基于树突状细胞的膀胱肿瘤疫苗的实验研究
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摘要
膀胱肿瘤是我国泌尿系统最常见的肿瘤,其中多数为非肌层浸润性膀胱癌。膀胱肿瘤的主要特点是复发率高,可能的原因是膀胱肿瘤患者膀胱粘膜局部免疫功能低下,使得残存肿瘤容易逃避免疫监视,得以生存和形成复发。因此,非肌层浸润性膀胱肿瘤术后常辅以膀胱灌注化疗或免疫疗法以预防肿瘤复发和进展。卡介苗(Bacillus Calmette-Guerin,BCG)膀胱灌注是目前投入临床应用最有效的免疫治疗方法,有效激活膀胱粘膜局部免疫应答是其抗肿瘤作用的主要机制,但BCG膀胱灌注等治疗仍不能完全预防肿瘤的复发和进展。预防膀胱肿瘤复发和进展仍是尚未完全解决的难题。
树突状细胞(DC)是目前发现的体内功能最强,也是唯一能激活初始型T淋巴细胞的抗原递呈细胞(APC)。DC在肿瘤免疫中的功能强大,但肿瘤组织及外周血中DC的数目、成熟及功能受到抑制。随着体外大量扩增DC和制备DC疫苗技术的日趋成熟,采用DC疫苗进行抗肿瘤治疗已成为当今肿瘤生物治疗领域倍受关注的焦点之一。
环氧化酶2(COX-2)是一种诱导型酶,正常组织几乎无表达,与多种肿瘤的发生发展有关,对肿瘤的分化、凋亡、血管生成等生物学行为产生影响,且与肿瘤分级分期、进展预后等有关。因此,近年来,COX-2抑制剂作为膀胱肿瘤治疗的新型途径,获得了广泛的关注。我们试验组也进行了大量相关的研究工作,发现COX-2抑制剂对膀胱肿瘤有明显的杀伤与抑制增殖的作用,同时对DC细胞具有很强的诱导作用。
CpG是卡介苗(BCG)中提取的DNA片段,以未甲基化胞嘧啶鸟嘌呤(CpG)二核苷酸为核心的六碱基序列为基础,具有强大的免疫刺激活性。人工合成的含CpG序列的寡脱氧核糖核苷酸(CpG-ODN)具有与细菌DNA相同的免疫活性,可与DC表面TLR-9受体特异性结合,激活DC,上调共刺激分子的表达、增强DC向T细胞递呈抗原的能力,可以替代BCG使DC成熟获得抗原递呈和刺激免疫的功能。
本实验组近年来在COX-2与膀胱肿瘤的相关研究以及CpG-ODN联合DC疫苗的研究都进行了大量的工作,本课题是本实验室前期相关研究延续和深入。课题研究的目的在于建立以DC为基础,以COX-2抑制剂和免疫佐剂CpG-ODN刺激诱导其成熟并发挥免疫功能的膀胱肿瘤疫苗,研究其对膀胱肿瘤的生物学效应,为寻找一种新的治疗膀胱癌和预防其复发的方法奠定实验基础。
本研究主要研究内容及结果如下:
第一部分基于DC的膀胱肿瘤疫苗的建立及其形态与功能的初步鉴定。
目的:建立基于DC的膀胱肿瘤疫苗并进行初步鉴定。
方法:
1.从人脐血中分离单个核细胞,体外诱导培养为树突状细胞(DC),加入COX-2抑制剂塞来昔布和免疫佐剂CpG-ODN,刺激使其成熟,制备膀胱肿瘤疫苗。观察其形态并对其表面标志进行检测。
2. ELISA法测定各组培养上清中Th1型细胞因子(IL-12)和Th2型细胞因子(IL-10)水平。
3. MTT法测定各组DC的同种异体混合淋巴细胞反应性。
结果和结论:
1.经鉴定建立的以DC为基础的膀胱肿瘤疫苗具有典型DC形态学和表型特征。
2.细胞因子检测结果显示BCG激活DC后IL-12和IL-10水平均较对照组显著升高,表明BCG既激活了Th1型,也激活了Th2型免疫应答,而加入塞来昔布的2组DC的细胞因子IL-12分泌显著增加而IL-10表达明显下降,证实了塞来昔布引起的免疫漂移现象。同时,应用佐剂CpG-ODN也可以见到IL-12的分泌明显增加,但对IL-10的分泌没有明显影响。
3.同种异体混合淋巴细胞反应性检测结果显示,实验组MLR明显比对照组增强。这些结果都初步显示了我们制备的DC疫苗具有更强的抗原递呈效应和诱导机体免疫的效应,为我们进一步验证DC疫苗的生物效应和动物试验打下了良好的基础。
第二部分DC疫苗对体外培养的膀胱肿瘤细胞株T24的免疫学效应研究。
目的:观察建立的DC疫苗对体外培养的膀胱肿瘤T24细胞的免疫治疗效应。
方法:
1.制备DC疫苗激活的细胞毒T淋巴细胞(CTL);复苏和培养人膀胱肿瘤T24细胞作为CTL的靶细胞。
2.将贴壁生长的T24细胞,分别与各组DC疫苗共培养,乳酸脱氢酶(LDH)释放法测定各组DC激活的细胞毒T淋巴细胞(CTL)对人膀胱肿瘤细胞株T24的细胞毒活性。
结果和结论:
1.成功复苏和扩增了靶细胞T24,成功获得了DC疫苗激活的细胞毒T淋巴细胞(CTL)。
2. LDH释放法测定各组DC激活CTL对人膀胱肿瘤细胞株T24的细胞毒活性,我们制备的DC疫苗激活CTL,产生很强的抗膀胱肿瘤细胞毒活性的效应。
随着效/靶比提高,细胞毒活性亦相应加强。由此可以推断我们制备的DC疫苗可能成为膀胱肿瘤的有效治疗方法。
第三部分负载肿瘤抗原的DC疫苗对T739小鼠体内膀胱肿瘤的免疫学效应研究。
目的:观察建立的DC疫苗对BTT739膀胱肿瘤荷瘤小鼠体内膀胱肿瘤的免疫治疗效应。
方法:
1.采用细胞悬液法建立BTT739膀胱肿瘤荷瘤小鼠模型,分别于接种肿瘤细胞后第7天和14天,应用负载肿瘤抗原的DC疫苗肿瘤周围注射,观察肿瘤的体积、瘤重、荷瘤小鼠的生存期等变化。
2.经负载肿瘤抗原的DC疫苗治愈的2只小鼠,30天后仍无肿瘤生长,再次皮下接种BTT739细胞,观察肿瘤生长情况。
结果和结论:
1.负载肿瘤抗原的DC疫苗实验组荷瘤鼠肿瘤平均体积和平均瘤重均显著低于对照组(P<0.01),生存期长于对照组,并且有2只小鼠无瘤长期存活;负载肿瘤抗原的DC疫苗联合CpG-ODN对膀胱肿瘤荷瘤小鼠具有抑瘤效应和生存期延长作用。
2.经DC疫苗实验组治愈的2只小鼠30天后无肿瘤生长,再次皮下接种BTT739细胞观察30天仍无肿瘤发生。我们建立的DC疫苗对膀胱肿瘤细胞再次攻击具有免疫保护作用,其机制可能是诱导机体产生了长期的肿瘤记忆性细胞免疫反应,在膀胱肿瘤复发预防中具有良好应用前景。
全文结论:
1.建立了以DC为基础,以COX-2抑制剂和免疫佐剂CpG-ODN刺激其诱导成熟的膀胱肿瘤疫苗,其具有和普通DC相同的细胞形态和表型特征。并能诱导明显的Th1/Th2应答平衡向Th1型极性漂移的现象。
2.我们建立的DC疫苗诱导的特异性CTL对体外培养的膀胱肿瘤T24细胞具有的很强的细胞毒活性。
3.负载肿瘤抗原的DC疫苗对膀胱肿瘤荷瘤小鼠具有显著的抑瘤效应和生存期延长作用。
4.负载肿瘤抗原的DC疫苗可诱导机体产生并维持长期的肿瘤记忆性细胞免疫反应,对膀胱肿瘤细胞再次攻击具有免疫保护作用,在膀胱肿瘤复发预防中具有良好应用前景。
Bladder tumor, the most frequent urinary malignancy in Chinese people, of which most is non-muscle invasive tumor is characterized of high recurrence, owing probably to the local immunodeficiency in mucous membrane of urinary bladder, which makes residual tumor cells be able to escape from immunological surveillance, surviving and forming recurrence. Therefore, intravesical chemotherapy or immunotherapy is used postoperatively for the prevention of tumor recurrence and progress usually. Bacillus Calmette-Guerin (BCG) is currently the most effective intravesical immunotherapeutic agent, which takes the anti-tumor effect by the mechanism of effective activation of local immune response in mucous membrane of urinary bladder. However, treatments like intravesical BCG therapy are still not able to completely control recurrence and progress of bladder cancer. Prophylaxis of recurrence and progress of bladder cancer is a tough problem unsettled.
Dendritic cells(DC) are believed to have the most powerful antigen presenting capacity(APC) and the native T cells can be only primed by DCs. But in tumor tissue and peripheral blood of tumor patients, amount of DCs decreases, of which maturity and function is inhibited. With the development of technique of DCs amplification vitro and DC vaccine constructed, Dendritic cell-based vaccine manifest a good clinical applications as a representative of new vaccines.
Cyclooxygenase 2 (COX-2) is a kind of enzyme which is almost not expressed in normal human urothelial cells, but it is significantly over expressed in human multiple cancer cells. COX-2 affects cell differentiation, apoptosis, vascularization of tumors, and is related with tumor grading and staging, tumor progress and prognosis. Therefore COX-2 has been supposed to be a new therapeutic target for the treatment of bladder cancer. Many studies,with which it was found that COX-2 inhibitor could restrain progress of bladder cancer and had powerful entrainment on DCs, were carried out by our group.
CpG, a DNA frag abstracted from Bacillus Calmette-Guerin Recent which unmethylated CpG motifs in bacterial DNA, was believed that had powerful immunostimulatory activity. Synthetic oligodeoxynucleotides(ODNs) containing CpG motifs have the same activity as bacterial DNA. CpG-ODNs are recognized by specific Toll-like receptor 9 on the surface of DCs and induce an activataion phenotype of DCs characterized by the expression of costimulatory molecules, enhanced antigen presentation to T cells.
In recent years, studies about the relation of COX-2 , DC, CpG-ODN and bladder cancer were progressed by our group, constantly. The purpose of this study is to construct bladder tumor vaccine based on DC which is inducted by COX-2 inhibitor and CpG-ODN and detect its biological effect on bladder cancer. It should be a experiment foundation of looking for the new method of bladder cancer therapy and prophylaxis.
Part I. Construction of bladder tumor vaccine based on DC and detection initially of its biological effect.
Purpose: To construc bladder tumor vaccine based on DC and detect its biological effect, initially.
Methods:
1. Mononuclear cells were isolated from human cord blood and induced to differentiate to dendrtic cells (DCs) in vitro. Then DCs were stimulated mature using COX-2 inhibitor and CpG-ODN. Then the morphous and surface markers were detected.
2. DCs were co-cultured with reagent concerted. Th1-type cytokine IL-12 and Th2-type cytokine IL-10 were measured in culture supernatant of each group by enzyme-linked immunosorbent assay (ELISA).
3. Allogenic mixed lymphocyte reactivity (MLR) was determined by MTT assay of DCs in each group.
Results:
1. Results of identification showed that the cultured cells were morphology- and phenotype-typical DCs.
2. Results of cytokine detection demonstrated that after BCG infection, both IL-12 and IL-10 production of DCs increased significantly compared with blank group, and no dominant immunological drift of the Th response occured. Celecoxib stimulated increased IL-12 and decreased IL-10 production. The results indicated that Celecoxib induced Th response drift to Th1, meanwhile CpG-ODN increased IL-12 and unchanged IL-10 production.
3. Results of MLR detection showed that experimental group had higher stimulating index than control group.
The effect of antigen presentation and inducing immune response of DC vaccine has been enhanced by combining celecoxib and CpG-ODN.
Part II: Immunotherapy Effects of dendritic cell vaccine on bladder tumor cell T24 in vitro.
Purpose: To investigate immunotherapy effects of dendritic cells vaccine on bladder tumor line T24 in vitro.
Methods:
1. The cytotoxic T lymphocyte activited by DC vaccine was prepared. Meanwhile as the target cell, bladder cancer cells T24 were resuscitated and cultivited.
2. Adherent T24 cells were respectively co-cultured with the DCs. Cytotoxicity of CTLs activted by DCs of each group was measured by LDH release assay.
Results:
1. As the target cell, bladder cancer cells T24 were resuscitated and cultivited, successfully. And The cytotoxic T lymphocyte activited by DC vaccine were gained.
2. Results of LDH release assay suggested that DC vaccine activated CTLs enhanced cytotoxicity on human bladder cancer cell line T24.
PartⅢImmunotherapy Effects of dendritic cell vaccine plused with tumor antigen on bladder tumor in vivo.
Purpose: To investigate immunotherapy effects of dendritic cells vaccine plused with tumor antigen on bladder tumor in T739 mice.
Methods:
1. After the BTT739 bladder tumor model in T739 mice were successfully established, DC vaccine were injected respectively into the margins of the tumor(peritumoral) on day 7 and day 14 after tumor cells inoculation. Tumor weight, volume and the living time of mice were recorded.
2. After two mice cured with DC vaccine were rechallenged with BTT739 cells, the development of tumor was monitored.
Results:
1. Tumor weight and volume inhibition effects of DC vaccine treatment experiment group were significantly higher than that of control group (P<0.01). The living time of experiment group mice was significantly longer than that of control group. DC vaccine plused with tumor antigen can inhibit tumor growth and prolong the living time of T739 mice with BTT739 bladder tumor.
2. Two mice cured with DC vaccine were tumor-free 30 days after being rechallenged with BTT739 cells. DC vaccine plused with tumor antigen can lead to complete rejection of tumor rechallenge and provide long-term maintenance of tumor immunity, It should be an appealing immunotherapeutic approach to preventing the recurrence of bladder tumor in clinic.
Summary and Conclusions:
1. Mononuclear cells were isolated from human cord blood and induced to differentiate to dendrtic cells (DCs) in vitro. Then DCs were stimulated mature using COX-2 inhibitor and CpG-ODN and were confirmed that the cultured cells were morphology- and phenotype-typical DCs. Meanwhile Th response drifting to Th1 was induced.
2. DC vaccine can induce effector cells to appear strong cytotoxicity and killing activity on bladder cell T24.
3. DC vaccine plused with tumor antigen can inhibit tumor growth and prolong the living time of T739 mice with BTT739 bladder tumor.
4. DC vaccine plused with tumor antigen can lead to complete rejection of tumor rechallenge, which shows that this method should be an appealing immunotherapeutic approach to preventing the recurrence of bladder tumor in clinic.
树突状细胞(DC)是目前发现的体内功能最强,也是唯一能激活初始型T淋巴细胞的抗原递呈细胞(APC)。DC在肿瘤免疫中的功能强大,但肿瘤组织及外周血中DC的数目、成熟及功能受到抑制。随着体外大量扩增DC和制备DC疫苗技术的日趋成熟,采用DC疫苗进行抗肿瘤治疗已成为当今肿瘤生物治疗领域倍受关注的焦点之一。
环氧化酶2(COX-2)是一种诱导型酶,正常组织几乎无表达,与多种肿瘤的发生发展有关,对肿瘤的分化、凋亡、血管生成等生物学行为产生影响,且与肿瘤分级分期、进展预后等有关。因此,近年来,COX-2抑制剂作为膀胱肿瘤治疗的新型途径,获得了广泛的关注。我们试验组也进行了大量相关的研究工作,发现COX-2抑制剂对膀胱肿瘤有明显的杀伤与抑制增殖的作用,同时对DC细胞具有很强的诱导作用。
CpG是卡介苗(BCG)中提取的DNA片段,以未甲基化胞嘧啶鸟嘌呤(CpG)二核苷酸为核心的六碱基序列为基础,具有强大的免疫刺激活性。人工合成的含CpG序列的寡脱氧核糖核苷酸(CpG-ODN)具有与细菌DNA相同的免疫活性,可与DC表面TLR-9受体特异性结合,激活DC,上调共刺激分子的表达、增强DC向T细胞递呈抗原的能力,可以替代BCG使DC成熟获得抗原递呈和刺激免疫的功能。
本实验组近年来在COX-2与膀胱肿瘤的相关研究以及CpG-ODN联合DC疫苗的研究都进行了大量的工作,本课题是本实验室前期相关研究延续和深入。课题研究的目的在于建立以DC为基础,以COX-2抑制剂和免疫佐剂CpG-ODN刺激诱导其成熟并发挥免疫功能的膀胱肿瘤疫苗,研究其对膀胱肿瘤的生物学效应,为寻找一种新的治疗膀胱癌和预防其复发的方法奠定实验基础。
本研究主要研究内容及结果如下:
第一部分基于DC的膀胱肿瘤疫苗的建立及其形态与功能的初步鉴定。
目的:建立基于DC的膀胱肿瘤疫苗并进行初步鉴定。
方法:
1.从人脐血中分离单个核细胞,体外诱导培养为树突状细胞(DC),加入COX-2抑制剂塞来昔布和免疫佐剂CpG-ODN,刺激使其成熟,制备膀胱肿瘤疫苗。观察其形态并对其表面标志进行检测。
2. ELISA法测定各组培养上清中Th1型细胞因子(IL-12)和Th2型细胞因子(IL-10)水平。
3. MTT法测定各组DC的同种异体混合淋巴细胞反应性。
结果和结论:
1.经鉴定建立的以DC为基础的膀胱肿瘤疫苗具有典型DC形态学和表型特征。
2.细胞因子检测结果显示BCG激活DC后IL-12和IL-10水平均较对照组显著升高,表明BCG既激活了Th1型,也激活了Th2型免疫应答,而加入塞来昔布的2组DC的细胞因子IL-12分泌显著增加而IL-10表达明显下降,证实了塞来昔布引起的免疫漂移现象。同时,应用佐剂CpG-ODN也可以见到IL-12的分泌明显增加,但对IL-10的分泌没有明显影响。
3.同种异体混合淋巴细胞反应性检测结果显示,实验组MLR明显比对照组增强。这些结果都初步显示了我们制备的DC疫苗具有更强的抗原递呈效应和诱导机体免疫的效应,为我们进一步验证DC疫苗的生物效应和动物试验打下了良好的基础。
第二部分DC疫苗对体外培养的膀胱肿瘤细胞株T24的免疫学效应研究。
目的:观察建立的DC疫苗对体外培养的膀胱肿瘤T24细胞的免疫治疗效应。
方法:
1.制备DC疫苗激活的细胞毒T淋巴细胞(CTL);复苏和培养人膀胱肿瘤T24细胞作为CTL的靶细胞。
2.将贴壁生长的T24细胞,分别与各组DC疫苗共培养,乳酸脱氢酶(LDH)释放法测定各组DC激活的细胞毒T淋巴细胞(CTL)对人膀胱肿瘤细胞株T24的细胞毒活性。
结果和结论:
1.成功复苏和扩增了靶细胞T24,成功获得了DC疫苗激活的细胞毒T淋巴细胞(CTL)。
2. LDH释放法测定各组DC激活CTL对人膀胱肿瘤细胞株T24的细胞毒活性,我们制备的DC疫苗激活CTL,产生很强的抗膀胱肿瘤细胞毒活性的效应。
随着效/靶比提高,细胞毒活性亦相应加强。由此可以推断我们制备的DC疫苗可能成为膀胱肿瘤的有效治疗方法。
第三部分负载肿瘤抗原的DC疫苗对T739小鼠体内膀胱肿瘤的免疫学效应研究。
目的:观察建立的DC疫苗对BTT739膀胱肿瘤荷瘤小鼠体内膀胱肿瘤的免疫治疗效应。
方法:
1.采用细胞悬液法建立BTT739膀胱肿瘤荷瘤小鼠模型,分别于接种肿瘤细胞后第7天和14天,应用负载肿瘤抗原的DC疫苗肿瘤周围注射,观察肿瘤的体积、瘤重、荷瘤小鼠的生存期等变化。
2.经负载肿瘤抗原的DC疫苗治愈的2只小鼠,30天后仍无肿瘤生长,再次皮下接种BTT739细胞,观察肿瘤生长情况。
结果和结论:
1.负载肿瘤抗原的DC疫苗实验组荷瘤鼠肿瘤平均体积和平均瘤重均显著低于对照组(P<0.01),生存期长于对照组,并且有2只小鼠无瘤长期存活;负载肿瘤抗原的DC疫苗联合CpG-ODN对膀胱肿瘤荷瘤小鼠具有抑瘤效应和生存期延长作用。
2.经DC疫苗实验组治愈的2只小鼠30天后无肿瘤生长,再次皮下接种BTT739细胞观察30天仍无肿瘤发生。我们建立的DC疫苗对膀胱肿瘤细胞再次攻击具有免疫保护作用,其机制可能是诱导机体产生了长期的肿瘤记忆性细胞免疫反应,在膀胱肿瘤复发预防中具有良好应用前景。
全文结论:
1.建立了以DC为基础,以COX-2抑制剂和免疫佐剂CpG-ODN刺激其诱导成熟的膀胱肿瘤疫苗,其具有和普通DC相同的细胞形态和表型特征。并能诱导明显的Th1/Th2应答平衡向Th1型极性漂移的现象。
2.我们建立的DC疫苗诱导的特异性CTL对体外培养的膀胱肿瘤T24细胞具有的很强的细胞毒活性。
3.负载肿瘤抗原的DC疫苗对膀胱肿瘤荷瘤小鼠具有显著的抑瘤效应和生存期延长作用。
4.负载肿瘤抗原的DC疫苗可诱导机体产生并维持长期的肿瘤记忆性细胞免疫反应,对膀胱肿瘤细胞再次攻击具有免疫保护作用,在膀胱肿瘤复发预防中具有良好应用前景。
Bladder tumor, the most frequent urinary malignancy in Chinese people, of which most is non-muscle invasive tumor is characterized of high recurrence, owing probably to the local immunodeficiency in mucous membrane of urinary bladder, which makes residual tumor cells be able to escape from immunological surveillance, surviving and forming recurrence. Therefore, intravesical chemotherapy or immunotherapy is used postoperatively for the prevention of tumor recurrence and progress usually. Bacillus Calmette-Guerin (BCG) is currently the most effective intravesical immunotherapeutic agent, which takes the anti-tumor effect by the mechanism of effective activation of local immune response in mucous membrane of urinary bladder. However, treatments like intravesical BCG therapy are still not able to completely control recurrence and progress of bladder cancer. Prophylaxis of recurrence and progress of bladder cancer is a tough problem unsettled.
Dendritic cells(DC) are believed to have the most powerful antigen presenting capacity(APC) and the native T cells can be only primed by DCs. But in tumor tissue and peripheral blood of tumor patients, amount of DCs decreases, of which maturity and function is inhibited. With the development of technique of DCs amplification vitro and DC vaccine constructed, Dendritic cell-based vaccine manifest a good clinical applications as a representative of new vaccines.
Cyclooxygenase 2 (COX-2) is a kind of enzyme which is almost not expressed in normal human urothelial cells, but it is significantly over expressed in human multiple cancer cells. COX-2 affects cell differentiation, apoptosis, vascularization of tumors, and is related with tumor grading and staging, tumor progress and prognosis. Therefore COX-2 has been supposed to be a new therapeutic target for the treatment of bladder cancer. Many studies,with which it was found that COX-2 inhibitor could restrain progress of bladder cancer and had powerful entrainment on DCs, were carried out by our group.
CpG, a DNA frag abstracted from Bacillus Calmette-Guerin Recent which unmethylated CpG motifs in bacterial DNA, was believed that had powerful immunostimulatory activity. Synthetic oligodeoxynucleotides(ODNs) containing CpG motifs have the same activity as bacterial DNA. CpG-ODNs are recognized by specific Toll-like receptor 9 on the surface of DCs and induce an activataion phenotype of DCs characterized by the expression of costimulatory molecules, enhanced antigen presentation to T cells.
In recent years, studies about the relation of COX-2 , DC, CpG-ODN and bladder cancer were progressed by our group, constantly. The purpose of this study is to construct bladder tumor vaccine based on DC which is inducted by COX-2 inhibitor and CpG-ODN and detect its biological effect on bladder cancer. It should be a experiment foundation of looking for the new method of bladder cancer therapy and prophylaxis.
Part I. Construction of bladder tumor vaccine based on DC and detection initially of its biological effect.
Purpose: To construc bladder tumor vaccine based on DC and detect its biological effect, initially.
Methods:
1. Mononuclear cells were isolated from human cord blood and induced to differentiate to dendrtic cells (DCs) in vitro. Then DCs were stimulated mature using COX-2 inhibitor and CpG-ODN. Then the morphous and surface markers were detected.
2. DCs were co-cultured with reagent concerted. Th1-type cytokine IL-12 and Th2-type cytokine IL-10 were measured in culture supernatant of each group by enzyme-linked immunosorbent assay (ELISA).
3. Allogenic mixed lymphocyte reactivity (MLR) was determined by MTT assay of DCs in each group.
Results:
1. Results of identification showed that the cultured cells were morphology- and phenotype-typical DCs.
2. Results of cytokine detection demonstrated that after BCG infection, both IL-12 and IL-10 production of DCs increased significantly compared with blank group, and no dominant immunological drift of the Th response occured. Celecoxib stimulated increased IL-12 and decreased IL-10 production. The results indicated that Celecoxib induced Th response drift to Th1, meanwhile CpG-ODN increased IL-12 and unchanged IL-10 production.
3. Results of MLR detection showed that experimental group had higher stimulating index than control group.
The effect of antigen presentation and inducing immune response of DC vaccine has been enhanced by combining celecoxib and CpG-ODN.
Part II: Immunotherapy Effects of dendritic cell vaccine on bladder tumor cell T24 in vitro.
Purpose: To investigate immunotherapy effects of dendritic cells vaccine on bladder tumor line T24 in vitro.
Methods:
1. The cytotoxic T lymphocyte activited by DC vaccine was prepared. Meanwhile as the target cell, bladder cancer cells T24 were resuscitated and cultivited.
2. Adherent T24 cells were respectively co-cultured with the DCs. Cytotoxicity of CTLs activted by DCs of each group was measured by LDH release assay.
Results:
1. As the target cell, bladder cancer cells T24 were resuscitated and cultivited, successfully. And The cytotoxic T lymphocyte activited by DC vaccine were gained.
2. Results of LDH release assay suggested that DC vaccine activated CTLs enhanced cytotoxicity on human bladder cancer cell line T24.
PartⅢImmunotherapy Effects of dendritic cell vaccine plused with tumor antigen on bladder tumor in vivo.
Purpose: To investigate immunotherapy effects of dendritic cells vaccine plused with tumor antigen on bladder tumor in T739 mice.
Methods:
1. After the BTT739 bladder tumor model in T739 mice were successfully established, DC vaccine were injected respectively into the margins of the tumor(peritumoral) on day 7 and day 14 after tumor cells inoculation. Tumor weight, volume and the living time of mice were recorded.
2. After two mice cured with DC vaccine were rechallenged with BTT739 cells, the development of tumor was monitored.
Results:
1. Tumor weight and volume inhibition effects of DC vaccine treatment experiment group were significantly higher than that of control group (P<0.01). The living time of experiment group mice was significantly longer than that of control group. DC vaccine plused with tumor antigen can inhibit tumor growth and prolong the living time of T739 mice with BTT739 bladder tumor.
2. Two mice cured with DC vaccine were tumor-free 30 days after being rechallenged with BTT739 cells. DC vaccine plused with tumor antigen can lead to complete rejection of tumor rechallenge and provide long-term maintenance of tumor immunity, It should be an appealing immunotherapeutic approach to preventing the recurrence of bladder tumor in clinic.
Summary and Conclusions:
1. Mononuclear cells were isolated from human cord blood and induced to differentiate to dendrtic cells (DCs) in vitro. Then DCs were stimulated mature using COX-2 inhibitor and CpG-ODN and were confirmed that the cultured cells were morphology- and phenotype-typical DCs. Meanwhile Th response drifting to Th1 was induced.
2. DC vaccine can induce effector cells to appear strong cytotoxicity and killing activity on bladder cell T24.
3. DC vaccine plused with tumor antigen can inhibit tumor growth and prolong the living time of T739 mice with BTT739 bladder tumor.
4. DC vaccine plused with tumor antigen can lead to complete rejection of tumor rechallenge, which shows that this method should be an appealing immunotherapeutic approach to preventing the recurrence of bladder tumor in clinic.
引文
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