CCDC8基因沉默对裸鼠乳腺癌移植瘤生长影响的研究

发布时间：2018-04-14 06:12

本文选题：CCDC8基因 + 乳腺癌细胞MDA-MB-231　；参考：《昆明医科大学》2017年硕士论文

【摘要】：[目的]近年来乳腺癌有年轻化的趋势,而其中三阴性乳腺癌预后较差。三阴性乳腺癌被认为是多阶段、多因素、多基因共同作用的结果。本课题选用三阴性乳腺癌细胞株MIDA-MB-231。通过慢病毒载体法沉默该细胞株的CCDC8基因并构建裸鼠乳腺癌移植瘤动物模型,观察CCDC8基因沉默后对乳腺癌MDA-MB-231细胞增殖能力的影响及对裸鼠乳腺癌移植瘤生长的影响。[方法]1、以KD1、KD2序列为作用靶点合成两条siRNA干扰片段并用慢病毒载体法稳定转染MDA-MB-231细胞。2、通过qRT-PCR筛选CCDC8基因敲减率高的慢病毒稳定转染细胞组。3、通过细胞克隆形成实验观察慢病毒稳定转染细胞组的MDA-MB-231细胞克隆形成能力。4、采用流式细胞术观察慢病毒稳定转染细胞组的MDA-MB-231细胞周期、凋亡率变化。5、采用MTT检测慢病毒稳定转染细胞组的MDA-MB-231细胞增殖能力。6、构建裸鼠乳腺癌移植瘤动物模型,观察瘤体体积及重量变化。[结果]1、qRT-PCR检测显示KD2-siRNA能有效抑制CCDC8基因表达(P0.01)。2、细胞克隆形成实验结果显示MDA-MB-231细胞经CCDC8基因沉默后细胞克隆形成能力降低(P 0.01)。3、流式细胞术检测结果显示MDA-MB-231细胞经CCDC8基因沉默后细胞周期阻滞于G1期(P0.01)且凋亡率增加(P0.01)。4、MTT检测显示MDA-MB-231细胞经CCDC8基因沉默后细胞增殖能力受到抑制(P 0.01)。5、裸鼠皮下注入CCDC8基因沉默后的MDA-MB-231细胞后,裸鼠乳腺癌移植瘤的生长减缓(P 0.01)。[结论]1.三阴性乳腺癌细胞MDA-MB-231经CCDC8基因沉默后其细胞增殖能力减低、细胞凋亡率增加。2. CCDC8基因沉默可以抑制裸鼠乳腺癌移植瘤的生长。
[Abstract]:Objective: breast cancer tends to be younger in recent years, but the prognosis of three negative breast cancer is poor.Triple-negative breast cancer is considered to be the result of multi-stage, multi-factor and multi-gene interaction.In this study, three negative breast cancer cell line MIDA-MB-231 was selected.The CCDC8 gene of the cell line was silenced by lentivirus vector method and an animal model of breast cancer transplantation in nude mice was established. The effects of CCDC8 gene silencing on the proliferation of breast cancer MDA-MB-231 cells and the growth of breast cancer transplanted tumor in nude mice were observed.[methods] 1. Two siRNA interference fragments were synthesized with KD1hkD2 sequence as the target and stably transfected into MDA-MB-231 cells by lentivirus vector method. The stable transfection cell group of lentivirus with high CCDC8 knockout rate was screened by qRT-PCR, and the cell clone was formed.The clone forming ability of MDA-MB-231 cells in lentivirus stable transfection group was observed. Flow cytometry was used to observe the MDA-MB-231 cell cycle in the lentivirus stable transfected cell group.MTT was used to detect the proliferative ability of MDA-MB-231 cells in lentivirus stable transfection group. The nude mice model of breast cancer transplantation was established and the changes of tumor volume and weight were observed.[results] 1qRT-PCR assay showed that KD2-siRNA could effectively inhibit the expression of CCDC8 gene, and the result of cell clone formation experiment showed that the cell clone formation ability of MDA-MB-231 cells silenced by CCDC8 gene was decreased by CCDC8 gene, and that of MDA-MB-231 cells by CCDC8 was decreased by flow cytometry.After gene silencing, cell cycle arrest in G1 phase P0.01) and apoptosis rate increased P0.01. 4MTT assay showed that the proliferation of MDA-MB-231 cells was inhibited by CCDC8 gene silencing. After subcutaneous injection of CCDC8 gene silenced MDA-MB-231 cells in nude mice, the cell proliferation was inhibited.The growth of breast cancer xenografts in nude mice was slowed down (P 0.01).[conclusion] 1.Three negative breast cancer cell line MDA-MB-231 was silenced by CCDC8 gene. The cell proliferation ability was decreased and the apoptosis rate was increased by 2. 2.CCDC8 gene silencing can inhibit the growth of breast cancer xenografts in nude mice.
【学位授予单位】：昆明医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.9

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