Notch1基因表达对T-ALL细胞增殖及硼替佐米敏感性的影响

发布时间：2018-02-12 01:47

本文关键词： Notch1 T-ALL RNA干扰硼替佐米药物敏感性　出处：《山西医科大学》2015年硕士论文　论文类型：学位论文

【摘要】：目的:通过检测干扰Notch1基因后Sup T1细胞的增殖、凋亡和Notch1受体基因及其下游靶基因表达水平的变化,探讨Notch信号通路与急性T淋巴细胞白血病发生、发展的关系。在下调Notch1基因后的Sup T1细胞中加入1ng/μl硼替佐米,检测对Sup T1细胞增殖、凋亡和Notch1受体基因及其下游靶基因表达水平的变化,探讨Notch1基因表达对硼替佐米敏感性的影响。方法:1.利用携带Notch1特异性和非特异性sh RNA的慢病毒载体包装成病毒颗粒感染Sup T1细胞,选取干扰效率高的感染细胞作为干扰组,分别于48、72、96个小时后,采用CCK-8法检测各组细胞增殖率;采用流式细胞术检测各组细胞凋亡率(Annexin V+/7-AAD-和Annexin V+/7-AAD+);采用实时荧光定量PCR法检测Notch1受体基因及下游靶基因基因Hes1、NF-κB、c-myc的m RNA表达水平。2.在感染Sup T1细胞后,分别在第24、48、72小时三个不同时间点加入1ng/μl硼替佐米继续孵育24小时后,采用CCK-8法、流式细胞术、实时荧光定量PCR法检测其在干扰Notch1基因后第48、72、96小时二者共同作用对Sup T1细胞增殖、凋亡及Notch1受体基因及下游靶基因基因Hes1、NF-κB、c-myc的m RNA表达水平的影响。结果:1.Notch1干扰组细胞的增殖较空白对照组、空载体组明显降低,细胞抑制率较空白对照组、空载体组明显升高(P均0.05),且细胞增殖的抑制作用呈时间依赖性。2.干扰组Annexin V-PE+/7-AAD-较空白对照组、空载体组明显升高(P均0.05);而各组细胞Annexin V-PE+/7-AAD+无明显变化(P0.05)。3.干扰组细胞中Notch1受体基因及其下游靶基因Hes1、c-myc、NF-κB表达较空白对照组及空载体组显著降低(P均0.05)。4.硼替佐米对Sup T1细胞的增殖有明显的抑制作用,且抑制作用呈浓度和时间依赖性;干扰加药组、空载加药组细胞抑制率均高于干扰组、空载体组(P均0.05)。5.干扰加药组细胞Annexin V-PE+/7-AAD-较空白对照组及空载体组明显升高(P均0.05);各组细胞Annexin V-PE+/7-AAD+细胞差异均无统计学意义(P0.05)。6.干扰加药组下游基因表达水平较干扰组明显降低(P0.05),且干扰组及干扰加药组的基因Notch1、Hes1、c-myc、NF-κB表达抑制呈时间依赖性。结论:1.特异性Notch1-sh RNA-4252可有效下调Notch1 m RNA的表达,并降低下游靶基因水平的表达;2.Notchl表达下调可以抑制Sup T1细胞增殖,且细胞抑制作用呈时间依赖性;促进Sup T1细胞的早期凋亡。3.sh RNA干扰后硼替佐米对Sup T1细胞增殖抑制、凋亡、Notch1受体基因及下游靶基因抑制更加明显,特异性sh RNA干扰Notchl基因与硼替佐米呈叠加作用。
[Abstract]:Objective: to investigate the relationship between Notch signaling pathway and acute T lymphocyte leukemia (AML) by detecting the proliferation, apoptosis and expression of Notch1 receptor gene and its downstream target gene in Sup T1 cells after interfering with Notch1 gene. The relationship of development. 1 ng / 渭 l bortezomil was added to Sup T1 cells after down-regulation of Notch1 gene. The changes of proliferation, apoptosis and expression of Notch1 receptor gene and its downstream target genes were detected in Sup T1 cells. To investigate the effect of Notch1 gene expression on the sensitivity of bortezomi.Methods 1. The lentivirus vector carrying Notch1 specific and nonspecific sh RNA was used to package virus particles to infect Sup T1 cells, and infected cells with high interference efficiency were selected as interference groups. The proliferation rate of each group was detected by CCK-8 assay after 72 hours and 96 hours respectively. Flow cytometry was used to detect the apoptosis rate of Annexin V / 7-AAD- and Annexin V / 7-AAD, and real-time fluorescence quantitative PCR was used to detect the expression of m RNA in Notch1 receptor gene and downstream target gene Hes1- 魏 Bc-myc. 1 ng / 渭 l borotezomil was added at 24 ~ 48h / 72h for 24 h, respectively. CCK-8 assay, flow cytometry and real-time fluorescence quantitative PCR were used to detect the proliferation of Sup T1 cells at 48h 7296 h after interfering with Notch1 gene. Apoptosis and the expression of m RNA in Notch1 receptor gene and downstream target gene Hes1- 魏 Bnc-myc. Results 1. The proliferation of cells in the Notch1 interference group was significantly lower than that in the blank control group, and the cell inhibition rate in the empty vector group was significantly lower than that in the blank control group. The inhibition of cell proliferation in the empty carrier group was time-dependent. The Annexin V-PE / 7-AAD- in the interference group was significantly higher than that in the blank control group. The expression of Notch1 receptor gene and its downstream target gene Hes1 c-myc NF- 魏 B in the interference group was significantly lower than that in the blank control group and the empty vector group. The expression of Notch1 receptor gene and its downstream target gene Hes1 c-myc NF- 魏 B was significantly lower than that of the blank control group and the empty vector group. The proliferation of T1 cells was inhibited obviously. The inhibitory effect was in a concentration-and time-dependent manner, and the cell inhibition rate of the interference plus group and the no-load group was higher than that of the interference group. Annexin V-PE / 7-AAD- was significantly higher in the empty carrier group than in the blank control group and empty carrier group, and there was no significant difference in Annexin V-PE / 7-AAD cells in each group. The downstream gene expression level of the interference plus drug group was higher than that of the interference group. The expression of Notch1Hes1 c-mycfon NF- 魏 B was inhibited in a time-dependent manner in the interference group and the interference plus group. Conclusion: 1. The specific Notch1-sh RNA-4252 can effectively down-regulate the expression of Notch1 m RNA. The down-regulation of Notchl expression can inhibit the proliferation of Sup T1 cells in a time dependent manner, and promote the early apoptosis of Sup T1 cells. 3. The effect of bortezomil on the proliferation of Sup T1 cells after the interference of RNA. The inhibition of apoptotic Notch1 receptor gene and downstream target gene was more obvious, and the specific sh RNA interference Notchl gene was superimposed with bortezomil.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R733.71

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