CN106047928A - 小鼠自发性胶质瘤免疫治疗动物模型的建立方法 - Google Patents
小鼠自发性胶质瘤免疫治疗动物模型的建立方法 Download PDFInfo
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Abstract
本发明提供一种小鼠自发性胶质瘤免疫治疗动物模型的建立方法，本发明应用SB载体局部转基因技术，利用AID基因诱导C57/BL6小鼠产生自发性胶质瘤从而建立了一种快速，自发的胶质瘤模型。所述SB是一种由两部分组成的系统，包含转座子DNA和转座酶，所述AID是在生理状态下可以在人类基因组中诱导DNA突变的酶，它能使DNA上的胞嘧啶脱氨基，转变成胸腺嘧啶。这种自发性胶质瘤模型能够应用生物发光技术监测肿瘤生长，其诱导肿瘤产生的速度等同于或优于病毒诱导的动物模型，而且没有病毒载体制备时间长，价格昂贵，致瘤率低等缺点。
Description
技术领域
本发明属于一种自发性胶质瘤免疫治疗动物模型的建立方法。
背景技术
胶质瘤是颅内最常见的原发性恶性肿瘤，约占成年人原发性脑肿瘤的一半,尽管手术治疗，放疗和化疗不断进步，胶质瘤患者的预后并未得到显著改善，恶性胶质瘤患者的平均生存期仅约一年左右。
近年来，国际上在胶质瘤的免疫治疗方面取得了较多的进展，这些研究所应用的胶质瘤动物模型大多是胶质瘤细胞系脑内移植瘤模型，然而实验证实胶质瘤细胞经过长时间的培养，已经出现多种蛋白表达的改变，不再具备最初肿瘤生物学性状。另外这些动物模型也不能体现胶质瘤的杂合性特征，不能反应胶质瘤与免疫系统之间的相互作用。
因此，本发明主要的主要目的是建立理想的胶质瘤免疫治疗动物模型，将能更好的模拟人恶性胶质瘤的杂合性和基因组不稳定状态。
发明内容
本发明要解决的技术问题是提供一种小鼠自发性胶质瘤免疫治疗动物模型的建立方法，能够有效能的模拟人恶性胶质瘤的杂合性和基因组不稳定状态。
本发明应用SB载体局部转基因技术，利用AID基因诱导C57/BL6小鼠产生自发性胶质瘤从而建立了一种快速，自发的胶质瘤模型。所述SB是一种由两部分组成的系统，包含转座子DNA和转座酶。SB转座酶能够切下转座子DNA并粘贴到目的基因组的TA双核苷酸中，编码转座酶的基因可以与转座子DNA在相同的或不同的质粒上。本研究中，我们采用的是转座酶和转座子在不同质粒上的方案。带有AID，或其他致瘤基因的转座子质粒以不同比率与编码转座酶的质粒混合，体内注射，诱导自发性胶质瘤的产生。SB局部转染操作简单，易于推广，可以建立理想的免疫治疗模型，但局部单纯转染SB质粒往往不能产生肿瘤，需要其他类型致瘤基因提供一个肿瘤产生的微环境，已经证实SV40-LgT和NRAS-G12V等基因可以与SB协作在局部产生自发性肿瘤。AID是目前为止唯一的生理状态下可以在人类基因组中诱导DNA突变的酶，它能使DNA上的胞嘧啶脱氨基，转变成胸腺嘧啶，这也是胶质瘤中最常见的突变类型，同时AID也可以产生其他类型突变。有大量实验证实AID可以在体内诱导肿瘤的产生。本研究应用SB载体局部转基因技术，利用AID基因诱导C57/BL6小鼠产生自发性胶质瘤从而建立了一种快速，自发的胶质瘤模型。这种自发性胶质瘤模型能够应用生物发光技术监测肿瘤生长，其诱导肿瘤产生的速度等同于或优于病毒诱导的动物模型，而且没有病毒载体制备时间长，价格昂贵，致瘤率低等缺点。
本研究中，我们证实了AID与SB合作将可以于局部诱导自发胶质瘤的产生。由于两者都能导致基因突变，因此所产生的肿瘤将能更好的模拟人恶性胶质瘤的杂合性和基因组不稳定状态。
该方法建立的小鼠脑胶质瘤动物模型稳定可靠, 符合恶性胶质瘤生物学特性。该模型可以用来建立理想的脑胶质瘤免疫治疗实验研究平台。
具体实施方式
为使本发明要解决的技术问题、技术方案和优点更加清楚，下面将结合具体实施例进行详细描述。
材料：pT2/C-luc/PGK-SB13质粒，pT/CAGGS-NRASV12质粒: 由John Ohlfest实验室惠赠。
实验动物及分组:健康C57/BL6小鼠由中国医科大学实验动物中心提供。小鼠配对，每天仔细监测直到胎鼠出生，所有小鼠均于SPF级实验动物饲养区饲养，小于两天的新生鼠用于进行自发胶质瘤诱导试验。36只雄性新生小鼠随机分成3组, 分别为PBS对照组，pT2/C-luc/PGK-SB13对照组和实验组，每组12只。
主要仪器设备: 微量进样器:上海。脑立体定向仪:江湾Ⅱ 型, 上海。
主要试剂: In vivo-jetPEI转染试剂 ：购自法国Polyplus-transfection公司，D-Luciferin, Potassium Salt（CAS:115144-35-9）：购自美国GOLDBIO公司。Maxiprep kit购自Invitrogen公司。
质粒载体的建立：克隆小鼠AID基因，克隆入pT3.5/CMV中，建立pT3.5/CMV-AID质粒。质粒体外扩增并应用Invitrogen的maxiprep kit纯化。
模型制作: 新生鼠首先腹腔注射20ul 25%甘露醇。然后将新生鼠置于冰上麻醉，固定于预冷的新生鼠立体定向架中，实验组应用显微注射泵向每只新生鼠右侧脑室内注入总体积为2 ul内含pT2/C-luc/PGK-SB13质粒0.1 ug、pT3.5/CMV-AID质粒0.2 ug和pT/CAGGS-NRASV12质粒0.2 ug以及in vivo-jetPEI试剂0.8ug的混合溶液，PBS对照组注射2ulPBS，pT2/C-luc/PGK-SB13对照组注射2ul pT2/C-luc/PGK-SB13和in vivo-jetPEI试剂混合液。新生小鼠局部注射，无需皮肤切口和磨开颅骨。坐标为相对于λ点：+1.5AP, 0.7ML,1.5DV。
肿瘤生长检测：生物发光技术监测肿瘤生长，生物发光检测时，动物腹腔注射avertin麻醉，然后注射luciferin，5分钟后应用小动物活体成像系统检测并应用相应软件分析。
HE染色：动物深度麻醉，PBS灌注后，应用4%的多聚甲醛固定。脑组织首先置于30%蔗糖溶液中48小时，然后石蜡切片，进行HE染色。
观察指标:每日观察小鼠活动及进食情况, 定期测量体重, 记录生存时间。
小鼠生存状态:接种瘤细胞后10天内, 实验组与对照组相比小鼠活动进食均无明显异常, 体重均有所增加。实验组小鼠多从12天左右逐渐出现进食减少,鼠毛失去光泽紊乱,发育迟缓，活动减少,接触反应减弱,偶有抽搐,肢体偏瘫,全身痉挛,进入濒死状态,既而死亡。平均生存31～46天(34.16±1.90天)。对照组均可长期存活。
小动物活体成像系统检测肿瘤生长:接种14天后小动物活体成像系统检测肿瘤提示PBS对照组和pT2/C-luc/PGK-SB13对照组均无肿瘤生长，实验组可见肿瘤明显生长。
病理组织形态学观察:实验组小鼠脑内可见肿瘤生长, 成瘤率83.3 %。肉眼观察肿瘤类似球形,边界较清楚,瘤内可见出血坏死。低倍镜下观察见肿瘤细胞呈巢状排列;肿瘤无包膜,瘤周有肿瘤细胞侵润,瘤周及瘤内新生血管丰富,瘤内常见出血,凝固性坏死灶。高倍镜下观察,见瘤细胞形态多样,分化程度低,核大, 多核,核质深染,异形性高。
理论上，免疫治疗可以激活患者的免疫系统，从而特异性的杀伤肿瘤细胞，是针对胶质瘤的理想治疗方法。近年来国际上在胶质瘤的免疫治疗方面取得了较多的进展，这些研究所应用的胶质瘤动物模型大多是胶质瘤细胞系脑内移植瘤模型，然而实验证实胶质瘤细胞经过长时间的培养，已经出现多种蛋白表达的改变，不再具备最初肿瘤生物学性状。另外这些动物模型也不能体现胶质瘤的杂合性特征，不能反应胶质瘤与免疫系统之间的相互作用。由于动物模型的局限，一些免疫疗法虽然在动物模型上取得了良好的疗效，但当应用到临床上时，却往往不能产生理想的治疗效果。因此建立理想的胶质瘤免疫治疗动物模型是当务之急。
免疫治疗研究对于动物模型的要求较高，1.动物模型应该能够模拟人胶质瘤病理特征，具有与人胶质瘤相似的杂合性。2.动物模型应该具备与人胶质瘤相似的疾病发生发展过程，能够体现胶质瘤与免疫系统的相互作用。3.模型动物必须具有完整的免疫系统。4、除肿瘤外，模型动物的其他系统（特别是免疫系统）不应具有基因突变。
以上所述是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明所述原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。
Claims (3)
1.一种小鼠自发性胶质瘤免疫治疗动物模型的建立方法，本发明应用SB载体局部转基因技术，利用AID基因诱导C57/BL6小鼠产生自发性胶质瘤从而建立了一种快速，自发的胶质瘤模型。
2.根据权利要求1所述的一种自发性胶质瘤免疫治疗动物模型，其特征在于，所述所述SB是一种由两部分组成的系统，包含转座子DNA和转座酶，SB转座酶能够切下转座子DNA并粘贴到目的基因组的TA双核苷酸中，编码转座酶的基因可以与转座子DNA在相同的或不同的质粒上。
3.根据权利要求1所述的一种自发性胶质瘤免疫治疗动物模型，其特征在于，所述AID是在生理状态下可以在人类基因组中诱导DNA突变的酶，它能使DNA上的胞嘧啶脱氨基，转变成胸腺嘧啶。
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