大肠癌患者外周血来源树突状细胞对T细胞活化作用的研究

目的 比较无血清培养液AIM—V和RPMI1640＋胎牛血清（FBS）培养液培养大肠癌患者外周血来源树突状细胞（DCs）的形态和免疫表型，检测DCs对T细胞的活化作用。方法对2003年3月至2004年10月首都医科大学附属北京朝阳医院消化内科收治的10例大肠癌患者，采集其外周血分离单核细胞．分别使用2种细胞培养液进行体外培养。观察细胞形态，检测细胞表面CD1aCD80CD86CD83CD14及HLA—DR的表达。再用丝裂霉素作用后的DCs作为刺激细胞，按照刺激细胞：反应细胞（S：R）1：1，1：10，1：20．1：40，1：80的比例进行同种异体混合淋巴细胞反应（MLR），检测DCs刺激同种异体T细胞增殖的能力。结果 大肠癌患者外周血单核细胞经2种细胞培养液培养后都可以诱导出DCs。细胞外型均呈毛刺状。细胞均表达CD1a、CD80、CD86和人类白细胞抗原（HLA—DR），不表达CD14、CD83，为不成熟DCs的特点。两组DCs细胞表型表达差异无显著性（P〉0.05）。按S：R不同比例进行同种异体MRL所得刺激指数分别为2．7，4．1，3．1，2．5，1．6，表明树突状细胞（DCs）在不同比例下都刺激T细胞增殖。结论使用AIM—V无血清培养液和传统的含FBS细胞培养液均可成功诱导培养大肠癌患者外周血来源的DCs，但使用AIM—V可以降低含血清培养液应用人体后引起的过敏反应等风险，更适合DCs的临床使用，可以替代含血清培养液。MLR结果显示．DCs在各种S：R比例下均有刺激T细胞增殖的能力，但S：R比为1：10时最佳。     

慢性乙型肝炎患者外周血树突状细胞HBV DNA载量与其功能的关系

目的:探讨乙型肝炎患者外周血DCs HBV DNA载量与DCs功能的关系.方法:采集20例慢性乙型肝炎患者和10例健康人的抗凝外周静脉血,分离外周血单个核细胞(PBMCs),IL-4和GM-CSF的作用下培养使DCs增殖、成熟,以流式细胞技术分别检测DCs表面HLA-DR、CD-1α、CD80及CD86的表达:ELISA检测DCs培养上清液IL-12的水平,实时荧光定量PCR法检测DCs HBV DNA的载量,同种异体混合淋巴细胞反应检测DCs刺激同种异体T细胞增殖能力.结果:慢性乙型肝炎患者外周血DCs内亦可检测至HBv DNA,患者DCs表面HLA-DR、CD-1α、CD80及CD86的表达水平、DCs分泌IL-12水平及DCs刺激同种异体T细胞增殖能力均显著低于健康对照组,其中DCs HBV DNA阳性组较DCs HBV DNA阴性组下降更为明显(39.17±6.02 VS 60.11±7.92,46.03±5.52 VS 58.77±4.12,20.95±6.32 VS 35.24±7.41,25.16±6.05 VS 45.30±8.01,19.67±7.32VS 30.74±8.39,0.32±0.08 VS 0.59±0.11,均P＜0.01或0.05);HLA-DR、CD-1α、CD80及CD86的表达、IL-12水平及DCs刺激同种异体T细胞增殖能力与DCs HBV DNA载量呈显著负相关(r=0.713,-0.713,-0.623,-0.702,-0.525,-0.841,均P＜0.001).结论:慢性乙型肝炎患者外周血DCs可被HBV DNA感染,患者DCs HBV DNA载量与DCs功能有密切关系,病毒栽量低者DCs功能好,病毒栽量高者DCs功能差.     

清热解毒法与疏肝健脾法对慢性乙型肝炎患者外周血树突状细胞功能的影响

目的:比较清热解毒法与疏肝健脾法对慢性乙型肝炎(CHB)患者外周血树突状细胞(DCs)成熟与功能的影响。方法:采集20例CHB患者和10例健康人的抗凝外周静脉血,分离外周血单个核细胞(PBMCs),体外培养使DCs增殖、成熟,大鼠清热解毒和疏肝健脾药物血浆干预;检测DCs表面HLA-DR、CD-1α、CD80及CD86的表达,DCs培养上清液IL-12的水平及DCs刺激同种异体T细胞增殖能力;比较干预前后DCs功能的变化。结果:清热解毒药物血浆、疏肝健脾药物血浆干预后CD80表达率升高,且疏肝健脾组大鼠CD80表达率的提高要超过清热解毒组,而HLA-DR、CD-1α、CD86的表达水平在干预前后差异无显著性意义。两组含药血浆干预后DCs刺激同种异体淋巴细胞增殖的能力提高,但两组药物血浆之间差异无显著性意义。两组含药血浆干预前后DCs分泌IL-12水平无明显变化。结论:清热解毒法和疏肝健脾法都能促进CHB患者DCs功能的恢复,疏肝健脾法对CHB患者DCs功能的影响更为显著。     

补肾法与健脾法对乙肝病毒感染免疫耐受期患者外周血树突状细胞功能的影响

目的:比较补肾法和健脾法对慢性乙肝病毒(HBV)感染患者免疫耐受状态外周血树突状细胞(DCs)的影响。方法:收集20例慢性HBV感染免疫耐受期患者和10例健康人的抗凝外周静脉血,分离外周血单个核细胞(PBMCs),体外培养使DCs增殖、成熟,采用大鼠补肾和健脾药物血浆进行干预。检测DCs表面HLA-DR、CD-1α、CD80及CD86的表达,观察DCs培养上清液IL-12的水平及DCs刺激同种异体T细胞增殖能力。结果:健脾药物血浆及补肾药物血浆干预后CD80表达率升高,且补肾组的CD80表达率的提高优于健脾组(P0.05),而HLA-DR、CD-1α、CD86的表达水平在干预前后无显著性差异(P0.05)。两组含药血浆干预后DCs刺激同种异体淋巴细胞增殖的能力提高,但两组药物血浆之间差异无显著性意义(P0.05)。两组含药血浆干预前后DCs分泌IL-12水平无明显变化(P0.05)。结论:补肾法和健脾法都能促进慢性HBV感染免疫耐受期患者DCs功能的恢复,补肾法对慢性HBV感染免疫耐受期患者DCs功能的影响更为显著。     

无血清培养液培养大肠癌病人外周血来源树突状细胞

目的使用无血清培养液培养大肠癌病人外周血来源树突状细胞（DC）。方法采集大肠癌病人外周血分离出单核细袍。加入分重组人白细胞介素-4和重组人粒-巨噬细胞集落刺激因子的无血清培养液进行体外诱导培养，培养7d后获得DC。显微镜下观察DC形态，流式细胞仪检测细胞表型CD1a，CD14，CD80，CD83，CD86和HLA—DR的表达。结果无血清培养液培养的细胞呈毛刺状，流式细胞仪检测细胞表达CD1a、CD80、CD86和HLA-DR，不表达CD14、CD83，符合DC的典型特征。结论无血清培养液可以从大肠癌病人外周血诱导培养出DC。     

胰腺癌MiaPaCa-2细胞总RNA体外转染树突细胞的方法探讨

目的 探讨将胰腺癌MiaPaCa-2细胞总RNA转染树突细胞(DCs)最优化的方法.方法 以rhGM-CSF、rhIL-4和TNF-α联合诱导外周血单核细胞以获得DCs,观察DCs的形态变化,流式细胞术检测DCs表面标志CD40、HLA-DR、CD83和CD86表达,混合淋巴细胞反应(MLR)测定DCs刺激异体T细胞增殖的能力.采用脂质体转染、电穿孔及被动转染三种方法将MiaPaCa-2总RNA转染DCs,应用实时定量PCR法检测MUC1 mRNA表达,MTT法测定DCs存活率.结果 所获细胞具有典型的成熟DCs形态特征,CD40、HLA-DR、CD83和CD86阳性表达率分别为34.3%、50.2%、89.2%和73.6%,对同种异体T淋巴细胞具有极强的刺激增殖作用.电穿孔法DCs转染48 h后,DCs的MUC1 mRNA表达量为45.39±9.33,明显高于脂质体法的31.68±7.25和被动转染法的18.53±3.26;DCs存活率为(80.36±2.43)%,较被动转染法的(91.48±5.42)%略低,但高于脂质体法的(67.44±2.51)%,且基本稳定在80%左右.结论 采用电穿孔法将胰腺癌MiaPaCa-2细胞总RNA体外转染DCs的效率较高,且较安全.     

胃癌患者外周血树突状细胞的体外诱导和扩增

目的探讨从胃癌患者外周血中体外诱导、扩增树突状细胞（DCs）的方法。方法从胃癌患者外周血中分离单个核细胞（PBMNCs）,在GM-CSF、IL-4和TNF-α的诱导下培养扩增DCs。用相差显微镜观察所得DCs细胞形态学特征,用扫描电镜和透射电镜观察细胞结构。用混合淋巴细胞反应法检测所得DCs刺激同种异体T细胞增殖的能力。结果经过GM-CSF、IL-4和TNF-α诱导培养12 d后,所得细胞具有DCs的典型形态学特征和结构,且具有极强的激发刺激同种异体T细胞增殖的能力。结论用GM-CSF、IL-4和TNF-α可以在体外诱导、扩增胃癌患者外周血中的DCs。     

HBV抗原基因修饰树突状细胞疫苗体外抗HBV的作用

目的：探讨腺病毒载体介导HBV抗原基因修饰的树突状细胞(DCs)诱导抗HBV特异性CTL反应方法：制备携带HBsAg、HBeAg和HBcAg基因的3种重组腺病毒Ad-HBs,Ad-HBe,Ad- HBc,分别转染自脐带血体外诱导培养的DCs,观察腺病毒转染DCs效率和DCs中HBV抗原的表达;混合淋巴细胞反应(MLR)测定HBV抗原基因修饰DCs刺激同种异体T淋巴细胞增殖能力;乳酸脱氢酶释放法检测特异性CTL细胞对HepG_222．1．5靶细胞的杀伤能力．结果：腺病毒载体能够高效介导HBV三个抗原基因在DCs中表达,90%以上DCs表达示踪基因EGFP,且DCs细胞形态完整：感染后72 h HBsAg和HBeAg含量分别为0．919和0．328(吸光度A值)．MLR实验显示,HBV抗原基因修饰DCs仍然具有刺激同种异体T细胞的增殖能力,Ad-HBs转染DCs组、Ad-HBe转染DCs组、Ad-HBc转染DCs组和未转染DCs组之间刺激T细胞的增殖水平无明显差异(F=1．194,P=0．389);在E：T比例为2：1,10：1和25：1时,Ad-HBs转染DC组、Ad-HBe转染DCs组和Ad-HBc转染DCs组对HepG_222．1．5细胞的杀伤率均明显高于未转染DCs组(P＜0．001);以Ad- HBc转染DC组对HepG_222．1．5细胞杀伤率最高．结论：HBV抗原基因修饰DCs疫苗具有刺激同种异体T细胞增殖能力,同时能增强抗HBV特异性CTL反应的能力,可能发展为一种新型抗病毒疫苗．     

系统性红斑狼疮患者树突状细胞CD200R下调意义

目的探讨系统性红斑狼疮(systemic lupus erythematosus,SLE)患者树突状细胞(dendritic cell,DC)表面CD200R表达及脂多糖(lipopolysaccharide,LPS)刺激单核细胞来源树突状细胞(monocytes derived dendritic cells,Mo DC)后其功能及CD200R表达的改变。方法收集SLE患者及健康对照各22例,利用流式细胞术、酶联免疫吸附法(enzyme linked immunosorbent assay,ELISA)及同种异体混合淋巴反应(mixed lymphocytes reaction,MLR)检测外周血DC表面CD200R的表达、LPS刺激对Mo DC活化及CD200R表达的影响。结果 SLE患者髓样树突状细胞(myeloid dendritic cell,m DC)及浆样树突状细胞(plasmacytoid dendritic cell,p DC)CD200R表达均低于健康对照(P0.05),且m DC的CD200R表达与红细胞沉降率呈负相关(r=-0.552,P=0.041)。Mo DC经LPS刺激活化后CD200R表达下调(P0.05),活化成熟标记HLA-DR、CD83、CD86表达上调(P0.05),白细胞介素(interleukin,IL)-6和IL-10分泌增加(P0.05),促CD4+T细胞增殖增加(P0.05),SLE和健康对照未见显著性差异。结论 SLE患者DC表面CD200R表达下调,可能与其疾病发生及炎症状态关系密切。     

HBsAg负载的慢性乙肝患者DCs诱导特异性Th1细胞分化的作用

目的:研究HBsAg负载的慢性乙肝患者外周血树突状细胞(dendritic cells,DCs)对自身Th1细胞分化的诱导作用.方法:Fico11密度梯度离心和贴壁法分离慢性乙肝患者外周血单核细胞,以含ThIL-4 rhGMCSF的完全培养基诱导DCs.流式细胞术检测各组DC表面CD80,CD86,CD40和HLA-DR分子的表达,CCK-8法检测各组DCs刺激同种异体淋巴细胞增殖的能力,ELISA法检测各组DCs培养液上清中IL-12的水平.免疫磁珠分选慢性乙肝患者外周血CD4 T细胞,分别与患者自身的DCs共培养,细胞内细胞因子染色,流式细胞仪检测共培养后CD4 T细胞内特征性细胞因子IFN-γ和IL-4,ELISA法检测共培养上清中IFN-γ/IL-4的水平.结果:与对照组相比,HBsAg、IFN-γ和HBsAg IFN-γ组DCs表面表达CD80,CD86,CD40和HLA-DR分子水平较高;刺激同种异体淋巴细胞增殖的能力较强;DCs分泌的上清液中、IL-12水平也较高.与对照组相比,HBsAg、IFN-γ和HBsAg IFN-γ组DCs与自身Th细胞共培养后,Th1细胞占CD4 T细胞的百分比升高(10.76%±3.98%,11.43%±4.32%,15.28%±4.73% vs 7.84%±3.10%,P＜0.01),Th2细胞占CD4 T细胞的百分比降低(1.43%±0.96%.1.68%±0.16%,0.92%±0.21% vs 2.61%±1.27%,P＜0.01),共培养上清中IFN-γ的水平增高(578±47 mg/L,496±92 mg/L,784±97 mg/L vs 342±34 meg/L,P＜0.05),IL-4的水平降低(187±52 mg/L,169±38 mg/L,89±37 mg/L vs 226±48 mg/L,P＜0.05),以HBsAg IFN-γ组最为明显.结论:经HBsAg负载的DCs可以改善患者体内因DCs功能低下而引起的Th1细胞分化不足的状态.     

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.