基于microcarrier 6人胃癌正常免疫小鼠模型的建立及病理特征探讨

发布时间：2018-02-23 19:33

本文关键词： 胃肿瘤微载体 MKN45细胞动物模型　出处：《山东大学》2017年硕士论文　论文类型：学位论文

【摘要】：研究目的基于新型微载体(microcarrier 6)复合人胃癌MKN45细胞接种于具有正常免疫功能的小鼠,以期建立新型人胃癌正常免疫小鼠移植瘤模型,并探讨其病理学特征。研究方法将60只雄性C57BL/6小鼠随机分成3组,2D组、对照组和3D组,每组20只;2D组只接种MKN45细胞,对照组只接种微载体,3D组接种细胞-微载体复合体。将人胃癌MKN45细胞与microcarrier 6共培养建立三维肿瘤细胞培养体系,采用皮下接种法将细胞-微载体复合体接种于C57BL/6小鼠皮下建立小鼠移植瘤模型。观察记录小鼠一般状况及瘤体生长情况,统计成瘤时间及成瘤率,分别于接种第10、20、30天分三批处死荷瘤小鼠并对瘤组织进行病理学检查。采用SPSS 13.0统计软件进行数据分析,计量资料数据以x士s表示,采用独立样本t检验进行组间比较,以P0.05为差异有统计学意义。研究结果1.基于microcarrier6体外建立MKN45细胞三维培养体系:2D环境下观察MKN45细胞贴壁,呈不规则形;microcarrier 6镜下观察为不规则团状或长梭状,质地疏松;MKN45细胞与microcarrier 6共培养24h后可以看到MKN45细胞可以很好的贴附在microcarrier6上,接种均匀,达到饱和。2.动物生存及成瘤情况:2D组和对照组小鼠食欲和活动度没有明显变化,第10天开始,3D组小鼠活动稍减少;各组食欲、毛发、体质量无明显差异;实验过程中没有小鼠死亡。对照组和2D组小鼠均未成瘤,3D组小鼠在接种后8～10 d即可触及皮下肿块,2周左右肉眼可观察到皮下有隆起包块,肿块生长迅速,3周左右即可达到0.5～1cm3体积,成瘤率为80%(16/20只)。3.病理学检查:HE染色后光镜下可见大量杂乱排列、大小不一、异形性明显的细胞,并向周围脂肪、肌肉组织浸润生长;伴有明显坏死,主要位于肿瘤中央,以20天处死组最为显著;10天处死组未被清除的部分microcarrier 6被包绕形成肉芽肿,20天时仍可见少许microcarrier 6,30天时microcarrier 6基本被清除干净;各组移植瘤组织中新生毛细血管丰富,多位于肿瘤边缘。免疫组化染色结果显示,CA199、CEA、COX-2、CK-7均为阳性表达,进一步证实异形性细胞为人肿瘤细胞。研究结论本实验成功基于microcarrier 6复合人胃癌MKN45细胞在正常免疫小鼠中建立了人胃癌小鼠移植瘤模型,且移植瘤的病理学特征符合人胃癌的特点,此模型可以更好的研究和进一步阐明胃癌在免疫功能正常机体中发生、发展机制,同时也为胃癌的精准化个体治疗及抗癌药的研发提供了较以往更有价值的新的动物模型。
[Abstract]:Objective to establish a xenograft tumor model of a new type of human gastric cancer immunized mice based on a novel microcarrier microcarrier 6 combined with human gastric cancer MKN45 cells inoculated into mice with normal immune function. Methods 60 male C57BL / 6 mice were randomly divided into 3 groups: 2D group, control group and 3D group. 20 mice in each group were inoculated with MKN45 cells only. Human gastric cancer MKN45 cells were co-cultured with microcarrier 6 to establish a three-dimensional tumor cell culture system. C57BL / 6 mice were inoculated subcutaneously with cell-microcarrier complex to establish transplanted tumor model. The general status and tumor growth of mice were observed and recorded, and the tumorigenesis time and tumorigenesis rate were counted. The tumor-bearing mice were killed in three batches on the 30th day of the 10th day of inoculation and the pathological examination of the tumor tissue was carried out. The data were analyzed by SPSS 13.0 statistical software. The measurement data were expressed as x + s, and the independent sample t test was used to carry out the inter-group comparison. Results 1. The three-dimensional culture system of MKN45 cells based on microcarrier6 was established in vitro to observe the adhesion of MKN45 cells under the environment of 1: 2D. The cells were observed to be irregular clusters or long fusiform cells under the microscope. 2. After co-culture of MKN45 cells with microcarrier6 for 24 hours, it was found that MKN45 cells could be well attached to microcarrier6, inoculated evenly and reached saturation. 2. There was no significant change in appetite and activity of mice in 2: 2D group and control group. On the 10th day, the activity of mice in 3D group decreased slightly, but there was no significant difference in appetite, hair and body mass in each group. No mice died in the experiment. In the control group and 2D group, the mice in the control group and the 2D group could reach the subcutaneous mass about 2 weeks after inoculation. The tumor could reach 0.5 ~ 1 cm ~ 3 volume in 3 weeks, and the tumorigenesis rate was 80 / 16 / 20. Pathological examination showed a large number of irregular cells with different size and obvious heteromorphism, and infiltrated and grew in the surrounding fat and muscle tissue under the light microscope after the pathological examination: 1. There was obvious necrosis, mainly located in the center of the tumor. The most significant part of microcarrier 6 in the death group on the 20th day was that microcarrier 6 was removed basically after 20 days of death, and the part of microcarrier 6 was wrapped around to form granuloma after 20 days of death. At 30 days, a little microcarrier 6 could still be removed. The new capillaries were abundant in the transplanted tumor tissues in each group, and most of them were located at the margin of the tumor. The immunohistochemical staining showed that the expression of CK-7 was positive in CA199CEACOX-2CK-7 cells. Conclusion the xenograft tumor model of human gastric cancer mice was successfully established in normal immunized mice based on microcarrier 6 combined with human gastric cancer MKN45 cells. The pathological characteristics of transplanted tumor accord with the characteristics of human gastric cancer. This model can better study and further elucidate the mechanism of gastric cancer occurrence and development in normal immune system. It also provides a more valuable animal model for the precise individual treatment of gastric cancer and the development of anti-cancer drugs.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.2;R-332

【相似文献】