PD-L1对人三阴性乳腺癌细胞增殖、凋亡的影响

发布时间：2019-01-18 07:38

【摘要】：背景乳腺癌属于全球高发癌症,在我国也是女性发病率最高的癌症,且近年来发病年龄愈趋向于年轻化,肿瘤本身呈高度异质性,病死率高。三阴性乳腺癌(Triple-negative breast cancer,TNBC)是其中一种乳腺癌亚型,该类型特点是癌细胞表面雌激素受体(estrogen receptor,ER)、孕激素受体(PR)和人表皮生长因子受体2(Her-2)免疫组化结果显示均为阴性,故而缺少特异性靶点,可行治疗手段单一,死亡率高,对该类癌细胞进展的分子机制研究有助于为三阴性乳腺癌治疗提供新靶点。程序性死亡配体1(PD-L1),又有B7-H1的称法,能和程序性死亡受体1(PD-1)互相识别并结合,在T细胞免疫反应过程中发挥负性调节作用。既往有研究表明各类型乳腺癌中三阴性乳腺癌细胞组织表面PD-L1表达较高,为研究PD-l1对三阴性乳腺癌细胞发生、发展的影响,本研究选取具有人三阴性乳腺癌特征的MDA-MB-231细胞株作为研究对象,探讨沉默PD-L1基因后三阴性乳腺癌细胞增殖及凋亡的情况。目的初步验证PD-L1在人三阴性乳腺癌细胞MDA-MB-231细胞株的表达情况;应用RNA干扰技术下调MDA-MB-231细胞系中PD-L1的表达,观察沉默PD-L1后对细胞增殖及凋亡的影响。方法1、Real-time PCR法检测三组乳腺细胞MDA-MB-231(ER、PR、Her-2均阴性)、MCF-7(ER、PR+;Her-2-)以及HBL-100(正常乳腺上皮细胞)中PD-L1的表达情况;2、应用RNA干扰技术构建稳定表达PD-L1 shRNA的MDA-MB-231细胞;3、转染后Real-time PCR检测PD-L1 mRNA;4、CCK-8法检测三组细胞增殖情况;流式细胞仪检测三组细胞凋亡及细胞周期。结果1、与正常乳腺上皮细胞(HBL-100)相比,PD-L1在乳腺癌中高表达(P0.01);且在三阴性乳腺癌细胞MDA-MB-231中表达高于MCF-7(P0.01);2、Real-time PCR检测转染后,相比较于空白组和对照组,PD-L1 sh RNA处理组中PD-L1 mRNA表达下降;3、转染PD-L1 shRNA后,MDA-MB-231细胞增殖能力(0.34±0.02)较阴性对照组(0.67±0.04)明显下降(F=120.342,P0.01);流式细胞仪结果显示PD-L1下调后实验组凋亡率(15.2%)较对照组明显升高(3.4%);G0/G1期的细胞比例(69.72±1.0)%较阴性对照组(58.34±1.8)%增加(F=58.071,P0.01)。结论PD-L1在人乳腺癌细胞中表达增高,尤其在三阴性乳腺癌细胞中表达明显;抑制细胞株MDA-MB-231 PD-L1的表达后,比之于空白组和对照组,三阴性乳腺癌细胞增值能力明显受到抑制,凋亡率增加,处于G0/G1期的细胞增多。
[Abstract]:Background Breast cancer is one of the most common cancers in the world and has the highest incidence among women in China. In recent years, the age of breast cancer tends to be younger, the tumor itself is highly heterogeneous and the mortality is high. Triple negative breast cancer (Triple-negative breast cancer,TNBC) is one of the subtypes of breast cancer, characterized by estrogen receptor (estrogen receptor,ER) on the surface of cancer cells. The results of immunohistochemical staining of progesterone receptor (PR) and human epidermal growth factor receptor 2 (Her-2) showed that both were negative. Molecular mechanisms for the progression of these cancer cells may provide new targets for triple-negative breast cancer therapy. Programmed death Ligand 1 (PD-L1), which has B7-H1, can be recognized and combined with programmed death receptor 1 (PD-1), and plays a negative role in T cell immune response. Previous studies have shown that there is a high expression of PD-L1 on the tissue surface of three-negative breast cancer cells in all types of breast cancer. In order to study the effect of PD-l1 on the occurrence and development of three-negative breast cancer cells, The aim of this study was to investigate the proliferation and apoptosis of three negative breast cancer cells with three negative PD-L1 gene silencing. Objective to investigate the expression of PD-L1 in MDA-MB-231 cell line of human triple-negative breast cancer cells. The expression of PD-L1 in MDA-MB-231 cell line was down-regulated by RNA interference technique, and the effect of PD-L1 silencing on cell proliferation and apoptosis was observed. Methods 1Real-time PCR assay was used to detect the expression of PD-L1 in breast MDA-MB-231 (ER,PR,Her-2 negative), MCF-7 (ER,PR; Her-2-) and HBL-100 (normal breast epithelial cells). (2) RNA interference technique was used to construct MDA-MB-231 cells expressing PD-L1 shRNA stably. 3. After transfection, the proliferation of three groups of cells was detected by PD-L1 mRNA;4,CCK-8 assay by Real-time PCR assay. Apoptosis and cell cycle were detected by flow cytometry. Results 1 the expression of PD-L1 was higher in breast cancer than in normal breast epithelial cells (HBL-100) (P0.01), and the expression of PD-L1 in MDA-MB-231 of three-negative breast cancer cells was higher than that of MCF-7 (P0.01). 2After transfection of real-time PCR, the expression of PD-L1 mRNA in PD-L1 sh RNA treated group was lower than that in blank group and control group. 3After transfection of PD-L1 shRNA, the proliferative ability of MDA-MB-231 cells (0.34 卤0.02) was significantly lower than that of negative control group (0.67 卤0.04). Flow cytometry showed that the apoptotic rate of the experimental group (15.2%) was significantly higher than that of the control group (3.4%) after down-regulation of PD-L1. The percentage of cells in G0/G1 phase (69.72 卤1.0)% was higher than that in negative control group (58.34 卤1.8%, P0.01). Conclusion the expression of PD-L1 is increased in human breast cancer cells, especially in triple-negative breast cancer cells. After inhibiting the expression of MDA-MB-231 PD-L1, the proliferation ability of triple-negative breast cancer cells was significantly inhibited, the apoptosis rate was increased and the number of cells in the G0/G1 phase was increased compared with the blank group and the control group.
【学位授予单位】：广东药科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.9

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