本文關(guān)鍵詞： Notch1 T-ALL RNA干擾 硼替佐米 藥物敏感性　出處：《山西醫(yī)科大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

【摘要】：目的:通過(guò)檢測(cè)干擾Notch1基因后Sup T1細(xì)胞的增殖、凋亡和Notch1受體基因及其下游靶基因表達(dá)水平的變化,探討Notch信號(hào)通路與急性T淋巴細(xì)胞白血病發(fā)生、發(fā)展的關(guān)系。在下調(diào)Notch1基因后的Sup T1細(xì)胞中加入1ng/μl硼替佐米,檢測(cè)對(duì)Sup T1細(xì)胞增殖、凋亡和Notch1受體基因及其下游靶基因表達(dá)水平的變化,探討Notch1基因表達(dá)對(duì)硼替佐米敏感性的影響。方法:1.利用攜帶Notch1特異性和非特異性sh RNA的慢病毒載體包裝成病毒顆粒感染Sup T1細(xì)胞,選取干擾效率高的感染細(xì)胞作為干擾組,分別于48、72、96個(gè)小時(shí)后,采用CCK-8法檢測(cè)各組細(xì)胞增殖率;采用流式細(xì)胞術(shù)檢測(cè)各組細(xì)胞凋亡率(Annexin V+/7-AAD-和Annexin V+/7-AAD+);采用實(shí)時(shí)熒光定量PCR法檢測(cè)Notch1受體基因及下游靶基因基因Hes1、NF-κB、c-myc的m RNA表達(dá)水平。2.在感染Sup T1細(xì)胞后,分別在第24、48、72小時(shí)三個(gè)不同時(shí)間點(diǎn)加入1ng/μl硼替佐米繼續(xù)孵育24小時(shí)后,采用CCK-8法、流式細(xì)胞術(shù)、實(shí)時(shí)熒光定量PCR法檢測(cè)其在干擾Notch1基因后第48、72、96小時(shí)二者共同作用對(duì)Sup T1細(xì)胞增殖、凋亡及Notch1受體基因及下游靶基因基因Hes1、NF-κB、c-myc的m RNA表達(dá)水平的影響。結(jié)果:1.Notch1干擾組細(xì)胞的增殖較空白對(duì)照組、空載體組明顯降低,細(xì)胞抑制率較空白對(duì)照組、空載體組明顯升高(P均0.05),且細(xì)胞增殖的抑制作用呈時(shí)間依賴性。2.干擾組Annexin V-PE+/7-AAD-較空白對(duì)照組、空載體組明顯升高(P均0.05);而各組細(xì)胞Annexin V-PE+/7-AAD+無(wú)明顯變化(P0.05)。3.干擾組細(xì)胞中Notch1受體基因及其下游靶基因Hes1、c-myc、NF-κB表達(dá)較空白對(duì)照組及空載體組顯著降低(P均0.05)。4.硼替佐米對(duì)Sup T1細(xì)胞的增殖有明顯的抑制作用,且抑制作用呈濃度和時(shí)間依賴性;干擾加藥組、空載加藥組細(xì)胞抑制率均高于干擾組、空載體組(P均0.05)。5.干擾加藥組細(xì)胞Annexin V-PE+/7-AAD-較空白對(duì)照組及空載體組明顯升高(P均0.05);各組細(xì)胞Annexin V-PE+/7-AAD+細(xì)胞差異均無(wú)統(tǒng)計(jì)學(xué)意義(P0.05)。6.干擾加藥組下游基因表達(dá)水平較干擾組明顯降低(P0.05),且干擾組及干擾加藥組的基因Notch1、Hes1、c-myc、NF-κB表達(dá)抑制呈時(shí)間依賴性。結(jié)論:1.特異性Notch1-sh RNA-4252可有效下調(diào)Notch1 m RNA的表達(dá),并降低下游靶基因水平的表達(dá);2.Notchl表達(dá)下調(diào)可以抑制Sup T1細(xì)胞增殖,且細(xì)胞抑制作用呈時(shí)間依賴性;促進(jìn)Sup T1細(xì)胞的早期凋亡。3.sh RNA干擾后硼替佐米對(duì)Sup T1細(xì)胞增殖抑制、凋亡、Notch1受體基因及下游靶基因抑制更加明顯,特異性sh RNA干擾Notchl基因與硼替佐米呈疊加作用。
[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.
【學(xué)位授予單位】：山西醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R733.71

【參考文獻(xiàn)】

相關(guān)碩士學(xué)位論文 前1條

1 黃燦;硼替佐米對(duì)T-ALL細(xì)胞株CCRF-CEM的作用及對(duì)Notch1通路的影響[D];山西醫(yī)科大學(xué);2012年

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本文編號(hào)：1504505