丙型肝炎高变区1合成肽对免疫小鼠脾淋巴细胞增殖作用的研究

目的探讨丙型肝炎病毒(HCV)高变区1(HVR1)模拟表位7肽(7P)在体外免疫小鼠后,小鼠脾淋巴细胞的增殖情况。方法20只小鼠随机分为实验组和对照组(n=10)。实验组小鼠皮下多点注射7P(500μl/只,含量5μg),对照组皮下多点注射等量生理盐水,单溶液细胞增殖法(MTS)检测前一天脾内直接注射7P(200μl/只,含量2.5μg)加强。收集脾淋巴细胞,分别加入不同浓度(6.25、12.50、25.0、50.0、100.0μg/ml)的7P,培养24、48、72h后光镜下观察细胞生长情况,应用MTS法检测小鼠脾淋巴细胞增殖活力,并计算增殖率。结果实验组小鼠脾淋巴细胞加入7P后第2天,镜下观察见细胞生长良好,可见散在的细胞增殖集落,对照组未见明显集落出现。与对照组比较,实验组经不同浓度7P诱导后,脾淋巴细胞的增殖活性均明显增强(P<0.01)。培养72h,7P诱导浓度为12.5μg/ml时,对脾淋巴细胞的增殖具有最佳促进效果,增殖率达到184.92%。在相同诱导浓度下,7P的最佳诱导效应随培养时间的延长而增加。结论合成的HVR1模拟表位7P在体外对免疫小鼠脾淋巴细胞具有一定的增殖作用。     

HCV HVR1相关基因诱导小鼠免疫反应的比较

目的 对小鼠进行两种基因免疫方法的比较:分别以真核质粒pcDN3.1( )为载体和以伤寒减毒活菌苗为载体,携带丙肝病毒高变区1(HVRI)相关模拟表位的DNA序列,诱导细胞免疫应答,以寻找较好的疫苗免疫途径.方法 根据HCV HVR1模拟表位的肽序列合成DNA序列,并将其连接到pcDN3.1( )(pcDN3.1-SP)真核质粒上,然后用重组质粒转化伤寒减毒活菌苗Ty21a(Ty21a-SP).Ty21a-SP和peDN3.1-SP分别经口服和肌注免疫小鼠后,杀鼠、分离脾细胞,脾细胞经混合肽刺激后,用流式细胞仪检测CD8 IFN-γ 细胞;用非放射性MTS法检测细胞增殖反应;以非放射性LDH法检测CTL反应.结果 对小鼠用pcDN3.1-SP和Tyr21a-SP免疫后,取脾淋巴细胞经混合肽刺激后,明显增殖,CD8 IFN-γ 淋巴细胞比例增高,并诱导较强的CTL反应,但pcDN3.1-SP免疫后的上述反应较Ty21a-SP免疫弱.结论 使用伤寒减毒活菌苗作为载体进行基因免疫有利于产生细胞免疫反应.     

剂量和佐剂对丙型肝炎病毒脱氧核糖核酸疫苗免疫效果的影响

目的研究几种佐剂对不同剂量丙型肝炎病毒脱氧核糖核酸疫苗免疫效果的影响.方法小鼠分别用脂质体溴化二甲基双＋烷基铵/卵黄卵磷脂和氢氧化铝为佐剂的丙型肝炎病毒脱氧核糖核酸疫苗免疫3次,用酶联免疫斑点测定方法观察脾淋巴细胞受丙型肝炎病毒蛋白抗原刺激后细胞因子的产生情况.结果溴化二甲基双＋烷基铵/卵黄卵磷脂组产生γ-干扰素和白细胞介素-4较多,用核心、E2或E1/E2刺激,显著高于裸脱氧核糖核酸和氢氧化铝组（P〈0.05）.以氢氧化铝为佐剂,用每种脱氧核糖核酸100 μg剂量免疫的小鼠,在受抗原刺激后,其脾淋巴细胞产生白细胞介素-4的能力显著高于相应的裸脱氧核糖核酸组（P＜0.05）.在多数情况下,接受每种脱氧核糖核酸100 μg剂量的小鼠产生γ-干扰素和白细胞介素-4的能力显著高于接种每种50 μg者 （P〈0.05）;与裸脱氧核糖核酸组及溴化二甲基双＋烷基铵/卵黄卵磷脂组比较,氢氧化铝组小鼠淋巴细胞产生的白细胞介素-4多于γ-干扰素.结论溴化二甲基双＋烷基铵/卵黄卵磷脂对丙型肝炎病毒脱氧核糖核酸疫苗有很强的免疫佐剂效应,为Ⅰ型辅助性T细胞佐剂,氢氧化铝是Ⅱ型辅助性T细胞佐剂,可将脱氧核糖核酸疫苗Ⅰ型辅助性T细胞为主的免疫特性转换为以Ⅱ型辅助性T细胞为主,50 μg的丙型肝炎病毒脱氧核糖核酸疫苗似乎不能诱导小鼠产生有效的免疫反应.     

丙型肝炎病毒高变区1合成肽抗原性验证

设计合成丙型肝炎病毒高变区1合成肽,并验证其抗原性.应用计算机软件设计抗原肽,合成后免疫小鼠,检测小鼠血中抗体滴度;计算机模建抗原肽结构,预测其抗原性.7条合成肽中,计算机模建显示第4、5、6、7号肽具有较好的抗原性,酶联免疫吸附实验表明第4、6、7号肽可使机体产生抗体,抗原性较强,二者结果基本相符.     

人参皂苷Rg1免疫佐剂作用的研究

目的：探讨人参皂苷Rg1的免疫佐剂效应。方法：分别将人参皂苷Rg1以及氢氧化铝胶（Alum）作为佐剂和卵清白蛋白（OVA）抗原混合免疫BALB/c小鼠,二次免疫后2周采血和取脾脏,分离血清,观察免疫前后血清OVA诱导特异性抗体及亚类含量变化,以及淋巴细胞在抗原刺激下的体外细胞增殖反应。结果：Rg1和抗原混合物免疫小鼠后,未见任何不良反应;Rg1组小鼠血清抗体IgG,IgG1和IgG2a水平显著高于抗原对照组（P〈0.05）;人参皂苷Rg1免疫小鼠受刀豆蛋白A（ConA）、脂多糖（LPS）和OVA刺激产生的淋巴细胞体外增殖能力显著高于OVA组（P〈0.05）,Rg1组小鼠脾淋巴细胞受OVA刺激后培养上清检测细胞因子IL-5和IFN-γ浓度显著高于OVA组。结论：Rg1是一种能同时激活Th1和Th2型免疫反应的免疫佐剂。     

per2对睡眠剥夺前后脾淋巴细胞功能影响的初步研究

目的 探讨生物钟基因per2对72 h睡眠剥夺(SD)前后脾淋巴细胞功能的影响.方法 将C57小鼠随机分为野生型小鼠对照组(WT)和野生型小鼠睡眠剥夺组(WT-SD);per2基因敲除小鼠(per2-/-)随机分为per2基因敲除小鼠对照组(per2-/-)和per2基因敲除小鼠睡眠剥夺组(per2-/--SD).SD组小鼠使用睡眠剥夺仪进行连续睡眠剥夺72 h.小鼠麻醉后摘脾,使用淋巴细胞分离液分离获得淋巴细胞悬液,利用流式细胞术检测各组别CD4+T、CD8+T、CD3+T以及B220+B比例变化,并通过脂多糖(LPS)刺激3 d体外培养进行流式染色检测上述各淋巴细胞的比例变化,同时使用CCK-8法检测各组小鼠脾B细胞的增殖情况.结果 与WT组小鼠相比,per2-/-组小鼠脾CD4+T、CD8+T以及B220+B细胞比例无明显改变;在进一步的睡眠剥夺实验中发现,per2基因敲除影响T细胞,尤其是CD8+T的组成变化.在LPS刺激实验中,与WT组相比,SD组小鼠脾淋巴细胞增殖更加明显,且与WT-SD组相比,per2-/--SD组B220+B细胞比例增加.同样睡眠剥夺应激条件下,小鼠脾淋巴细胞增殖无明显变化,但与WT-SD组相比,per2-/--SD组脾B细胞降低同时对应T细胞比例升高.结论 per2对脾淋巴细胞的组成影响较小,但在一定程度上参与调节睡眠剥夺后B淋巴细胞对LPS的增殖反应.     

中药蒲公英对小鼠脾淋巴细胞增殖的影响

目的　观察中药蒲公英对小鼠脾淋巴细胞增殖的影响。方法　利用氢化可的松导致小鼠免疫异常 ,观察蒲公英对小鼠脾淋巴细胞增殖的影响。结果　小鼠服用蒙古及碱地蒲公英的水煎剂 2 0g(生药 ) /kg后 ,可明显对抗氢化可的松所致的免疫抑制作用 ;两者对小鼠脾T淋巴细胞有显著的增殖作用 ,与模型组相比有显著性差异 (P <0 .0 1) ,而对B淋巴细胞虽有一定的增加 ,但无显著性差异 (P >0 .0 5 )。结论　蒲公英具有明显增加小鼠脾淋巴细胞增殖的作用 ,对氢化可的松所致的免疫抑制有保护作用 ,主要作用于T淋巴细胞 ,可增强细胞免疫功能。     

普氏立克次体120×103表面抗原的N端和C端重组蛋白免疫原性的研究

目的:评价普氏立克次体120×103表面抗原的N端和C端重组蛋白的免疫原性。方法:将纯化的N端和C端重组蛋白分别免疫小鼠,采用酶联免疫吸附试验和间接免疫荧光试验分析免疫小鼠的体液免疫应答水平;用MTT法评价小鼠脾淋巴细胞的增殖情况,分析免疫小鼠的细胞免疫应答水平,并采用RT-PCR方法检测免疫小鼠脾淋巴细胞被重组蛋白刺激后细胞因子的表达。结果:N端和C端重组蛋白均诱导小鼠产生较高水平的特异性IgG抗体,诱导高水平的小鼠脾淋巴细胞增殖;经两种重组蛋白刺激的小鼠脾淋巴细胞均表达了IFN-γ和IL-2。结论:普氏立克次体120×103表面抗原的N端和C端重组蛋白具有良好的免疫原性,能够诱导机体产生高水平的体液免疫和细胞免疫应答,可作为流行性斑疹伤寒亚单位疫苗的候选蛋白。     

猴头菌多糖促进同基因骨髓移植小鼠免疫功能重建

同基因骨髓移植55d后,受体小鼠免疫功能严重缺损,脾细胞产生IL—2的能力等细胞免疫功能明显低下。经腹腔连续注射猴头菌多糖15d后,小鼠脾细胞产生IL—2的能力,对ConA刺激的增殖反应和混合淋巴细胞反应均显著增强;抗羊红细胞抗体(IgM)分泌细胞数明显增加。实验结果提示:猴头菌多糖能明显促进同基因骨髓移植小鼠免疫功能的早期恢复。     

重组免疫毒素hIL-2-LuffinP1对淋巴细胞体外增殖的影响

目的检测基因工程免疫毒素hIL-2-LuffinP1的体外生物学活性。方法采用分离的C57和BALB/c小鼠脾细胞进行混合淋巴细胞反应,刀豆球蛋白A(ConA)刺激BALB/c小鼠脾细胞进行淋巴细胞增殖实验,以LuffinP1毒素分子作为对照。3H-TdR核素掺入法检测hIL-2-LuffinP1对淋巴细胞增殖的抑制作用。以CTLL-2细胞检测hIL-2-LuffinP1对IL-2依赖细胞株的杀伤作用,实验共设6组:PBS、IL-2、LuffinP1、LuffinP1 IL-2、hIL-2-LuffinP1、hIL-2-LuffinP1 IL-2,CTLL-2细胞加入各组试剂培养12h,台盼蓝染色,计数每100个细胞中的死亡细胞数。结果随剂量增加,hIL-2-LuffinP1对淋巴细胞增殖的抑制作用逐渐增强,在10-6mol/L浓度时,抑制率可达到97%,而LuffinP1对淋巴细胞增殖无明显抑制作用。hIL-2-LuffinP1对Con A刺激的脾细胞活化也有抑制作用,与在混合淋巴细胞体系中一致,而LuffinP1对上述两个反应体系中的淋巴细胞增殖均无明显抑制作用。加入hIL-2-LuffinP1 IL-2和hIL-2-LuffinP1后CTLL-2细胞的死亡率分别为29.6%和47.4%,明显高于IL-2组(5.6%,P<0.01)。结论重组免疫毒素hIL-2-LuffinP1在体外能够抑制淋巴细胞增殖,对表达IL-2R的活化淋巴细胞产生定向杀伤作用。     

The late effects of radiation on lifespan,lymphocyte proliferation and p53 haplodeficiency in mice

Purpose:?We investigated the effect of irradiation on the lifespan of eight-week-old mice, the number of lymphocytes in bone marrow and the levels of p53 protein expression in the splenocytes.

Methods and materials:?Eight-week-old mice, wild-type p53 (p53^+/+) and heterozygous p53 (p53^+/?), were irradiated with 3 Gy. The cell numbers and cell cycle phases of bone marrow cells were determined by flow cytometry. The splenocyte proliferation was evaluated by a fluorescent cell viability assay. The p53 expression was evaluated by Western blotting.

Results:?The lifespan of the irradiated mice was shorter than that of the non-irradiated mice. In irradiated 72-week-old p53^+/+ mice and 56-week-old p53^+/? mice, the number of lymphocytes in bone marrow decreased as compared to that in the non-irradiated mice. In 56-week-old p53^+/? mice, the S- and G2/M-phases of lymphocytes in the irradiated mice were increased compared to that in the non-irradiated mice. The splenocyte proliferation in p53^+/+ mice decreased with age, and the proliferation in the irradiated mice was much lower than that in the non-irradiated mice. In 72-week-old p53^+/+ mice after re-irradiation, the p53 protein expression in the splenocytes of the irradiated mice was delayed as compared to those from the non-irradiated mice.

Conclusion:?We suggest that the decrease in the number of lymphocytes in bone marrow and the delayed p53 expression in splenocytes from the irradiated mice are related to the shortened lifespan after irradiation at a young age.     

新生小鼠视网膜光感受器前体细胞的体外培养及鉴定

目的 对新生小鼠视网膜光感受器前体细胞进行分离和鉴定.方法同窝新生小鼠14只,随机分为7组(P1-P7),每组2只,出生后连续7d每天取1组幼鼠,分离视网膜并进行体外培养,观察其生长状况.传代后培养2周,然后采用细胞荧光免疫组化法检测细胞中的5-溴-2'脱氧尿嘧啶(BrdU)、细胞神经上皮干细胞蛋白(Nestin)、神经视网膜亮氨酸拉链(Nrl)和视蛋白(Opsin)的表达情况,并对视网膜光感受器前体细胞性质进行分析.结果 在特定的培养基中,部分P1-P7各组新生小鼠视网膜细胞贴壁生长,荧光免疫组化标记显示传代后生长细胞大部分为BrdU阳性细胞.P1-P7组Nestin阳性率分别为31.0%、31.6%、32.3%、30.2%、31.2%、30.9%、29.5%.Nrl阳性细胞数出生后3d开始明显增多,随后小幅增加,P1-P7组阳性率分别20.6%、35.2%、65.5%、68.6%、71.6%、73.O%、73.3%.P1-P4组细胞不表达Opsin,P5-P7组少量细胞表达Opsin.结论 新生小鼠视网膜存在视网膜光感受器前体细胞,该细胞具有分裂增殖能力及向视网膜光感受器细胞分化和表达视蛋白的潜能,并于生后3d明显增多,此后逐渐分化为成熟的光感受器细胞.     

OK-432增加肿瘤局部IL-12阳性细胞浸润

目的 :探讨溶血链球菌制剂OK - 432的抗肿瘤机理。方法 :B16黑色素瘤细胞接种于C5 7BL/ 6小鼠皮下 ,3d后腹腔注射1KE的OK - 432 ,每周 1次 ,连续 3周。观察肿瘤生长体积 ,并采用ELISA法和ABC免疫染色法分别测定OK - 432治疗后荷瘤小鼠脾细胞IL - 12的分泌及肿瘤局部IL - 12阳性细胞的表达。结果 :OK - 432治疗能显著抑制B16黑色素瘤的生长 (P <0 .0 5 ) ;刺激荷瘤小鼠脾细胞IL - 12的分泌 (P <0 .0 5 ) ;诱导肿瘤局部IL - 12阳性细胞的表达。结论 :OK - 432可刺激荷瘤小鼠脾细胞IL - 12的分泌 ;增加肿瘤局部IL - 12阳性细胞的浸润 ,从而增强宿主的抗肿瘤免疫功能     

Increased (18)F-FDG uptake in a model of inflammation: concanavalin A-mediated lymphocyte activation.

The aim of this project was to study a mechanism that might explain the increased uptake of (18)F-labeled FDG seen in inflammation. The approach chosen was to examine the effect on (18)F-FDG uptake of acute activation of murine lymphocytes by concanavalin A (Con A). METHODS: Immunocompetent BALB/c mice and nude mice received an intravenous injection of 10 mg/kg Con A. Twenty-four hours later, the mice received an intravenous injection of 0.74 MBq (20 microCi) (18)F-FDG. One hour later, biodistribution was determined. The distribution of the radiolabel in the liver was also evaluated by autoradiography. In vitro (18)F-FDG uptake to splenocytes from BALB/c mice with and without Con A pretreatment were determined 30, 60, and 120 min after the splenocytes were mixed with (18)F-FDG (0.74 MBq [20 microCi]/200 microL). RESULTS: In immunocompetent BALB/c mice, pretreatment with Con A significantly increased (18)F-FDG uptake in the spleen and liver. Autoradiographs of the liver showed that pretreatment with Con A produced a specific localization of (18)F-FDG at periportal areas, where numerous sites of cellular infiltration were observed. In vitro binding of (18)F-FDG to the splenocytes was significantly higher for Con A-pretreated BALB/c mice than for control mice. CONCLUSION: This study showed that Con A increased (18)F-FDG uptake. Con A-activated lymphocytes actively took up (18)F-FDG both in vitro and in vivo, and (18)F-FDG specifically accumulated in Con A-mediated acute inflammatory tissues.     

多次低剂量照射对雄性糖尿病大鼠脾细胞凋亡和免疫因子的影响

目的 观察多次低剂量辐射(LDR)对糖尿病(DM)大鼠脾细胞凋亡和免疫因子的影响。方法 大鼠随机分为对照组、DM组和DM+LDR组;剂量分别为25、50和75 mGy,共照射15次;照射后4周,采用流式细胞术检测脾细胞凋亡和TCRα β百分数的变化,酶联免疫吸附法(ELISA)检测血清和脾细胞培养上清IL-2含量的变化。结果 与对照组相比,DM和DM + LDR两组大鼠体重均下降,尤以DM组明显。DM+LDR组大鼠血糖水平虽明显高于对照组(t₂₅=23.321、 t₅₀=18.329、 t₇₅=9.23,P<0.01),但显著低于DM组(t₂₅=3.574、 t₅₀=4.593、 t₇₅=5.577,P< 0.01)。同时发现:与对照组相比,DM+LDR各组脾细胞凋亡增加,其中DM+50 mGy组增加明显(t₅₀=4.102,P<0.01)。血清IL-2含量也增加,但是差异均无统计学意义。脾细胞培养上清IL-2含量明显下降(t₂₅=7.778、 t₅₀=7.411、 t₇₅=8.325,P<0.01)。与DM组相比,DM+LDR各组脾细胞凋亡和TCRα β百分数均明显降低(凋亡:t₂₅=4.772、 t₅₀=3.346、 t₇₅=6.778;TCRα β:t₂₅=3.381、 t₅₀=5.807、 t₇₅=2.356,P<0.05～P<0.01)。血清IL-2含量呈下降趋势;脾细胞培养上清IL-2含量均有升高趋势。结论 多次LDR能够削弱糖尿病造成的大鼠体重减轻和血糖升高,降低糖尿病所致的脾细胞凋亡,并能调节脾脏免疫因子,改善其失衡状态。     

Augmentation in mitogen-induced proliferation of rat splenocytes by low dose whole body X-irradiation

The hypothesis of radiation hormesis has been proposed. To elucidate the hormetic effect on the immune system, we studied the effect of low dose whole body irradiation on the in vitro mitogen-induced proliferation of rat thymocytes and splenocytes. The rats were irradiated with low doses (0.01-2 Gy) of X-ray and the cells were cultivated in the presence of various mitogens. The cell proliferation was evaluated by the incorporation of 3H-thymidine into the cells. Concanavalin A (Con A)-induced proliferation of splenocytes prepared at 4 hr after irradiation was augmented with 0.05 Gy, whereas that of thymocytes was not affected. Irradiation of rats with 0.05 Gy also induced the enhanced proliferation of splenocytes stimulated by phytohemagglutinin or lipopolysaccharide, though their responses were lower than that by Con A. This augmentation in mitogen-induced proliferation of splenocytes was observed within a few hours after irradiation, being a temporary effect. These results suggest that very low dose whole body irradiation possibly induce a hormesis-like effect on the immune splenocytes.     

Possible role of elevation of glutathione in the acquisition of enhanced proliferation of mouse splenocytes exposed to small-dose gamma-rays

PURPOSE: To examine the relation between the induction of an increased glutathione level and the elevated proliferative response of mouse splenocytes by a small dose of gamma-rays. MATERIALS AND METHODS: Male ICR strain mice, 7 weeks of age, were divided into irradiated and non-irradiated control groups. Irradiation was done with gamma-rays from a 137Cs source at a dose of 50 cGy (1.11 Gy/min). Glutathione content in the splenocytes was measured using a modified spectrophotometric technique. Concanavalin A (Con A)-induced proliferative response of the splenocytes after whole-body gamma-ray irradiation was estimated from the 3H-thymidine incorporation into the cells. RESULTS: The glutathione level in mouse splenocytes increased 2 h after whole-body y-ray irradiation at 50cGy, peaked at 4h and thereafter decreased almost to the zero-time level by 12-h postirradiation. A significant enhancement of Con A-induced proliferation was observed in the splenocytes obtained from the whole-body-irradiated animals between 2h and 6h post-irradiation. Glutathione exogenously added to splenocytes obtained from normal mice enhanced the Con A-induced proliferation of splenocytes in a dose-dependent manner. This enhancement was completely blocked by buthionine sulfoximine, a specific inhibitor of the de novo pathway of glutathione synthesis. CONCLUSIONS: The induction of endogenous glutathione immediately after low-dose gamma-ray irradiation is at least partially responsible for the enhancement of immune function, and may throw light on the mechanisms of carcinostatic effects induced by low dose ionizing radiation.     

Possible role of elevation of glutathione in the acquisition of enhanced proliferation of mouse splenocytes exposed to small-dose γ-rays

Purpose : To examine the relation between the induction of an increased glutathione level and the elevated proliferative response of mouse splenocytes by a small dose of γ-rays. Materials and methods : Male ICR strain mice, 7 weeks of age, were divided into irradiated and non-irradiated control groups. Irradiation was done with γ-rays from a 137 Cs source at a dose of 50cGy (1.11Gy/min). Glutathione content in the splenocytes was measured using a modified spectrophotometric technique. Concanavalin A (Con A)-induced proliferative response of the splenocytes after whole-body γ-ray irradiation was estimated from the 3 H-thymidine incorporation into the cells. Results : The glutathione level in mouse splenocytes increased 2h after whole-body γ-ray irradiation at 50 cGy, peaked at 4h and thereafter decreased almost to the zero-time level by 12-h postirradiation. A significant enhancement of Con A-induced proliferation was observed in the splenocytes obtained from the wholebody-irradiated animals between 2h and 6h post-irradiation. Glutathione exogenously added to splenocytes obtained from normal mice enhanced the Con A-induced proliferation of splenocytes in a dose-dependent manner. This enhancement was completely blocked by buthionine sulfoximine, a specific inhibitor of the de novo pathway of glutathione synthesis. Conclusions : The induction of endogenous glutathione immediately after low-dose γ-ray irradiation is at least partially responsible for the enhancement of immune function, and may throw light on the mechanisms of carcinostatic effects induced by low dose ionizing radiation.