CN105879017A - Tnfr1/map4k4/arp3对裸鼠胶质瘤生长影响的脑内模型构建方法 - Google Patents
Tnfr1/map4k4/arp3对裸鼠胶质瘤生长影响的脑内模型构建方法 Download PDFInfo
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Abstract
TNFR1/MAP4K4/ARP3对裸鼠胶质瘤生长影响的脑内模型构建方法,其特征为包括以下步骤:步骤1,分别构建TNFR1、MAP4K4与ARP3过表达、小干扰的稳定细胞株,以1×108密度在脑立体定位仪下注射至裸鼠皮质内,构建裸鼠胶质瘤脑内模型;步骤2,4周后收集脑组织,检测肿瘤形成的大小、重量等情况,并比较其与裸鼠生存率的关系,HE染色检测肿瘤侵袭情况。
Description
技术领域
本发明涉及一种医学领域的疾病动物模型的构建方法,具体涉及TNFR1/MAP4K4/ARP3对裸鼠胶质瘤生长影响的脑内模型构建方法。
背景技术
长期以来对于胶质瘤侵袭性生长的研究主要着重于胶质瘤细胞内相关癌基因的作用机制,而忽略了炎症微环境的作用。1863年,现代病理学奠基人Rudolf Virchow就提出假说:炎症微环境是导致包括肿瘤在内的多种慢性疾病的原因之一。最近越来越多的实验和临床研究已经证实了这一假说,而这也是当前肿瘤研究的热点。组织损伤,无论是物理,化学或感染所致,均会发生炎症反应,是机体对伤害刺激的防御反应的一部分,但机体免疫功能未能控制的炎症反应将会扰乱细胞的微环境,从而导致癌症相关基因和细胞信号蛋白的翻译后修饰的改变;同时巨噬细胞、肥大细胞和中性粒细胞等炎症细胞可导致包括肿瘤坏死因子α(tumor necrosis factor-α,TNF-α)、白介素-6(interleukin-
6,IL-6)、转化生长因子-β(transforming growth factor-β,TGF-β)等在内的非特异性炎性细胞因子上调,从而支持肿瘤的发生发展。在恶性胶质瘤中,肿瘤相关巨噬细胞的浸润往往提示肿瘤患者预后的不良,说明炎症微环境在胶质瘤的发生发展中起关键作用。因此需要构建TNFR1/MAP4K4/ARP3胶质瘤生长影响的动物模型。
发明内容
本发明提供TNFR1/MAP4K4/ARP3对裸鼠胶质瘤生长影响的脑内模型构建方法。
TNFR1/MAP4K4/ARP3对裸鼠胶质瘤生长影响的脑内模型构建方法,其特征为包括以下步骤:步骤1,分别构建TNFR1、MAP4K4与ARP3过表达、小干扰的稳定细胞株,以1×108密度在脑立体定位仪下注射至裸鼠皮质内,构建裸鼠胶质瘤脑内模型;
步骤2 ,4周后收集脑组织,检测肿瘤形成的大小、重量等情况,并比较其与裸鼠生存率的关系,HE染色检测肿瘤侵袭情况;
步骤3,获取裸鼠肿瘤组织Western
Blot及免疫组化检测TNFR1、MAP4K4与ARP3的表达情况;
步骤4,组织消化,原代细胞培养,分析并验证TNFR1/MAP4K4/ARP3三聚体对裸鼠原代胶质瘤细胞肌动蛋白聚合、细胞伪足形成及细胞迁移、侵袭能力的影响。
进一步的,在步骤1中,分别构建TNFR1过表达、小干扰及不与MAP4K4相互作用的稳定细胞株;
在步骤3中,获取裸鼠肿瘤组织Western
Blot及免疫组化检测TNFR1及MAP4K4的表达情况;
在步骤4,验证并分析TNFR1与MAP4K4的相互作用对裸鼠原代胶质瘤细胞迁移、侵袭能力的影响。
进一步的,在步骤1中,分别构建MAP4K4过表达、小干扰及不与ARP3相互作用的稳定细胞株;
在步骤3中,获取裸鼠肿瘤组织Western
Blot及免疫组化检测MAP4K4及ARP3的表达情况;
在步骤4中,验证并分析MAP4K4与ARP3的相互作用对裸鼠原代胶质瘤细胞迁移、侵袭能力的影响。
构建裸鼠胶质瘤脑内模型,探讨干预TNFR1/MAP4K4/ARP3相互作用对胶质瘤发生发展的影响。将有助于揭示肿瘤微环境炎性因子与胶质瘤侵袭性生长的关系,为胶质瘤的治疗提供新的靶点。
具体实施方式
实施例1:
步骤1,分别构建TNFR1过表达、小干扰及不与MAP4K4相互作用的稳定细胞株,以1×108密度在脑立体定位仪下注射至裸鼠皮质内,构建裸鼠胶质瘤脑内模型;
步骤2,4周后收集脑组织,检测肿瘤形成的大小、重量等情况,并比较其与裸鼠生存率的关系,HE染色检测肿瘤侵袭情况;
步骤3,获取裸鼠肿瘤组织Western
Blot及免疫组化检测TNFR1及MAP4K4的表达情况。
步骤4,组织消化,原代细胞培养,验证并分析TNFR1与MAP4K4的相互作用对裸鼠原代胶质瘤细胞迁移、侵袭能力的影响。
实施例2:
步骤1,分别构建MAP4K4过表达、小干扰及不与ARP3相互作用的稳定细胞株,以1×108密度在脑立体定位仪下注射至裸鼠皮质内,构建裸鼠胶质瘤脑内模型;
步骤2,4周后收集脑组织,检测肿瘤形成的大小、重量等情况,并比较其与裸鼠生存率的关系,HE染色检测肿瘤侵袭情况;
步骤3,获取裸鼠肿瘤组织Western
Blot及免疫组化检测MAP4K4及ARP3的表达情况;
步骤4,组织消化,原代细胞培养,验证并分析MAP4K4与ARP3的相互作用对裸鼠原代胶质瘤细胞迁移、侵袭能力的影响。
实施例3:
步骤1,分别构建TNFR1、MAP4K4与ARP3过表达、小干扰的稳定细胞株,以1×108密度在脑立体定位仪下注射至裸鼠皮质内,构建裸鼠胶质瘤脑内模型;
步骤2 ,4周后收集脑组织,检测肿瘤形成的大小、重量等情况,并比较其与裸鼠生存率的关系,HE染色检测肿瘤侵袭情况;
步骤3,获取裸鼠肿瘤组织Western
Blot及免疫组化检测TNFR1、MAP4K4与ARP3的表达情况;
步骤4,组织消化,原代细胞培养,分析并验证TNFR1/MAP4K4/ARP3三聚体对裸鼠原代胶质瘤细胞肌动蛋白聚合、细胞伪足形成及细胞迁移、侵袭能力的影响。
Claims (3)
1.TNFR1/MAP4K4/ARP3对裸鼠胶质瘤生长影响的脑内模型构建方法,其特征为包括以下步骤:步骤1,分别构建TNFR1、MAP4K4与ARP3过表达、小干扰的稳定细胞株,以1×108密度在脑立体定位仪下注射至裸鼠皮质内,构建裸鼠胶质瘤脑内模型;
步骤2 ,4周后收集脑组织,检测肿瘤形成的大小、重量等情况,并比较其与裸鼠生存率的关系,HE染色检测肿瘤侵袭情况;
步骤3,获取裸鼠肿瘤组织Western Blot及免疫组化检测TNFR1、MAP4K4与ARP3的表达情况;
步骤4,组织消化,原代细胞培养,分析并验证TNFR1/MAP4K4/ARP3三聚体对裸鼠原代胶质瘤细胞肌动蛋白聚合、细胞伪足形成及细胞迁移、侵袭能力的影响。
2.如权利要求1所述TNFR1/MAP4K4/ARP3对裸鼠胶质瘤生长影响的脑内模型构建方法,其特征为在步骤1中,分别构建TNFR1过表达、小干扰及不与MAP4K4相互作用的稳定细胞株;
在步骤3中,获取裸鼠肿瘤组织Western Blot及免疫组化检测TNFR1及MAP4K4的表达情况;
在步骤4,验证并分析TNFR1与MAP4K4的相互作用对裸鼠原代胶质瘤细胞迁移、侵袭能力的影响。
3. 如权利要求1所述TNFR1/MAP4K4/ARP3对裸鼠胶质瘤生长影响的脑内模型构建方法,其特征为在步骤1,分别构建MAP4K4过表达、小干扰及不与ARP3相互作用的稳定细胞株;
在步骤3中,获取裸鼠肿瘤组织Western Blot及免疫组化检测MAP4K4及ARP3的表达情况;
在步骤4中,验证并分析MAP4K4与ARP3的相互作用对裸鼠原代胶质瘤细胞迁移、侵袭能力的影响。
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CN109694852A (zh) * | 2019-02-22 | 2019-04-30 | 武汉赛尔朗灵科技有限公司 | 一种人胶质瘤原代细胞及其体外分离培养方法和应用 |
CN110129445A (zh) * | 2019-05-29 | 2019-08-16 | 山东大学齐鲁医院 | 一种与胶质瘤相关的脑脊液外泌体miRNA标志物及其应用 |
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CN103627673A (zh) * | 2012-08-21 | 2014-03-12 | 上海睿智化学研究有限公司 | 一种人脑胶质瘤细胞系及其建立方法和应用 |
CN104582477A (zh) * | 2012-06-21 | 2015-04-29 | 社会福祉法人三星生命公益财团 | 制备患者特异性胶质母细胞瘤动物模型的方法及其用途 |
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CN104582477A (zh) * | 2012-06-21 | 2015-04-29 | 社会福祉法人三星生命公益财团 | 制备患者特异性胶质母细胞瘤动物模型的方法及其用途 |
CN103627673A (zh) * | 2012-08-21 | 2014-03-12 | 上海睿智化学研究有限公司 | 一种人脑胶质瘤细胞系及其建立方法和应用 |
Non-Patent Citations (2)
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唐万忠等: "《NF-kB与脑胶质瘤的研究进展》", 《中国临床神经外科杂志》 * |
马常慧等: "《肿瘤坏死因子受体1的信号传导与肿瘤的关系》", 《汕头大学医学院学报》 * |
Cited By (2)
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CN109694852A (zh) * | 2019-02-22 | 2019-04-30 | 武汉赛尔朗灵科技有限公司 | 一种人胶质瘤原代细胞及其体外分离培养方法和应用 |
CN110129445A (zh) * | 2019-05-29 | 2019-08-16 | 山东大学齐鲁医院 | 一种与胶质瘤相关的脑脊液外泌体miRNA标志物及其应用 |
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