负载肝癌抗原的人树突状细胞生物学特性的研究

目的　探讨负载肝癌抗原的树突状细胞 (DC)对自身CIK细胞的诱导增殖能力和杀伤能力 .方法　从人外周血分离获得单核细胞 ,体外经重组细胞因子 (GM -CSF)、白细胞介素 - 4 (IL - 4 )、肿瘤坏死因子α(TNF -α)培养获得DC ,电镜观察其形态 ,流式细胞仪检测其表面抗原 ,混合淋巴细胞反应检测负载肝癌抗原DC诱导CIK细胞增殖活性 ,检测激活的CIK对肝癌细胞的杀伤作用 .结果　经体外培养 ,从外周血分离获得的大量成熟DC ,检测表明高表达DC的抗原标志 ,负载抗原的DC具有很强的激发同种CIK增殖的能力 ,激活的CIK对肝癌细胞系具有很强的杀伤作用.结论　实验结果为肝癌的临床免疫治疗提供了理论基础 .     

卵巢癌细胞株冻融抗原负载的树突状细胞诱导CTL抗卵巢癌免疫应答的体外研究

目的研究卵巢癌冻融抗原负载的树突状细胞(dendriticcells,DC)诱导细胞毒性T淋巴细胞(CTL)体外杀伤卵巢癌细胞的细胞毒性效应。方法利用免疫磁珠分离法(MACS)分离纯化脐血CD34 细胞并在体外诱导分化为DC,用反复冻融法从卵巢癌细胞系SKOV3中提取的可溶性相关抗原负载DC。流式细胞学检测负载抗原后DC表面各种分化相关抗原的表达,ELISA法检测DC上清中IL12的表达,混合淋巴细胞反应(MLR)测定DC体外刺激T细胞增殖的能力,MTT法检测抗原负载DC激活的抗原特异性CTL对卵巢癌细胞的杀伤作用。结果与未经抗原负载的DC相比,经卵巢癌抗原负载的DC不仅能更高地表达各种DC分化相关抗原CD1α(73.35%±2.94%vs34.1%±2.35%)、CD83(73.9%±8.46%vs54.68%±3.26%)、CD80(91.95%±2.48%vs52.53%±3.18%)、HLADR(70.05%±2.35%vs48.7%±2.07%)以及CD54(88.9%±5.52%vs71.45%±2.29%),同时具有更强的刺激同种异体T淋巴细胞增殖和IL12分泌的能力(P均<0.05)。此外,卵巢癌细胞SKOV3冻融抗原负载DC激活的CTL在体外对SKOV3的杀伤率为77.35%,显著高于未经抗原负载的DC(P=0.0001)。结论经卵巢癌细胞冻融抗原负载DC激活的CTL在体外具有更强的增殖能力和杀伤卵巢癌细胞的作用。     

HBsAg脉冲的树突状细胞对CIK细胞功能的影响

目的:探讨HBsAg脉冲的树突状细胞(DC),对细胞因子诱导的杀伤细胞(CIK)的增殖和杀伤作用的影响。方法:选择慢性乙肝患者23例,用常规方法分离外周血单个核细胞(PBMC),经HBsAg脉冲后培养为特异性的DC,用3H-TdR掺入法检测该细胞对CIK细胞增殖的刺激作用,用乳酸脱氢酶释放法检测特异性CIK细胞对HepG2215细胞的特异性杀伤作用。结果:HBsAg脉冲的DC对CIK细胞的增殖具有刺激作用。由HBsAg脉冲的DC所诱导的特异性CIK细胞对HepG2215细胞的特异性杀伤作用增强。结论:HBsAg脉冲的DC可增强CIK细胞的杀伤活性。     

抗原负载的DC诱导CIK对MDA-MB-231细胞的杀伤活性研究

探讨乳腺癌相关抗原负载后树突状细胞(DC)联合细胞因子诱导的杀伤细胞(CIK)对MDA-MB-231乳腺癌细胞株的杀伤效应。采用密度梯度离心法获取外周血单个核细胞(PBMC),常规法诱导DC、CIK细胞;流式细胞技术分析测定细胞表型;用反复冻融法制备MDA-MB-231细胞的相关抗原,并测定抗原浓度;MTT法分别测定在四个不同效靶比时CIK、DC+CIK和抗原负载的DC+CIK三组效应细胞对MDA-MB-231乳腺癌细胞的杀伤活性。结果:CIK细胞分别与DC和Ag-DC共培养后,可见CD3+CD56+双阳性表达细胞较培养前显著增加(P<0.05);将负载肿瘤相关抗原的DC与CIK共培养后,杀瘤活性明显提高(P<0.05)。提示,抗原负载的DC与CIK细胞共同孵育培养后,在相同效靶比时,对MDA-MB-231乳腺癌细胞杀伤效应与CIK细胞和DC-CIK两组效应细胞相比有显著提高,表明抗原负载后可提高CIK细胞对肿瘤细胞的杀伤活性,为乳腺癌临床生物免疫治疗提供实验室基础。     

脐血来源的树突状细胞增强CIK细胞的杀伤作用

为确认体外诱导出的成熟脐血来源树突状细胞 (CBDC )可以引发强大抗肿瘤免疫反应 ,CBDC培养 12d后用电镜分析形态 ,用流式细胞仪检测其表型。在不同培养时间 ,用3 H TdR掺入法检测CBDC和细胞因子诱导杀伤细胞 (CIK )的扩增功能 ;用MTT法检测被CBDC激活的CIK抗肿瘤活性。结果表明 ,体外诱导出形态典型的DC ,高表达主要组织相容性抗原I、II类分子、CD5 4 ,也表达CD80、CD83、CD1a,不表达CD6 8。体外培养 12d成熟的CBDC ,能使CIK以最高的比率扩增 ,2种细胞最佳混合比为 1∶10时被激活的CIK杀伤BEL 74 0 2肝癌细胞的功能最强 ,最佳效靶比为 80∶1     

体外构建的HSP70-肝癌抗原肽诱导抗原肽特异性免疫反应1

目的研究体外构建的HSP70-肝癌抗原肽复合物诱导针对肝癌的特异性免疫反应能力,为该复合物的临床应用奠定基础.方法在体外构建HSP70-肝癌抗原肽复合物,联合应用粒/巨细胞集落刺激因子(GM-CSF)及白介素-4(IL-4)直接从志愿者外周血中培养出DC;以HSP70、HSP70-肝癌抗原肽、抗原肽分别刺激DC,DC激活同源的T淋巴细胞产生细胞毒性T淋巴细胞(CTL),检测其杀伤T2细胞和肝癌细胞系的能力.结果HSP70-抗原肽、抗原肽均可诱导CD8+的抗原肽特异性CTL,而前者的诱导效果更强.结论体外构建的HSP70-抗原肽复合物具有免疫原性,HSP70可以增强抗原肽诱导特异性免疫反应的能力,HSP70-抗原肽复合物有可能作为肽疫苗用于临床肿瘤免疫治疗.     

两种白血病细胞抗原负载DC的体外诱导特异性CTL应答的比较

目的: 比较树突状细胞 (DC)与白血病细胞融合及白血病细胞冻融抗原负载DC两种白血病DC疫苗诱导抗原特异性CTL应答的能力。方法: 采用羟乙基淀粉 Ficoll两步法分离外周血单个核细胞(PBMC), 通过贴壁 2h获得黏附的单核细胞, 用GM- CSF加IL- 4诱导培养 5d收获细胞。将细胞分为 4组: A组将K562细胞或原代慢性粒细胞白血病(CML)细胞在 500g/LPEG 100mL/LDMSO诱导下与DC融合; B组加入相应细胞数量的白血病细胞冻融抗原; C组将K562细胞或CML细胞与DC共培养; D组: 单独DC培养组。融合前以红色荧光染料PKH26标记K562细胞, 采用流式细胞仪(FCM)检测PKH26 /FITC 抗HLA ABC抗体双标细胞并评估融合率。培养的第 6天, 加入TNF -α诱导DC成熟, 然后分别与自体T细胞共培养, 用MTT比色法检测各组CTL对靶细胞的杀伤活性。结果: GM- CSF加IL -4和TNF- α依次诱导成熟的DC具有经典的DC的形态和表型特征, 在PEG -DMSO的介导下, DC与白血病细胞的融合率为 ( 17. 33 ~29. 94 )%。两种抗原负载方案激活的CTL均对表达K562抗原的细胞具有特异性的细胞毒作用。在效靶比相同时, 融合组诱导CTL的杀伤活性强于冻融抗原致敏DC组。结论: 与冻融抗原致敏的DC相比, DC与白血病细胞融合细胞递呈抗原的效率更高, 体外诱导的CTL特异性杀伤靶     

树突状细胞体外诱导抗肿瘤血管内皮细胞特异性免疫的研究

目的:探讨肿瘤内皮细胞抗原负载的树突状细胞(DC)诱导的细胞毒性T淋巴细胞(CTL)的特异性杀伤效应。方法:采用肿瘤细胞的培养上清诱导人脐静脉血管内皮细胞(HUVEC)增殖,制备肿瘤血管衍生的内皮细胞(TdEC)。用RTPCR检测肿瘤内皮标志物(TEM)的表达。制备TdEC的冻融抗原,负载从外周血中扩增的DC,用MTS比色法检测DC刺激自体淋巴细胞增殖的效应;用LDH法检测DC诱导的CTL的特异性杀伤效应。结果:TdEC可表达TEM1和TEM8。负载TdEC抗原的DC,可显著刺激自体淋巴细胞增殖。由其诱导的CTL对TdEC具有特异性的杀伤作用。在效靶比为20∶1和10∶1时,杀伤率分别为33%和27%,高于对照组的14%和10%。结论:TdEC抗原负载的DC,在体外可有效地诱导CTL产生,并特异性地杀伤TdEC。     

自体肿瘤抗原致敏的树突状细胞联合细胞因子诱导的杀伤细胞治疗晚期肾癌的疗效观察

目的探讨负载自体肿瘤抗原的树突状细胞(DC)联合细胞因子诱导的杀伤细胞(CIK细胞)在晚期肾癌治疗中的临床疗效,并监测患者的免疫状态及不良反应。方法回顾分析2011-01/2013-12期间在我科治疗的82例晚期肾癌患者,单采其外周血单个核细胞(PBMC),其中贴壁细胞经体外诱导产生DC,并负载自体肿瘤细胞裂解物(Ag)制备Ag-DC;T淋巴细胞经体外诱导产生CIK细胞,将Ag-DC与CIK细胞共培养制备Ag-DC-CIK瘤苗,检测DC表面分子的表达及IL-12的分泌,监测CIK细胞增殖情况,并将Ag-DC-CIK分次回输给其中41名患者,以41例单纯的CIK细胞治疗作为对照,治疗2疗程后,检测患者外周血细胞因子水平及T细胞亚群变化,结合影像学等检查综合评价疗效,同时观察不良反应。结果负载肿瘤细胞冻融抗原的DC,高表达CD11c、CD83、CD86、HLA-DR表面分子,IL-12的分泌显著增加。Ag-DC与CIK细胞共培养后,可明显刺激CIK细胞的增殖,同时CD3+CD8+细胞及CD3+CD56+细胞的含量明显增加,此外,Ag-DC-CIK瘤苗的临床疗效明显高于单纯CIK细胞治疗组,2组均未发生严重的不良反应。结论晚期肾癌PBMC来源的DC负载自身肿瘤细胞冻融抗原能提高晚期肾癌患者的免疫状态。     

慢性粒细胞白血病患者DC对细胞因子诱导的杀伤细胞细胞毒作用的影响

目的 观察慢性粒细胞白血病（CGL）患者树突状细胞（DC）对自身细胞因子诱导的杀伤细胞（CIK）细胞毒的影响。方法 从CGL患者和正常人末梢血中分离单个核细胞（PBMC），用细胞因子（rhGM-CSF,rhIL-4和rHTNF-α)联合定向诱导培养DC。用CGL细胞抗原致敏DC，再将致敏的DC与CIK共同培养后，检测CIK对不同靶细胞的杀伤作用。结果 CGL患者和正常人的PBMC经细胞因子联合诱导培养后，DC占20．78％－56．0％。CGL患者的CIK和经CGL细胞抗原致敏的DC作用后的CIK，对自身白血病细胞的杀伤活性分别为56．0％和83．4％；正常人对照组则分别为30％和62％。经CGL患者CGL细胞抗原致敏的DC作用后的CIK, K562和SGC－7901的杀伤活性较未致敏的CIK活性低，而正常人对照组则无此现象。结论 从CGL患者的PBMC中能定向诱导扩增出DC。经CGL细胞抗原致敏的DC，能明显增强CIK对自身CGL细胞的杀伤活性。     

Topical organization of the projections of neurons of the substantia nigra and the ventral field of the tegmentum of the midbrain to the head of the caudate nucleus of the cat

Neuroscience and Behavioral Physiology -     

Asymmetry of the internal connections of the striate cortex of the cat in the projection zone of the center of the field of vision

Studies were carried out on the organization of the internal connections of the striate cortex in cats in the projection zone of the center (0–5°) of the field of vision by microintophoretic application of horseradish peroxidase to electrophysiologically identified orientational columns. The area containing neurons showing retrograde labeling in most cases extended in the mediolateral direction. Labeled cells were located in the upper (II, III) and lower (V, VI) layers of the cortex, and the shapes and orientations of the areas containing labeled neurons in these layers coincided. Spatial asymmetry was detected in the distribution of labeled neurons relative to the orientational column studied. Labeled cells were located predominantly medial to the columns, regardless of the distance from the projection of the area centralis. Considering the visuotopical map of field 17, the asymmetry detected here provides evidence that neurons in orientational columns have more extensive connections with neurons of the peripheral part of the cortex. An asymmetrical distribution of “silent” zones around the receptive fields of neurons in orientational columns is suggested, and that these appear to receive influences from the periphery of the visual field. Laboratory of Visual Physiology and Laboratory of Central Nervous System Morphology, I. P. Pavlov Institute of Physiology, Russian Academy of Sciences, 6 Makarov Bank, 199034 St. Petersburg, Russia. Translated from Fiziologicheskii Zhurnal imeni I. M. Sechenova, Vol. 82, No. 12, pp. 23–29, December, 1996.     

Characteristics of the topographic interrelation of the head of the pancreas and the duodenum and pathology of the organs of the pancreatoduodenal area

Seventy pancreatoduodenal complexes of 55 patients with chronic pancreatitis and tumours of this zone and 15 patients died from other diseases are studies histotopographically . The pieces of the pancreatic head tissue in the medial wall of the duodenum were found in 12 cases of the first group and in 4 control cases. The pancreatic tissue consisted either of all elements of this organ or cystically dilated ducts and seemed to infiltrate different layers of the duodenum wall. Three variants of the pancreatic head structure are suggested on the basis of anatomo-topographical interrelationships of the pancreatic head and duodenum. In 12 out of 14 cases chronic pancreatitis and carcinoma of organs of this zone were combined with the variants of the pancreatic head structure, in 2 cases there was a true heterotopy . Pathogenetic significance of these variants for the development of chronic pancreatitis is discussed.     

Influence of the level of the transmembrane potential on the dynamics of the extinction of the amplitude of the synaptic reactions of the snail under voltage clamping conditions

The influence of the level of the transmembrane potential on the dynamics of the extinction of the amplitudes of summary excitatory postsynaptic currents arising in identified giant parietal neurons in response to rhythmic stimulation of the intestinal nerve was investigated in a preparation of the isolated CNS of the common snail in order to identify the possibility of the participation of the postsynaptic element in synaptic plasticity. It was demonstrated that, at a greater value of the transmembrane potential, the decrease in the amplitudes of the postsynaptic currents which have been induced by rhythmic stimulation takes place more rapidly. It was also demonstrated that at a higher frequency of stimulation of the nerve, the effect of the influence of the membrane potential level on the dynamics of the synaptic reactions is more prominent. The data obtained may be regarded as an argument in favor of the possibility of the participation of the post-synapse in plasticity. This study was partially supported by the Russian Basic Research Fund (project No. 94-04-12209). Institute of Higher Nervous Activity and Neurophysiology, Russian Academy of Sciences, Moscow. Translated from Zhurnal Vysshei Nervnoi Deyatel'nosti imeni I. P. Pavlova, Vol. 45, No. 1, pp. 171–179, January–February, 1995.     

Effects of the lesion of the postcommissural part of the septum on the behavior of the rat.

The effects of the lesion of the postcommissural part of the septum on behavior of the rat has been studied. Results may be summarized as follows. An increase in the exploratory behavior in the open field which decreases rapidly; a decrease in the number of defecations in this test and a decrease in time leaving a dark environment for exploration. In the shuttle box test, no facilitation of the acquisition, but a permanent and quite significant increase in the intertrial activity has been found. We conclude that the lesions tend to decrease the emotivity of the subjects. An interpretation on the basis of the species -- specific defensive reactions explains the transitory and permanent effects of the lesions on the spontaneous activity.     

Effect of the conditions of adaptation of the optic analyzer to the function of the thermoreceptors of the human skin

Summary A discussion is presented of the effect produced by light stimulation applied to the optic analyzer alone on the function of the temperature analyzer. The cutaneous-temperature analyzer was kept on constant illumination and temperature. The light effects from the retinal photoreceptors on the cutaneous thermoceptor system led to reflex changes of the functional rate of the latter. Conditions of the optic analyzer of light adaptation caused reflex adjustment of cold receptors as to heating. Dark adaptation of the eyes changed the functional state of the cold receptors, corresponding to their adjustment to low temperatures.(Presented by Active Member AMN SSSR V. V. Parin) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 57, No. 3, pp. 3–6, March, 1964