间充质干细胞联合胰岛移植对受体鼠T淋巴细胞亚群的影响

目的研究骨髓间充质干细胞(MSCs)与胰岛共移植时,探讨MSCs如何通过T淋巴细胞亚群发挥免疫抑制作用。方法 Balb/c小鼠骨髓MSCs,与C57LB/6小鼠脾脏分离的T淋巴细胞和Balb/c小鼠脾淋巴细胞共培养,监测对T淋巴细胞增殖能力和细胞周期影响。与C57LB/6小鼠来源的T淋巴细胞共培养,流式细胞术分析T淋巴细胞亚群的变化。用Balb/c小鼠的骨髓MSCs和胰岛联合移植到C57BL/6糖尿病模型鼠,流式细胞术分析受体鼠T淋巴细胞亚群的变化。结果 MSCs可明显抑制T淋巴细胞增殖能力,使T细胞滞留在G0/G1期。使Th1/Th2和Tc1/Tc2比值向Th2和Tc2倾斜,对初始和记忆T细胞具有明显的抑制作用。MSCs联合胰岛移植可使免疫排斥反应减轻。结论 MSCs可抑制T淋巴细胞的增殖,Th1/Th2和Tc1/Tc2比值向Th2和Tc2倾斜,下调初始和记忆T细胞,以发挥免疫抑制作用。     

微波消融对小鼠黑色素瘤动物模型中T淋巴细胞亚群比例和功能的影响

目的研究微波消融（MA）对小鼠黑色素瘤动物模型中T淋巴细胞亚群的比例和功能的影响。方法建立C57BL／6J小鼠B16．BL6黑色素瘤皮下移植模型;流式细胞术、免疫荧光法分别检测微波消融后荷瘤小鼠瘤内浸润T淋巴细胞比例变化和主要组织相容性复合体-I（MHC-I）的表达;ELISA法检测微波消融对肿瘤内T细胞细胞因子干扰素γ（IFN-γ）、白细胞介素-2（IL-2）、肿瘤环死因子-α（TNF-α）分泌的影响。结果微波消融组小鼠肿瘤内辅助性T细胞（Th）、杀伤性T细胞（CTL）比例显著增高,且肿瘤内细胞因子IFN-γ、IL．2、TNF-α、MHC—1分泌显著上调。结论微波消融有效增加CTL比例,并增强局部淋巴细胞抗肿瘤效应。     

日本血吸虫感染小鼠肠系膜淋巴结T细胞亚群的变化

目的:观察C57BL/6小鼠感染日本血吸虫(Schistosome japonicum,Sj)4～6周肠系膜淋巴结T细胞亚群的改变。方法:用Sj尾蚴腹贴法建立Sj感染的小鼠模型。4～6周后取肠系膜淋巴结做淋巴细胞计数,使用细胞内细胞因子染色的方法,利用流式细胞仪检测肠系膜淋巴结淋巴细胞中分泌不同细胞因子的T细胞亚群含量的变化。结果:Sj感染C57BL/6小鼠4～6周后,肠系膜淋巴结细胞数量明显增多;流式细胞仪检测发现肠系膜淋巴结中CD4+T细胞中分泌IFN-γ的Th1细胞增多1倍,分泌IL-4和IL-5的Th2细胞增多近20倍,Th1/Th2轴发生偏移;分泌IL-17的Th17细胞也增多近5倍;分泌IFN-γ的CD8+T细胞也增多1倍。结论:日本血吸虫感染C57BL/6小鼠4～6周肠系膜淋巴结细胞增多,并向Th2和Th17型细胞极化。     

PTD-HBcAg融合蛋白经树突状细胞诱导特异性CTL抑制HBV的体外研究

目的:探讨经PTD-HBcAg融合蛋白致敏的树突状细胞(DCs)体外诱导特异性细胞毒T淋巴细胞(CTLs)对HBV的抑制作用.方法:体外分离培养小鼠髓源性DC,加入融合蛋白刺激DC成熟后与T淋巴细胞共培养,ELISA 法检测T淋巴细胞上清中IL-2、IL-4、IL-10和INF-γ的分泌水平,流式细胞术检测胞内细胞因子水平,乳酸脱氢酶释放试验检测特异性CTL活性,并对HepG2.2.15细胞上清中HBsAg及HBV DNA水平进行检测.结果:经不同融合蛋白刺激的DCs能有效促进T淋巴细胞的细胞因子分泌,同时融合蛋白PTD-HBcAg组中IL-2(552.7±117.5 ng/L)和INF-γ(150.6±7.945 ng/L)明显高于HBcAg组中IL-2(420±12.47 ng/L)和INF-γ(107.5±12.19 ng/L)分泌.流式细胞计数术检测的PTD-HBcAg融合蛋白诱导CTL细胞水平明显高于对照组.经PTD-HBcAg融合蛋白诱导的CTL比HBcAg有明显的特异性杀伤作用(P＜0.05),同时对HBsAg及HBV DNA水平有明显的抑制作用.结论:经PTD-HBcAg融合蛋白致敏的DCs能有效刺激T淋巴细胞分泌细胞因子及增加细胞毒T淋巴细胞的表达,并增强特异性CTL活性及对HepG2.2.15细胞上清中HBsAg及HBV DNA水平的抑制.     

Vγ1+γδ T淋巴细胞对气道变应性炎症和气道高反应性的调节

目的:探讨消除Vγ1+γδ T淋巴细胞的哮喘C57BL/6小鼠中观察气道高反应性和支气管灌洗液中细胞因子浓度变化、肺组织中CD4+ CD25+ T淋巴细胞变化.方法:T淋巴细胞消除是以OVA首次致敏和第二次致敏1周前注射anti-Vγ1 mAbs、anti-Vγ4 mAbs、nonspecific hanmster IgG来完成.对C57BL/6小鼠测定气道高反应性后留取支气管灌洗液,分离肺组织.采用ELISA法测定支气管灌洗液中IL-10和TGF-β的浓度,利用FACS分析肺组织中提取的CD4+ CD25+ T淋巴细胞.结果:(1)诱导哮喘时消除Vγ1+γδ T淋巴细胞的C57BL/6小鼠气道高反应性比注射hanmster IgG的对照组减弱.但是消除Vγ4+γδ T淋巴细胞的C57BL/6小鼠气道高反应性与对照组相比无差别.(2)消除Vγ1+γδ T淋巴细胞的C57BL/6小鼠支气管灌洗液中IL-10和TGF-β浓度比对照组增加.(3)消除Vγ1+γδ T淋巴细胞的C57BL/6小鼠肺组织中CD4+ CD25+ T淋巴细胞比对照组明显增加.结论:消除Vγ1+γδ T淋巴细胞的哮喘C57BL/6小鼠气道高反应性减弱与支气管灌洗液中细胞因子IL-10和TGF-β的增加有关,并且与肺组织中的调节T淋巴细胞CD4+ CD25+ T淋巴细胞增加有关.     

不同大鼠胶质瘤抗原致敏的DC体外诱导CTL活性的研究

目的: 比较大鼠骨髓来源的树突状细胞(dendritic cells, DC)体外经由大鼠C6胶质瘤细胞由不同方式制备的不同抗原致敏后,对特异性细胞毒性T淋巴细胞(cytotoxic T lymphocyte, CTL)的诱导作用。方法: 自大鼠骨髓分离DC前体细胞,经重组大鼠粒细胞巨噬细胞集落刺激因子(rrGM-CSF)+白细胞介素4(rrIL-4)诱导培养、扩增;由C6胶质瘤细胞经由反复冻融、煮沸灭活及超声破碎细胞抽提其总蛋白的方法制备各种不同抗原致敏DC,致敏的DC与T淋巴细胞进行共培养诱导CTL;以ELISA法检测CTL诱导过程中淋巴细胞趋化因子(lymphocyte chemoattractant factor)及细胞因子IFN-γ分泌水平:以 -TdR掺入法检测DC诱导T细胞增殖及其特异性CTL杀伤活性。 结果: 体外应用煮沸灭活瘤细胞制备的肿瘤抗原致敏DC,能诱导更强的刺激T细胞增殖的能力、并且可以诱导杀伤活性更强的CTL。结论: 应用煮沸灭活的瘤细胞制备瘤抗原负载DC获得瘤苗可获得更强的抗肿瘤保护作用。     

CTLA4Ig修饰树突状细胞对实验动物淋巴细胞增殖及胞毒效应的影响

目的：探讨CTLA4Ig修饰的DC对实验动物免疫功能的影响。方法：将经CTLA4Ig基因修饰或未修饰Dc腹腔注射C57BL／6致敏小鼠，以致敏或未致敏C57BL／6单个核细胞作为反应细胞，以未修饰DC细胞及修饰DC为刺激细胞，共培养6天，采用MTT比色法检测细胞增殖，乳酸脱氢酶法测定细胞毒活性。结果：CTLA4Ig融合蛋白对未修饰DC致敏或未致敏小鼠的同种细胞刺激的增殖反应有明显的抑制作用。CTLA4Ig修饰DC诱导不同组小鼠淋巴细胞增殖反应均明显降低，CTLA4Ig融合蛋白对CTL细胞毒活性有显著抑制作用。CTLA4Ig修饰DC对不同组小鼠CTL细胞毒活性均具有抵抗作用，未修饰DC细胞对未致敏小鼠以及未修饰DC对致敏小鼠CTL细胞毒活性敏感。结论：稳定表达CTLA4Ig融合蛋白的DC诱导显著降低同种小鼠淋巴细胞的增殖反应和对CTL细胞毒活性的抵抗。     

旋毛虫成虫可溶性虫体蛋白对葡聚糖硫酸钠诱导的小鼠急性结肠炎的影响

本文探讨旋毛虫成虫可溶性虫体蛋白(Soluble adult proteins,SAP)对葡聚糖硫酸钠(Dextran sulphate sodium,DSS)诱导的C57 BL/6小鼠结肠炎的影响.将36只小鼠随机均分为3组:对照组(PBS 组)、肠炎组(PBS+ DSS组)、虫体蛋白治疗组(SAP+ DSS组).小鼠连续饮用3％DSS溶液7d诱发急性肠炎,对照组给予双蒸水.诱发肠炎的同时,治疗组每天经腹腔注射25μg虫体蛋白,肠炎组注射PBS.每天观察对照组和实验组小鼠的临床表现,并进行临床疾病活动指数(DAI)评分.第7d将小鼠处死,观察结肠肉眼或大体病变及组织病理改变.同时提取小鼠脾淋巴细胞和肠系膜淋巴结细胞,用酶联斑点免疫试验检测细胞因子水平.结果显示,与肠炎组相比,虫体蛋白治疗组的小鼠肠炎症状明显缓解,结肠病理变化较轻,脾淋巴细胞和肠系膜淋巴结细胞的Th1型细胞因子IFN-γ分泌水平下降,Th2型细胞因子IL-4和调节性细胞因子IL-10分泌水平显著升高.表明旋毛虫可溶性成虫虫体蛋白对DSS诱导的C57BL/6小鼠结肠炎有缓解作用.     

B细胞连接蛋白的表达抑制小鼠实验性自身免疫性脑脊髓炎

目的研究B细胞连接蛋白BLNK(B-cell linker protein)的表达对小鼠实验性自身免疫性脑脊髓炎(experimentalautoimmune encephalomyelitis,EAE)的影响并初步探讨其可能的机制。方法 MOG35-55多肽免疫BLNK缺陷(BLNK-deficient,BLNK-/-)鼠及C57BL/6鼠制备EAE小鼠模型,观察实验动物的临床症状及中枢神经系统的病理学变化;应用ELISA方法检测血清中的MOG特异性IgG和IgM抗体水平;采用流式细胞术检测脾脏中调节性T细胞的变化;分选的脾脏B淋巴细胞采用RT-PCR技术评价B细胞所分泌的细胞因子的变化。结果 BLNK-/-鼠临床症状评分明显高于C57BL/6鼠(P0.01);HE染色及LFB染色结果显示,BLNK-/-鼠与C57BL/C鼠比较炎症感染灶及脱髓鞘病灶明显增多。BLNK-/-鼠血清中的MOG特异性IgG和IgM抗体水平显著低于C57BL/C鼠(P0.005)。BLNK-/-鼠与C57BL/6鼠相比,脾脏中调节性T细胞百分比及B细胞所分泌的IL-10的表达明显减少,而IL-2的表达显著增高(P0.05);IFN-γ的表达水平在2组小鼠之间无统计学差异(P0.05)。结论 BLNK的表达抑制EAE,其机制可能是通过对B细胞分泌的Th1/Th2细胞因子的调节来实现。     

化学修饰的糖脂4′″-dh-iGb3对NKT细胞分泌Th1/Th2型细胞因子的影响

为研究两种i Gb3类似物(化学修饰的糖脂4′″-dh-i Gb3和4-HO-i Gb3)对NKT细胞Th1/Th2型细胞因子分泌的影响。采用流式细胞术检测经腹腔注射两种i Gb3类似物后C57BL/6小鼠脾脏NKT细胞数量的变化以及NKT细胞胞内IFN-γ和IL-4的表达水平;用Real-ti me PCR方法检测体外培养的脾脏淋巴细胞与i Gb3类似物共孵育后IFN-γ和IL-4的mRNA表达水平,并用ELISA方法检测孵育上清中IFN-γ和IL-4的含量。结果显示:与i Gb3组相比,经两种i Gb3类似物体内刺激后脾脏NKT细胞的数量都没有显著性变化。糖脂4-dh-i Gb3能够较i Gb3更强地诱导脾脏NKT细胞胞内IFN-γ的表达,也能够上调体外培养的脾脏淋巴细胞IFN-γ的mRNA水平及IFN-γ的分泌,而IL-4在所检测的各个水平上都没有显著性变化。提示化学修饰的糖脂4′″-dh-i Gb3能够诱导C57BL/6鼠脾脏NKT细胞Th1型细胞因子的分泌,而并不显著影响Th2型细胞因子的分泌,从而诱导Th1/Th2型细胞因子平衡向Th1方向偏移。     

Pathogenicity of PMV-3/parakeet/Netherlands/449/75 for chickens

Infection of 1-day-old chicks with PMV-3/parakeet/Netherlands/449/75 (449) by intramuscular, intranasal or contact routes resulted in severe impairment of growth in all groups compared to uninfected control birds. In the group infected intramuscularly with 449 virus 16/22 birds died within 14 days of infection. No clinical signs were seen in 6-week-old chickens infected with 449 by intramuscular, intranasal or contact routes. One-day-old chicks infected with a large dose of NDV-B(1) and one-day-old chicks placed in contact with these birds also showed significant impairment of growth compared to uninfected controls.     

Nucleotide sequence of RNA segment 3 of the avian influenza A/FPV/Rostock/34 and its comparison with the corresponding segment of human strains A/PR/8/34 and A/NT/60/68

The nucleotide sequence of RNA segment 3 of A/FPV/Rostock/34 (H7N1), an avian strain of influenza A virus, has been determined from a cloned DNA copy. Segment 3 codes for the PA polypeptide and the sequence specifies an acidic polypeptide of 716 amino acid residues. Comparison of the sequence with the corresponding segment of two human strains A/PR/8/34 and A/NT/60/68 indicates significant divergence of the avian sequence from the human sequences at the nucleotide level. At the amino acid level there is considerably greater homology between the avian and human strains. This presumably reflects a constraint on divergence of the PA polypeptide imposed by a common functional requirement of PA in all influenza virus strains.     

Determination of the rpoB gene sequences of Bartonella henselae and Bartonella quintana for phylogenic analysis

Using the Genome Walker™ procedure, which allows PCR amplification of genomic DNA using a single gene-specific primer and direct automated sequencing methodology, we obtained the nucleotide sequence of the RNA polymerase β subunit (rpoB) from Bartonella henselae and Bartonella quintana. A phylogenetic tree constructed from these data and other rpoB sequences available in GenBank is, in part, consistent with those previously derived from 16S rRNA gene sequences and confirms the position of Bartonella within the α subdivision of Proteobacteria. In fact, this analysis showed that rpoB data are similar to 16S rRNA data for the α, β and γ subdivisions of Proteobacteria. In contrast, concerning other bacteria included in our study, the topologies of phylogenetic trees were different. Based on the bootstrap values derived from rpoB phylogenic analysis, we believe that this molecule should contribute to better understanding the evolutionary process.     

Evaluation of the A/Seal/Mass/1/80 virus in squirrel monkeys

An influenza A virus isolated from seals [A/Seal/Mass/1/80 (H7N7)] and an isolate of this virus obtained from a human conjunctiva were evaluated for replication and virulence in squirrel monkeys. When the seal virus was administered intratracheally, it replicated in lungs and nasopharynges and induced illness almost to the same extent that a human influenza A virus [A/Udorn/72 (H3N2)] did. In one monkey that died of pneumonia, the seal virus was recovered from spleen, liver, and muscle as well as lung. After conjunctival administration in monkeys, the seal virus replicated to a peak titer in the conjunctivae 30-fold greater than that attained by the human virus, but this difference was not statistically significant. In contrast, the seal virus replicated less well than the human virus in the tracheae and nasopharynges when administered by the conjunctival route. These results indicate that the seal virus can replicate efficiently in primates, that it can spread systemically, and that it might differ from human virus in being able to replicate slightly better in primate conjunctival tissue.     

An evaluation of the genetic stability and pathogenicity of the Russian cold-adapted influenza A donor strains A/Leningrad/134/17/57 and A/Leningrad/134/47/57 in ferrets

The influenza A components of live attenuated vaccines used in Russia have been prepared as reassortants of the cold-adapted (ca) H2N2 viruses, A/Leningrad/134/17/57-ca (Len/17) and A/Leningrad/134/47/57-ca (Len/47), and virulent epidemic strains. The lesions responsible for attenuation within the six internal genes of each donor strain have been sequenced and described, but relatively little is known as to their stability before and after passage in susceptible hosts. In the work reported in this paper, RT-PCR restriction analysis and limited sequencing of individual genes were used to evaluate the stability of lesions in stocks of the both donor strains after passage in ferrets, which have been used widely as susceptible hosts for assessment of the virulence of influenza strains. Len/47 was shown to possess expected lesions by RT-PCR and restriction analysis. Substitution at position 1066 of the NP gene, which has been previously reported to be unique to Len/47 [Klimov et al., Virology 186 (1992) 795], was also shown to be present in all clones of Len/17. This change was confirmed by limited sequence analysis and was shown to be retained in progeny viruses isolated from the lungs and turbinates of inoculated ferrets. Two other changes in the PB2 and PB1 genes that were present in Len/47 were detected by limited sequence analysis alone. Further previously unreported minor changes were shown to be present for Len/17 and Len/47, but not both, and their significance is unknown. Limited replication of each donor strain occurred in ferrets and minimal clinical signs and histopathology were present. By contrast, the parental strain Len/57 and the recent epidemic strain A/Sydney/6/97 induced clinical signs and histopathology that were typical of influenza disease.     

Cold-Adapted Variants of Influenza A Virus: Evaluation in Adult Seronegative Volunteers of A/Scotland/840/74 and A/Victoria/3/75 Cold-Adapted Recombinants Derived from the Cold-Adapted A/Ann Arbor/6/60 Strain

Influenza A/Scotland/74 (H3N2) and A/Victoria/75 (H3N2) cold-adapted (ca) recombinant viruses, prepared by mating the A/Ann Arbor/6/60 (H2N2) ca donor virus and influenza A wild-type virus, were evaluated in adult seronegative volunteers (serum hemagglutination-inhibiting antibody titer, 相似文献   

A comparison of the biochemical properties of the human diaphorase (DIA3) isozymes determined by the common alleles DIA13, DIA23 and DIA33

(1) Various buffer systems for the starch gel electrophoresis of human diaphorase isozymes have been explored. Electrophoresis in a Tris/Borate system at pH 8.6 which includes 70 micron NADH in the gel and cathodal electrode buffers, provides good resolution of the six DIA3 phenotypes previously resolved by isoelectric focusing. (2) The variant genes DIA13, DIA23 and DIA33 occur with frequencies of about 0.76, 0.23 and 0.01 respectively in the English population. (3) The isozymes determined by the least common gene, DIA33, are markedly different from the isozymes determined by DIA13 and DIA23 in their relatively low heat stability, high affinity for Blue Sepharose and slow anodal electrophoretic mobility in buffer systems containing borate. The DIA3 1 and DIA3 2 isozymes are similar to one another in these characteristics.