【作者】 姚伟荣；

【导师】 徐方云；

【作者基本信息】 江西医学院 ， 病理生理学， 2003， 硕士

【摘要】 肿瘤疫苗是肿瘤主动免疫治疗的重要手段，其核心问题是肿瘤抗原的免疫原性。为提高肿瘤抗原的免疫原性，对抗原的修饰非常重要。脂质体作为一种无毒、无免疫原性的生物载体已得到广泛的应用，以脂质体负载抗原是增强肿瘤抗原免疫原性的有效方法之一。本课题采用阴离子脂质体制备宫颈癌U14细胞抗原瘤苗，实验观察其免疫效应和抑瘤作用，以探讨脂质体瘤苗用作肿瘤免疫治疗的可能性。正丁醇法提取U14细胞表面抗原（CBE），以PC，PG，Chol制成阴离子脂质体，运用薄膜水合法和超声粉碎、过膜相结合的方法，将CBE包裹入阴离子脂质体制成CBE-脂质体瘤苗。瘤苗（含CBE 50μg）腹腔接种免疫Km小鼠，每周一次，共2次。分别在末次免疫后的1、2、3、4周取血，用间接ELISA法检测血清抗CBE特异性抗体。此后，同法免疫Km小鼠，在末次免疫一周后，再腹腔接种104 U14细胞/每鼠，记录荷瘤动物的生存时间，观察瘤苗对后续U14细胞负荷攻击的免疫保护作用。实验发现：经CBE-脂质体瘤苗免疫的小鼠血清中有显著的抗CBE抗体生成，抗体滴度在末次免疫后的第2周开始升高，第3周达到高峰，此后保持一段时间。单纯CBE免疫也可产生抗CBE抗体，但各时间抗体滴度均明显低于瘤苗组（P<0.05）。抑瘤实验结果证实，瘤苗免疫组的荷瘤小鼠平均生存时间为25.50±2.08天，明显长于空白对照组（18.00±0.00天）、单纯脂质体组（18.33± 0.52天）和单纯CBE组（19.33±1.75天），t检验表明有非常显著的差异（P<0.01）；单纯CBE组的生存时间较对照组有所延长，但差异无显著性（P>0.05）。 瘤苗免疫组尚有33%的动物无瘤生存期超过60天。 本研究结果表明，脂质体包裹抗原能显著提高肿瘤抗原的免疫原性，激发出更强的体液免疫反应，并对后续腹腔攻击的相关肿瘤细胞产生明显的生长抑制作用。脂质体包被的肿瘤抗原是一种安全、有效的瘤苗，其在肿瘤主动免疫治疗中的作用值得进一步研究。更多还原

【Abstract】 Tumor vaccine is an important method of tumor active immunotherapy. Its core question is the immunogenicity of tumor antigen. In order to improve the immunogenicity of tumor antigen, modification is very essential. As a non-toxic and non-immunogenic biological carrier, liposome was widely used. Encapsulating antigen into liposome was an effective method to improve the immunogenicity of tumor antigen. In this experiment the antigenic vaccine of an cervical cancer U14 cells was producted using negative liposome and the immunologic and antitumor effect was observed in order to discuss the possibility of this vaccine used in tumor immunotherapy.The crude butanol extraction of U14 (U14-CBE) were prepared by 2.5% 1-butanol, and negative liposomes made up of PC, PG and Chol (7:1:2,molar ratio) were producted. The liposomal vaccine encapsulating U14-CBE was prepared by a combination of the thin-film hydration method, ultrasonication and extrusion through polycarbonate filters with a pore size of 220nm. The Km mice were immunized i.p. on day -14 and day -7. Each immunization consisted of 50μg CBE. From day 0, a group of mice were massacred every seven days(day 0,day 7,day 14,day 21). The production of anti-CBE antibody in serum was determined by an indirect Enzyme Linked Immunoabsorbant assay (ELISA). In another side, on day 0, 104 U14 cells/mouse were injected i.p. into those immunized mice, and surviving time was recorded for observing the protective immunity against the succesive challenged U14 cells accused by this vaccine.In this experimental the anti-CBE antibodies in the serum of the mice immunized with the liposomal vaccine were found significantly. The anti-CBE antibody began to appear on the second week after the latest administration, and reached a peak on the third week after latest administration .And it would retain for a certain time. Immunized with CBE alone could also product anti-CBE antibody, while the titre was lower than the former. In tumor challenge experiment the surviving time of mice immunized with liposomal vaccine is 25.50 ±2.08days, which was significantly<WP=6>longer than PBS control group (surviving time was 18.00±0.00 days), liposome-alone group (18.33 ±0.52days) and CBE-alone group (19.33 ±1.75days) (P<0.01). And about 33% mice immunized with liposomal vaccine could survive longer than 60 days without tumor appearence. It seems immunization with CBE alone could not prolong the surviving time (P>0.05).The present study indicates that liposome, when encapsulates tumor antigen, can improve the immunogenicity of this antigen. Liposomal antigen-baced vaccine can induce stronger humoral immunity responses and inhibit the growth of succesive challenged related tumor cells. The antigen incorporated in liposome is a safe and useful vaccine and its effects in tumor active immunotherapy are worthy to be studied deeply.更多还原