慢病毒介导的TSC2表达降低对白血病U937细胞系的影响及作用机制

发布时间：2018-09-05 13:17

【摘要】：目的:研究抑癌基因TSC2(Tuberous Sclerosis Complex 2)表达降低对白血病U937细胞系的生长、增殖、分化和凋亡的影响及其对mTORC1(mammalian target of rapamycin,mTOR complex 1)通路蛋白激酶及其靶分子活性的影响,揭示急性白血病发生的新机制。方法:选择TSC2高表达的U937细胞,通过慢病毒介导的RNA干扰技术下调TSC2基因表达,筛选出GFP阳性率高的细胞培养至对数生长期,采用CCK-8法和细胞克隆形成能力实验检测细胞的增殖活力;用PI(碘化丙啶)标记流式细胞术检测细胞周期;用流式细胞仪检测细胞分化;采用AnnexinV-PE/7-AAD双染流式细胞术检测细胞凋亡;通过蛋白印记实验,检测TSC2表达下调对mTORC1信号通路蛋白活性的影响;采用实时定量PCR法检测TSC2基因低表达对mTORC1通路下游靶基因表达的影响。结果:1.慢病毒感染U937细胞后,实时定量PCR方法检测TSC2 mRNA的表达,干扰效率为71.7%;Western blot方法检测蛋白表达,可见TSC2蛋白的表达量明显下调。2.下调TSC2基因表达对U937细胞的生物学作用:CCK-8细胞增殖实验结果表明,TSC2基因表达下调后,细胞增殖活性增强,与未感染组细胞(空白对照)相比,实验组增殖活性增强了(5.15±0.72)倍,空载病毒组增殖活性增强了(1.92±0.63)倍,两组之间差异具有统计学意义(P0.05);细胞克隆形成能力实验表明,与空载病毒对照组相比,实验组的细胞集落形成能力明显增强(P0.05);细胞周期实验结果表明:与空病毒组相比,实验组细胞G1期比例明显降低(P0.05),G2/M期细胞比例增高(P0.05),S期比例增高(P0.05);流式细胞仪检测细胞分化结果表明,与空载病毒对照组相比,实验组细胞CD13、CD33、CD11b、CD15、CD14、CD64表达无明显变化;流式细胞仪检测细胞凋亡结果表明,TSC2表达降低对细胞凋亡无明显改变(P0.05)。3.TSC2基因表达下调对mTORC1通路蛋白的影响:Western blot方法检测mTOR及其下游分子的表达结果表明,TSC2表达下调后,mTOR总蛋白未见明显变化,而磷酸化p-mTOR(Ser2448)表达升高;底物激酶P70S6K和4EBP1总蛋白未见明显变化,但是活性形式p-P70S6K(Thr389)和p-4EBP1(Thr37/46)的表达明显升高;AKT总蛋白及活性形式的p-AKT的表达未见明显变化;实时定量PCR结果表明,与细胞增殖相关的基因cyclinD1、c-myc和PTEN表达量明显升高,P27kip1基因、凋亡相关基因BCL-XL表达量没有明显变化。结论:TSC2基因表达下调可以通过调节mTORC1信号通路的活性促进白血病细胞的增殖能力,为急性白血病的分子发病机制以及靶向治疗提供了进一步的分子理论基础。
[Abstract]:AIM: To investigate the effects of TSC2 (Tuberous Sclerosis Complex 2) down-regulation on the growth, proliferation, differentiation and apoptosis of leukemia U937 cell line and the activity of protein kinase and its target molecule in the mTORC1 (mammalian target of rapamycin, mTOR complex 1) pathway, and to reveal the new mechanism of acute leukemia. U937 cells with high expression of TSC2 were selected, and TSC2 gene expression was down-regulated by lentivirus-mediated RNA interference. The cells with high positive rate of GFP were screened out and cultured to logarithmic growth stage. Cell proliferation activity was detected by CCK-8 assay and cell cloning ability assay. Cell cycle was detected by PI (propidium iodide) labeling flow cytometry. Cell differentiation was detected by the instrument; apoptosis was detected by Annexin V-PE/7-AAD double staining flow cytometry; the effect of TSC2 down-regulation on the activity of mTORC1 signaling pathway protein was detected by Western blotting; and the effect of TSC2 down-regulation on the expression of target genes downstream of mTORC1 pathway was detected by real-time quantitative PCR. The expression of TSC2 mRNA was detected by real-time quantitative PCR, and the interference efficiency was 71.7%. The expression of TSC2 protein was significantly down-regulated by Western blot. 2. The biological effect of TSC2 gene down-regulated on U937 cells: CCK-8 cell proliferation experiment showed that TSC2 gene down-regulated cell proliferation activity increased. Compared with the uninfected group (blank control), the proliferative activity of the experimental group increased (5.15 + 0.72) times, and that of the viral group increased (1.92 + 0.63) times. The difference between the two groups was statistically significant (P 0.05). Cell cloning ability test showed that compared with the viral control group, the colony forming ability of the experimental group was obvious. Cell cycle test showed that compared with the empty virus group, the G1 phase ratio decreased significantly (P 0.05), G2 / M phase ratio increased (P 0.05), S phase ratio increased (P 0.05); Flow cytometry showed that compared with the empty virus control group, the experimental group cells CD13, CD33, CD11b, CD15, CD14, CD64 expression. The results of flow cytometry showed that there was no significant change in TSC2 expression (P 0.05). 3. The effect of TSC2 down-regulation on mTORC1 pathway protein: Western blot assay showed that the total protein of mTOR did not change significantly after TSC2 down-regulation, but phosphorus. The expression of acidified p-mTOR (Ser2448) was elevated, the total protein of substrate kinase P70S6K and 4EBP1 was not changed significantly, but the expression of active forms p-P70S6K (Thr389) and p-4EBP1 (Thr37/46) were significantly increased, the expression of total protein and active form of AKT were not changed significantly, and the results of real-time quantitative PCR showed that cyclinD1, c-myc genes related to cell proliferation were not changed. Conclusion: Downregulation of TSC2 gene expression can promote the proliferation of leukemic cells by regulating the activity of mTORC1 signaling pathway, which provides a further theoretical basis for the molecular pathogenesis and targeted therapy of acute leukemia. Foundation.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R733.7

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