血小板冻干再水化的形态结构与凋亡

目的探讨冷冻干燥法对血小板的细胞形态、超微结构和细胞凋亡的影响。方法以新鲜血小板为对照,以保存30d后再水化的冻干血小板为实验对象,分别在倒置相差显微镜和透射电镜下观察血小板的细胞形态和超微结构,用流式细胞仪（FCM）检测血小板凋亡率,并以1×10^3U/L凝血酶为诱导剂检测再水化血小板的聚集活性。结果与新鲜血小板相比,冻干再水化后的血小板在形态上为圆盘状,未见有明显的细胞聚集,仅细胞透光性相对较弱;超微结构上也无明显变化,其内部基本结构清晰可辨,细胞膜、细胞器及分泌颗粒的包膜保持完整。新鲜血小板的细胞早期凋亡率为（0.44±0.19）%,显著低于冻干血小板的细胞凋亡率（3.25±1.68）%（P〈0.05）。冻干再水化血小板的最大聚集率为（93.28±2.3）%。结论冻干再水化的血小板形态结构完整,细胞内超微结构完好,为本实验室成功建立血小板冻干保存方法提供了依据。     

血小板冻干再水化的形态结构与凋亡

目的 探讨冷冻干燥法对血小板的细胞形态、超微结构和细胞凋亡的影响.方法 以新鲜血小板为对照,以保存30 d后再水化的冻干血小板为实验对象,分别在倒置相差显微镜和透射电镜下观察血小板的细胞形态和超微结构,用流式细胞仪(FCM)检测血小板凋亡率,并以1×103U/L凝血酶为诱导剂检测再水化血小板的聚集活性.结果 与新鲜血小板相比,冻干再水化后的血小板在形态上为圆盘状,未见有明显的细胞聚集,仅细胞透光性相对较弱;超微结构上也无明显变化,其内部基本结构清晰可辨,细胞膜、细胞器及分泌颗粒的包膜保持完整.新鲜血小板的细胞早期凋亡率为(0.44±0.19)%,显著低于冻干血小板的细胞凋亡率(3.25±1.68)%(P<0.05).冻干再水化血小板的最大聚集率为(93.28±2.3)%.结论 冻干再水化的血小板形态结构完整,细胞内超微结构完好,为本实验室成功建立血小板冻干保存方法提供了依据.     

轮状病毒脂质体DNA疫苗体内诱导免疫应答研究

目的:通过对轮状病毒脂质体DNA疫苗体内免疫应答研究,寻找轮状病毒DNA疫苗理想的免疫佐剂.方法:脂质体包被的轮状病毒基因疫苗pcDNA1/VP7及裸DNA经肌肉注射及鼻粘膜两种途径免疫BALB/c小鼠,利用ELISA方法对其诱导产生的体液免疫应答进行测定.结果:经裸DNA及脂质体包被的质粒DNA免疫小鼠后,滴鼻组和肌注组血清特异性IgG水平较对照组明显升高.值得注意的是,脂质体包被的质粒DNA经肌注接种后血清特异性IgA水平也较对照组明显升高,而其余各实验组均未能诱导血清特异性IgA水平的升高.结论:脂质体在DNA接种中不仅作为质粒DNA疫苗的载体,而且起到免疫佐剂的作用.经该脂质体包被的质粒DNA免疫小鼠后,肌注组血清IgA水平较对照组明显升高,提示该脂质体包被的DNA经肌肉注射途径接种可能对机体产生免疫保护.     

新型免疫佐剂的研究及其在人兽共患病疫苗中的应用

随着许多人兽共患病的突发和流行，使得新疫苗的发展非常急迫。但由基因工程重组抗原或化学合成多肽组成的新一代疫苗存在免疫原性弱等缺陷，需要新型免疫佐剂来增强其作用，对适用于临床应用的新佐剂的研究与开发势在必行。目前研究较多的有黏膜佐剂、天然佐剂和纳米粒子佐剂等，因此分析阐述细胞因子、CpGDNA、脂质体、免疫刺激复合物和纳米粒子佐剂的特点、作用机制及在人兽共患病疫苗中的应用、佐剂应用的安全性和发展趋势很有必要。     

胸腺素α原作为约氏疟原虫疫苗免疫佐剂的初步研究

目的:研究胸腺素α原(Prothymosinα,ProTα)作为约氏疟原虫疫苗免疫佐剂的作用。方法:提取P.yoelii-17XNL全蛋白作为抗原,用胸腺素α原作为免疫佐剂,免疫小鼠。具体方案为:昆明小鼠分为4组,每组6只,A组免疫P.yoelii-17XNL全蛋白+ProTα;B组免疫P.yoelii-17XNL全蛋白;C组只注射ProTα;D组为空白对照,以相同体积的生理盐水代替。免疫结束后感染致死的P.yoelii-17XL,1×107个虫/只小鼠。结果:感染后的前10天A组小鼠疟原虫血症平均值要低于其他三组,且最终有3只小鼠存活下来,存活率50%,C组有一只小鼠存活,B、D组小鼠全部死亡。结论:用P.yoelii-17XNL全蛋白做抗原,用ProTα作为佐剂,比单独用P.yoelii-17XNL全蛋白对小鼠有更好的免疫保护作用,提示了ProTα可以成为一种有潜力的蛋白疫苗。     

脂质体介导pcDNA3/hVEGF165转染骨髓基质干细胞复合冻干骨的体内成骨和血管化

背景：以往的研究表明血管内皮生长因子、碱性成纤维细胞生长因子可以促进移植物的存活和体内生长。 目的：观察脂质体介导的pcDNA3/hVEGF165转染骨髓基质干细胞后复合冻干松质骨在体内的成骨和血管化效果。 方法：取同种异体新西兰大白兔的耾骨和股骨制备冻干骨,用脂质体将血管内皮生长因子转染入体外培养扩增新西兰大白兔骨髓基质干细胞中,使其附着于同种异体冻干松质骨。将新西兰大白兔分为3组,于兔竖脊肌分别植入单纯冻干骨、单纯骨髓间充质干细胞复合冻干骨组、转染有血管内皮生长因子的骨髓基质干细胞复合冻干松质骨。 结果与结论：植入后8周时可见到转染血管内皮细胞生长因子的骨髓基质干细胞复合冻干松质骨标本的表面有软骨和骨质形成,有成骨和破骨细胞出现,并形成类骨质,在移植骨周围组织中有大量血管形成,其他两组仅有大量纤维包裹。说明,脂质体介导的pcDNA3/hVEGF165转染后骨髓基质干细胞复合冻干骨具有较好成骨活性,优于单纯骨髓基质干细胞复合冻干松质骨和单纯冻干骨。     

rhGM-CSF作为成人乙肝疫苗无(弱)应答者免疫佐剂初探

目的 探讨重组人粒细胞.巨噬细胞集落刺激因子(rhGM-CSF)对提高成人乙肝疫苗无(弱)应答免疫的作用.方法 将两年内完成1～2个标准乙肝疫苗接种程序、复查HBV标志物均阴性的健康人群随机分为A、B两组.A组:33人,按标准免疫程序予10μg乙肝疫苗;B组:34人,先予rhGM-CSF 300μg皮下注射,次日始按A组方案予乙肝疫苗.接种首针后第1、2、8个月(T1、T2、T8)采血检测抗-HBs.结果 A、B组T8抗-HBs阳性率分别为39.39%和64.71%(P=0.038);A组三次抗体滴度检测结果 无显著性变化;B组升高明显,分别为(113.85±198.56)mIU/ml,(312.40±349.44)mIU/ml,(427.74±411.58)mIU/ml(P=0.001).A组和B组T8抗体水平差异有统计学意义(P=0.010).结论 rhGM-CSF联合乙肝疫苗复种对无(弱)应答者免疫效果优于单纯复种,GM-CSF具有提高机体对乙肝疫苗免疫应答的能力.     

rhGM-CSF作为成人乙肝疫苗无(弱)应答者免疫佐剂初探

Objective To investigate the effect of recombinant human granulocyte macrophage colony stimulating factor (rhGM-CSF) as adjuvant on immune response in adults of non-and hyporesponders to hepatitis B vaccine. Methods Those who were once immunized with recombined yeast gene hepatitis B vaccine more than one standard scheme in two years and negative for hepatitis B markers were randomly sorted as group A and group B. 33 adults of group A were given hepatitis B vaccine 10 μg each time. The immune procedure was O, 1 and 6month. 34 adults of group B were given rhGM-CSF 300 μg for the first day, then 10 μg each time for routine immune. The blood samples were collected before the first injection and in 1, 2 and 8 months (T1, T2, TS)following the first injection to test Anti-HBs. Results Anti-HBs positive conversion rates of group A and B at T8 was 39.39% and 64.71% respectively(P=0.038). Anti-HBs levels of group B at TI, T2, T8 were(113.85±198.56) mIU/ml, (312.40±349.44) mIU/ml, (427.74±411. 58) mIU/ml (P=0.001). There was significant difference between group A and B in T8 Anti-HBs levels(P=0.010). Conclusion Better immune response was found in the group of rhGM-CSF with hepatitis B vaccine. So rhGM-CSF can induce the immune respond to hepatitis B vaccine.     

rhGM-CSF作为成人乙肝疫苗无(弱)应答者免疫佐剂初探

Objective To investigate the effect of recombinant human granulocyte macrophage colony stimulating factor (rhGM-CSF) as adjuvant on immune response in adults of non-and hyporesponders to hepatitis B vaccine. Methods Those who were once immunized with recombined yeast gene hepatitis B vaccine more than one standard scheme in two years and negative for hepatitis B markers were randomly sorted as group A and group B. 33 adults of group A were given hepatitis B vaccine 10 μg each time. The immune procedure was O, 1 and 6month. 34 adults of group B were given rhGM-CSF 300 μg for the first day, then 10 μg each time for routine immune. The blood samples were collected before the first injection and in 1, 2 and 8 months (T1, T2, TS)following the first injection to test Anti-HBs. Results Anti-HBs positive conversion rates of group A and B at T8 was 39.39% and 64.71% respectively(P=0.038). Anti-HBs levels of group B at TI, T2, T8 were(113.85±198.56) mIU/ml, (312.40±349.44) mIU/ml, (427.74±411. 58) mIU/ml (P=0.001). There was significant difference between group A and B in T8 Anti-HBs levels(P=0.010). Conclusion Better immune response was found in the group of rhGM-CSF with hepatitis B vaccine. So rhGM-CSF can induce the immune respond to hepatitis B vaccine.     

以壳聚糖为佐剂的幽门螺杆菌疫苗诱导的细胞免疫反应

目的:探讨以壳聚糖为佐剂的幽门螺杆菌(Hp)疫苗诱导的细胞免疫反应及其在免疫保护中的作用。方法:BALB/c小鼠随机分为空白对照组(PBS溶液)、壳聚糖酸溶液组、壳聚糖颗粒组、Hp抗原组、Hp抗原 壳聚糖酸溶液组、Hp抗原 壳聚糖颗粒组、Hp抗原 霍乱霉素(CT)组、Hp抗原 壳聚糖酸溶液 CT组及Hp抗原 壳聚糖颗粒 CT组,各组于第0、7、14、21天灌胃各免疫1次,末次免疫后4周给予1×1012CFU/L的SS1Hp菌液0·5mL/只进行攻击,隔日1次,共2次。在攻击前后分批处死小鼠,采用Hp培养和病理改良Giemsa染色法检测胃黏膜内Hp感染。用定量ELISA法检测胃黏膜内IL-2、IL-4和IL-10含量;HE染色进行胃黏膜病理学检测。结果:①以壳聚糖为佐剂的Hp疫苗的免疫保护率达60%,与以CT为佐剂的Hp疫苗的免疫保护率(58·33%)相似,显著高于单纯Hp抗原组及其他不含Hp抗原组(P<0·05)。②胃黏膜内IL-2的水平攻击后含佐剂组显著高于对照组(P<0·05)。IL-10水平攻击前以壳聚糖为佐剂组高于无佐剂组,攻击后以CT 壳聚糖颗粒为佐剂组高于其他组(P<0·05)。IL-4水平攻击前以壳聚糖为佐剂组高于无佐剂组(P<0·05),攻击后以壳聚糖颗粒为佐剂组高于以CT为佐剂组(P<0·05),以壳聚糖溶液为佐剂组高于对照组、无佐剂组及佐剂中含CT组(P<0·05)。③胃黏膜炎症程度在单纯壳聚糖组和以壳聚糖颗粒为佐剂组显著低于以CT为佐剂组(P<0·05)。结论:①以壳聚糖为佐剂的Hp疫苗对Hp感染具有免疫保护作用。②以壳聚糖为佐剂的Hp疫苗可促进Th1和Th2的混合免疫反应,并逆转Hp感染所致Th2反应的抑制,使Th1和Th2反应达到平衡,从而发挥其免疫保护作用。③以壳聚糖为佐剂的Hp疫苗所致的免疫后胃炎显著低于以CT为佐剂的Hp疫苗。     

新型佐剂SWZY对弱免疫原性小鼠黑色素瘤瘤苗的免疫增强作用

目的:评价佐剂SWZY对弱免疫原性黑色素瘤瘤苗的免疫增强作用。方法:C57BL/6小鼠分为6组,实验组分别用5种不同配方的佐剂(FCA,FCA IL-2 GM-CSF,FIA IL-2 GM-CSF,FIA SWZY,FIA SWZY IL-2 GM-CSF)和照射灭活的小鼠黑色素瘤细胞株D5制成瘤苗免疫小鼠,对照组免疫用不加任何佐剂的灭活D5黑色素瘤细胞。末次免疫后3d各组取半数动物检测DTH反应、脾细胞的杀伤活性以及免疫小鼠血清及脾细胞培养上清中IFN-γ和IL-10的水平,剩余半数动物接种未灭活的D5黑色素瘤细胞,3周后再次检测上述各免疫学参数。结果:与对照组小鼠比较,各实验组小鼠的DTH反应及脾细胞的杀伤活性均明显升高(P<0.05),但成瘤后随着肿瘤的增大,则呈下降趋势。成瘤前各实验组小鼠血清及脾细胞培养上清中IFN-γ的水平均高于对照组小鼠(P<0.05),但IL-10的水平均低于对照组小鼠。成瘤后各实验组及对照组小鼠血清及脾细胞培养上清中IFN-γ的水平均下降,而IL-10的水平均明显上升,其中FCA瘤苗组和FIA SWZY瘤苗组免疫小鼠的血清及脾细胞培养上清中IFN-γ的水平仍高于对照组小鼠(P<0.05),IL-10的水平仍低于对照组小鼠(P<0.05)。结论:用5种佐剂配方制成的瘤苗免疫小鼠均能诱导对弱免疫原性肿瘤的细胞免疫应答,并增强Th1型细胞免疫的应答,但随着肿瘤的形成和逐渐进展,细胞免疫应答的效应逐渐减弱。其中佐剂SWZY与FCA的作用相当,但前者的毒副作用较小,有可能成为一种新型的人用肿瘤疫苗的佐剂。     

Character of the immune response in rats and mice with adjuvant disease

Phasic changes in the immune response were observed in rats and mice with adjuvant disease: stimulation of antibody formation on the seventh day after injection of Freund's complete adjuvant (FCA) and inhibition on the 21st day. Inhibition of production of normal antibodies against 0- and Vi-typhoid antigens also was demonstrated.Laboratory for the Study of Nonspecific Resistance of the Organism and Immunity, Institute of General Pathology and Pathophysiology, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR A. M. Chernukh.) Translated from Byulleten' Éxperimental'noi Biologii i Meditsiny, Vol. 86, No. 10, pp. 462–465, October, 1978.     

Inhibition of immune reactions in vivo by liposome associated transforming growth factor (TGF) type beta 1.

In view of its potent inhibitory capacity on immune cells in culture, we wished to determine the ability of transforming growth factor (TGF) beta 1 to down-regulate immune responses in vivo. Preliminary experiments suggested that, at the doses used, systemic injection of soluble TGF beta 1 could not affect bacterial-induced spleen enlargement in mice. Therefore, we sought to utilize a physiochemical property of this molecule, namely its high pI, to determine possible association between the ligand and preformed liposomes possessing an opposite charge. TGF beta 1 was preferentially associated with negatively charged, but not with neutral, liposomes. These TGF beta 1 associated liposomes were able to deliver a suppressive signal to indicator cells in vitro. Intravenous injection of TGF beta 1, associated with liposomes possessing an opposite charge, into mice immunized with heat-killed Corynebacterium parvum significantly reduced the size of the spleen as well as the number of splenocytes. Systemically administered TGF beta 1 associated liposomes could also inhibit delayed type hypersensitivity reactions to Listeria monocytogenes. These data suggest that appropriately administered, TGF beta 1 can inhibit immune responses in vivo.     

Enhancement of humoral immune responses to a human cytomegalovirus DNA vaccine: adjuvant effects of aluminum phosphate and CpG oligodeoxynucleotides

A human cytomegalovirus (HCMV) glycoprotein B (gpUL55) DNA vaccine has been evaluated in BALB/c mice. Intramuscular immunization of these mice with pRc/CMV2-gB resulted in the generation of high levels of gpUL55-specific antibody (geometric mean titer [GMT] 1:8900) and neutralizing antibody (GMT 1:74) after 2 booster doses given 5 and 10 weeks after primary inoculation. Emulsifying the construct with the aluminum phosphate gel adjuvant Adju-Phos before immunization enhanced gpUL55-specific antibody responses (GMT 1:17800, P = 0.04). Co-immunization with CpG oligodeoxynucleotides was shown to enhance levels of neutralizing antibodies generated by immunization of mice with a pRc/CMV2-gB/Adju-Phos emulsion (P = 0.04). The results provide a rationale for evaluating combinations of other HCMV proteins for incorporation into a multi-target DNA vaccine, and for the optimization of adjuvant usage, to elicit enhanced levels of neutralizing antibodies. 2003.     

Protection against influenza virus infection by intranasal vaccine with surf clam microparticles (SMP) as an adjuvant

A safe and effective adjuvant is necessary to enhance mucosal immune responses for the development of an inactivated intranasal influenza vaccine. The present study demonstrated the effectiveness of surf clam microparticles (SMP) derived from natural surf clams as an adjuvant for an intranasal influenza vaccine. The adjuvant effect of SMP was examined when co-administered intranasally with inactivated A/PR8 (H1N1) influenza virus hemagglutinin vaccine in BALB/c mice. Administration of the vaccine with SMP induced a high anti-PR8 haemagglutinin (HA)-specific immunoglobulin A (IgA) response in the nasal wash and immunoglobulin G (IgG) response in the serum, resulting in protection against both nasal-restricted infection and lethal lung infection by A/PR8 virus. In addition, administration of SMP with A/Yamagata (H1N1), A/Beijing (H1N1), or A/Guizhou (H3N2) vaccine conferred complete protection against A/PR8 virus challenge in the nasal infection model, suggesting that SMP adjuvanted vaccine can confer cross-protection against variant influenza viruses. The use of SMP is suggested as a new safe and effective mucosal adjuvant for nasal vaccination against influenza virus infection.     

Evaluation of properties of chitosan as an adjuvant for inactivated influenza vaccines administered parenterally

Studies on mice showed that chitosan as an adjuvant for H5 inactivated influenza vaccines administered intramuscularly enhances significantly antibody titers and protective efficiency not only against homologous influenza viruses, but also against drift variants. Chitosan adjuvanted vaccines induced high antibody titers after a single immunization and with a low dose of antigen. High antibody titers remained for at least 6 months. Chitosan adjuvanted vaccine stored at 4°C preserves its adjuvant properties for at least 8 months. Chitosan stimulates proliferative and cytotoxic activity of splenic mononuclear leukocytes in mice and promotes an increase in the numbers of CD3, CD3/NK, I‐AK (MHC II), and H‐2Db (MHC I) cells. After intramuscular immunization, chitosan did not induce IgE antibodies and antibodies against chitosan itself. Chitosan is a promising adjuvant candidate for inactivated influenza vaccines administered parenterally. J. Med. Virol. 81:494–506, 2009. © 2009 Wiley‐Liss, Inc.     

破伤风类毒素非磷脂脂质体疫苗的实验研究

以二氧乙烯十六烷醚、胆固醇和油酸为原料制备非磷脂脂质体 ,试验证明其包裹率高、稳定性好。与破伤风类毒素(TT )铝佐剂疫苗相比较 ,TT非磷脂脂质体疫苗二针免疫小鼠产生的抗体应答水平高一倍左右 ,抗体亚类以IgG1为主 ,加入IFN γ可增加IgG2a亚类应答。但免疫脾细胞IL 2产量不高。此外 ,该疫苗鼻粘膜接种可诱导显著的血清抗体应答。因而这种新型脂质体值得疫苗工作者重视。     

PIKA as an adjuvant enhances specific humoral and cellular immune responses following the vaccination of mice with HBsAg plus PIKA

An adjuvant is usually used to enhance the immune response induced by vaccines. The choice of adjuvant or immune enhancer determines the effectiveness of the immune response. Currently, aluminium （Alum, a generic term for salts of aluminium） is the only FDA-approved adjuvant. Alum predominantly induces the differentiation of Th2 cells and thus mediates an antibody immune response. Therefore, there is an urgent need for new adjuvants that enhance not only humoral but also cellular immune responses. In the present study, we demonstrates that PIKA （a stabilized dsRNA） as an adjuvant directly induces the activation and the proliferation of both B and NK cells in vitro. Injection of PIKA into mice results in the production of cytokines in vivo. In addition, the study demonstrates that PIKA promotes the maturation of bone marrow-derived dendritic cells （BMDCs） including up-regulation of the co-stimulatory molecules CD80, CD86 and CD40, and the induction of cytokines such as IL-12p70, IL-12p40 and IL-6. Importantly, after immunization of mice with HBsAg plus PIKA, the presence of PIKA enhances the titers of HBsAg-specific IgG and HBsAg-specific IFN-γ production. These results demonstrate that PIKA as an adjuvant can promote both humoral and cellular immune responses. These might have an implication in applying PIKA as an adjuvant to be used in the design and development of both therapeutic and preventive vaccines, and used in the clinical study.     

Circulating immune complexes as "tumor marker" in hepatoma-bearing rats (Yoshida AH 130)

The presence of circulating immune complexes (CIC) in a large number of malignancies was investigated in relation to the activity, the extent and the clinical course of the tumor clone. The aim of this study was to explore the relation between CIC serum levels and tumor growth in rats bearing the 130 Yoshida ascites hepatoma. For the assay of C3biCIC levels all samples were tested by a new competitive immunoenzymatic test. Experiments carried out on normal rats and on hepatoma-bearing ones, showed that during tumor growth values of CIC serum levels expressed as percent coefficient of inhibition are significantly higher in hepatoma-bearing rats (mean = 60.2) than in the normal controls (mean = 29.5). Furthermore CIC level and tumor proliferation are strictly correlated r = 0.9559. In this view sequential measurements of CIC levels may be valuable in establishing diagnosis, estimating prognosis and monitoring the behavior of neoplasia.