰老.     

miR-626下调HepG2细胞载脂蛋白(a)的表达

目的筛选高效调控LPA基因表达的miRNA。方法用在线预测工具预测作用于LPA基因3'UTR的miRNA,RT-PCR和Western blot检测转染预测所得miRNA mimic后HepG2细胞载脂蛋白(a)[Apo(a)]mRNA和蛋白表达水平。实验分为对照组、miR-626 mimic组、miR-626 mimic+miR-626 inhibitor组和miR-626 inhibitor组共4组。使用荧光素酶报告系统进行miR-626靶标验证实验。结果可作用于LPA基因3'UTR的miRNA有9个。mimic转染组与对照组相比,Apo(a)的mRNA表达水平差异不显著,但miR-626能显著下调Apo(a)蛋白的表达。使用miR-626 inhibitor后,miR-626对Apo(a)蛋白的下调作用减弱。转染miR-626后细胞裂解液的荧光强度显著下降。结论miR-626能显著下调Apo(a)蛋白表达水平,其通过与LPA mRNA 3'UTR序列结合,抑制LPA mRNA翻译实现的。     

miR-1284过表达对胃癌细胞中EIF4A1的影响

目的通过验证miR-1284可能的靶基因,从而探讨miR-1284影响胃癌发生发展可能的分子作用机制。方法采用生物信息学软件预测miR-1284可能的靶基因。以慢病毒介导miR-1284过表达转染胃癌MGC-803细胞为miR-1284组(LV-miR-1284组),转染空载体慢病毒载体的细胞为阴性对照组,未转染慢病毒载体的细胞为空白对照组。运用荧光定量PCR检测各组EIF4A1基因的表达,Western blot法检测各组EIF4A1蛋白的表达,通过双荧光素酶对EIF4A1进行靶基因验证。结果与阴性对照组和空白对照组比较,miR-1284组的EIF4A1基因和蛋白的表达量下降,双荧光素酶示miR-1284能与靶基因EIF4A1的3'UTR的结合。结论 miR-1284通过作用其靶基因EIF4A1发挥生物功能学作用。     

结肠癌SW480细胞中miR-142-3p靶基因的确定及意义

目的 确定结肠癌SW480细胞中miR-142-3p的靶基因,探讨miR-142-3p与其靶基因的相互作用.方法 通过miRNA数据库预测可能与miR-142-3p相互作用的靶基因,构建miR-142-3p及阴性对照序列;将miR-142-3p、对照序列、TCF7的3'非翻译区(3'UTR)以及突变的TCF7 3'UTR分别克隆到表达载体上,共转染SW480细胞,36 h后检测绿色荧光蛋白的表达水平;Trizol抽提RNA,RT-PCR检测TCF7 mRNA表达水平;Western blot检测TCF7蛋白表达水平.结果 生物信息学方法预测TCF7可能为miR-142-3p的靶基因,进一步双荧光素酶报告实验验证了miR-142-3p可以与TCF7的3'UTR结合,阻断TCF7蛋白翻译过程,从而下调TCF7的蛋白水平.结论 TCF7可能为miR-142-3p的靶基因,miR-142-3p通过转录后负调控靶基因TCF7蛋白的表达.     

miR-145-3p在肝癌中的表达及其对肝癌细胞生长的调控作用

背景miR-145-3p在头颈癌、肺癌和胃癌等肿瘤可发挥抑癌的作用,而其在肝癌中的表达和作用并不清楚.目的探讨miR-145-3p在肝癌中的表达及其对肝癌细胞的增殖与凋亡的调控作用.方法用RT-q PCR法检测肝癌组织、癌旁组织、肝细胞株(Hep3B、Huh-7、Hep G2和SMMC-7721)与正常肝细胞株L-02中miR-145-3p的表达水平;将miR-145-3pmimic或miR-145-3pinhibitor转入Hep3B细胞,用CCK-8法、流式细胞术、TUNEL法和Westernblot法分别检测细胞活力、细胞周期、细胞凋亡和4型黏蛋白(mucin4, MUC4)表达.用荧光素酶报告基因实验鉴定miR-145-3p的靶基因.将pc DNA-MUC4转入已转染miR-145-3p mimic的细胞,用CCK-8法和TUNEL法分别检测细胞活力与细胞凋亡.结果miR-145-3p在肝癌组织和细胞中呈低表达.过表达miR-145-3p能抑制细胞增值和G0/G1期到S期转换,促进凋亡和降低MUC4表达;而敲减miR-145-3p则作用相反. MUC4是miR-145-3p的靶基因.过表达MUC4能逆转miR-145-3p mimic对细胞存活的抑制作用.结论miR-145-3p在肝癌低表达,且其表达与T分期呈负相关.过表达miR-145-3p可抑制肝癌细胞存活与生长,而敲减miR-145-3p能促进肝癌细胞存活与生长.     

炎症条件下miR-222-3 p靶向CD4对肺泡巨噬细胞IL-6分泌水平的影响

目的研究miR-222-3p靶向CD4对肺泡巨噬细胞炎症因子分泌水平的影响,探讨其具体作用机制。方法 CCK8法检测0、1、2、4、8μg/mL的炎症诱导剂LAMPS对MH-S细胞的毒性影响。LAMPS处理MH-S细胞0、30、60、90、120 min后,RT-qPCR检测miR-222-3p和IL-6的表达水平的变化,Western blot检测CD4的蛋白表达水平。miRDB软件预测和双荧光素酶报告实验检测miR-222-3p对CD4基因的靶向作用。siR-222-3p转入MH-S细胞后,Western blot检测CD4的表达水平变化,RT-qPCR检测细胞内的IL-6的mRNA表达水平。结果 LAMPS对MH-S细胞的生长具有抑制作用,具有浓度依赖性。2μg/mL的LAMPS作用于MH-S细胞后,miR-222-3p和IL-6上调表达,CD4下调表达,具有时间依赖性。miRDB软件预测CD4为miR-222-3p的靶基因,双荧光素酶实验验证了靶向关系。siR-222-3p能够促进CD4的表达,降低IL-6的表达水平。结论 miR-222-3p可通过靶向CD4来调控肺泡巨噬细胞炎症因子的分泌水平。     

miR-146b对人单核细胞株THP-1细胞COX2表达的影响及可能机制

目的探讨微小RNA-146b(miR-146b)对人单核细胞株(THP-1)细胞p38分裂素原活化蛋白激酶(p38MAPK)表达的影响。方法血管紧张素Ⅱ(AngⅡ)刺激THP-1建立细胞模型,慢病毒感染pre-miR-146b-3p或anti-miR-146b-3p进入THP-1细胞中,检测p38MAPK与环氧化酶2(COX2)表达的变化。转入p38MAPK siRNA,检测p38MAPK与COX2表达的变化。结果 AngⅡ刺激THP-1细胞p38MAPK与COX2表达升高,pre-miR-146b-3p可以放大这种增高效应,当转染p38MAPK siRNA后,能逆转pre-miR-146b-3p升高COX2的趋势(P0.01)。结论在AngⅡ刺激的THP-1细胞中,miR-146b可能通过p38MAPK调控COX2表达水平,在心血管疾病的诊治中发挥重要作用。     

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.