ESCC组织SNHG17表达及转染si-SNHG17质粒的人食管鳞癌细胞株增殖、凋亡、放射敏感性观察

目的 观察食管鳞癌（ESCC）组织小核仁RNA宿主基因17(SNHG17)表达情况及转染si-SNHG17质粒的人ESCC细胞株ECA109增殖、凋亡、放射敏感性。方法 取ESCC肿瘤组织与癌旁组织标本各89例份;另取ECA109细胞,并随机分为对照组（Control组,空白培养基处理）、si NC组（转染si NC质粒）、si-SNHG17组（转染siSNHG17质粒）、si-SNHG17+inhibitor NC组（si-SNHG17与inhibitor NC共转染）、si-SNHG17+miR-375 inhibitor组（si-SNHG17与miR-375 inhibitor共转染）;采用RT-qPCR法检测ESCC组织、癌旁组织和各组细胞SNHG17、miR-375、USP14 mRNA,CCK-8法检测各组细胞增殖能力（OD值）,流式细胞术及Western blot法检测各组细胞凋亡能力[凋亡率及凋亡相关蛋白（Bax、Bcl-2、Caspase3、USP14蛋白）],克隆形成实验检测各组细胞放射敏感性。双荧光素酶报告基因实验分别验证SNHG17、miR-375及miR-37...     

微小RNA-101对胰腺癌PANC1细胞增殖、凋亡和侵袭的影响及其作用机制

目的探讨微小RNA-101(miR-101)对胰腺癌PANC1细胞增殖、凋亡和侵袭的影响及其可能的作用机制。方法取对数生长期PANC1细胞分为5组, 除对照组外, 分别为转染miRNA模拟体的阴性对照(mimic-NC)组、转染miRNA-101模拟体(miR-101 mimic)组、转染miRNA抑制剂的阴性对照(inhibitor-NC)组、转染miRNA-101抑制剂(miR-101 inhibitor)组。采用qRT-PCR检测miR-101相对表达水平, MTT法检测细胞增殖率, 流式细胞仪检测细胞凋亡率, Transwell实验检测细胞侵袭能力, qRT-PCR和蛋白质免疫印迹法检测IL-6、JAK2、p-JAK2、STAT3、p-STAT3 mRNA蛋白的相对表达水平, 双荧光素酶报告基因法检测miR-101与IL-6的靶向关系。结果转染48 h后, 对照组、mimic-NC组、miR-101 mimic组、inhibitor-NC组、miR-101 inhibitor组PANC1细胞miR101表达水平分别为1.98±0.12、2.01±0.18、6.73±0.23、2...     

miR-675调控低氧诱导肺动脉平滑肌细胞的增殖

目的探讨miR-675对低氧诱导大鼠肺动脉平滑肌细胞(PASMCs)增殖的调控作用及相关机制。方法低氧(1%O2)处理PASMCs 48 h,利用Real time-PCR检测0、6、12、24和48 h时各个时相点miR-675的表达水平,应用Western blot检测低氧处理PASMCs 48 h后靶基因REPS2蛋白的表达情况。先合成miR-675抑制剂(inhibitor)再将其和阴性对照(NC)转染PASMCs细胞,24 h后再进行低氧处理从0h至48 h,应用MTT法检测0、12、24、48 h时细胞的活力,并检测REPS2蛋白的表达状况。构建野生型和突变型REPS2 3'UTR插入p MIR-REPORTTMluciferase vector载体,并将其与pRL-TK质粒共转染PASMCs细胞,之后将miR-675 inhibitor和NC分别转染PASMCs细胞,利用双荧光素酶报告基因检测活性。在常氧下先分别转染miR-675 inhibitor或模拟物(minic)48 h,再检测PASMCs中REPS2的表达水平。结果随着低氧处理时间的延长,miR-675在PASMCs细胞中的表达水平逐渐增高(P0.05)。而与对照组比较,REPS2蛋白在低氧处理的PASMCs细胞中表达显著下调(P0.05)。miR-675 inhibitor组在24 h和48 h的细胞活力显著低于对照组和miR-NC组(P0.01)。荧光素酶报告基因结果说明REPS2是miR-675的靶基因。在常氧环境上调miR-675可显著下调REPS2蛋白表达,而在常氧和低氧环境下抑制miR-675的水平可明显升高REPS2蛋白表达。结论 miR-675通过下游靶基因REPS2调控低氧诱导的PASMCs的异常增殖,可能是低氧肺动脉高压、肺源性心脏病等疾病治疗的潜在靶点。     

心房颤动相关微小RNA1266调控钠通道蛋白

目的采用双荧光素酶报告基因分析微小RNA 1266(miR-1266)与心房颤动相关离子通道蛋白的靶向关系。方法利用TargetScan、miRanda和miRDB数据库预测出miR-1266的4种靶基因,分别为电压门控钠通道α亚基(SCN5A)、电压门控钾通道eag相关H家族2型(KCNH2)、电压门控钾通道E家族β1亚基(KCNE1)和内向整流钾通道J家族5型(KCNJ5),并针对靶基因构建3非编码区的重组荧光素酶报告质粒,4种靶基因(SCN5A、KCNH2、KCNE1和KCNJ5)分别分为miR-1266转染组、阴性对照组和空白对照组,miR-1266转染组和阴性对照组分别将SCN5A、KCNH2、KCNE1和KCNJ5的重组荧光素酶报告质粒与miR-1266及阴性对照质粒共转染于人胚肾HEK293细胞,48h后检测各组荧光素酶活性。结果 SCN5A转染中,与阴性对照组比较,miR-1266转染组相对荧光素酶活性显著降低(0.100±0.007 vs 0.209±0.011,P=0.002);而KCNH2、KCNE1和KCNJ5转染中,与空白对照组和阴性对照组比较,miR-1266转染组相对荧光素酶活性无明显变化(P=0.1269,0.0652,0.2326)。结论 SCN5A是miR-1266的直接靶基因,而KCNH2、KCNE1和KCNJ5并非miR-1266的直接靶基因,miR-1266可能通过抑制SCN5A表达参与房颤电重构,有可能成为未来干预治疗靶点。     

miR-125b靶向Smad4调控非创伤性股骨头坏死骨髓基质干细胞成骨分化与增殖的相关性

目的探讨miR-125靶向Smad4调控对非创伤性股骨头坏死骨髓基质干细胞成骨分化与增殖的影响。方法培养人骨髓基质干细胞系HMSC-bm,将miR-125b模拟物(mimics)、miR-125b抑制物(inhibitor)和对照(miR-NC)转染到细胞内,qRT-PCR和Western印迹检测过表达对Smad4表达的影响,构建野生型和突变型的Smad4的3'-UTR荧光素酶报告载体,分别命名为Wt-Smad4和Mut-Smad4,将构建好的质粒做三组共转染,即Wt-Smad4与miR-125b mimics、Mut-Smad4和miR-125b mimics以及miR-NC与miR-125b mimics,检测荧光素酶的活性;BMP-2诱导HMSC-bm分化,将miR-125b mimics和si-Smad4转染到分化的细胞内,以不转染的细胞作为参照,检测miR-125b靶向Smad4对细胞分化的影响;将miR-125b mimics、miR-125b inhibitor和miR-NC转染到对数生长期的HMSC-bm细胞系,胆囊收缩素(CCK)8检测过表达对细胞增殖的影响;将miR-125b mimics与pc DNA3.1-Smad4质粒共转染;miR-125b mimics;miR-NC组;miR-NC与pc DNA3.1-Smad4组转染到HMSC-bm细胞系,检测miR-125b靶向Smad4对细胞增殖和分化的影响。结果转染miR-125b mimics组Smad4的相对表达量显著低于miR-125b NC组,转染miR-125b inhibitor组Smad4的相对表达量显著高于miR-125b NC组(P<0.01),Western印迹的检测结果与其一致,荧光素酶结果显示,Wt-Smad4与miR-125b mimics组荧光素酶活性显著低于miR-NC与miR-125b mimics组(P<0.01),结果证实Smad4是miR-125b的靶基因;miR-125b mimics和si-Smad4组Runx2和Osterix mRNA表达量显著低于对照组(P<0.01);CCK8检测结果显示,miR-125b mimics组在24 h、48 h、72 h 3个时间点细胞的增殖率显著高于对照组(P<0.01),miR-125b inhibitor组在3个时间点的细胞增殖率显著低于对照组(P<0.01);miR-125b靶向Smad4对细胞增殖分化的影响试验结果显示,miR-125b mimics组细胞增殖显著高于miR-NC组(P<0.01),miR-125b mimics与pc DNA3.1-Smad4质粒共转染组细胞增殖显著高于miR-NC(P<0.05),miR-NC与pc DNA3.1-Smad4组细胞增殖显著低于miR-NC组(P<0.05),miR-125b mimics组和miR-125b mimics+pc DNA3.1-Smad4组细胞中Runx2和Osterix的mRNA相对表达量显著高于miR-NC组(P<0.01);miR-NC+pc DNA3.1-Smad4组细胞中Runx2和Osterix的mRNA相对表达量显著低于miR-NC组(P<0.01)。结论 Smad4是miR-125b的靶基因,上调miR-125b促进细胞增殖,下调miR-125b减弱细胞增殖,miR-125b靶向Smad4调控HMSC-bm细胞增殖和分化。     

MiR-32对非小细胞肺癌细胞增殖、迁移和侵袭的影响及机制

目的探讨miR-32靶向CCNE2调控非小细胞肺癌(NSCLC)细胞的增殖、迁移和侵袭的机制。方法实时荧光定量-聚合酶链反应(qRT-PCR)检测30例NSCLC患者癌组织及其匹配癌旁组织和支气管上皮细胞株16-HBE及NSCLC细胞株A549、H460和H1299中miR-32 mRNA表达水平;选择A549和H1299细胞为研究对象,将细胞随机分8组:NC组(转染miR-NC)、miR-32组(转染miR-32 mimics)、NC inhibitor组(转染miR-32-NC)、miR-32 inhibitor组(转染miR-32 inhibitor)、si-NC组(转染对照干扰RNA)、si-CCNE2组(转染CCNE2干扰RNA)、miR-32+vector(共转染miR-32 mimics和pcDNA3.1)、miR-32+CCNE2组(共转染miR-32 mimics和pcDNA3.1-CCNE2),转染按照Lipofectamine^TM2000试剂进行。qRT-PCR检测转染效果,qRT-PCR和Western印迹分别检测CCNE2 mRNA和蛋白...     

人乳腺癌上皮细胞miR-217对EZH2表达的调控作用

目的 验证乳腺癌上皮细胞中miR-217是否通过直接作用于homolog2 zeste基因增强子（EZH2）3＇UTR调控EZH2表达.方法 利用RT-PCR技术检测人正常乳腺上皮细胞系HBL-100及乳腺癌上皮细胞系MCF-7、MDA-MB-231细胞株中的miR-217,在低表达miR-217细胞株中转染miR-217模拟物（miR-217 minics）,在高表达miR-217细胞株中转染miR-217抑制剂（miR-217 inhibitor）;Western blot法检测各实验细胞中的EZH2蛋白.用含有miR-217野生型及突变型识别位点的EZH2 3＇UTR载体（UTREZH2-UTR-WT和EZH2-UTR-MUT）质粒分别转染人胚肾细胞293T,检测其相对荧光素酶活性.结果 miR-217在MDA-MB-231细胞中低表达,转染miR-217 minics后,其EZH2蛋白表达下降;miR-217在HBL-100中高表达,转染miR-217 inhibitor后,其EZH2蛋白表达增高.人胚肾细胞293T中EZH2-UTR-WT的相对荧光素酶活性低于EZH2-UTR-MUT,两者相比,P＜0.05.结论 在乳腺癌上皮细胞中,miR-217对EZH2的表达具有调控作用,且miR-217通过直接靶向EZH2 3＇UTR调控EZH2表达.     

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.     

心房颤动相关微小RNA4279靶向调控钙通道蛋白

目的采用双荧光素酶报告基因等手段,探讨心房颤动(房颤)相关微小RNA4279(miR-4279)与房颤相关离子通道蛋白的靶向调控关系。方法利用在线数据库Targetscan,miRanda,miRDB预测可能的靶基因后,分别将靶基因的重组荧光素酶报告质粒与miR-4279及阴性对照质粒共转染于人胚胎肾HEK293细胞,实验分为转染组,阴性组和空白组检测各组相对荧光素酶的活性。并将miR-4279模拟体导入大鼠胚胎心肌H9c2细胞,观察其对靶基因的表达调控。结果 CACNA1C基因转染中,与阴性组比较,转染组相对荧光素酶活性降低18%(0.300±0.012 vs 0.366±0.011,P<0.01),转染组CACNA1C基因mRNA表达水平下调31%(0.820±0.058 vs 1.193±0.060,P<0.01),miR-4279模拟体转入H9c2细胞可抑制CACNA1C基因的表达。结论电压门控L型钙通道α亚基CACNA1C基因可能是miR-4279的直接靶基因,miR-4279可能通过抑制CACNA1C表达参与房颤电重构,有可能成为未来房颤干预治疗的靶点。     

miR-185通过靶向BRCA1抑制三阴乳腺癌细胞的增殖与侵袭

目的探讨miR-185是否通过靶向人类乳腺癌易感基因(BRCA)1抑制三阴乳腺癌细胞的生长与侵袭及其抗肿瘤机制。方法采用Target Scan软件预测miR-185靶基因、将野生型或突变型E2F6的3'-UTR区域克隆到荧光素酶报告基因的下游(E2F6-wt或E2F6-mut)并分别与miR-185模拟物(miR-185 mimics)及其无关对照寡核苷酸序列(scramble)、抗miR-185寡核苷酸序列(miR-185-LNA)及其无关对照寡核苷酸序列(control)共转染,荧光素酶报告基因实验验证所预测的靶基因E2F6、qRT-PCR检测转染后靶基因的mRNA表达水平、Western印迹检测转染后靶基因的蛋白表达水平。MTS和Transwell验证不同转染后,三阴乳腺癌细胞MDA-MB-231的细胞生长增殖活性和侵袭能力。结果在共转染miR-185mimics和E2F6-wt、scramble和E2F6-wt两组中,miR-185组荧光素酶活性强度降低了约41.3%(P<0.05)。转染miR-185后,E2F6的蛋白和mRNA水平均显著下调,BRCA1的蛋白和mRNA水平均显著上调(P<0.05)。在细胞增殖试验中,转染miR-185后,三阴乳腺癌细胞MDA-MB-231的生长速度显著低于scramble组(P<0.05);与vector组比,BRCA1组可显著抑制三阴乳腺癌细胞MDA-MB-231的生长(P<0.05),且miR-185+BRCA1组比BRCA1组对细胞生长的抑制作用更加明显(P<0.05)。Transwell侵袭实验中,转染miR-185和BRCA1组在36 h后穿过基底膜的细胞数分别为(100±6)和(100±9),而对照组即转染scramble和转染vector组穿膜细胞数分别为(210±13)和(180±14),即miR-185和BRCA1能明显抑制三阴乳腺癌细胞MDA-MB-231的穿膜能力,差异有统计学意义(P<0.05)。结论 miR-185通过靶向调控BRCA1表达而抑制三阴乳腺癌细胞的生长与侵袭。     

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.