乙脑病毒prME蛋白与BALB/c鼠IgG Fc段编码基因联合构建DNA免疫研究

目的 研究IgG Fc编码基因对流行性乙型脑炎(Japanese encephalitis,JE)DNA疫苗免疫增强效应的影响.方法 巢式RT-PCR法从BALB/c鼠脾组织获取IgG Fc段编码基因,用限制性内切酶从含流行性乙型脑炎病毒(Japanese encephalitis virus,JEV)prME蛋白基因重组子获取prME蛋白基因,分别插入同-真核表达载体pcDNA3.1(+)不同酶切位点,构建蘑组子pJME/IgG Fc并经酶切及DNA测序分析.脂质体法将pJMrY/IgG Fc转染CHO细胞.免疫荧光、Western blot法检测转染的CHO细胞中融合蛋白分布与表达.将pJME/IgG Fc肌注免疫BALB/c鼠,检测小鼠脾特异性细胞毒T细胞(CTL)杀伤活性和中和抗体滴度.结果 pJME/IgG Fc经BamH Ⅰ/EcoR Ⅰ和BamH Ⅰ/Not Ⅰ酶切释出的插入子大小,(2001 bp,2730 bp)分别与预期结果相符合.所编码的融合蛋白相对分子质量(Mr)为101×103,主要分布于胞浆,少最分布于胞膜,pJME/IgG Fc转染CHO细胞经32次传代仍可表达融合蛋白.pJME/IgGFc免疫组中和抗体滴度与CTL活性较pJME及灭活疫苗组均升高(P<0.05).结论 pJME/IgG Fc成功构建,转染的CHO细胞可稳定表达融合蛋白,IgG Fc段编码基因能够增强JEV DNA疫苗的细胞和体液免疫应答.     

HPV多肽致敏的树突状细胞体外激发特异性CTL能力的研究

目的 观察以高危型人乳头瘤病毒16型(HPV16) E6和E7基因编码的多肽E613-21(KLPDLCTEL)和E786-94(TLGIVCPI)为抗原负载的树突状细胞(DC)体外诱导特异性CTL的能力.方法 采集HPV+HLA-A2+宫颈癌患者外周血,分离单核细胞(Mo)与外周淋巴细胞(PBL);将Mo诱导成DC后负载E6和E7多肽,用其反复致敏PBL(3次);ELISA法检测致敏细胞上清细胞因子分泌水平;流式细胞标记法检测致敏细胞中特异性CTL的比例;MTT法检测致敏细胞杀伤靶细胞的能力.结果 11例HPV 16+ HLA-A2+宫颈癌患者DC平均得率为(10.79±0.88) ×l06(每100 ml外周血),CD11c+ HLA-DR+为(97.15±2.41)％,其中CD80+为(84.28±5.39)％,CD83+为(85.17+5.06)％,CD86+为(97.74±0.87)％.PBLs致敏3次后,平均增殖(15.4±1.5)倍;致敏第21天上清细胞因子水平分别为:IL-2(2551.9±195.3) pg/ml、IL-12(554.9士64.0)pg/ml、IFN-γ(2416.9±281.7)pg/ml,TNF-α(632.4±71.1) pg/ml,明显高于未致敏组(P＜0.05),IL-10(235.1+34.7) pg/ml与对照组比较无显著差异;特异性CTL的平均比例为(6.32±1.54)％,明显高于对照组(P＜0.05),对负载E6、E7多肽的T2细胞株杀伤率明显高于对照组(P＜0.05).结论 HPV E6和E7混合多肽负载的DC体外能有效地诱导特异性CTL,刺激Thl型细胞因子的分泌,为治疗型宫颈癌疫苗的研制提供科学依据.     

荷载人生存素的多表位树突状细胞疫苗的抗肿瘤活性研究

目的:观察荷载人生存素(survivin)的多表位树突状细胞(DC)疫苗的抗肿瘤活性.方法:分别将含4个sur-vivin的HLA-A2类限制性CD8+ CTL表位和1个CD4+ Th细胞表位的重组真核表达质粒pPIRESneo3.0-survivin(4)/Th,以及含4个CD8+ CTL表位的重组质粒pPIRESneo3.0-survivin (4),转染人DC并制备DC疫苗.实验分为survivin(4)/Th组、survivin (4)组、空质粒组、未转染DC与T细胞共培养组和单独T淋巴细胞组.DC疫苗作用后,MCF-7细胞的凋亡率明显高于survivin(4)组(P＜0.05),亦明显高于空质粒组、未转染DC与T细胞共培养组和单独T淋巴细胞组(P＜0.05).运用流式细胞术(FCM)分别检测DC表面CD83、CD86、T淋巴细胞表面CD4、CD8a的表达以及DC疫苗作用后MCF-7乳腺癌细胞的凋亡.用ELISA法检测上清中IFN-γ的含量.用四甲基偶氮唑蓝(MTT)比色法检测DC疫苗诱导的CTL对MCF-7细胞的抑制率.结果:流式细胞术检测显示,人DC高表达CD83、CD86;人外周血T淋巴细胞高表达CD4、CD8a;survivin (4)/Th组IFN-γ的含量[(66.50±3.34) ng/L]明显高于survivin(4)组[(46.10±1.35)ng/L]、空质粒组[(25.17±0.32) ng/L]、未转染DC与T细胞共培养组[(25.47±0.95)ng/L]和单独T淋巴细胞组[(23.73 +0.50)ng/L],P＜0.05.survivin (4)/Th组中MCF-7细胞的抑制率明显高于survivin (4)组、空质粒组、未转染DC与T细胞共培养组和单独T淋巴细胞组(P＜0.05).DC疫苗作用后,MCF-7细胞的凋亡率[ (10.63±0.29)％]明显高于survivin(4)组(P＜0.05),亦明显高于空质粒组、未转染DC与T细胞共培养组和单独T淋巴细胞组(P＜0.05).结论:荷载多个survivin的CD8+ CTL表位的树突状细胞肿瘤疫苗具有很强的抗肿瘤活性,CD4+ Th细胞对CD8+ CTL的抗肿瘤方面有明显的促进作用.     

MUC1模拟表位肽致敏DC治疗宫颈癌荷瘤小鼠

目的 研究黏蛋白1(MUC1)模拟表位肽致敏树突状细胞(DC)对宫颈癌荷瘤小鼠生长和T淋巴细胞亚群的影响。方法 培养小鼠骨髓细胞，诱导并刺激DC成熟，将MUC1模拟表位肽加入成熟DC，形成负载MUC1模拟表位肽的DC。建立稳定表达MUC1的宫颈癌U14细胞株荷瘤BALB/c小鼠模型。空白对照组注射0.2 mL生理盐水；DC组注射2×10⁵个未加MUC1模拟表位肽的DC;DC+MUC1组注射2×10⁵个经过负载MUC1模拟表位肽的DC。免疫1周后，进行二次免疫。结果 肿瘤体积与治疗前比较，空白对照组、DC组和DC+MUC1组均升高(P<0.05)；治疗前，各组间比较差异无统计学意义(P>0.05)；二次免疫1周后，DC+MUC1组低于空白对照组和DC组(P<0.05)，空白对照组和DC组间差异无统计学意义(P>0.05)；小鼠抑瘤率DC组和DC+MUC1组均高于空白对照组(P<0.05),DC+MUC1组高于DC组(P<0.05)；小鼠外周血CD3⁺、CD4⁺、CD...     

登革病毒抗原肽免疫小鼠诱导特异性CD4^＋ T细胞反应

目的:探讨4条登革病毒抗原肽在不同遗传背景小鼠中的免疫原性.方法:4条登革病毒抗原肽(C45-57KLVMAFIAFLRFL,E396-408SSIGKMFEATARG,NS323-35YRILQRGLLGRSQ 和 NS3141-155NREGKIVGLYGNGVV) 中每条肽分别免疫BALB/c小鼠和C57BL/6小鼠;3周后,处死小鼠并制备脾细胞悬液;不刺激或同样抗原肽刺激脾细胞后,采用细胞内细胞因子染色流式细胞术(ICS) 检测小鼠脾细胞CD4+ T细胞中肽特异性产生IFN-γ或IL-4的CD4+ T 细胞的百分比.结果:肽C45-57可诱导BALB/c小鼠产生特异性的IFN-γ+ CD4+ T细胞(0.72%±0.04% vs 0.04%±0.02%,P＜0.05)而肽E396-408 则诱导产生特异性的IL-4+ CD4+ T细胞(0.09%±0.01% vs 0.01%±0.01%,P＜0.05);肽E396-408、NS323-35和NS3141-155均可诱导C57BL/6小鼠产生特异性的IFN-γ+ CD4+ T细胞(分别为0.31%±0.03% vs 0.02%±0.01%,P＜0.05;0.21%±0.03% vs 0.04%±0.01%,P＜0.05;0.44%±0.04% vs 0.02%±0.01%,P＜0.05),而肽C45-57可诱导产生特异性的IL-4+ CD4+ T细胞(0.45%±0.05% vs 0.02%±0.02%,P＜0.05).结论:肽C45-57和E396-408在BALB/c小鼠中具有免疫原性而肽C45-57、E396-408、NS323-35和NS3141-155在C57BL/6小鼠中具有免疫原性.     

重组疫苗E.coli LLO/OVA对小鼠CD4^＋ CD25^＋ Treg细胞调节作用的研究

目的:探讨重组E.coli LLO/OVA对小鼠CD4+CD25+Treg细胞的调节作用.方法:E.coli LLO/OVA和E.coli OVA分别免疫小鼠后,磁珠分离脾脏CD11c、CD4+CD25+Treg和CD4+CD25-T细胞,比较两组CD11c细胞对CD4+CD25+Treg细胞分泌IL-10的影响,以及CD4+CD25+Treg细胞抑制CD4+CD25-T细胞增殖的作用;流式细胞术分析荷瘤小鼠OVA特异性CD8+T细胞比率,观察去除CD4+CD25+Treg细胞前后两组黑色素瘤B16-OVA荷瘤小鼠肺转移情况.结果:E.coli LLO/OVA免疫组小鼠脾脏CD4+CD25+Treg细胞产生IL-10水平明显低于E.coli OVA免疫组小鼠(P＜0.05),CD4+CD25+Treg细胞对CD4+CD25-T 细胞增殖的抑制作用明显减弱(P＜0.05),且OVA特异性CD8+T细胞数量明显增多(P＜0.05).在去除CD4+CD25+Treg细胞前后,E.coli LLO/OVA免疫的荷瘤小鼠肺转移结节数无明显减少(P＞0.05).结论:重组E.coli LLO/OVA可通过下调小鼠CD4+CD25+Treg细胞数量、抑制其功能而促进机体特异性抗肿瘤免疫.     

卵清蛋白免疫耐受因子转移免疫耐受活性的研究

目的:探讨卵清蛋白免疫耐受因子(OVA immune tolerance factors,OVA ITFs)转移抗原特异性外周免疫耐受的特性.方法:分别从OVA耐受鼠或空白小鼠的脾淋巴细胞裂解液中分离分子量小于3 kD的组分,即为OVA ITFs或OVA ITFscontorol.试验BALB/c小鼠分成5组,A、B、C、D组经尾静脉分别注射OVA ITFs、OVA ITFs control、OVA耐受鼠脾淋巴细胞或PBS给正常BALB/c小鼠,E组不做任何处理.流式细胞术检测转移前后受体鼠脾脏CD4+CD25+T细胞亚群的比例变化,MTT法检测受体鼠OVA特异性T淋巴细胞的增殖情况,夹心ELISA法测定培养上清中IL-10、TGF-β1的水平.结果:转移OVA ITFs或OVA耐受鼠脾淋巴细胞后,受体鼠脾脏CD4+CD25+T细胞亚群在总CD4+T细胞的比例分别为(15.32±1.03)%和(15.35±0.62)%,与转移前(9.97±1.38)%相比显著上升(P＜0.05);OVA特异性淋巴细胞增殖刺激指数(Stimulation Index,SI)分别为0.699±0.05和0.704±0.03,与PBS对照组(1.356±0.07)相比明显受到抑制(P＜0.05);培养上清中TGF-β1的分泌量分别为129.15±6.14和106.71±2.89 pg/ml,显著高于PKS对照组(52.82±3.68 pg/ml,P＜0.01),IL-10分泌量未检出.转移OVA ITFscontrol后,受体小鼠的CD4+CD25+T细胞比例、OVA特异性淋巴细胞增殖及TGF-β1、IL-10分泌量均无显著变化.结论:OVAITFs具有将OVA特异性免疫耐受传递给正常受体小鼠的特性.     

补体调节蛋白Crry对树突状细胞的调控及诱导同种移植免疫低反应

目的:探讨补体调节蛋白Crry对树突状细胞(DC)的调节作用及诱导同种移植免疫低反应性的机制。方法:分离BALB/c小鼠的骨髓来源树突状细胞,试验分为2组:①脂多糖(LPS)刺激组,即培养结束前加入外源性脂多糖(1 mg/L)刺激24小时;②Crry处理组,加入抗小鼠Crry抗体5μg/ml,结束前加入外源性脂多糖(1 mg/L)刺激24小时,流式细胞仪检测树突状细胞表面分子CD86、CD40、MHCⅡ的表达,ELISA法检测其细胞上清液中IFN-γ、IL-10和IL-12的水平,ELISA法检测细胞上清液中C3、C5、C3a和C5a的含量,并以BALB/c小鼠的DC作为刺激细胞,以C57BL/6小鼠的淋巴细胞作为反应细胞进行同种混合淋巴细胞培养,MTT法检测淋巴细胞增殖活性。结果:经LPS刺激后,培养的DC表面共刺激分子CD40、CD86和MHCⅡ分子的表达显著增加(P<0.05);Crry处理组DC表面共刺激分子CD86和MHCⅡ分子的表达较LPS刺激组显著降低(P<0.05),而CD40的表达无显著性差异;Crry处理组培养上清液中C3和C5的含量无明显变化,但C3a、C5a的含量较LPS刺激组显著减少(P<0.05);培养DC上清液中IFN-γ和IL-12的含量较LPS刺激组显著降低(P<0.05),IL-10的含量较LPS刺激组显著增加(P<0.05);培养的DC加入Crry抗体后,其淋巴细胞增殖活性显著降低(P<0.05)。结论:补体调节蛋白Crry可以对DC具有重要的调控作用,可以影响其共刺激分子的表达、补体的生成以及细胞因子的合成等,从而诱导同种移植免疫低反应性,丰富先天免疫对获得性免疫的调节作用。     

MUC1模拟表位肽致敏DC对宫颈癌荷瘤小鼠肿瘤生长及免疫微环境的影响

目的：研究黏蛋白1(MUC1)模拟表位肽致敏树突状细胞(DC)对宫颈癌荷瘤小鼠生长和免疫微环境的影响。方法：培养小鼠骨髓细胞,诱导并刺激DC成熟,MUC1模拟表位肽处理构建负载MUC1模拟表位肽的DC。构建稳定表达MUC1的宫颈癌U14细胞株的荷瘤BALB/c小鼠模型,随机分为3组：空白对照组(注射0.2 ml生理盐水)、DC组(注射2×10⁵个未加MUC1模拟表位肽的DCs)和DC+MUC1组(注射2×10⁵个负载MUC1模拟表位肽的DCs),免疫1周后进行二次免疫。治疗前和二次免疫1周后测量肿瘤体积;二次免疫1周后取小鼠肿瘤组织称重并计算抑瘤率;取小鼠外周血,流式细胞术检测CD3⁺T、CD4⁺T、CD8⁺T细胞、CD4⁺CD25⁺Foxp3⁺Treg水平;ELISA检测小鼠血清IL-6、IL-10、TGF-β、VEGF浓度。结果：与治疗前比较,空白对照组、DC组和DC+MUC1组肿瘤体积增大(P<0.0...     

D-半乳糖所致衰老小鼠抗原提呈细胞的改变及其意义

目的:研究D-半乳糖所致衰老小鼠专职性抗原提呈细胞的改变及其意义。方法:取健康昆明雌性小鼠,2月龄,运用D-半乳糖建立衰老小鼠模型并进行生物学鉴定后,采用免疫荧光和流式细胞术测定脾脏中巨噬细胞CD11b、树突状细胞CD11c、B淋巴细胞CD40的表达;采用免疫组化法测定脾脏组织Fas表达的改变。结果:模型组小鼠巨噬细胞CD11b+/MHC-Ⅱ+CD11b+的百分率为9.78±1.42,与对照组10.76±1.93相比下降(P<0.05);模型组小鼠树突状细胞CD11c+/MHC-Ⅱ+CD11c+、B淋巴细胞CD40+的百分率分别为5.34±0.74、43.60±8.06,与对照组7.88±1.73、56.70±10.97相比明显下降(P<0.01)。模型组小鼠脾脏组织Fas表达增加。结论:D-半乳糖所致衰老小鼠脾脏各种专职性抗原提呈细胞减少,脾脏组织Fas的表达出现渐进性增加。     

Recovery of responsiveness of cells of lateral geniculate nucleus of rat

1. Recovery of responsiveness of single cells in lateral geniculate nucleus of rat has been determined in both P and I cells. There are three types of recovery curve among P cells; (a) early recovery, (b) early partial recovery followed by depression and then complete recovery, (c) prolonged depression followed by cyclic recovery. Type (c) is by far the commonest recovery curve. In contrast to the spike in a P cell, the synaptic potential recovers to its full amplitude in about 20 msec. All I cells exhibit similar rapid recovery curves after a prolonged depression.2. Conditioning stimuli applied to visual cortex also produce a prolonged depression in most P cells but I cells can be re-excited at short intervals from cortex. Decortication does not prevent the prolonged depression of the multineuronal response produced by optic nerve stimulation.3. A neuronal model is proposed to explain these observations. It is supposed that I cells (interneurones) are innervated by axon collaterals of the P cells (principal cells, projecting to visual cortex) and that the I cells exert an inhibitory influence on the P cells.     

Modes of Inheritance of Errors of Refraction

Eighteen families in which both parents had refractions within the range of +4·0 D to −4·0 D and axial lengths seen in emmetropia (22·3-26·0 mm) showed coefficients of correlation of the order 0·5 indicative of polygenic inheritance. Such coefficients were seen for axial length (0·407) and for the cornea (0·487), but not for the lens (which is known to be yoked to the axial length). No such coefficients were seen in 19 families in which one of the parents had axial length outside the emmetropic range (nine families with long axes and 10 with short axes).

The pattern of polygenic inheritance for emmetropia (completely correlated optical components) and errors of refraction up to 4·0 D (inadequately correlated components: correlation ametropia) follows that seen in stature and other measurable characters. In contrast the high refractive errors with their abnormal axial lengths (component ametropia) are—like the extremes in stature—pathological anomalies with monofactorial inheritance.

     

Level of functioning of siblings and parents of probands of varying degrees of retardation

A further analysis of already published data supports the position that retardates of low ability level less frequently have retarded siblings, retarded parents, and parents low in occupational level than do retardates higher in ability level. The analysis supports the position that there are two types of retarded individuals, persons retarded as a result of gene or chromosomal anomalies, brain injury, etc., who more frequently occur in the lower-level retardate group, and persons whose retardation represents polygenic segregation, who more frequently occur in the higher-level group.     

Analysis of two types of dopaminergic responses of neurons of the spinal ganglia of rats

It was established, in experiments on isolated spinal ganglia of adult rats in concluons of intracellular recording, that dopamine (1 M/liter) elicits depolarized responses in 61% of neurons, hyperpolarized in 20% of neurons, and depolarized-hyperpolarized in 19% of neurons. The depolarized responses are associated with the activation of D₁ dopamine receptors, and are governed by the shift of cAMP-dependent cation (sodium) channels to the conducting state. The hyperpolarized responses are triggered by the activation of D₂ dopamine receptors, which by means of HTP-binding protein convert the potassium channels to the conducting state. The change in the polarization of neurons with the action of dopamine influences their electrical excitability variously.Translated from Fiziologicheskii Zhurnal SSSR imeni I. M. Sechenova, Vol. 76, No. 6, pp. 739–745, June, 1990.