联合应用Ag85A和GM-CSF DNA疫苗对小鼠移植性膀胱癌BTT739细胞瘤免疫治疗的研究

目的：探讨联合应用Ag85A与GM-CSF DNA疫苗对小鼠移植性膀胱癌BTT739细胞瘤免疫治疗的效果。方法：选用小鼠膀胱癌BTT739细胞株,构建T739小鼠移植瘤模型。随机数字表法分为5组：生理盐水组、空载体组、Ag85A组、联合应用组和BCG组。荷瘤后第7天、14天和21天各肌注1次,末次注射后第7天检测各组小鼠脾脏的CD4＋和CD8＋T细胞亚群数量及CD4＋/CD8＋比值、血清IFN-γ和肿瘤重量。结果：与生理盐水组对比,单独应用Ag85A和联合应用Ag85A与GM-CSF DNA疫苗均能够升高CD4＋和CD8＋T细胞的百分比及CD4＋/CD8＋的比值,提高血清IFN-γ浓度,降低移植瘤重量,差异具有统计学意义（P〈0.05）。联合应用的效果要明显优于单独应用组,差异具有统计学意义（P〈0.05）,但尚不及单纯应用BCG的免疫治疗效果。结论：联合应用Ag85A与GM-CSF DNA疫苗能够提高荷瘤小鼠的免疫功能,从而达到了抑制肿瘤生长的作用,其效果明显优于单独应用Ag85A DNA疫苗,二者联用具有协同增强免疫效应。     

联合应用Ag85A和GM-CSF DNA疫苗对小鼠移植性膀胱癌BTT739细胞瘤免疫治疗的研究

目的:探讨联合应用Ag85A与GM-CSF DNA疫苗对小鼠移植性膀胱癌BTT739细胞瘤免疫治疗的效果。方法:选用小鼠膀胱癌BTT739细胞株,构建T739小鼠移植瘤模型。随机数字表法分为5组:生理盐水组、空载体组、Ag85A组、联合应用组和BCG组。荷瘤后第7天、14天和21天各肌注1次,末次注射后第7天检测各组小鼠脾脏的CD4+和CD8+T细胞亚群数量及CD4+/CD8+比值、血清IFN-γ和肿瘤重量。结果:与生理盐水组对比,单独应用Ag85A和联合应用Ag85A与GM-CSF DNA疫苗均能够升高CD4+和CD8+T细胞的百分比及CD4+/CD8+的比值,提高血清IFN-γ浓度,降低移植瘤重量,差异具有统计学意义(P<0.05)。联合应用的效果要明显优于单独应用组,差异具有统计学意义(P<0.05),但尚不及单纯应用BCG的免疫治疗效果。结论:联合应用Ag85A与GM-CSF DNA疫苗能够提高荷瘤小鼠的免疫功能,从而达到了抑制肿瘤生长的作用,其效果明显优于单独应用Ag85A DNA疫苗,二者联用具有协同增强免疫效应。     

活体荧光成像评估Ag85A和Ag85B DNA疫苗对小鼠膀胱癌移植瘤的疗效

目的：运用活体红色荧光成像技术及影像学特征评估Ag85A DNA 疫苗和Ag85B DNA 疫苗对膀胱癌的免疫治疗效果。方法：构建稳定转染香菇珊瑚红色荧光蛋白（discosomasp red fluorescent protein, DsRed）基因的小鼠膀胱癌BTT细胞（BTTDsRed）,建立BTTDsRed细胞移植瘤小鼠模型,将建模成功的24 只小鼠随机分为pVAX1Ag85A DNA 疫苗组、pVAX1Ag85B DNA 疫苗组和生理盐水治疗组,各组分别于肿瘤细胞接种后的第6天肌内注射pVAX1Ag85A、pVAX1Ag85B及生理盐水,然后用活体荧光成像系统检测移植瘤的生长和转移情况。结果：成功制备了稳定转染DsRed基因的BTTDsRed细胞,BTTDsRed接种小鼠建立可视化红色荧光膀胱癌移植瘤模型。重组质粒pVAX1Ag85A和pVAX1Ag85B治疗后的第2周,活体荧光成像显示pVAX1Ag85B 治疗组小鼠肿瘤荧光强度明显低于生理盐水组（P<0.05）;治疗后第3周,pVAX1Ag85A 和pVAX1Ag85B组小鼠肿瘤荧光强度都明显低于生理盐水组（P<0.01）,但pVAX1Ag85B 与pVAX1Ag85A组无明显差别（P>0.05）;pVAX1Ag85B 组小鼠淋巴结转移率（25.0%）明显低于生理盐水组（87.5%,P<0.01）和pVAX1Ag85A组（625%,P<0.05）。结论：应用活体红色荧光成像技术能够动态、灵敏、可视化地评估 DNA 疫苗对小鼠膀胱癌移植瘤的疗效;Ag85A和Ag85B DNA 疫苗均具有抗肿瘤免疫疗效。     

重组表皮生长因子受体噬菌体疫苗的抗肿瘤效果

目的构建重组表皮生长因子受体(EGFR)噬菌体疫苗,并观察其抗肿瘤效果。方法将鸡源EGFR膜外部分的5个基因片段插入T7噬菌体表达系统中,EGFR蛋白以融合蛋白的形式表达在噬菌体的外壳蛋白10B上,从而构建成噬菌体疫苗。Western blot方法检测噬菌体壳蛋白的EGFR表达,ELISA方法检测免疫小鼠血清中抗EGFR抗体。分离免疫小鼠脾细胞,检测淋巴细胞对靶细胞A431的细胞毒作用。C57BL／6J小鼠经过4次免疫,接种Lewis肺癌细胞,记录肿瘤的生长情况,评价疫苗的抗肿瘤效果。结果成功构建5种鸡源EGFR噬菌体疫苗REPV-C1、REPV-C2、REPV-C3、REF-C4和REPV-C5。Western blot结果显示,噬菌体壳蛋白上有EGFR表达。经免疫的小鼠血清中可检测到抗EGFR抗体。免疫后的小鼠脾细胞对EGFR受体高表达的A431细胞具有杀伤作用,效靶比为10:1时,最大杀伤率可达45．74％±7．21％。肿瘤生长曲线显示,免疫后的C57BL／6J小鼠Lewis肺癌生长受到明显抑制。结论重组EGFR噬菌体疫苗具有抗肿瘤效果,利用噬菌体表达技术构建噬菌体疫苗是一种有效的抗肿瘤方法。     

基因疫苗在肿瘤免疫研究中的进展

 基因疫苗技术自从20世纪90年代问世以来被迅速应用到传染病、免疫缺陷、肿瘤等重大疾病的预防和治疗的研究中,有一部分已经进入临床试验阶段。肿瘤基因疫苗可以打破免疫耐受,增强免疫原性,诱导机体产生针对肿瘤的体液和细胞反应,既有预防又有治疗肿瘤的作用。能够防治肿瘤的基因疫苗发展迅猛,主要包括与肿瘤相关抗原（TAA）有关的全长、表位、独特型（Id）和融合DNA疫苗,能够自主复制的RNA疫苗,与树突细胞（DC）相关的肿瘤基因疫苗等。现对基因疫苗在肿瘤免疫研究中的进展进行综述。     

Immunotherapeutic effect of tumor necrosis after cryosurgery, electrocoagulation, and ligation.

Tumor necrosis in situ by cryosurgery, electrocoagulation, or ligation generally induces or augments some increase in tumor-specific transplantation immunity (TSTI) as compared with excision. Excision of the tumor 24 hours after it has been rendered ischemic by either ligation or cryosurgery seems to produce TSTI that is significantly greater than that after excision of viable tumor without prior ligation or cryosurgery; the degree of this immunity is similar to that seen after the tumor is left in situ indefinitely after treatment. These data suggest that most of the immunization following tumor necrosis in situ occurs within 24 hours of treatment. The experimental findings support the clinical reports of putative immunologic potentiation after tumor necrosis in situ.     

膀胱癌组织中B7-H1表达水平及其对经尿道膀胱肿瘤电切术后膀胱灌注治疗效果的影响

目的 探讨膀胱癌组织中B7-H1表达水平及其对经尿道膀胱肿瘤电切术后膀胱灌注治疗效果的影响.方法 选取20例膀胱癌患者作为研究对象,采用回顾性分析法分析所有患者的临床资料,所有膀胱癌患者均行经尿道膀胱肿瘤电切术且术后均行膀胱灌注治疗,将膀胱癌组织作为A组,将癌旁正常组织作为B组.根据资料结果比较所有研究对象的B7-H1水平以及所有膀胱癌手术患者的术后治疗情况及效果.结果 A组膀胱癌组织中B7-H1表达水平明显高于B组正常膀胱组织(P﹤0.05);不同临床分期和病理分级膀胱癌患者间B7-H1表达水平比较,差异有统计学意义,且随着临床分期和病理分级越高其B7-H1表达水平越高(P﹤0.05);膀胱癌组织B7-H1表达水平与临床分期和病理分级呈正相关(r=0.684、0.762,P﹤0.05).结论 B7-H1在膀胱癌组织中以高水平表达,且临床分期和病理分级越高B7-H1表达水平越高,可考虑作为预测膀胱癌病情发展的指标.     

Preparation of murine B7.1-glycosylphosphatidylinositol and transmembrane-anchored staphylococcal enterotoxin. A dual-anchored tumor cell vaccine and its antitumor effect

BACKGROUND: The authors have previously reported a tumor cell vaccine modified with superantigen staphylococcal enterotoxin A (SEA) and its antitumor effect. The tumor cell vaccines modified with multiple immune activators frequently elicited stronger immune responses against established tumors than single-modified vaccines. METHODS: The authors explored the effectiveness of a tumor cell vaccine transduced with immune activators, dual-modified using the protein transfer technique. First, a glycosylphosphatidylinositol (GPI)-anchored murine B7.1 (mB7.1-GPI) and a transmembrane-anchored SEA (TM-SEA) were genetically generated. Then, the murine lymphoma EL4 cells were dual modified with the incorporation of mB7.1-GPI and TM-SEA onto the cell surface. Flow cytometry and laser confocal microscopy showed that the incorporation of B7.1 and SEA molecules onto EL4 cells was quite stable. RESULTS: The dual-modified tumor cell vaccine EL4/mB7.1-GPI + TM-SEA elicited significantly stronger antitumor immune responses both in vitro and in vivo when compared with the single-modified tumor cell vaccines EL4/mB7.1-GPI and EL4/TM-SEA. CONCLUSIONS: The results of the current study validated the novel approach for preparing tumor cell vaccines modified with dual immune active molecules using the protein transfer technique, and supported the feasibility and effectiveness of the dual-modified tumor cell vaccine.     

莪术醇修饰的肿瘤疫苗抗MFC效应的研究

目的:研究中药莪术提取的有效成分-莪术醇生物修饰构建的肿瘤细胞疫苗对荷瘤(MFC)小鼠的免疫功能变化及治疗效应。方法:用现代生物学技术修饰构建瘤苗,观察莪术醇-MFC细胞瘤苗体外激活脾淋巴细胞的能力,体内成瘤性变化及作为瘤苗对荷瘤胃癌的小鼠治疗作用。结果:莪术醇-MFC细胞瘤苗在体外能有效诱导免疫细胞活化增殖和特异性CTL活性的产生,体内的成瘤能力明显下降,免疫接种瘤苗后对荷瘤鼠胃癌小鼠有较好的治疗效果。结论:莪术醇修饰的肿瘤细胞瘤苗能明显增强其免疫原性、对荷瘤胃癌小鼠有较好的治疗效应。     

Detection of circulating tumor cells and of tumor DNA in plasma during tumor progression in rats

The role and clinical significance of circulating tumor cells and of tumor DNA in the plasma have not yet been clarified. In the present study, we compared rates of detection of tumor-derived DNA in the buffy coat to those in plasma from tumor-bearing rats, and we attempted to correlate these rates with the progression of tumors. We injected DHD/K12-PROb cancer cells subcutaneously into BD-IX rats and divided the animals into six groups according to the time between the injection of tumor cells and euthanasia. After euthanasia, macroscopic metastases were assessed and samples of blood and lung were collected. We used mutant allele-specific amplification by PCR to detect tumor-derived DNA. We detected tumor DNA in lung samples from the first week after inoculation, in plasma from the third week and in the buffy coat from the fifth week. All animals analyzed on the 11th week had macro- or micrometastases in their lungs. Regardless of group, the rate of PCR-positive plasma samples was significantly higher than that of circulating tumor cells (P=0.005). In animals with metastases, this difference was also statistically significant (P=0.008). However, neither the detection of tumor DNA in the plasma nor the presence of circulating tumor cells was strongly correlated with the presence of metastases. Thus, cell-free tumor DNA was detected sooner and more frequently than circulating tumor cells and the dissemination of tumor DNA in the plasma seems to be much more common than detectable hematogenic tumor cells during the spread of colorectal cancer.