本文選題：程序性死亡分子1　切入點(diǎn)：(PD-L1)　出處：《中國人民解放軍醫(yī)學(xué)院》2017年碩士論文

【摘要】：目的:多項研究發(fā)現(xiàn),腎透明細(xì)胞癌(clear cell renal cell carcinoma, ccRCC)中異常高表達(dá)的程序死亡性分子1 (programmed death ligand 1,PD-L1)與其較差預(yù)后密切相關(guān)。目前正在進(jìn)行的Atezolizumab (抗PD-L1抗體)治療晚期及轉(zhuǎn)移性腎透明細(xì)胞癌的臨床試驗取得了符合預(yù)期的階段性結(jié)果,但內(nèi)源性PD-L1的表達(dá)調(diào)控機(jī)制及其對腫瘤細(xì)胞增殖和侵襲能力改變的作用尚不完全清楚。Notch1信號通路在ccRCC的發(fā)生發(fā)展過程中發(fā)揮重要作用,但其與PD-L1之間是否存在相互調(diào)控關(guān)系亦不清楚。所以本實驗主要探索Notch1及內(nèi)源性PD-L1之間是否存調(diào)控與被調(diào)控的關(guān)系以及PD-L1對ccRCC腫瘤生物學(xué)功能的作用。方法:1、首先驗證Notch1及PD-L1在37對腎透明細(xì)胞癌樣本及其瘤旁樣本、HKC、786-0和769-P等三種細(xì)胞系中的表達(dá)情況;2、利用Notch1和PD-L1特異性小干擾RNA(short interfering RNA,siRNA)或過表達(dá)質(zhì)粒分別在786-0及769-P細(xì)胞中構(gòu)建敲低或過表達(dá)模型,并檢測兩者表達(dá)水平的變化以探索兩者之間的調(diào)控關(guān)系;4、通過細(xì)胞周期分析、MTS及平板克隆評估干預(yù)后的腫瘤細(xì)胞的增殖能力。同時,通過Transwell及劃痕實驗對上述細(xì)胞的遷移和侵襲能力進(jìn)行分析。P0.05時差異具有統(tǒng)計學(xué)意義。結(jié)果:1、在37對樣品中,Notch1和PD-L1在腫瘤組織中同時異常高表達(dá)者共26對(70.3%),同時表達(dá)降低者共8對(21.6%),而3對(8.1%)樣品分析結(jié)果為Notch1和PD-L1表達(dá)變化方向不一致。同時,Notch1及PD-L1在腫瘤細(xì)胞系中的mRNA水平與蛋白水平表達(dá)均高于正常細(xì)胞系。2、敲低Notch1后,PD-L1在mRNA及蛋白質(zhì)水平的表達(dá)均同時降低;而敲低PD-L1卻沒有引起Notch1表達(dá)水平的變化。3、過表達(dá)Notch1后,PD-L1表達(dá)同時增高。4、在研究內(nèi)源性PD-L1在腫瘤細(xì)胞生物學(xué)行為中發(fā)揮的作用時,我們將786-0和769-P細(xì)胞株分別分為四組:實驗組:PD-L1敲低組和Notch1+PD-L1共敲低組;對照組:陰性組和空白組。MTS實驗提示,實驗組腫瘤細(xì)胞增殖顯著減低,Notch1+PD-L1共敲低組細(xì)胞增殖能力最低(P0.05)。細(xì)胞周期分析提示,實驗組細(xì)胞受抑制主要原因在于細(xì)胞被阻滯在G1期。平板克隆實驗結(jié)果與上述兩個實驗結(jié)果相一致。5、Transwell實驗提示:實驗組腫瘤細(xì)胞的遷移和侵襲能力較對照組顯著減低,其中Notch1+PD-L1共敲低組細(xì)胞的轉(zhuǎn)移侵襲能力最低(P0.05);其結(jié)果與劃痕實驗相一致。結(jié)論:在腎透明細(xì)胞癌中,內(nèi)源性PD-L1表達(dá)水平的變化受到Notch1的調(diào)控。異常高表達(dá)的內(nèi)源性PD-L1促進(jìn)腫瘤細(xì)胞增殖、遷移和侵襲。聯(lián)合抑制Notch1和PD-L1的抗腫瘤效果可能優(yōu)于單獨(dú)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.
【學(xué)位授予單位】：中國人民解放軍醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R737.11

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