Ag85A及Ag85B-DNA疫苗对移植性小鼠膀胱肿瘤免疫治疗的实验研究

目的：比较研究Ag85A-DNA疫苗和Ag85B-DNA疫苗对移植性小鼠膀胱肿瘤的免疫作用。方法：采用小鼠膀胱癌BTT739细胞株，构建可移植性T739小鼠膀胱肿瘤模型。随机分为生理盐水组、卡介苗（BCG）组、空白质粒（pcDNA3．1）组、pcDNA—Ag85A组、pcDNAAg85B组，荷瘤后第7天、14天和21天各肌注1次，末次注射后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、小鼠血清IFN-γ、肿瘤体积重量并作组织病理学检查。结果：Ag85B-DNA疫苗能够显著提高荷瘤小鼠CD4^＋T细胞亚群数量、CD8^＋T细胞亚群数量和CD4^＋T细胞与CD8^＋T细胞的比值，明显抑制肿瘤生长，促进IFN-γ分泌。肿瘤组织细胞坏死明显，瘤体周围及瘤体内有大量炎性细胞浸润，与各组相比差异有显著统计学意义（P〈0．01），但低于BCG组；Ag85A—DNA疫苗组与生理盐水组和空白质粒组比较，差异无统计学意义（P〉0．05）。结论：单独应用Ag85B-DNA疫苗可以诱导Th1免疫反应，增加IFN-γ等细胞因子的分泌，提高荷瘤小鼠的细胞免疫功能，抑制肿瘤生长，但效果不及BCG。尚不能认为单独应用Ag85ADNA疫苗会诱发有效的抗膀胱肿瘤免疫反应。     

活体红色荧光成像评估Ag85A／BDNA疫苗联合抑瘤效应的实验研究

目的：探讨可视、直观评估Ag85A／BDNA疫苗联合抑瘤效应的科学方法。方法：采用红色荧光蛋白基因DsRed—Express标记小鼠膀胱癌细胞BTT739,经G418抗性筛选获得稳定表达RFP的细胞克隆BTT739-RFP,将BTT739～RFP细胞接种到615小鼠皮下,建立可视化膀胱癌移植瘤模型。将建模成功的32只小鼠随机分为pVAX1-Ag85A／B联合组：BCG组;空白质粒（pVAX1）;生理盐水（NS）。建模后第7、14、21天注射相应的药物。分别于给药前即第7和第28天进行活体荧光成像,进行统计学处理。并于第28天检测血清特异性抗体水平和脾淋巴细胞的增殖活性。结果：构建了稳定表达RFP的BTT739细胞;成功建立可视化膀胱癌模型,成瘤率100％;经治疗后,pVAX1-Ag85A／B组的荧光强度明显低于pVAX1组和NS组,与BCG组无统计学差异。pVAX1-Ag85A／B组的抗体滴度为1：1000,BCG免疫组的抗体滴度为1：800。pVAX1-Ag85A／B组和BCG组均可提高淋巴细胞增殖活性,二者差异无统计学意义。结论：应用Ag85A／BDNA疫苗可提高膀胱癌小鼠的免疫功能,其效果与BCG差异无统计学意义;活体红色荧光体外成像的评估体系与细胞水平的分析一致。     

pVAX1-Ag85B的构建及其免疫原性的初步探索

目的构建以Ag85B基因为基础的DNA疫苗,并对其在小鼠体内的免疫原性进行初步分析。方法以pET28a-Ag85B基因组DNA为模板,PCR扩增获得Ag85B全长基因;将PCR产物构建成pUCm-Ag85B亚克隆;经限制性内切酶消化后克隆入pVAX1载体中构建真核表达质粒pVAX1-Ag85B,酶切、DNA测序鉴定。并将构建的DNA疫苗经肌肉免疫荷瘤小鼠,检测血清特异性抗体水平和脾淋巴细胞的增殖活性,并与BCG组、空白质粒组、生理盐水组相比较。结果经NheⅠ和HindⅢ双酶切、DNA测序鉴定后证实,Ag85B基因定向克隆入pVAX1载体,碱基无突变,序列完全正确。免疫荷瘤小鼠后,pVAX1-Ag85B组和BCG组均可提高淋巴细胞增殖活性,但效果不及卡介苗;pVAX1-Ag85B组的抗体滴度为1∶400,BCG免疫组的抗体滴度为1∶800。结论成功地构建了pVAX1-Ag85B质粒,为膀胱肿瘤的基因免疫治疗提供可靠的实验依据。免疫小鼠后,可提高小鼠免疫水平,但效果不及卡介苗。     

pVAX1-Ag85B质粒的构建及其免疫原性的可视化研究

构建以Ag85B基因为基础的DNA疫苗,并用活体红色荧光成像探讨其在动物体内免疫原性。方法：以pET28a—Ag85B基因组DNA为模板,用PCR扩增获得Ag85B全长基因;将PCR产物构建成pUCm—Ag85B亚克隆;经限制性内切酶消化后克隆人pVAX1载体中构建pVAX1-Ag85B,酶切、DNA测序鉴定。并将构建的DNA疫苗经肌肉免疫可视化膀胱癌移植瘤模型,通过活体红色荧光成像,从整体上直接、可视地观测Ag85B的免疫原性。结果：经NheI和HindⅢ双酶切、DNA测序鉴定后证实,Ag85B基因定向克隆人pVAX1载体,碱基无突变,序列完全正确。免疫小鼠后,pVAX1-Ag85B组和BCG组均可提高淋巴细胞增殖活性,但效果不及卡介苗。结论：成功地构建了pVAX1-Ag85B质粒,为膀胱肿瘤的基因免疫治疗提供可靠的实验方法。免疫小鼠后,可提高膀胱癌小鼠免疫水平,但效果不及卡介苗。     

分支杆菌Ag85B蛋白对小鼠膀胱癌抑癌效应的研究

目的:探讨分支杆菌Ag85B蛋白对小鼠膀胱癌的抑癌效应。方法:采用T739 可移植性膀胱癌模型,于移植瘤处局部注射分支杆菌Ag85B蛋白,并用卡介苗(BCG)和生理盐水(NS)作对照,观察治疗后各组的肿瘤重量和存活期。结果:分支杆菌Ag85B蛋白治疗组小鼠肿瘤瘤重(4.12±0.85) g,显著低于NS组(7.01±1.18) g(P<0.01),与BCG组(3.85±1.18) g比较差异无统计学意义(P>0.05),抑瘤率为41.23%;分支杆菌Ag85B蛋白治疗组小鼠存活天数为(28.75±2.71) d,显著长于NS组(23.50±2.45) d(P<0.05),与BCG组的(29.63±3.25) d比较差异无统计学意义(P>0.05),存活延长率为22.34%。结论:分支杆菌Ag85B蛋白对小鼠膀胱癌具有抑制作用,并能延长膀胱癌荷瘤鼠的生存期。     

分枝杆菌Ag85B与膀胱癌的治疗

Ag85B是分枝杆菌最主要的分泌蛋白,能够诱导机体强烈的Th1免疫反应,并在分枝杆菌细胞壁合成中发挥重要作用,还具有与纤维粘连素(FN)结合的特性。Ag85B蛋白能够诱导膀胱癌患者PBMC的增殖反应,并能诱导Th1细胞分泌细胞因子IFN-γ和IL-2;Ag85B基因修饰的膀胱肿瘤细胞疫苗具有抗膀胱肿瘤效应;利用Ag85B的外分泌特性构建分泌细胞因子的BCG在治疗膀胱癌方面可能更具有优势;Ag85B基因编码的DNA疫苗为膀胱癌的基因免疫治疗提供了新的思路。     

分枝杆菌Ag85B与膀胱癌的治疗

Ag85B是分枝杆菌最主要的分泌蛋白，能够诱导机体强烈的Th1免疫反应，并在分枝杆菌细胞壁合成中发挥重要作用，还具有与纤维粘连素(FN)结合的特性。Ag85B蛋白能够诱导膀胱癌患者PBMC的增殖反应，并能诱导Th1细胞分泌细胞因子IFN-γ和IL-2；AdB5B基因修饰的膀胱肿瘤细胞疫苗具有抗膀胱肿瘤效应；利用Ag85B的外分泌特性构建分泌细胞因子的BCG在治疗膀胱癌方面可能更具有优势；Ag85B基因编码的DNA疫苗为膀胱癌的基因免疫治疗提供了新的思路。     

白细胞介素-7促进CD8+T细胞干扰素-γ的分泌增强小鼠抗乳腺肿瘤的免疫效应

目的 观察白细胞介素(IL)-7是否通过促进CD8+T细胞干扰素-γ(IFN-γ)分泌来增强小鼠抗乳腺肿瘤的免疫效应.方法 荷瘤小鼠瘤体内直接注射IL-7真核表达质粒(pcDNA3-IL-7);免疫磁珠分选CD4+T、CD8+T细胞并体外培养;流式细胞仪检测细胞内干扰素(IFN)-γ的分泌量;酶联免疫吸附试验(ELISA)检测培养上清IFN-γ的量;噻唑蓝(MTT)法检测CD8+T细胞体外杀伤活性;小鼠体内预先注射anti-CD8抗体阻断活化.结果 pcDNA3-IL-7注射组CD8+T细胞内IFN-γ(38.6±4.4)％、CD8+T细胞培养上清IFN-γ( 136.0±11.2) ng/L,与对照组比较,pcDNA3-IL-7明显促进CD8+T细胞IFN-γ表达量(P＜0.05);IL-7处理过的荷瘤小鼠CD8+T细胞杀伤活性显著增强,尤其效靶比为100∶1;小鼠体内预先注射anti-CD8抗体阻断CD8+T细胞活化,显著抑制IL-7抗瘤效应(P＜0.05).结论 IL-7通过促进小鼠体内CD8+T细胞分泌IFN-γ介导抗瘤效应,从而增强小鼠机体抗乳腺肿瘤的免疫反应.     

重组hIFN-α-2b-BCG对小鼠原位膀胱肿瘤免疫效应的研究

目的 探讨重组hIFN-α-2b-BCG对原位膀胱肿瘤小鼠体内抗肿瘤免疫反应及其作用机制.方法 构建C57BL/6原位膀胱肿瘤小鼠模型,采用流式细胞仪对小鼠膀胱灌注治疗后的淋巴细胞亚群进行分析,并测定小鼠血清中mTNF-α和mIL-12的水平,了解小鼠全身免疫状况.肿瘤组织冰冻切片进行T细胞亚群的免疫组织化学分析,免疫组化检测肿瘤Fas表达,了解膀胱局部免疫反应. 结果 重组BCG灌注治疗后小鼠外周血CD4+ T细胞比例明显增高,CD8+ T细胞比例无明显变化,CD4+ /CD8+比例为2.63,与野生BCG组(2.10)比较差异有统计学意义(P＜0.05).荷瘤小鼠外周血中Th1型细胞因子mTNF-α和mIL-12灌注治疗后比PBS对照组大幅提高,重组BCG组mTNF-α为806 pg/ml,mIL-12为860 pg/ml,与野生BCG组及野生BCG联合IFN组比较差异无统计学意义(P＞0.05).重组BCG组瘤组织内CD3、CD4和CD5检测强阳性,显著高于PBS对照组(P＜0.05),重组BCG组和野生BCG加IFN组瘤组织CD4+、重组BCG组CD8+检测值显著高于野生BCG组(P＜0.05).BCG灌注后荷瘤小鼠膀胱肿瘤表达Fas明显高于PBS对照组(P＜0.05). 结论 重组hIFN-α-2b-BCG具有在小鼠体内调节系统及局部免疫能力的作用,纠正荷瘤小鼠淋巴细胞亚群的比例失调,增强局部淋巴细胞浸润,具有增强Th1型细胞因子产生作用,上调荷瘤小鼠Fas的表达,诱导对膀胱肿瘤细胞的免疫攻击.

Abstract：

Objective To study local and systemic immune response in an animal model treated with recombinant hIFN-α-2b-BCG instillation. Methods The MB49 orthotopic bladder cancer model in C57BL/6 mice was established and treated separately with rBCG, wild BCG, wild BCG combined with IFN-α-2b and PBS as the control. The changes of lymphocyte subgroups in peripheral blood were analyzed with FCM, and mTNF-α and mIL-12 in peripheral blood of mice were detected with ELISA.Immunohistochemistry was carried out to detect the local immune reaction, T cell subsets and FAS, in bladder cancer after being treated with rBCG or wBCG. Results The content of CD4+ T lymphocyte was up-regulated in the rBCG group. The CD4+/CD8+ ratio of 2. 63 was up-regulated than pretreatment, significantly different than that of wBCG group(P＜0.05). ELISA assay showed that BCG significantly up-regulated the level of mTNF-α and mIL-12 in serum of orthotopie murine bladder cancer mice. The mTNF-α 806 pg/ml, mIL-12 860 pg/ml in rBCG group, was not significantly higher than those in wBCG group and combination group. The immunocompetent cell numbers with CD3, CD4,CD8 phenotype increased significantly in the tumor tissue of BCG treated group than the control(P＜0.05). The results of CD4+ in rBCG group and the combination group, and CD8+ in rBCG group were significantly higher than that of the wBCG(P＜0.05). The expression of Fas in tumor tissues treated with intravesical BCG was increased(P＜0. 05). Conclusions The recombinant IFN-α-2b-BCG can retrieve the disproportion of systemic lymphocyte subgroups, and increases Th1-type factors and local Fas expression in orthotopic murine bladder cancer. The recombinant IFN-α-2b-BCG is effective in regulating local and systemic immune reaction in orthotopic murine bladder cancer model.     

卡介苗穿梭表达质粒pMS肿瘤坏死因子的构建与鉴定

目的构建分泌性表达肿瘤坏死因子(TNF)-α的重组卡介苗。方法分别以卡介苗(BCG)和TNF-α cDNA为模板，通过PCR扩增得到117bp的BCG—Ag85B信号肽序列和702bp的TNF-α基因序列。将BCG—Ag85B信号肽序列插入大肠杆菌一卡介苗穿梭质粒pMV261，得到重组质粒pMS。再将TNF—α基因序列克隆至pMS中，得到重组质粒pMSTNF。结果质粒pMSTNF用双酶切和PCR扩增及测序鉴定证实，克隆基因BCG—Ag85B和TNF-α。正确插入载体pMV261。结论重组质粒pMSTNF可望在BCG中分泌性表达细胞因子TNF-α从而协同加强对肿瘤的杀伤作用，该质粒的成功构建为改造卡介苗、发展新型抗膀胱肿瘤疫苗提供了依据。     

微波消融联合树突状细胞瘤内注射对荷瘤小鼠T淋巴细胞亚群的影响

目的 观察微波消融联合树突状细胞(DC)瘤内注射对荷瘤小鼠T淋巴细胞亚群的影响.方法 建立BALB/C小鼠皮下骨髓瘤模型,分为对照组、微波消融组、微波消融联合不成熟DC组、微波消融联合成熟DC组,分别于微波消融后第1、3、5、7、9及11天采用流式细胞仪检测各组小鼠外周血T淋巴细胞亚群.结果 对照组小鼠的CD3~+、CD4~+及CD4~+/CD8~+值逐渐下降(P<0.05);微波消融组小鼠的CD3~+、CD4~+及CD4~+/CD8~+无明显改变(P>0.05);微波消融联合不成熟DC组小鼠的CD3~+、CD4~+及CD4~+/CD8~+在术后第3、5、7天明显减低(P<0.05),第9、11天恢复;微波消融联合成熟DC组小鼠CD3~+、CD4~+及CD4~+/CD8~+则在术后第3、5、7天明显增高(P<0.05),第9、11天下降.结论 微波消融联合成熟DC在一定时间内(约1周)能够增强实验小鼠的抗肿瘤免疫反应.     

Inhibitory effects of IFN on N-butyl-N-(hydroxybutyl) nitrosamine induced rats bladder tumors]

Bladder tumors were induced in rats by the oral administration of 0.025% N-butyl-N-(hydroxybutyl) nitrosamine (BBN). In order to study the suppressive effects of rat interferon-alpha (IFN-alpha) on induction of carcinogenesis in vivo, rats were treated with IFN-alpha i.m. twice a week. The treatment began at 5th week of BBN administration. The bladder mucosa was observed macroscopically and microscopically, and NK activity was examined. The results were as follows. 1) There was no significant difference in the bladder to body weight ratio between the BBN + IFN-alpha and BBN groups in during stage A (10th-14th week when changes in the mucous membrane such as hypertrophy in the bladder wall or vascular formation are observed). This ratio in the BBN + IFN-alpha group was less than that in the BBN group in during stages B and C (15th-19th week and 20th-30th week respectively when tumors are visually recognizable). 2) The rate of carcinogenesis in the BBN + IFN-alpha group was less than that in the BBN group in stages A and C. 3) The pathological grade and stage of the bladder cancer in the BBN + IFN-alpha group were lower than those in the BBN group in stages B and C. 4) There was no significant difference in NK activity between the two groups in stage A, but NK activity of the BBN + IFN-alpha group was higher than that of the BBN group in stage B. 5) These findings substantiated the hypothesis that IFN-alpha can suppress tumor-growth in BBN induced bladder carcinoma.     

PTEN真核表达质粒对人膀胱癌裸鼠移植瘤的抑制效应

目的:探讨PTEN真核表达质粒对人膀胱癌裸鼠移植瘤的抑制作用。方法:将人膀胱癌细胞株BIU-87裸鼠背部皮下接种,成瘤后于瘤内多点注射重组真核表达质粒pBp-PTEN,设pBp空质粒和生理盐水为对照,观察肿瘤生长情况、PTEN表达的变化。结果:pBp-PTEN治疗组肿瘤变小,PTEN表达阳性率提高,与对照组比较,差异有统计学意义(P<0.05)。结论:PTEN真核表达质粒pBp-PTEN瘤内注射对人膀胱癌裸鼠移植瘤有一定的抑制作用。     

The sensitivity and clinical implications of periodical bladder biopsy following transurethral resection of superficial bladder transitional cell carcinoma

The role of the periodical bladder biopsy after transurethral resection (TUR-Bt) of superficial bladder cancer (sBT) was evaluated. Sixty-four patients (85 TURs) with sBT who underwent TUR-Bt between 1993 and 1998 were divided into 14 (22 TURs) who had carcinoma in situ (CIS) at the first TUR (group A), and 50 (64 TURs) who had papillary tumors without concomitant CIS (group B). Post-TUR intravesical instillation was performed with bacillus Calmette-Guerin for the majority of group A, and mitomycin C for the majority of group B. The first biopsy was performed at 3 months postoperatively, and the second biopsy was done at 8 to 12 months postoperatively. The mean observation time was 4 years and 6 months. Residual cancer was detected in 7 out of 34 biopsies (20.6%) in group A, and 19 out of 94 (20.2%) in group B. Every residual lesion in group A was CIS with negative cytology. In group B, with exclusion of 11 recurrent papillary tumors, the detection rate was only 8/83 (9.6%). In both groups, even in the cases with no sign of disease in biopsies, the recurrence immediately after the termination of the biopsy protocol was common. The progression of the cancer was more frequent in group A (4 patients), than in group B (2 patients) (p < 0.01, log-rank test), and no case in group B showed local progression. The periodical biopsy may have a certain, but limited advantage over conventional examinations. A less invasive and more sensitive method in awaited.     

荷人膀胱癌严重联合免疫缺陷鼠自发性转移模型的建立

目的建立荷人膀胱癌SCID鼠模型并观察其自发性转移情况。方法体外侵袭筛选膀胱癌细胞株T24,完成三次筛选的子代细胞记作T24-3;选取24只雌性SCID鼠,随机分为膀胱原位模型组和皮下异位模型组各12只,原位模型组采用酸预处理膀胱黏膜经尿道灌注3×106个T24-3细胞,同样剂量细胞注射接种于异位模型组鼠右后肢内侧皮下。通过成瘤性试验及鼠外周血中细胞角蛋白20（CK20）mRNA RT-PCR检测判定肿瘤模型及自发转移的发生。结果膀胱原位组64%成瘤,皮下组100%成瘤,肿瘤组织病理类型为移行细胞癌;且至6周膀胱原位组部分鼠出现肉眼可见多处远隔转移灶;不同周次部分鼠外周血中表达人CK20mRNA,至6周分别为5/11例和4/12例阳性表达。结论用经过侵袭筛选的高转移亚群T24-3细胞,行原位或异位种植可以成功制备荷瘤SCID鼠模型,并实现自发性转移。