SPAG6与MDS细胞凋亡的关系及机制研究

发布时间：2018-05-04 19:19

本文选题：骨髓增生异常综合征 + SPAG6基因　；参考：《重庆医科大学》2017年硕士论文

【摘要】：目的通过pGC-SPAG6-shRNA重组慢病毒载体靶向沉默SPAG6基因后,探讨SPAG6基因与人骨髓增生异常综合征(MDS)细胞凋亡的关系及其机制,为MDS的靶向治疗奠定基础。方法1.明确SPAG6-sh RNA慢病毒载体对人SPAG6基因干扰效率:将SPAG6-sh RNA及NC-sh RNA慢病毒转染人MDS细胞株SKM-1细胞后,利用荧光显微镜观察其表达情况,流式细胞术检测其转染效率,q RT-PCR及western blot验证SPAG6基因是否被成功干扰。2.探索SPAG6基因的表达对TRAIL信号通路活化的影响:慢病毒转染SKM-1细胞后,利用流式细胞术检测细胞凋亡,western blot检测TRAIL通路相关蛋白的表达情况。3.研究SPAG6基因与TRAIL凋亡通路之间的量的依赖性:同时利用不同浓度的r TRAIL蛋白处理SKM-1细胞,相同浓度r TRAIL蛋白分别处理SKM-1、U937及K562三种细胞,利用流式细胞术检测细胞凋亡情况,CCK-8法检测各组细胞增殖活性,western blot检测TRAIL通路活化情况。4.探索SPAG6在TRAIL凋亡信号通路中的作用途径及关键分子:采用q RT-PCR及western blot检测TRAIL通路相关受体及蛋白的表达水平,免疫沉淀检测二者的结合情况。结果1.SPAG6-sh RNA慢病毒成功转染人SKM-1细胞。在荧光显微镜下观察到大量的绿色荧光,流式细胞术检测得其转染率分别为(97.19±3.21)%与(88.31±12.81)%,QRT-PCR及Western blot结果显示SKM-1细胞中SPAG6基因在m RNA及蛋白层面的表达均被成功抑制。2.SPAG6基因表达降低后,TRAIL凋亡通路被活化。干扰组的凋亡率明显高于对照组(NC-sh RNA 8.65%±2.07%vs.SPAG6-sh RNA20.20%±2.11%,P0.05)。而且,SPAG6干扰组cleaved PARP、caspase-8以及cleaved caspase-8表达水平均升高,PARP表达则无明显差异。同时,BAK、BAX、BID和caspase-3较对照组表达也明显升高。3.SPAG6基因能抵抗r TRAIL蛋白引起的凋亡。随着r TRAIL蛋白浓度的增加,SKM-1细胞凋亡率增加,增殖被抑制,且TRAIL通路活化的相关蛋白表达增加。而利用相同浓度的r TRAIL蛋白处理SKM-1、U937及K562时,在U937及K562中,细胞凋亡及增殖均无明显区别。4.SPAG6能影响FADD与TRAIL通路受体间的相互作用。SPAG6基因表达被干扰后,FADD的表达较对照组明显升高,而DR4及DR5的表达则无明显区别。免疫沉淀的结果则表明,降低SPAG6表达后,FADD与DR4和DR5之间的相互作用明显增加。结论SPAG6基因能够通过影响TRAIL信号通路的活化调控SKM-1细胞的凋亡。其具体机制可能是细胞内SPAG6基因的高表达能够影响FADD的表达,进而致使FADD与TRAIL通路凋亡受体结合减少。
[Abstract]:Objective to investigate the relationship between SPAG6 gene and apoptosis of human myelodysplastic syndromes (MDS) cells by targeting SPAG6 gene silenced by pGC-SPAG6-shRNA recombinant lentivirus vector, and to lay a foundation for the targeted therapy of MDS. Method 1. To determine the interference efficiency of SPAG6-sh RNA lentivirus vector to human SPAG6 gene: after SPAG6-sh RNA and NC-sh RNA lentivirus were transfected into SKM-1 cell line of human MDS cell line, the expression of SPAG6-sh RNA lentivirus vector was observed by fluorescence microscope. The transfection efficiency was detected by flow cytometry. Q RT-PCR and western blot were used to verify whether the SPAG6 gene was successfully interfered with. 2. 2. To explore the effect of SPAG6 gene expression on the activation of TRAIL signaling pathway: after lentivirus transfection into SKM-1 cells, apoptosis was detected by flow cytometry and the expression of TRAIL pathway related protein was detected by western blot. To study the quantitative dependence between SPAG6 gene and TRAIL apoptosis pathway: SKM-1 cells were treated with different concentrations of r TRAIL protein, and SKM-1 U937 and K562 cells were treated with the same concentration of r TRAIL protein, respectively. Cell apoptosis was detected by flow cytometry and the activation of TRAIL pathway was detected by western blot with CCK-8 assay. To explore the role and key molecules of SPAG6 in TRAIL apoptotic signaling pathway, Q RT-PCR and western blot were used to detect the expression of TRAIL receptor and protein, and immunoprecipitation was used to detect their binding. Results 1.SPAG6-sh RNA lentivirus was successfully transfected into human SKM-1 cells. A large amount of green fluorescence was observed under a fluorescence microscope, The results of flow cytometry showed that the transfection rates were 97.19 卤3.21% and 88.31 卤12.81%, respectively. The results of QRT-PCR and Western blot showed that the expression of SPAG6 gene at m RNA and protein level in SKM-1 cells was successfully inhibited. 2. The apoptosis pathway of trail was activated after the expression of SPAG6 gene decreased. The apoptosis rate of interference group was significantly higher than that of NC-sh RNA 8.65% 卤2.07%vs.SPAG6-sh 20.20% 卤2.11 in control group. Moreover, there was no significant difference in the expression of cleaved PARP caspase-8 and cleaved caspase-8 in SPAG6 interference group. At the same time, the expression of BAXBID and caspase-3 was significantly higher than that of control group. 3. SPAG6 gene could resist the apoptosis induced by r TRAIL protein. With the increase of the concentration of r TRAIL protein, the apoptosis rate of SKM-1 cells increased, the proliferation was inhibited, and the expression of related proteins activated by TRAIL pathway increased. However, when SKM-1U937 and K562 were treated with the same concentration of r TRAIL protein, there was no significant difference in apoptosis and proliferation between U937 and K562. 4. SPAG6 could affect the interaction between SPAG6 and the receptor of TRAIL pathway. The expression of SPAG6 gene in SKM-1U937 and K562 was significantly higher than that in the control group. However, the expression of DR4 and DR5 had no significant difference. The results of immunoprecipitation showed that the interaction between DR4 and DR5 increased significantly after the decrease of SPAG6 expression. Conclusion SPAG6 gene can regulate the apoptosis of SKM-1 cells by affecting the activation of TRAIL signaling pathway. The specific mechanism may be that the high expression of SPAG6 gene in cells can affect the expression of FADD, which leads to the reduction of FADD binding to the apoptotic receptor of TRAIL pathway.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R551.3

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