表达SDF-1/HOXB4融合基因的间充质干细胞联合脐血CD34~+干细胞共移植对辐射损伤小鼠的影响

目的观察表达SDF-1/HOXB4融合基因的间充质干细胞(mesenchymal stem cells,MSCs)联合脐血CD34+造血干细胞(hematopoietic stem cells,HSCs)共移植对辐射损伤小鼠的影响。方法表达SDF-1、HOXB4和SDF-1/HOXB4基因的3个腺病毒载体分别转染正常人骨髓MSCs,将其联合脐血CD34+细胞经尾静脉移植到辐射损伤的NOD/SCID小鼠体内(MSCs 8×105细胞/只,CD34+1×105细胞/只),分别为SDF-1/MSCs+CD34+组(SDF-1组)、HOXB4/MSCs+CD34+组(HOXB4组)、SDF1-HOXB4/MSCs+CD34+组(S-H组),另外3组为未转染MSCs+CD34+组(MSC-HSC组)、单纯CD34+组(HSC组)、单纯辐照组(IR组)。检测移植后各组小鼠存活率、外周血象恢复、骨髓病理变化及人源CD45+细胞植入率。结果 S-H组小鼠存活率高,且外周血WBC、HGB、PLT和骨髓造血功能恢复快。在移植后6周骨髓CD45+细胞植入率(47.43%±8.89%)较其余各组高。结论表达SDF-1/HOXB4融合基因的间充质干细胞(MSCs)联合脐血CD34+造血干细胞(HSCs)共移植能促进造血重建及植入,提高移植成功率。     

按不同时间方案注射的乙肝免疫球蛋白与乙肝疫苗对小鼠脾脏淋巴细胞 亚群的影响

目的：观察按不同时间方案注射的乙肝免疫球蛋白(HBIG)与乙肝疫苗对小鼠脾脏淋巴细胞亚群的影响。方法：饲养BALB/c小鼠120只(雌雄各60只),将小鼠按HBIG与第1针乙肝疫苗接种时间间隔分为三组(0周组、2周组、4周组),每组小鼠按接种的乙肝疫苗剂量分为2个亚组(1μg组、2μg组)。每只小鼠于第0周注射50 IU HBIG。所有小鼠于第3针乙肝疫苗接种后4周分离脾脏淋巴细胞,使用流式细胞仪检测淋巴细胞亚群(CD3~+、CD3~+CD4~+、CD3~+CD8~+、CD3~-CD19~+)比例。结果：0、2、4周组比较,两亚组(1μg、2μg)显示,与体液免疫相关的CD3~-CD19~+B淋巴细胞所占比例在雄、雌鼠4周组中均分别高于0、2周组,差异有统计学意义;与细胞免疫相关的T淋巴细胞及其亚群(CD3~+、CD3~+CD4~+、CD3~+CD8~+)均未显示4周组高于其他两组。乙肝疫苗1μg组、2μg组比较,在雄、雌鼠中均显示4周组中2μg组CD3~-CD19~+B淋巴细胞所占比例高于1μg组(P<0.05)。与细胞免疫相关的T淋巴细胞亚群未显示出2μg组高于1μg组的趋势...     

GM-CSF作为维持性血液透析患者乙肝疫苗接种的辅助剂

维持性血液透析患者 ( MHD)乙肝疫苗接种后的阳转率很低 ,本文探讨使用粒 -噬细胞刺激因子 ( GM- CSF)作为 MHD患者乙肝疫苗接种的辅助剂 ,以增强疫苗接种阳转率的可行性。资料与方法　将实验分为两组 : 组 12例为血清学HBs Ag、HBs Ab均阴性的 MHD患者 ,6例仅接受乙肝疫苗接种 ( a) ,其余 6例接种前 2 4h皮下注射 GM- CSF 15 0～2 5 0μg( 4～ 5μg/ kg) ( b) ,接种后 6个月血清学 HBs Ab滴度>10 IU/ m l考虑为阳性。 组 16例 ,均为接受乙肝疫苗接种后 6个月血清 HBs Ab未能阳转的 MHD患者。其中 8例仅接受加强剂量乙肝疫…     

口服重组β2糖蛋白1的实验性抗磷脂综合征小鼠CD4~+CD25~+调节性T细胞及Foxp3 mRNA表达变化

目的:观察口服重组人β2糖蛋白1(rhβ2GP1)的实验性抗磷脂综合征(EAPS)小鼠CD4+CD25+调节性T细胞(CD4+CD25+Treg)数量和功能变化,探讨EAPS口服耐受机制。方法:以rhβ2GP1主动免疫BALB/c小鼠建立EAPS模型,口服耐受组在EAPS小鼠免疫10天前经胃肠道给予rhβ2GP1,在免疫12周后检测各组小鼠抗β2糖蛋白1抗体(anti-β2-GP1)、抗心磷脂抗体(aCL)、流产率(%)、活化部分凝血活酶时间(aPTT)、血小板计数(PLTPBC);以流式细胞术检测各组小鼠PBMC中CD4+CD25+Treg细胞百分率;RT-PCR检测转录因子Foxp3mRNA的表达。结果:耐受组小鼠与模型组相比,anti-β2-GP1、aCL水平明显降低,aPTT缩短,PLTPBC升高,流产率降低,均具有显著性差异(P0.05);耐受组小鼠PBMC中Foxp3mRNA表达在第4、8、12周均高于对照组(P0.05),此后下降,20周时与正常对照组无明显差异(P0.05),4周时与模型组无差异(P0.05),8周后模型组逐渐降低,12周后降低明显(P0.05);耐受组PBMC中CD4+CD25+Treg细胞百分率与对照组相比,未见显著性差异(P0.05)。结论:CD4+CD25+Treg在口服耐受的诱导中发挥作用。     

CⅡTA基因修饰树突状细胞增强荷肝癌小鼠抗瘤效应

目的:探讨MHC-Ⅱ类分子反式激活因子(MHCclassⅡtransactivator)CⅡTA基因对树突状细胞(Dendriticcell,DC)表面MHC-Ⅰ/Ⅱ类分子及共刺激分子CD80、CD86的上调作用,为肝癌生物治疗提供新的思路。方法:从小鼠骨髓中大量扩增DC,并用脂质体转染法将小鼠CⅡTA(mCⅡTA)基因转入DC中,用流式细胞术观察转染前、后DC表面MHC-Ⅰ/Ⅱ类分子及CD80、CD86的表达。建立小鼠肝癌模型,将荷肝癌小鼠分为4组,第1组:分别于肝癌组织块周围注入PBS;第2组:未转入mCⅡTA基因的DC;第3组:转入mCⅡTA基因的DC;第4组:转入mCⅡTA基因并经H22肿瘤抗原刺激的DC。每周1次,连续接种3wk,连续7wk动态测量肝癌的大小,观察转染mCⅡTA基因对荷肝癌小鼠抗瘤效应的影响。结果:转染mCⅡTA基因使DC上MHC-Ⅰ/Ⅱ分子的表达率由74.2%/66.7%明显上调为93.6%/91.4%;CD80/CD86的表达率由52.3%/60.5%明显上调为89.7%/91.5%(P<0.05)。免疫接种后,第4组荷肝癌小鼠肝癌组织块的平均面积明显小于第1、2、3组(在第3、4、5、6、7周时,与第1、2组相比较,P<0.05;在第5、6、7周时与第3组相比较,P<0.05)。结论:转入mCⅡTA后,DC表面MHC-Ⅰ/Ⅱ分子及CD80/CD86的表达率明显上调,DC的抗瘤效应增强。本研究为将DC应用于肿瘤的生物治疗提供了新的途径。     

乙型肝炎病毒感染小鼠肝组织视黄醇结合蛋白4表达降低

目的观察乙型肝炎病毒(HBV)感染对小鼠体内视黄醇结合蛋白4(RBP4)表达的影响。方法将C57BL/6小鼠随机分为正常组,HBV感染组(尾静脉高压注射rAAV8-1.3HBV病毒载体1×10~(11) vg/只),HBV感染+拉米夫定喂养组(拉米夫定150 mg/kg),分别于6周、12周和18周麻醉后处死6只小鼠。荧光定量PCR(RT-qPCR)检测血清HBV DNA含量;全自动生化仪以及ELISA检测血清相关生化指标以及RBP4含量;ELISA检测肝脏组织中肿瘤坏死因子α(TNF-α)以及白细胞介素6(IL-6)炎性因子水平;RT-qPCR、Western blot分别检测肝脏组织中RBP4 mRNA以及蛋白的表达。结果 HBV感染组小鼠血清中HbsAg均保持较高水平且阳性率稳定在80%以上,小鼠血清HBV DNA含量显著高于对照组(P0.05),HBV感染小鼠模型构建成功;感染组小鼠肝脏组织中TNF-α以及IL-6水平均随时间的增加而升高,其中第12周、18周时要显著高于正常组(P0.05);此外,感染组小鼠肝脏组织中RBP4蛋白以及RBP4 mRNA的表达水平均随感染时间的增加而下降,其水平在第6周、12周、18周均要低于正常组以及拉米夫定喂养组(P0.05)。结论 HBV感染小鼠后能够显著降低小鼠肝脏组织中视黄醇结合蛋白4的表达水平。     

兵（学员）预防接种20 μg重组乙型肝炎疫苗（酿酒酵母）的免疫效果分析

目的:分析评价军队某部乙型肝炎表面抗体(Antibody to hepatitis B virus surface antigen,抗-HBs)阴性/ 弱阳性的新入伍人员,接种剂型为20 g/ ml 的酿酒酵母重组乙型肝炎疫苗(Recombinant hepatitis B vaccine derived Saccharomyces Cerevisiae Yest,HepB-SCY)后的免疫效果。方法:利用酶联免疫吸附法(Enzyme-linked immunosorbent assay,ELISA)和放射免疫法(Radioimmunoassay,RIA),筛选抗-HBs 为阴性的3 251 名和弱阳性的1 090 名新兵(学员)为实验观察对象,按照0、1 和6的接种程序,接种20 g/ ml 剂型的HepB-SCY,分别在每针接种后第28 天采血,用化学粒子发光免疫分析法(Chemilumines-cence immunoassay,CLIA)检测血清抗体的几何平均浓度(Geometric mean titer,GMT)。结果:观察对象全程免疫后,在弱阳性新兵(学员)中,第1、2 和3 针接种后阳转率分别为90.24%、97.82%和99.52%,第2 针免疫后的阳转率明显优于1 针免疫后阳转率(字2 =25.65,P<0.01),而第2 与3 针免疫后的阳转率之间无统计学差异(字2 =4.66,P>0.01);第1 和第2 针免疫后乙肝表面抗体的GMT 值分别为1 575.92 mU/ ml(374.94 ~4 606.45)和2 144.592 mU/ ml(757.078 ~6 089.4),二者的GMT 水平存在统计学差异(Z =-2.372,P<0.01)。在阴性人群中,第2 和第3 针接种后阳转率分别为86.13% 和98.72%,其中,高应答反应阳转率分别为56.43%和87.53%,第3 针免疫后的阳转率明显优于第2 针(字2 =118.5,P<0.05),第3 针免疫后的高应答反应率也明显高于第2 针(字2 = 231.07,P<0.05);第1、2 和3 针免疫后的GMT 值分别为101.352 mU/ ml(2.11 ~ 409.23)、155.812 mU/ ml(26.76 ~840.78)和747.312 mU/ ml(228.60 ~ 1893.6),第2 和3 针的GMT 水平之间差异存在统计学意义(Z =-23.042,P<0.01),第1 和2 针的GMT 水平之间差异也存在统计学意义(Z =-3.099,P<0.01)。结论:接种20 g/ ml 剂型的HepB-SCY 可激发机体产生较高水平的抗-HBs,并可有效提高群体的阳转率和高应答反应率。     

CIK对SGC-7901胃癌移植瘤小鼠模型体内抗肿瘤作用的实验研究

目的探讨细胞因子诱导杀伤细胞(CIK)对胃癌移植瘤小鼠模型的抗肿瘤作用并探讨其作用机制。方法体内实验选取60只C57BL/6小鼠分为模型对照组、5-Fu组、CIK大剂量、中剂量、小剂量组和正常对照组(n=10),分别治疗周期为4周,比较各组胃癌小鼠治疗期间移植瘤生长情况,测定各组小鼠血清和肿瘤组织MMP-9、VEGF及bFGF水平。结果 1)CIK中剂量组小鼠瘤体积于治疗后1周、2周、3周和4周时均较CIK小剂量组显著减少(P0.05),CIK大剂量组小鼠瘤体积治疗后1周、2周、3周和4周时均较CIK中剂量组显著减少(P0.05);2)治疗4周时CIK中剂量组小鼠血清MMP-9、VEGF和bFGF水平均较小剂量组小鼠显著降低(P0.05),CIK大剂量组小鼠血清MMP-9、VEGF和bFGF水平均较中剂量组小鼠显著降低(P0.05);3)治疗4周时CIK中剂量组小鼠移植瘤组织MMP-9、VEGF和bFGF mRNA和蛋白水平均较小剂量组小鼠显著降低(P0.05),CIK大剂量组小鼠移植瘤组织MMP-9、VEGF和bFGF mRNA和蛋白水平均较中剂量组小鼠显著降低(P0.05)。结论细胞因子诱导杀伤细胞可显著抑制SGC-7901胃癌小鼠模型瘤组织的生长,并且降低血液及癌组织中MMP-9、VEGF和bFGF的表达。     

新兵(学员)预防接种20μg重组乙型肝炎疫苗(酿酒酵母)的免疫效果分析

目的:分析评价军队某部乙型肝炎表面抗体(Antibody to hepatitis B virus surface antigen,抗-HBs)阴性/弱阳性的新入伍人员,接种剂型为20μg/ml的酿酒酵母重组乙型肝炎疫苗(Recombinant hepatitis B vaccine derived Saccharomyces Cerevisiae Yest,Hep B-SCY)后的免疫效果。方法:利用酶联免疫吸附法(Enzyme-linked immunosorbent assay,ELISA)和放射免疫法(Radioimmunoassay,RIA),筛选抗-HBs为阴性的3 251名和弱阳性的1 090名新兵(学员)为实验观察对象,按照0、1和6的接种程序,接种20μg/ml剂型的Hep B-SCY,分别在每针接种后第28天采血,用化学粒子发光免疫分析法(Chemiluminescence immunoassay,CLIA)检测血清抗体的几何平均浓度(Geometric mean titer,GMT)。结果:观察对象全程免疫后,在弱阳性新兵(学员)中,第1、2和3针接种后阳转率分别为90.24%、97.82%和99.52%,第2针免疫后的阳转率明显优于1针免疫后阳转率(χ~2=25.65,P0.01),而第2与3针免疫后的阳转率之间无统计学差异(χ~2=4.66,P0.01);第1和第2针免疫后乙肝表面抗体的GMT值分别为1 575.92 m U/ml(374.94~4 606.45)和2 144.592 m U/ml(757.078~6 089.4),二者的GMT水平存在统计学差异(Z=-2.372,P0.01)。在阴性人群中,第2和第3针接种后阳转率分别为86.13%和98.72%,其中,高应答反应阳转率分别为56.43%和87.53%,第3针免疫后的阳转率明显优于第2针(χ~2=118.5,P0.05),第3针免疫后的高应答反应率也明显高于第2针(χ~2=231.07,P0.05);第1、2和3针免疫后的GMT值分别为101.352 m U/ml(2.11~409.23)、155.812 m U/ml(26.76~840.78)和747.312 m U/ml(228.60~1893.6),第2和3针的GMT水平之间差异存在统计学意义(Z=-23.042,P0.01),第1和2针的GMT水平之间差异也存在统计学意义(Z=-3.099,P0.01)。结论:接种20μg/ml剂型的Hep B-SCY可激发机体产生较高水平的抗-HBs,并可有效提高群体的阳转率和高应答反应率。     

胰岛素腹腔注射配合饮食干预对自发性2型糖尿病KKAy小鼠血糖调控效果的研究

目的研究胰岛素腹腔注射配合饮食干预对自发性2型糖尿病KKAy小鼠血糖调控的效果。方法建立2型糖尿病动物模型, 选取健康C57BL/6J小鼠作为正常对照组, 健康KKAy小鼠作为未发病组, 将建模成功的KKAy小鼠随机分为皮下胰岛素治疗组、腹腔胰岛素治疗组和发病不治疗组。未发病组给予维持饲料, 其他组全部采用高脂高糖饲料。每日喂养时间为08:00至20:00, 间隔4 h喂养1次, 共4次。皮下和腹腔胰岛素治疗组在喂养前分别进行胰岛素皮下和腹腔注射, 第1次喂养前注射重组甘精胰岛素注射液(皮下组:0.125 IU/g;腹腔组:0.250 IU/g), 间隔0.5 h后注射生物合成人胰岛素注射液(皮下组:0.075 IU/g;腹腔组:0.125 IU/g);其余3次喂养前, 注射生物合成人胰岛素注射液(皮下组:0.075 IU/g;腹腔组:0.125 IU/g), 共给药4周。定期检测各组小鼠饮食量、体质量及空腹血糖、餐后1、2 h血糖, 并进行口服葡萄糖耐受实验。结果腹腔胰岛素治疗组小鼠的总饮食量低于皮下胰岛素治疗组;与初始体质量相比, 第4周皮下和腹腔胰岛素治疗组体质量分别下降5.05...     

Mechanism and therapeutic potential of DNA-based immunization against the envelope proteins of hepatitis B virus in normal and transgenic mice

Two plasmid DNA vectors, pCAGGS(S) encoding the genes of the major envelope protein of hepatitis B virus (HBV), and pCAGGS(S + preS2) encoding the genes of the middle envelope protein were used to study the mechanism and therapeutic potential of DNA-based immunization. Injection of these plasmids into the regenerating bilateral tibialis anterior muscle (TA) of normal C57BL/6 mice induced hepatitis B surface antigen (HBsAg)-specific humoral and cellular immune responses. Seventy-two hours after injection of pCAGGS(S), infiltrating cells including antigen-presenting dendritic cells (DC) were localized around the injection site and HBsAg was expressed by both muscle cells and infiltrating cells. Spleen DC from the mice were exposed to HBsAg for up to 32 weeks after a single injection of pCAGGS(S), because these DC induced the proliferation of HBsAg-specific memory lymphocytes in culture without exogenous HBsAg. A single injection of pCAGGS(S) or pCAGGS(S + preS2) resulted in the clearance of HBsAg in 28 out of 30 HBV-transgenic (Tg) mice. In contrast, more than 7 monthly injections of an HBsAg-based vaccine were required for the clearance of HBsAg in 6 out of 29 HBV-Tg mice. Infiltrating DC at the DNA vaccine injection site may have a role in initiating HBsAg-specific immune response, whereas the persistence of HBsAg exposed spleen DC may contribute to long-lasting immunity. This study also suggested that DNA-based vaccines may be a potent tool for treating chronic HBV carriers.     

骨骼肌注射乙型肝炎表面抗原基因在小鼠体内诱发乙肝表面抗原特异的免疫反应

将含有乙型肝炎表面抗原（ＨＢｓＡｇ）基因的重组质粒ｐＡｃｔ－ＭＳ直接注入小鼠骨骼肌中，２周后加强免疫１次。免疫后４～９周间，每周检测小鼠血清中ＨＢｓ。结果表明实验组６／８只小鼠ＨＢｓ为阳性，对照组８只小鼠均未检测到ＨＢｓ。本实验证实直接注射质粒ＤＮＡ外源基因能获得表达，并能引起免疫反应。     

几种不同基因免疫途径的比较研究

以乙肝表面抗原基因疫苗为模型, 采用肌肉注射、腹腔注射、表皮划痕三种免疫途径和不同剂量的裸露质粒ＤＮＡ免疫小鼠, 以ＥＬＩＳＡ方法检测小鼠血清中抗ＨＢｓＡｇ 抗体变化。实验结果表明表皮划痕方式免疫小鼠免疫反应阳性率高,且在相同剂量下其抗体滴度水平最高; 肌肉注射方式免疫小鼠, 抗体反应可维持较长时间; 腹腔注射方式免疫小鼠, 抗体反应产生快, 但维持时间较短。     

Antigen-antibody complex as therapeutic vaccine for viral hepatitis B

In a previous study, hepatitis B surface antigen (HBsAg) complexed to human anti-HBs immunoglobulins (HBIG) in excess of HBsAg was used as therapeutic vaccine to treat chronic hepatitis B patients and promising results were obtained. To study the mechanisms of this approach, mice were immunized with HBsAg or IC (immunogenic complex, i.e. HBsAg complexed with mouse polyclonal anti-HBs). Studies indicate that IC induced enhanced immune responses by increasing uptake of HBsAg through Fc receptors on antigen presenting cells and modulated HBsAg processing and presentation. This modulation led to stimulation of T cell responses, and increased production of IL-2 and IFN-gamma. Assay for antibody subclasses showed that higher ratio of IgG 2a was observed in the IC immunized group, which correlated with the production of lymphokine pattern. When alum was used as the adjuvant, though antibody response was enhanced, production of cytokines decreased. When DNA from a recombinant plasmid was added to IC as an adjuvant, the titer of anti-HBs was significantly higher than those in mice immunized only with the DNA or the IC. Since DNA immunization can induce both cellular and humoral immune responses, combined immunization using IC and DNA might serve as another type of therapeutic vaccine for viral hepatitis B.     

Antigen-Antibody Complex as Therapeutic Vaccine for Viral Hepatitis B

In a previous study, hepatitis B surface antigen (HBsAg) complexed to human anti-HBs immunoglobulins (HBIG) in excess of HBsAg was used as therapeutic vaccine to treat chronic hepatitis B patients and promising results were obtained. To study the mechanisms of this approach, mice were immunized with HBsAg or IC (immunogenic complex, i.e. HBsAg complexed with mouse polyclonal anti-HBs). Studies indicate that IC induced enhanced immune responses by increasing uptake of HBsAg through Fc receptors on antigen presenting cells and modulated HBsAg processing and presentation. This modulation led to stimulation of T cell responses, and increased production of IL-2 and IFN-γ. Assay for antibody subclasses showed that higher ratio of IgG 2a was observed in the IC immunized group, which correlated with the production of lymphokine pattern. When alum was used as the adjuvant, though antibody response was enhanced, production of cytokines decreased. When DNA from a recombinant plasmid was added to IC as an adjuvant, the liter of anti-HBs was significantly higher than those in mice immunized only with the DNA or the IC. Since DNA immunization can induce both cellular and humoral immune responses, combined immunization using IC and DNA might serve as another type of therapeutic vaccine for viral hepatitis B.     

Improved Humoral and Cellular Immune Responses Against the gp120 V3 Loop of HIV-1 Following Genetic Immunization with a Chimeric DNA Vaccine Encoding the V3 Inserted into the Hepatitis B Surface Antigen

The gp120-derived V3 loop of HIV-1 is involved in co-receptor interaction, it guides cell tropism, and contains an epitope for antibody neutralization. Thus, HIV-1 V3 is an attractive vaccine candidate. The V3 of the MN strain (MN V3) contains both B- and T-cell epitopes, including a known mouse H-2^d-restricted cytotoxic T lymphocyte (CTL) epitope. In an attempt to improve the immunogenicity of V3 in DNA vaccines, a plasmid expressing MN V3 as a fusion protein with the highly immunogenic middle (pre-S2 + S) surface antigen of hepatitis B virus (HBsAg) was constructed. Epidermal inoculation by gene gun was used for genetic immunization in a mouse model. Antibody and CTL responses to MN V3 and HBsAg were measured and compared with the immune responses obtained after vaccination with plasmids encoding the complete HIV-1 MN gp160 and HBsAg (pre-S2 + S), respectively. DNA vaccination with the HIV MN gp160 envelope plasmid induced a slow and low titred anti-MN V3 antibody response at 12 weeks post-inoculation (p.i.) and a late appearing (7 weeks), weak and variable CTL response. In contrast, DNA vaccination with the HBsAg-encoding plasmid induced a rapid and high titred anti-HBsAg antibody response and a uniform strong anti-HBs CTL response already 1 week p.i. in all mice. DNA vaccination with the chimeric MN V3/HBsAg plasmid elicited humoral responses against both viruses within 3–6 weeks which peaked at 6–12 weeks and remained stable for at least 25 weeks. In addition, specific CTL responses were induced in all mice against both MN V3 and HBsAg already within the first 3 weeks, lasting at least 11 weeks. Thus, HBsAg acts as a ‘genetic vaccine adjuvant’ augmenting and accelerating the cellular and humoral immune response against the inserted MN V3 loop. Such chimeric HIV–HBsAg plasmid constructs may be useful in DNA immunizations as a ‘carrier’ of protein regions or minimal epitopes which are less exposed or poorly immunogenic.     

Long-term persistence of hepatitis B surface antigen and antibody induced by DNA-mediated immunization results in liver and kidney lesions in mice

DNA-mediated immunization has been recognized as a new approach for prevention and treatment of hepatitis B virus (HBV) infection. However, the side effects of this approach have not been well described. Here we report that DNA-mediated immunization by intramuscular injection of plasmid DNA encoding HBV surface antigen (HBsAg) induced long-term persistence of HBsAg and HBsAg-specific antibody (anti-HBs) in the sera of the immunized BALB/c mice and resulted in liver and kidney lesions. The lesions persisted for 6 months after injection. Lesions were also found in normal mice injected with the sera from immunized mice, and in HBV-transgenic mice injected with anti-HBs antibody, or sera from immunized mice. Furthermore, lesions were accompanied by deposition of circulating immune complex (CIC) of HBsAg and anti-HBs antibody in the damaged organs. These results indicate that long-term persistence of HBsAg and anti-HBs in the immunized mice can result in deposited CIC in liver and kidney, and in development of lesions. The use of DNA containing mammalian replication origins, such as the plasmids used in this study, is not appropriate for human vaccines due to safety concerns relating to persistence of DNA; nevertheless, the safety of DNA-mediated immunization protocols still needs to be carefully evaluated before practical application.     

HCV/HBV复合DNA免疫的初步研究

目的 探讨HCV/HBV复合抗原PCX/S免疫原性。方法 将已证实具有良好免疫原性的人工合成的HCV复合多表位抗原基因PCX与HBV的S抗原基因融合于真核表达载体pcDNA3.0,构建真核表达载体pcDNA-PCXS。大量提取质粒并免疫小鼠,用ELISA的方法检测抗-HBs和抗-HCVAb;~3H-TdR渗入法检测免疫小鼠T淋巴细胞增殖;用~(51)Cr释放法检测免疫小鼠特异性CTLs杀伤作用。结果 免疫后均能检测到抗-HBsAb和抗-HCVAb,抗体水平随着时间的推移逐渐升高,但后者OD_(450nm)平均值低于抗-HBsAb的OD_(450nm)。免疫小鼠诱发针对PCX或S抗原的CTL反应和淋巴细胞转化。结论 复合型PCX/S抗原基因可诱发特异性免疫应答,为HCV/HBV双价疫苗的研究提供一定实验基础。     

Antigenicity and immunogenicity of hepatitis B virus particles produced by mouse cells transfected with cloned viral DNA

Antigenicity and immunogenicity of the hepatitis B surface antigen (HBsAg) 22-nm particles produced by mouse cells transfected with HBV DNA were studied. Both the a group and the y subtype determinants were present on the particles. Injected into mice the particles induced formation of anti-HBs. An antibody titer of 400 IU/mP, 7 weeks after the primary injection was obtained in one experiment. The affinity constant of these antibodies to human HBsAg particles was about 1 × 10^8? mol/liter. The anti-HBs antibodies react specifically with both the a group and the y subtype HBV determinants. These results show that these particles have antigenic and immunogenic properties similar to those of the 22-nm particles present in human serum.     

大剂量HBsAg疫苗对小鼠细胞免疫应答的调节作用

目的:探讨大剂量HBsAg疫苗对机体细胞免疫应答的影响。方法:不同剂量HBsAg疫苗两次免疫小鼠后, 用氚-胸腺嘧啶核苷(-[H³]TdR)掺入法, ELISA方法分别测定免疫鼠T淋巴细胞增殖能力, 细胞培养上清中白细胞介素-2(IL-2), γ干扰素(IFN-γ)含量及血清中抗HBsIgG2a阳转率。结果:大剂量HBsAg疫苗单次免疫后, 小鼠T淋巴细胞增殖能力显著强于对照组(P＜0.05);Th1型细胞因子IL-2、IFN-γ显著多于对照组(P<0.05);抗HBsIgG2a阳转率显著高于小剂量组(P＜0.01)。加强免疫后, 各项指标增高更显著, 小剂量组才呈现显著性增高。结论:大剂量HBsAg疫苗可诱生特异性细胞免疫应答, 并使细胞免疫趋向Th1型。