【作者】 任淑萍；

【导师】 王丽颖；

【作者基本信息】 吉林大学 ， 生物化学与分子生物学， 2005， 博士

【摘要】 MUC1 蛋白在多种肿瘤细胞呈高水平表达。这些肿瘤细胞包括:乳腺癌细胞、卵巢癌细胞、胰腺癌细胞、前列腺癌细胞、膀胱癌细胞、黑色素瘤细胞和喉癌细胞等。MUC1 能够刺激机体产生抗肿瘤的特异性细胞毒效应。在乳腺癌、卵巢癌、胰腺癌患者体内存在MUC1 特异性的抗体和受MHC 限制的MUC1 特异性CTLs,这说明,MUC1 可以刺激机体产生针对MUC1 的抗肿瘤细胞免疫应答。外源性的蛋白类肿瘤抗原在免疫人体后,通常被抗原提呈细胞摄取、加工,进入MHC II 类提呈途径,激发体液免疫反应,但不能有效激活肿瘤特异性的CTL 的产生。因此,需要一种能够将肿瘤抗原携带入APC 内,并使肿瘤抗原与MHCI 类分子结合后被递呈在APC 的表面,进而激活CD8+T 淋巴细胞。热休克蛋白(heat shock protein)恰具有这种能力和作用。HSP 是存在于多种生物体内的一个分子伴侣蛋白质家族。在免疫应答的过程中,HSP 可以协助外源性的肿瘤抗原进入包括树突状细胞在内的抗原提呈细胞并进入MHC I 类抗原提呈途径,进而激活肿瘤抗原特异性细胞毒性T 淋巴细胞。为了提高HSP65-MUC1 激活特异性CTL 的效率,开发出HSP65-MUC1 的高效佐剂,本文研究了BCG(Bacillus Calmette Guerin,BCG)对HSP65-MUC1 特异性抑瘤效果的影响,并对BCG 发挥佐剂作用的机制进行了探讨。体外实验结果表明:BCG 和HSP65-MUC1 联合应用,能够显著上调DC 表面CD86分子的表达,其上调程度明显高于BCG 和HSP65-MUC1 的单独作用。体内实验结果证实:BCG+HSP65-MUC1 组小鼠的肿瘤体积和重量均显著低于BCG 组和HSP65-MUC1 组。BCG 免疫小鼠后,体内会产生HSP65 抗体。小鼠体内HSP65 抗体的存在,并没有影响HSP65-MUC1 的抑瘤效果。小鼠体内已存在HSP65 抗体后,接受HSP65-MUC1注射后,会在小鼠体内形成抗原抗体复合物(Immune complex,IC)。体外实验结果表明:IC 能够显著地使DC 表面的CD86 分子表达上调,其上调效果明显高于HSP65-MUC1 的作用。体内抑瘤试验结果表明:体内HSP65 抗体的存在组小鼠肿瘤的重量和体积均显著小于PBS 组。以上的实验结果清楚表明:BCG 可以作为HSP65-MUC1 的高效佐剂。更多还原

【Abstract】 At present, stratigies employed to treat cancers are operation, chemotherapy, and radiotherapy etc. But each of them has its drawbacks. Recent years, immunotherapy based on tumor vaccines has been developed. Accumulating data demonstrates that cytotoxic T lymphocytes (CTL) are the most powerful tumor cell killers in the immune system, therefore a key consideration of developing an effcient tumor vaccine is how to select a tumor antigen with specific CTL generating properties. MUC1 is a tumor-specific antigen which is highly expressed in tumor cells of breast cancer, prostate cancer, colon cancer, lung cancer and ovarian cancer etc while non or very low level of MUC1 is expressed in normal cells. Several MUC1 based tumor vaccines have been developed and their efficacies are testing in Phase I, Phase II and Phase III clinical trials with promising results. But exogenous antigens fail to generate CTL in vivo because they are taken up, processed and loaded with MHCII pathway in antigen presenting cells and subsequently activate humoral immune response, but can not induce effectively the development of tumor-specific CTL, and therefore can not be tumor preventive and therapeutic. So, conferring exogenously applied MUC1 with activities of specific CTL generating is crucial for the development of the most effective MUC1-based recombinant protein that will induce preventive and therapeutic CTLs to human MUC1 expressing carcinomas including breast cancer, ovarian cancer, colon cancer, prostate cancer and lung cancer. Several experiments have demonstrated that heat-shock proteins (HSP) can confer an antigenic peptide with CTL generating properties and thereby activate human tumor-specific CTL to kill tumor cells in an effective way. Heat shock protein 65 was fused to MUC1 to produce a recombinant fusion protein HSP65-MUC1, is injected into the mice, it can stimulate specific cytotoxic T lymphocytes(CTL) which are targeting to tumor cells which express high level of MUC1, the elicited CTL will not kill those cells which express no MUC1 or express low level of MUC1.BCG has been used for many years in prevention of lung tubeculosis. It is discovered that BCG stimulatre antigen presenting cells including dendritic cells to induce the expression of costimulatory molecules such as CD86 which is a crucial signal for the activation of CTL and activate TOLL-LIKE receptor mediated signaling pathways. To explore whether BCG enhances HSP65-MUC1 to generate MUC1 specific CTLs which kills MUC1 expressing tumor cells, tumor suppression experiments were conducted in mice using BCG and HSP65-MUC1. To determine whether BCG can enhance the tumor suppressionelicited by HSP65-MUC1, mice were injected by BCG and HSP65-MUC1 in BCG+HSP65-MUC1 group and mice in HSP65-MUC1 group were injected by HSP65-MUC1. BCG and PBS group were also set up. Mice were injected by MUC1 transfected B16 cells, two days later, the mice were injected with BCG and/or HSP65-MUC1 respectively. Mice were sacrificed on day 33 after the mice were injected with MUC1 transfected B16 cells and the tumor were removed and weighed. The tumor size in mice injected by BCG and HSP65-MUC1 was significantly smaller than that in mice injected by HSP65-MUC1, indicating that BCG can enhance the tumor suppression elicited by HSP65-MUC1. To explore how BCG enhance the tumor suppression elicited by HSP65-MUC1, effects of BCG and HSP65-MUC1 on the antigen presenting cells DC were elucidated. BCG and HSP65-MUC1 can upregulate the CD86 molecules expression on the surface of DC more significantly than HSP65-MUC1 alone and BCG alone, suggesting that BCG and HSP65-MUC1 can work synergically to upregulate the expression of CD86 molecules on the surface of DC. Since the HSP65 gene is part of the BCG genome, most people have been immunized with BCG when they were born, HSP65-MUC1 injected may be eliminated by a secondary immune reaction specific to BCG or elicits allergic reactions. To testify whether BCG immunization in infants will affect the tumor suppression elicited by HSP65-MUC1, mice of aged 4-5 weeks wereimmunized with BCG, when the mice grew up to 6-7 weeks, based on different groups, they were injected BCG and or HSP65-MUC1 The tumor volume in mice injected with BCG and HSP65-MUC1 was much smaller than that in mice injected with BCG alone and HSP65-MUC1 alone, suggesting that infant mice were immunize with BCG did not have negative effects on the efficacy of tumor suppression elicited by HSP65-MUC1.The efficacy of tumor suppression in mice immunized with BCG and HSP65-MUC1 was significantly greater than that immunized with BCG alone and HSP65-MUC1 alone(P<0.05). It was demonstrated that mice can produce HSP65 antibodies after immunized with BCG. When the mice were injected with HSP65-MUC1, HSP65-MUC1 antigen/HSP65 antibody immune complex (IC) would be formed in the mice. To elucidate the effects of HSP65-MUC1/HSP65 antibodies IC on the tumor suppression elicited by HSP65-MUC1, mice were immunized with BCG three times to produce HSP65 antibodies in mice and then the mice were injected with HSP65-MUC1 to test the tumor suppression elicited by HSP65-MUC1. The tumor weight and volume in mice injected with HSP65-MUC1 was much smaller than that in mice not injected with HSP65-MUC1. Effects of IC on the expression of CD86 molecules on the surface of DC were conducted to explore the mechanisms of IC enhancing tumor suppression elicited by HSP65-MUC1. HSP65-MUC1/HSP65 IC was a better activator of DC compared with HSP65-MUC1, suggesting that IC can activating DC more effectively than antigen HSP65-MUC1. Mechanisms that BCG can enhance the efficacy of tumorsuppression elicited by HSP65-MUC1 are that BCG can stimulate dendritic cells and upregulate the expression of costimulatory molecules CD 86. The activation of specific cytotoxic T lymphocytes depends on the availability of two signals. The first signal is the complex of MHC class I molecules with peptides. The first signal is recognized by T cell receptor(TCR). Another is costimulatory molecules expressed by antigen presenting cells, the second signal is recognized by CD28 molecules of T lymphocytes. Only when the T lymphocytes receive the two signals at the same time, can T lymphocytes become activated. Recognition of the MHC-peptide complex by the TCR in the absence of CD28 ligation by B7 molecules leads to clonal inactivation of T cells. Activation of a given T cell depends, therefore, on the availability of the specific antigenic peptides and on the expression of B7 molecules on the surface of antigen-presenting cells that present these peptides in complex with MHC molecules. Importantly, the cell surface expression of the B7 molecules on the antigen-presenting cells is inducible by infection. Thus, in the absence of infection, antigen-presenting cells express self-peptides (the only peptides available in the absence of infection) but not B7 molecules. Therefore, T cells specific for the self-peptides (autoreactive T cells) are not activated. Upon infection, however, antigen-presenting cells begin to express B7 molecules and present pathogen-derived peptides to T cells. Thus T cells specific to pathogen-derived peptides become activated because they receive both signals necessary for activation. B7 molecules, therefore, function as signals that "flag" pathogen-derivedpeptides as foreign. The results from the activation of DC by BCG and HSP65-MUC1 demonstrated: BCG+HSP65-MUC1 can significantly upregulate the expression of CD86(B7.2)molecules on the surface of DC to a higher level than that of BCG alone and HSP65-MUC1 alone. The results from Shoutaro et al’s research showed that the cell wall of BCG can activate DC through TLR2 and TLR4, upregulating the expression of CD80, CD83, CD86 on the surface of DC and enhancing the antigen presenting ability of DC, in addition, BCG can still stimulate the expression of cytokines which will assist the activation and maturation of DC. The content of cytokine IL-6, IL-12 and TNFαis significantly higher in the culture supernatant of DC in BCG+HSP65-MUC1 group than those in BCG, HSP65-MUC1, PBS alone. The function of IL-12 is to induce lymphocytes to differentiate into T lymphocytes. TNFαis the most important cytokine which induce DC to mature. Therefore,the combination of BCG with HSP65-MUC1 can increase the activation efficiency and intensity of cytotoxic T lymphocytes to a stronger extent. From the discussion above, it is clear that BCG can act as an efficient adjuvant to HSP65-MUC1. Furthermore, BCG is very safe in clinical application. The presence of HSP65 antibodies did not affect the tumor suppression elicited by HSP65-MUC1. The results from the present study showed that the presence of HSP65 in mice could enhance the tumor suppression elicited by HSP65-MUC1 because the tumor weight and volume was significantly lighter and smaller in the mice which HSP65 antibodies already present. The reasons that the presence of HSP65 antibodies can strengthen the tumor suppression elicited by HSP65-MUC1 may be that IC can activate strongerly specific T lymphocytes, since the results from the present research showed that IC could upregulate the expression level of CD86 molecules on the surface of DC to a higher extent compared with HSP65-MUC1 alone and BCG alone. The results from Danita H and Armelle R’s research suggested that IC could activate CD8 + T lymphocytes through cross-presentation. The reason that IC can be easilier presented may be that IC could bind to Fcγreceptor which can mediate endocytosis on the DC surface through Fcγchain of HSP65 antibodies, so IC will be easily endocytosed by APC, like DC, and then processed, loaded with MHC class I molecules and presented on the surface of DC, which can be recognized by TCR of T lymphocytes. Thus,IC is easilier to be endocytosed compared to soluble antigen. From the results and discussion above, we can draw some conclusions. 1. BCG can act as efficacious adjuvant to HSP65-MUC1. 2. BCG can enhance the tumor suppression elicited by HSP65-MUC1. 3. BCG can induce the expression of costimulatory molecules on the surface of DC; in addition, BCG can also induce the excretion of cytokines such as TNFα, IL-12. 4. The presence of HSP65 antibodies and formation of IC did not affect the tumor suppression elicited by HSP65-MUC1, on the更多还原