恶性肿瘤基因工程纳米疫苗抗肿瘤转移与复发作用的研究

发布时间：2018-05-03 15:43

本文选题：纳米脂质体 + MAGE3　；参考：《第四军医大学》2007年硕士论文

【摘要】： 恶性肿瘤危害巨大,但恶性肿瘤的三大传统疗法-手术、放疗和化疗,治疗效果不尽人意。随着对肿瘤免疫理论的深入认识和一些新的实验方法的涌现,肿瘤疫苗已成为预防和治疗肿瘤转移、复发的有力手段。但由于机体免疫网络的复杂性、肿瘤异质性、疫苗生物利用度差等问题,使得现有肿瘤疫苗的免疫效果并不理想。黑色素瘤抗原(Melanoma Antigen,MAGE)是第一个被发现的人类肿瘤特异性抗原,MAGE3是MAGE家族的重要成员之一,在多种不同类型恶性肿瘤中均有表达,在正常组织细胞(除睾丸、胎盘外)不表达;且MAGE3可与人类白细胞抗原(Human Leukocytes Antigen,HLA)分子结合形成抗原复合物,此抗原复合物可被细胞毒性T淋巴细胞(Cytotoxic T Lymphocyte,CTL)的T细胞受体(T cell receptor,TCR)所识别,诱导特异性CTL的杀伤作用,是肿瘤特异性免疫治疗理想的靶分子。热休克蛋白(Heat Shock Protein,HSP)具有分子伴侣作用,参与肿瘤抗原的加工递呈,在诱导肿瘤免疫反应中发挥着重要作用;本室研究表明,HSP70与MAGE3融合蛋白能提高MAGE3蛋白疫苗的免疫效果。纳米载药系统与传统载药系统相比有很多优点,可有效解决蛋白质药物的生物利用度差的问题,目前已经有许多深入而广泛的研究。本研究以纳米脂质体包裹肿瘤特异性抗原MAGE3与热休克蛋白HSP70的融合蛋白(MH),制备出纳米脂质体疫苗NL(MH),着重对其抗肿瘤转移与复发作用进行研究,为该纳米疫苗临床前研究奠定基础。一纳米脂质体疫苗制备目的:制备无明显毒性、高效的恶性肿瘤基因工程纳米疫苗。方法:纯化融合蛋白MH,薄膜分散-超声法制备包裹MH的纳米脂质体肿瘤疫苗NL(MH),并评价其粒径、包裹率及稳定性;观察NL(MH)免疫组小鼠的急性毒性反应,及重要脏器的病理学改变。结果:①成功制备出粒径为80±25 nm的纳米脂质体,其药物包裹率为30%,于4℃放置6个月后或3000rpm 10分钟离心后无分层,证明其稳定性良好;②与PBS对照组相比,免疫组小鼠未见明显毒性反应。结论:该恶性肿瘤基因工程纳米疫苗具有良好的理化性质,未见毒性反应。二纳米疫苗预防和治疗肿瘤转移和复发效应研究目的:在小鼠体内观察纳米疫苗预防和治疗肿瘤肺转移及预防肿瘤复发的作用。方法:以PBS、空白脂质体、MH、MH /NL、NL(MH)免疫C57BL/6小鼠3次后,IFN-γELISPOT和LDH杀伤试实验检测纳米疫苗激活小鼠特异性细胞免疫反应的情况;以实验性肺转移模型和自然肺转移模型评价纳米疫苗预防和治疗肿瘤转移作用;以肿瘤攻击实验和肿瘤切除后复发实验来评价纳米疫苗预防肿瘤复发作用。结果:与其它组相比,NL(MH)组小鼠脾淋巴细胞中分泌IFN-γ的T细胞数量明显增多(P0.05),CTL对B16-MAGE3细胞具有显著的特异性杀伤作用;在预防和治疗肿瘤肺转移实验中,与其它组小鼠相比,NL(MH)免疫组小鼠B16-MAGE3肿瘤肺脏转移结节数明显减少(P0.05);在肿瘤攻击实验中,NL(MH)组B16-MAGE3肿瘤成瘤时间长、成瘤率低;肿瘤切除后复发实验中,NL(MH)组B16-MAGE3肿瘤复发时间延迟,复发率明显降低。结论: NL(MH)能够刺激机体产生强烈的MAGE3特异性的细胞免疫反应,对表达MAGE3的肿瘤细胞具有显著的杀伤作用,能有效预防和治疗B16-MAGE3肿瘤转移及预防肿瘤切除后复发。综上所述,本研究成功制备出粒径为80±25nm的纳米脂质体疫苗NL(MH),其稳定性良好;NL(MH)能诱导机体产生强烈的MAGE3特异性CTL,对表达MAGE3肿瘤转移和复发具有良好的治疗和预防作用,对表达MAGE3肿瘤复发具有良好的预防作用。本研究为研制使用安全、高效的肿瘤疫苗展示了良好的前景,为本疫苗的临床前研究奠定了基础。
[Abstract]:Malignant tumor is very harmful, but the three traditional therapies of malignant tumor - surgery, radiotherapy and chemotherapy are unsatisfactory. With the deep understanding of the theory of tumor immunity and the emergence of some new experimental methods, the tumor vaccine has become a powerful means to prevent and treat tumor metastasis and relapse. But the complexity of the immune network of the body is complicated. Melanoma Antigen (MAGE) is the first human tumor specific antigen found. MAGE3 is one of the important members of the MAGE family, which is expressed in many different types of malignant tumors and in the normal group. The cells (except the testis, the placenta) are not expressed, and MAGE3 can be combined with the Human Leukocytes Antigen (HLA) molecules to form an antigen complex, which can be identified by the T cell receptor of the cytotoxic T lymphocyte (Cytotoxic T Lymphocyte, CTL), and induces the killing effect of the specific antigen. It is an ideal target molecule for tumor specific immunotherapy. Heat Shock Protein (HSP) has the role of molecular chaperone, participates in the processing of tumor antigen and plays an important role in inducing tumor immune response. This laboratory study shows that the fusion of egg white with HSP70 and MAGE3 can improve the immune effect of the MAGE3 protein vaccine. Compared with the traditional drug carrying system, the system has many advantages, which can effectively solve the problem of poor bioavailability of protein drugs, and there have been a lot of in-depth and extensive research.
In this study, nano liposome specific antigen MAGE3 and heat shock protein HSP70 fusion protein (MH) was used to prepare nano liposome vaccine NL (MH), focusing on its anti-tumor metastasis and recurrence effect, which lay the foundation for the pre clinical study of the nano vaccine.
Preparation of a nano liposome vaccine
Objective: to prepare the nano vaccine of malignant tumor gene engineering without obvious toxicity and high efficiency. Methods: to purify the fusion protein MH and to prepare the nano liposome tumor vaccine NL (MH) coated with MH by thin film dispersion ultrasonic method and evaluate its size, encapsulation rate and stability. The acute toxicity of NL (MH) immunized mice and the pathological changes of important organs were observed. Results: (1) the nano liposomes with a diameter of 80 + 25 nm were successfully prepared. The drug encapsulation rate was 30%. After 6 months at 4 C or after 3000rpm 10 minutes without stratification, it proved that the stability was good. 2. Compared with the PBS control group, the immune group had no obvious toxic reaction. No toxic reaction was found in physical and chemical properties.
Two nano vaccine for prevention and treatment of tumor metastasis and recurrence
Objective: To observe the effect of nano vaccine in preventing and treating tumor lung metastasis and preventing tumor recurrence in mice. Methods: after 3 doses of PBS, blank liposomes, MH, MH /NL, NL (MH) immunized C57BL/6 mice, IFN- gamma ELISPOT and LDH killing test test was used to detect the specific cell immune response of mice activated by nano vaccine; the experimental lung transfer model was used. The effect of nano vaccine on the prevention and treatment of tumor metastasis was evaluated by the model and natural lung metastasis model. The effect of nano vaccine on tumor recurrence was evaluated by tumor attack experiment and recurrence experiment after tumor resection. Results: compared with other groups, the number of T cells secreted IFN- gamma in spleen lymphocyte of NL (MH) mice increased significantly (P0.05), CTL to B16-MAGE3 In the prevention and treatment of tumor lung metastasis, the number of metastatic nodules of lung in the NL (MH) immune group was significantly reduced in the NL (MH) immunization group than in the other mice (P0.05). In the tumor attack experiment, the NL (MH) group of B16-MAGE3 tumors had a long tumorigenesis and a low tumor rate; NL in the recurrence experiment after tumor resection, NL (MH) group B16-MAGE3 tumor recurrence time delay, recurrence rate obviously decreased. Conclusion: NL (MH) can stimulate the body to produce a strong MAGE3 specific cellular immune response, the expression of MAGE3 tumor cells have a significant killing effect, the effective prevention and treatment of B16-MAGE3 swollen tumor metastasis and the prevention of recurrence after tumor resection.
To sum up, the nano liposome vaccine NL (MH) with a particle size of 80 + 25nm was successfully prepared, and its stability was good. NL (MH) could induce a strong MAGE3 specific CTL in the body, and had a good therapeutic and preventive effect on the metastasis and recurrence of MAGE3 tumor, and had a good preventive effect on the recurrence of the MAGE3 tumor. The use of a safe and highly effective tumor vaccine shows good prospects and lays the foundation for preclinical research of this vaccine.

【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R392;R73-36

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