肾透明细胞癌中Notch1对内源性PD-L1表达调控机制的研究

发布时间：2018-03-30 21:46

本文选题：程序性死亡分子1　切入点：(PD-L1)　出处：《中国人民解放军医学院》2017年硕士论文

【摘要】：目的:多项研究发现,肾透明细胞癌(clear cell renal cell carcinoma, ccRCC)中异常高表达的程序死亡性分子1 (programmed death ligand 1,PD-L1)与其较差预后密切相关。目前正在进行的Atezolizumab (抗PD-L1抗体)治疗晚期及转移性肾透明细胞癌的临床试验取得了符合预期的阶段性结果,但内源性PD-L1的表达调控机制及其对肿瘤细胞增殖和侵袭能力改变的作用尚不完全清楚。Notch1信号通路在ccRCC的发生发展过程中发挥重要作用,但其与PD-L1之间是否存在相互调控关系亦不清楚。所以本实验主要探索Notch1及内源性PD-L1之间是否存调控与被调控的关系以及PD-L1对ccRCC肿瘤生物学功能的作用。方法:1、首先验证Notch1及PD-L1在37对肾透明细胞癌样本及其瘤旁样本、HKC、786-0和769-P等三种细胞系中的表达情况;2、利用Notch1和PD-L1特异性小干扰RNA(short interfering RNA,siRNA)或过表达质粒分别在786-0及769-P细胞中构建敲低或过表达模型,并检测两者表达水平的变化以探索两者之间的调控关系;4、通过细胞周期分析、MTS及平板克隆评估干预后的肿瘤细胞的增殖能力。同时,通过Transwell及划痕实验对上述细胞的迁移和侵袭能力进行分析。P0.05时差异具有统计学意义。结果:1、在37对样品中,Notch1和PD-L1在肿瘤组织中同时异常高表达者共26对(70.3%),同时表达降低者共8对(21.6%),而3对(8.1%)样品分析结果为Notch1和PD-L1表达变化方向不一致。同时,Notch1及PD-L1在肿瘤细胞系中的mRNA水平与蛋白水平表达均高于正常细胞系。2、敲低Notch1后,PD-L1在mRNA及蛋白质水平的表达均同时降低;而敲低PD-L1却没有引起Notch1表达水平的变化。3、过表达Notch1后,PD-L1表达同时增高。4、在研究内源性PD-L1在肿瘤细胞生物学行为中发挥的作用时,我们将786-0和769-P细胞株分别分为四组:实验组:PD-L1敲低组和Notch1+PD-L1共敲低组;对照组:阴性组和空白组。MTS实验提示,实验组肿瘤细胞增殖显著减低,Notch1+PD-L1共敲低组细胞增殖能力最低(P0.05)。细胞周期分析提示,实验组细胞受抑制主要原因在于细胞被阻滞在G1期。平板克隆实验结果与上述两个实验结果相一致。5、Transwell实验提示:实验组肿瘤细胞的迁移和侵袭能力较对照组显著减低,其中Notch1+PD-L1共敲低组细胞的转移侵袭能力最低(P0.05);其结果与划痕实验相一致。结论:在肾透明细胞癌中,内源性PD-L1表达水平的变化受到Notch1的调控。异常高表达的内源性PD-L1促进肿瘤细胞增殖、迁移和侵袭。联合抑制Notch1和PD-L1的抗肿瘤效果可能优于单独抑制PD-L1。
[Abstract]:Objective: a number of studies have found, Clear cell renal cell carcinomas (ccRCCs) are associated with poor prognosis. Atezolizumab (anti-#en6# antibody) is currently being used in the treatment of advanced and metastatic renal clear cell carcinoma. The results of clinical trials were in line with the expected results. However, the regulatory mechanism of endogenous PD-L1 expression and its effect on the proliferation and invasion of tumor cells are not fully understood. Notch1 signaling pathway plays an important role in the pathogenesis and development of ccRCC. But it is not clear whether there is a mutual regulation relationship between Notch1 and PD-L1. Therefore, this experiment mainly explores the relationship between Notch1 and endogenous PD-L1 and the effect of PD-L1 on the biological function of ccRCC tumor. Method: 1, first. The expression of Notch1 and PD-L1 in 37 cell lines of renal clear cell carcinoma and its adjacent samples was tested. Notch1 and PD-L1 specific small interfering RNA(short interfering siRNAs or overexpression plasmids were constructed in 786-0 and 769-P cells, respectively. Knock down or over-express the model, In order to explore the regulatory relationship between them, the cell cycle analysis of MTS and plate clone were used to evaluate the proliferative ability of tumor cells after intervention. Transwell and scratch test were used to analyze the migration and invasion ability of the above cells. The results showed that there were 26 pairs of cells with abnormal expression of Notch1 and PD-L1 in tumor tissues in 37 pairs of samples. The results of analysis showed that the expression of Notch1 and PD-L1 were not the same. The expression of mRNA and PD-L1 in tumor cell line was higher than that in normal cell line. The expression of mRNA and PD-L1 in tumor cell line was higher than that in normal cell line. The expression of PD-L1 in tumor cell line was higher than that in normal cell line. After knocking down Notch1, the expression of PD-L1 in tumor cell line was higher than that in normal cell line. The expression of mRNA and protein decreased at the same time. However, knockout of PD-L1 did not cause the change of Notch1 expression level. However, the expression of PD-L1 increased simultaneously after overexpression of Notch1, which was important in the study of the role of endogenous PD-L1 in the biological behavior of tumor cells. We divided 786-0 and 769-P cell lines into four groups: the experimental group: the 1: PD-L1 knockout group and the Notch1 PD-L1 co-knockout group; the control group: negative group and blank group. The proliferation of tumor cells in the experimental group was significantly reduced, and the proliferation ability of the cells in the co-knockout group was the lowest (P0.05N). The cell cycle analysis suggested that the tumor cell proliferation was significantly decreased in the experimental group. The main reason for the inhibition of the cells in the experimental group was that the cells were blocked in G1 phase. The results of the plate cloning assay were in agreement with the results of the above two experiments. The results showed that the migration and invasion ability of the tumor cells in the experimental group was significantly lower than that in the control group. The metastatic and invasive ability of Notch1 PD-L1 co-knockout group was the lowest, and the result was consistent with scratch test. Conclusion: in renal clear cell carcinoma, The changes of endogenous PD-L1 expression were regulated by Notch1. The abnormal high expression of endogenous PD-L1 promoted the proliferation, migration and invasion of tumor cells. The anti-tumor effect of combined inhibition of Notch1 and PD-L1 may be better than that of PD-L1 alone.
【学位授予单位】：中国人民解放军医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.11

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