HCV HVR1模拟肽诱导小鼠骨髓树突状细胞免疫应答机制的初步研究

目的探讨与丙型肝炎病毒相关的7肽诱导小鼠骨髓树突状细胞（DC）在体外分泌细胞因子的能力。方法采用＂Bulk培养法＂从小鼠骨髓获得DC,经7肽负载;取经7肽免疫10天的小鼠脾细胞,分选获得CD4＋T和CD8＋T细胞;将脾细胞与DC共孵育24小时,收集上清,应用流式细胞术检测细胞因子。结果培养的细胞表面CD11c的表达率超过80%,具有典型的DC形态;实验组CD4T细胞上清中TNF-α降低I,L-5I、L-2和INF-γ升高;实验组CD8T细胞上清中IFN-γ、TNF-αI、L-10和IL-6较对照组有所升高,均具有统计学差异。结论 7肽能诱导以Th1及CD8T细胞为主的细胞免疫反应。     

HCV HVR1模拟肽诱导小鼠树突状细胞免疫应答及机制的初步研究

目的探讨HCV HVR1模拟肽(7肽)诱导小鼠树突状细胞免疫应答模式及其机制。方法用"Bu lk培养法"制备树突状细胞,经7肽免疫10天的小鼠,取脾细胞,分选CD8+T细胞与经过7肽负载并成熟树突状细胞共孵育24 h,收集上清,应用流式细胞技术方法检测上清细胞因子。结果此方法培养的细胞表面CD11 c的表达率超过80%,具有典型的DC形态;实验组中CD8+T上清中IFNγ-、TNF-α、IL-10及IL-6较对照组有所升高,均具有统计学意义。结论利用此法能在体外培养出大量的未成熟DC;7肽能诱导以CD8+T细胞为主的细胞免疫。     

胸腺基质淋巴细胞生成素对效应T细胞分化功能的影响

目的：探讨体外氧化型低密度脂蛋白（ ox-LDL）诱导人血管平滑肌细胞产生的胸腺基质淋巴细胞生成素（ TSLP）对效应T细胞分化功能的影响。方法分离并培养人血管平滑肌细胞、人树突状细胞（ DC）、初始CD4＋T细胞,随机分为4组,每组设5个样本。对照组、实验组人血管平滑肌细胞分别经PBS、ox-LDL处理后,取其上清液与DC、初始CD4＋T细胞共培养;TSLP中和抗体组、中和抗体对照组在实验组的基础上分别加入TSLP中和抗体、TSLP非特异性中和抗体与DC、初始CD4＋T细胞共培养。采用ELISA法检测各组细胞培养上清液中TSLP及Th17细胞因子IL-17、IL-22、TNF-α水平,流式细胞术检测Th17细胞构成比。结果与对照组比较,实验组培养上清液TSLP、IL-17、IL-22、TNF-α水平及Th17细胞构成比升高（ P均＜0．01）。与实验组、中和抗体对照组比较,TSLP中和抗体组TSLP、IL-17、IL-22、TNF-α水平及Th17细胞构成比降低（ P均＜0．01）。结论 Ox-LDL体外诱导人血管平滑肌细胞产生的TSLP可促进CD4＋T细胞向Th17细胞分化。     

体外培养的树突状细胞与细胞因子诱导的杀伤细胞相互作用的研究

目的 观察同源的树突状细胞（DC）与细胞因子诱导的杀伤（CIK）细胞于体外同一培养体系共同培养时的相互影响,为临床联合应用DC和CIK细胞进行肿瘤生物治疗提供依据.方法 用无血清培养基进行DC和CIK细胞的体外培养制作,共同培养1 w后,检测CIK细胞免疫表型、杀瘤活性的变化以及DC分泌IL-12的变化.结果 DC与CIK细胞的共培养会增加CIK细胞的CD3CD56双阳性细胞的比例和非特异性增强对K562细胞的杀伤活性;同时增强DC分泌IL-12的能力.结论 体外共培养DC与CIK细胞可相互增强抗肿瘤免疫活性.     

猪囊尾蚴排泄分泌抗原对仔猪DC成熟活化的影响北大核心CSCD

目的探讨猪囊尾蚴排泄分泌抗原(excretory secretory antigen,ESA)对仔猪体外培养的树突状细胞(dendritic cell,DC)成熟活化的影响。方法建立囊虫病猪动物模型,收集囊尾蚴体外培养并制备其ESA。实验分为control组、LPS阳性对照组、ESA及LPS+ESA实验组,体外用囊尾蚴ESA分别刺激仔猪DC。流式细胞术检测DC表面标志物MHC-II、CD80、CD86的表达量;ELISA检测细胞培养上清IL-6、IL-10、IL-12、TNF-α分泌水平。结果与control组相比,LPS及LPS+ESA组DC表面标志物表达量及细胞因子分泌水平均显著升高,差异具有统计学意义(P<0.05);ESA组MHC-II、CD80、CD86表达量及IL-6、TNF-α分泌水平均显著升高,差异具有统计学意义(P<0.05),IL-10及IL-12分泌水平差异无统计学意义(P>0.05)。与LPS组相比,ESA组CD86、MHC-II表达及IL-6、IL-10、IL-12、TNF-ɑ分泌均较低,差异具有统计学意义(P<0.05);LPS+ESA组CD80、CD86、MHC-II表达及IL-6、IL-10、IL-12、TNF-α分泌水平均显著升高,差异具有统计学意义(P<0.05),且细胞因子分泌水平均高于LPS组及ESA组,差异具有统计学意义(P<0.05)。结论猪囊尾蚴ESA可刺激DC成熟活化并分泌IL-6及TNF-α,本研究为进一步探讨DC参与的囊虫病免疫发病机制提供了实验基础。     

肺癌胸腔积液中树突状细胞的诱导培养及功能分析

目的 证实癌性胸腔积液中存在树突状细胞(dendritic cell,DC)的前体细胞,体外培养可获得DC。方法 双层Ficoll密度梯度离心法获得胸腔积液中的单个核细胞,在贴壁的单个核细胞中加入粒细胞/巨噬细胞集落刺激因子(GM-CSF)、白细胞介素4(IL-4)、肿瘤坏死因子α(TNF-α)。应用光镜、电镜观察细胞形态;用流式细胞仪对DC进行表型鉴定;四甲基偶氮唑蓝法检测DC的功能;酶联免疫吸附法(ELISA)检测致敏的DC刺激T淋巴细胞分泌干扰素γ(IFN-γ)的含量。结果GM-CSF和IL-4是DC体外增殖、分化的必要条件,TNF-α能促进DC的成熟及功能表达。培养的DC显示其特有的形态特征,并且高表达CD86(84.6±6.1)％、HLA-DR(81.1±13.O)％、CD40(42.0±21.7)％、CD1a(20.O±9.5)％,低表达CD14(4.8±3.5)％。DC具有强大的刺激同种异体淋巴细胞增殖的能力和明显的诱导T淋巴细胞分泌IFN-γ的功能。结论 恶性胸腔积液中可以获得具备正常形态、表型、功能的成熟DC。     

胆汁酸膜受体TGR5在小鼠骨髓来源的树突状细胞中的表达及其相关功能

目的 通过TGR5天然激动剂石胆酸(LCA)刺激小鼠树突状细胞(DC),探讨TGR5受体在DC成熟活化及吞噬功能方面的作用.方法 体外诱导小鼠骨髓细胞形成DC,LCA 30 μmol/L预处理2h后,用CFSE标记的胸腺凋亡细胞测试其吞噬功能;加入脂多糖(LPS)1 μg/mL诱导其成熟,6h后提取细胞总RNA,PCR法检测IL-12、TNF-α和IL-10等细胞因子;48 h后以流式细胞术检测细胞表面分子MHCII、CD80、CD86表达水平.组间比较采用t检验.结果 (1)LCA可以上调DC表达TGR5,并增强DC吞噬凋亡细胞功能(P＜0.05);(2)LCA可以降低LPS引起的DC表面MHCⅡ、CD80和CD86表达水平(均P＜0.05),提示LCA可抑制DC成熟;(3)LCA可抑制LPS诱导的DC促炎性细胞因子IL-12、IL-6、TNF-α mRNA表达,而IL-10表达水平升高(均P＜0.05).结论 TGR5受体激动剂可抑制DC成熟并增强DC吞噬功能,发挥免疫调节作用.     

高浓度胰岛素对人树突状细胞分化、成熟及免疫功能的影响

目的:研究高浓度胰岛素对树突状细胞(Dendritic Cell,DC)分化、成熟及免疫功能的影响。方法:采用连续贴壁法分离正常人外周血单核细胞,在含重组人粒-巨噬细胞集落刺激因子(GM-CSF,100ng/ml)和重组人白细胞介素-4(IL-4,100ng/ml)的完全培养基中培养。5天后收集细胞,重新铺板后继续在胰岛素浓度分别为0nmol/L、10nmol/L及100nmol/L的培养基中培养48小时,收集细胞和上清液,采用流式细胞术检测细胞表面CD40、CD80和CD83的表达;用ELISA法检测上清液中细胞因子IL-12、IL-10、TNF-α的浓度;用倒置显微镜动态观察DC形态变化。结果:胰岛素浓度为10nmol/L、100nmol/L组的DC表面标成CD40、CD80和CD83阳性表达率较含胰岛素0nmol/L的对照组升高,培养上清液中细胞因子IL-12、TNF-α的浓度也较对照组升高,而细胞因子IL-10的浓度则较对照组降低。结论:高浓度胰岛素促进了树突状细胞表型CD40、CD80和CD83的表达;促进了DC对细胞因子IL-12和TNF-α的分泌;对IL-10的分泌则起抑制作用。高浓度胰岛素通过促进树突状细胞免疫功能的成熟,可能是其参与动脉粥样硬化免疫炎症反应的发生、发展的机制之一。     

K562细胞可溶性抗原负载树突状细胞体外诱导CTL作用的研究

目的探讨经K562细胞裂解物冲击致敏的外周血单个核细胞衍生的树突状细胞（DC）的生物特性及体外诱导抗原特异性CTL应答的能力。方法采集健康人抗凝外周血分离单个核细胞，贴壁细胞用含rhGM—CSF、rhIL-4、TNF—α的RPM1640＋10％FBS培养基体外诱导培养产生DC，5天收获细胞并将细胞分组：A组：未负载抗原DC；B组：加入K562细胞裂解液脉冲DC。7天后用流式细胞仪检测成熟DC免疫表型，并将非贴壁细胞（淋巴细胞）作为效应细胞与各组DE共育，以产生细胞毒性T淋巴细胞（CTL）。12天用LDH释放试验测定对K562细胞的杀伤活性。并用ELISA方法测定细胞上清液中IL-12的含量。结果（1）经细胞因子联合体外诱导的各组DC较培养前在数量，形态及免疫表型上差异有统计学意义，CD86、CD83、CD40、CD1a表达增加，其中经K562细胞裂解液冲击的DC的CD83CD86表达率明显升高。（2）效应细胞与K562细胞混合培养时，负载K562细胞裂解液的DC刺激后的T细胞比单独DC刺激后的T细胞对K562细胞的杀伤作用更明显。（3）负载K562细胞裂解液的DC细胞培养上清液中产生IL-12含量较未负载抗原的DC明显增加。结论用GM—CSF、IL-4以及TNF—α诱导培养健康人外周血单个核细胞可以得到成熟的DC，且经K562细胞裂解液致敏可以进一步促进DC的成熟并体外诱导特异性杀伤靶细胞的CTL。     

基因1型戊型肝炎病毒 ORF3对人外周血来源的树突状细胞成熟和活化功能的影响

目的:探讨基因1型戊型肝炎病毒(HEV)开放读码框3(ORF3)对人外周血来源的树突状细胞(DC)成熟和活化功能的影响。方法:抽取健康志愿者的外周血分离单个核细胞,经体外培养、诱导为DC。然后分别用含有ORF3的慢病毒载体以及空载载体感染DC 48h,流式细胞仪检测ORF3组、空载组和空白组细胞表面CD_(80)、CD_(83)、CD_(86)的表达,ELISA法检测DC上清中IFN-β、IL-4、IL-10和IL-12的分泌。之后再加入TNF-α进一步刺激DC成熟,流式细胞仪检测各组细胞CD_(80)、CD_(83)、CD_(86)以及IFN-β、IL-4、IL-10、IL-12的表达。最后将DC与T淋巴细胞共培养,CCK8法检测T淋巴细胞增殖。结果:含有ORF3组的DC与空载组相比,加入TNF前后,其细胞表面CD_(80)、CD_(83)、CD_(86)以及细胞上清中IFN-β、IL-4、IL-10和IL-12的表达都明显较低(P0.05)。ORF3组与空载组相比其刺激T细胞增殖的能力也较弱(P0.05)。结论:基因1型HEV ORF3可以抑制人外周血来源DC的成熟,抑制其分泌IFN-β、IL-4、IL-10和IL-12等细胞因子,抑制DC对T淋巴细胞的增殖刺激作用,从而削弱机体对HEV的免疫反应,实现免疫逃逸,可能是导致戊型肝炎慢性化的原因之一。     

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.     

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.