阿克拉霉素B,阿霉素和小蘖碱对人外周血NK细胞活性影响的初步研究

本文研究了阿克拉霉素B,阿霉素和小蘖碱对人外周血NK细胞活性的影响。NK细胞活性由测定51cr从NK细胞作用的标记K562细胞的释放率来决定。阿克拉霉素B(0.5和1.0μg/ml),阿霉素(0.5和1.0μg/ml)和小蘖碱(1.0和10μg/ml)作用1小时后,未     

瑞香狼毒甲醇提取物抗瘤免疫机理初探

有报道瑞香狼毒复方或提取物用于恶性肿瘤治疗有一定疗效[1 3 ] ,但有关其抗瘤免疫机理研究资料较少。本文用瑞香狼毒甲醇提取物 ,在体内外对其抗瘤免疫机理进行了初步探讨。1　材料与方法1 1　试剂与动物　瑞香狼毒甲醇提取物 (醇提物 )为白色粉末 ,不能直接水溶 ,用时以少量二甲基亚砜(DMSO)溶解 ,再用完全培养液稀释供细胞培养用或生理盐水稀释供腹腔注射用 ,DMSO终浓度均为1%。KM小鼠 ,18～ 2 2g。1 2　醇提物体外对脾细胞生长的影响　在小鼠脾细胞 (96孔板 ,每孔 5× 10 5个细胞 )中加不同剂量醇提物作为实验孔 ,对照孔加等量完全…     

超抗原SEA活化淋巴细胞抗肿瘤活性的实验研究

纯化SEA在 10 -12 ～ 10 -5g/ml浓度范围对体外培养的BALB/c鼠脾细胞表现了细胞增殖诱导能力 ,并呈剂量依赖关系 ,其中 10 -7～ 10 -5g/mlSEA的作用强于最适量 ( 2 5 μg/ml)PHA。在E/T为 5∶1～ 2 0∶1条件下 ,10 -5g/mlSEA活化 48h的BALB/c鼠脾细胞对YAC 1细胞的杀伤活性高于NK细胞 ,但SEA未能增强BALB/c鼠脾细胞对B16细胞的杀伤活性。 5 μg和5 0 μgSEA体内用药对L615白血病无治疗作用 ,但转输经SEA活化的同系小鼠脾细胞对L615白血病有一定疗效。实验结果提示 ,SEA活化淋巴细胞对NK敏感的淋巴瘤、白血病有杀伤及治疗作用 ,这种作用依赖于较大数量级SEA活化淋巴细胞的存在 ,并且与SEA活化T细胞分泌的细胞因子有关     

双表达外源性4-1BBL/IL-15的K562细胞活化外周血淋巴细胞的研究

为肿瘤过继免疫治疗开发体外激活T细胞、NK细胞的高效途径,研究双表达外源性4-1BBL和IL-15的K562细胞刺激外周血淋巴细胞活化的能力。采用分子克隆技术,分别将4-1BBL和IL-15基因插入双表达载体pVITRO-2,命名为pV4-1BBL-IL-15。经测序鉴定后,利用脂质体介导的转染及潮霉素筛选,获得稳定双表达4-1BBL、IL-15分子的K562细胞(K562/4-1BBL/IL-15)。经流式细胞仪(FACS)分选后,K562/4-1BBL/IL15细胞和K562细胞分别用丝裂霉素C处理,与外周血淋巴细胞孵育24 h,FACS检测淋巴细胞表面活化性受体CD69的表达。对NK细胞不仅同时检测活化性受体NKG2D的表达,还用乳酸脱氢酶释放法观察NK细胞受不同刺激细胞作用后,细胞毒活性的变化。结果显示受K562/4-1BBL/IL15细胞刺激后,T细胞CD69的表达无明显变化。γδT细胞CD69表达增长5倍。NK细胞CD69表达增长6倍,而NKG2D的表达增加1.5倍;NK细胞受K562/4-1BBL/IL15细胞作用72 h后,细胞毒活性明显提高。提示双表达4-1BBL/IL-15的K562细胞能够高效激活γδT细胞及NK细胞,有望用于肿瘤的过继免疫治疗。     

玉屏风散对荷瘤小鼠肿瘤生长及IL-2与IL-2R基因表达的影响

目的 探讨玉屏风散对荷瘤鼠肿瘤生长及脾淋巴细胞IL-2与IL-2K基因表达的影响.方法 采用体外培养S180肉瘤细胞,接种健康小鼠,建立荷瘤小鼠模型.成瘤后给予玉屏风散治疗,观察计算抑瘤率,MTT比色法检测脾脏T淋巴细胞增殖能力,实时定量RT-PCR技术检测脾淋巴细胞IL-2与IL-2R基因表达情况.结果 荷瘤后小鼠的T细胞增殖能力及IL-2与IL-2R基因表达受到明显抑制;玉屏风散治疗组小鼠的平均瘤重明显低于荷瘤对照组,T细胞增殖能力及IL-2与IL-2R基因表达水平明显升高.结论 玉屏风散可能通过促进荷瘤小鼠T淋巴细胞的免疫功能进而抑制肿瘤生长.     

重组人IL-18在大肠杆菌中的表达及其抗肿瘤作用

目的：获取rhIL-18原核表达产物并研究其活性。方法：用本室构建的含基因重组表达载体PBV220-IL-18的大肠杆菌DHSot，经42℃热诱导表达和包涵体提取纯化后获取rhIL-18蛋白；采用ELISA试剂盒检测rhIL-18刺激PBMC分泌IFN-γ的活性及MTT法检测其对NK细胞杀伤K562细胞自然杀伤活性；同时用荷瘤小鼠模型检测其体内抗瘤功能。结果：诱导含重组表达载体PBV220-IL-18的大肠杆菌D145ct后，蛋白电泳显示出一条相对分子量（Mτ）为18000的蛋白条带；10μg rhIL-18蛋白体外刺激PBMC后，其分泌IFN-γ的能力与对照组相比提高了8倍，细胞毒性提高3倍；同时rhIL-18蛋白能明显抑制肿瘤生长和延长荷瘤鼠生存期。结论：获得了有免疫调节功能和抗肿瘤活性的rhIL-18蛋白。为今后IL-18重组产物的研制和开展肿瘤的生物治疗提供了实验依据。     

K652重组表达IL-6基因对NK细胞表型和功能的影响

目的:为了研究表达IL-6的重组K562细胞对自然杀伤细胞(NK)细胞的扩增数量、表型和功能的影响。方法:根据人类IL-6 c DNA的5'端序列,设计PCR引物以K562细胞c DNA文库的DNA为模板进行扩增,表达,转染,在K562细胞上表达IL-6基因,构建重组的K562工程细胞作为刺激细胞,以人外周血单个核细胞(PBMC)为扩增培养对象,使NK细胞在体外培养条件下得到大量的扩增。流式细胞仪分析NK细胞表型。将NK细胞作用到K562细胞,对其杀伤性进行功能分析,51Cr释放实验检测NK细胞对K562细胞杀伤水平的影响。结果:成功构建了表达IL-6的重组K562细胞,和PBMC共同孵育后,培养体系经过21 d的刺激后,IL-6重组K562细胞诱导PBMC扩增,CD56+CD16+CD3-细胞数量比诱导前扩增了(760±18)倍,CD56+CD16+CD3-细胞的纯度从培养前占PBMC的6%±0.4%,扩增后第3组结果比未扩增的多了91%±2%。细胞毒实验表明,在NK效应细胞∶K562靶细胞为5∶1时,扩增的NK细胞的杀伤率达到了92%±2%。结论:本方法以重组K562为刺激细胞,能够实现NK细胞体外的大规模制备,建立了优化的NK细胞体外扩增方法,且扩增的细胞杀伤K562细胞的活性较好。对于NK的大量扩增和应用于临床具有重要的指导意义。     

蒙药材瑞香狼毒体外细胞毒性的研究

目的研究蒙药瑞香狼毒炮制前后的体外细胞毒性和抗肿瘤活性。方法利用石油醚、醋酸乙酯和正丁醇分别萃取获得瑞香狼毒药材生品、奶制品、煎膏制品的各溶剂提取物,再通过萃取、分离纯化方法分别获得瑞香狼毒的9个单体化合物、总蛋白质和6个相对分子质量不同的蛋白质组分(Pr.1~Pr.6)。将各溶剂提取物及单体化合物制成浓度为2.0mg/ml的溶液,总蛋白质制成浓度分别为0.5、1.0、2.0、4.0、6.0、8.0mg/ml的系列溶液,各蛋白质组分制备成浓度均为10mg/ml的溶液,分别作为样品溶液,采用MTT法分别测定各样品对人肝癌SMMC-7721细胞、狗肾MDCK细胞的生长抑制作用。结果瑞香狼毒总蛋白质抗肿瘤活性和细胞毒性作用随其浓度增加而增强,其中4个蛋白质组分呈现较强的抗肿瘤活性,其活性由强至弱依次为Pr.4≥Pr.6≥Pr.3=Pr.2,3个蛋白质组分具有细胞毒性,其毒性由强至弱依次为Pr.4≥Pr.3≥Pr.1。瑞香狼毒单体化合物中,西瑞香素、狼毒色原酮、异狼毒素呈现不同程度的抗肿瘤活性,而狼毒色原酮、异狼毒素同时具有较弱的细胞毒性。除石油醚和多糖提取物外,瑞香狼毒药材生品各溶剂提取物均呈现不同程度的抗肿瘤活性和细胞毒性,并呈药物剂量依赖性;而炮制后,在保留一定程度抗肿瘤活性的基础上,其相应各溶剂提取物的细胞毒性均降低。结论瑞香狼毒的细胞毒性主要源自多成分的协同作用,其蛋白质可能是瑞香狼毒的主要毒性位点之一,而炮制方法可降低瑞香狼毒药材的毒性且保留其生物活性。     

重组可溶性MHC Ⅰ类相关蛋白A对NK细胞生物学活性的影响

目的:研究体外重组可溶性MHC Ⅰ类相关蛋白A(sMICA)对NK细胞杀伤靶细胞活性、分泌IFN-γ、增殖和凋亡的影响.方法:将重组sMICA蛋白与人外周血NK细胞相互作用过夜后,流式细胞仪检测NK细胞杀伤K562靶细胞的能力;ELISA检测培养上清IFN-γ浓度;MTS/PMS法检测sMICA对NK细胞增殖的影响;给NK细胞标记Annexin V和碘化丙啶检测凋亡情况.结果:可溶性MICA抑制NK细胞杀伤K562细胞的活性,下调IFN-γ的分泌,却对NK细胞的增殖和凋亡没有影响.结论:肿瘤细胞表面脱落的sMICA抗原可通过抑制NK细胞活性而逃逸机体的免疫监视功能.     

小鼠脾淋巴细胞可能存在5HT_3受体

本文用3H TdR掺入法和MTT法观察 5HT3受体激动剂 1 phenylbiguanide(PBG )、 5HT3受体拮抗剂格拉司琼 (granisetron )和托烷司琼 (tropisetron )对体外培养ICR小鼠脾淋巴细胞ConA ,LPS刺激的增殖和NK细胞活性的影响。结果表明PBG ( 10 6～ 10 4mol/L )抑制ConA刺激的脾细胞增殖反应和脾细胞IL 2的生成 (P <0 0 5 ) ;增强LPS剌激的脾细胞增殖反应 (P <0 0 5 ) ;格拉司琼和托烷司琼双相影响 ,即在低浓度 ( 10 7～ 10 6 mol/L )促进、在较高浓度 ( 10 5～ 10 4mol/L )抑制ConA刺激的增殖反应 (P <0 0 5 ) ;二药均浓度依赖抑制LPS刺激的脾细胞增殖效应。PBG对脾淋巴细胞增殖的影响被同时加入格拉司琼或托烷司琼拮抗 ,格拉司琼和托烷司琼减弱PBG 10 5mol/L对脾细胞增殖反应的影响。本实验 5HT3受体激动剂或拮抗剂浓度不明显影响脾NK细胞活性和无丝裂原刺激的增殖反应。结果提示小鼠脾T细胞和B细胞表面可能存在对淋巴细胞增殖有不同影响的 5HT3受体     

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.     

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.     

The development of the ligament of the head of the femur

The hip joints of 30 human male and female fetuses and stillborns between 20 mm and 350 mm crown-rump length were studied by light microscopy. The ligament of the head of the femur developed in situ as a condensation of mesenchyme at the end of the second month of intra-uterine life (IUL), and was vascularized by branches of acetabular vessels early in the fourth month. In the majority of fetuses older than 5.5 months IUL, vessels in the ligament passed a short way into the femoral head within cartilage canals, to supply a small region around the fovea capitis. The remainder of the head was supplied by vessels in canals from around the upper part of the neck. The ligament changed from predominantly cellular to fibrous during the last 4 months of IUL. This increase in strength suggested significant mechanical functions in utero: limitation of adduction-flexion and opposition to postero-superior dislocation were the most likely.     

Counterpointing the functional role of the forebrain and of the brainstem in the control of the sleep-waking system

This paper reviews the lifetime contributions of the author to the field of sleep-wakefulness (S-W), reinterprets results of the early studies, and suggests new conclusions and perspectives. Long-term cats with mesencephalic transection show behavioral/polygraphic rapid eye movement sleep (REMS), including the typical oculo-pupillary behavior, even when the section is performed in kittens prior to S-W maturation. REMS can be induced as a reflex. Typical non-rapid eye movement S (NREMS) is absent and full W/arousal is present only after a precollicular section. The isolated forebrain (IF) rostral to the transection exhibits all features of W/arousal and NREMS [with electroencephalographic (EEG) spindles and delta waves], arousal to olfactory stimuli, and including the appropriate oculo-pupillary behaviors. These features also mature normally after neonatal transection. REMS is absent from the IF. After deprivation there is NREMS pressure and rebound in the IF, but the decerebrate cat only shows pressure for REMS. Most IF reactions to pharmacologic agents are within expectations, except for the tolerance/withdrawal effects of long-term morphine use which are absent. In contrast, these effects are supported by the brainstem (i.e. seen in the decerebrate cat). In cats with ablation of the telencephalon, or diencephalic cats, delta waves are absent in the thalamus. EEG thalamic spindle waves are seen triggering S for only 4-5 days after ablation. Therefore, true NREMS is absent in chronic diencephalic cats although pre- and postsomniac behaviors persist. These animals are hyperactive and show a pronounced, permanent insomnia; however, a low dose of barbiturate triggers a dramatic REMS/atypical NREMS rebound. Cats without the thalamus (athalamic cats), initially show a dissociation between behavioral hyperactivity/insomnia and the neocortical EEG, which for 15-20 days exhibits only delta and slower oscillations. Fast, low-voltage W rhythms appear later on, first during REMS, but spindle waves and S postures are absent from the start, such that these cats also display only atypical NREMS. Athalamic cats also show barbiturate-sensitive insomnia. Cats with ablation of the frontal cortices or the caudate nuclei remain permanently hyperactive. They also show a mild, but significant hyposomnia, which is permanent in afrontal cats, but lasts for about a month in acaudates. The polygraphic/behavioral features of their S-W states remain normal. We conclude and propose that: (a) the control of the S-W system is highly complex and distributed, but is organized hierarchically in a well-defined rostro-caudal manner; the rostral-most or highest level (telencephalon), is the most functionally complex/adaptative and regulates the lower levels; the diencephalic/basal forebrain, or middle level, has a pivotal role in inducing switching between S and W and in coordinating the lowest (brainstem) and highest levels; (b) W can occur independently in both the forebrain and brainstem, but true NREMS- and REMS-generating mechanisms exist exclusively in the forebrain and brainstem, respectively; (c) forebrain and brainstem S-W processes can operate independently from each other and are preprogrammed at birth; this helps understanding normal and abnormal polygraphic/behavioral dissociations in humans and normal dissociations/splitting in aquatic mammals; (d) NREMS homeostasis is present in the IF, but only REMS pressure after deprivation persists in the decerebrate cat; (e) the thalamus engages in both NREMS and W; (f) insomnia in diencephalic cats is the result of an imbalance between antagonistic W- and S-promoting cellular groups in the ventral brain (normally modulated by the telencephalon); (g) the EEG waves, which are signature for each S-W state, appear to truly drive the concomitant behaviors, e.g. a hypothetical human IF could alternate between behavioral NREMS and W/arousal/awareness; (h) a role for REMS is to keep the individual sleeping at the end of the self-limiting NREMS periods. The need for accelerating research on telencephaling NREMS periods. The need for accelerating research on telencephalic S-W processes and downstream control of the lower S-W system levels is emphasized.     

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 -     

Involvement of the striatum in the central regulation of the hormonal functions of the gonads

Studies reported here show that intrastriatal administration of corticoliberin to rats decreases the blood testosterone level. However, in conditions of chemical deficiency of dopaminergic transmission in the dorsal striatum induced by injection of 6-hydroxydopamine, the effect of this neurohormone did not appear. It is concluded that extrahypothalamic corticoliberin is involved in regulating the hormonal reproductive system acting via dopaminergic mechanisms. Translated from Rossiiskii Fiziologicheskii Zhurnal imeni I. M. Sechenova, Vol. 85, No. 4, pp. 594–597, April, 1999.     

Ultrastructure of the endothelium of the drainage system of the eye

The endothelium of the ocular drainage system (Schlemm’s canal, collector tubules, and aqueous veins) in primary juvenile glaucoma undergoes degenerative dystrophic changes with compensatory hypertrophy and proliferation at the initial stages of the glaucomatous process and atrophy and desquamation at advanced and terminal stages. Progressive decrease in the pinocytous function of endotheliocytes, reduction of the protein-synthesizing and mitochondrial compartments of the cytoplasm, and formation of autophagosomes reflect the process of endotheliocyte degeneration in general. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 145, No. 5, pp. 574–577, May, 2008