沉默Musashi-2基因对B型急淋白血病细胞的抑制作用以及对LEF1表达的影响

发布时间：2018-04-11 12:37

本文选题：Msi2基因 + 小干扰RNA　；参考：《宁波大学》2017年硕士论文

【摘要】：背景与目的:急性淋巴细胞白血病(Acute lymphoblastic leukemia,ALL)是儿童和成人常见的造血系统恶性肿瘤之一。随着医疗技术和水平不断发展,儿童ALL治疗效果稳步提高,其5年无病生存率可达80%左右;但目前的成人治愈情况仍不乐观,如不移植,5年无病生存率不超过20%。因此,为了提高成人ALL的治愈率,必须进一步明确其发病机制、并且寻找有效分子靶点来完善其临床预后评价体系。Musashi-2(Msi2)基因首先被KHARAS等发现:其在造血系统内的原始细胞呈高水平表达,并随原始细胞的分化成熟而快速下降,提示Msi2是骨髓干细胞维持自我更新和保持多潜能性的重要调节因子。随后人们陆续在各类白血病患者中发现Msi2的高表达,但其分子作用机制仍不是很明确。有研究在造血干细胞和髓系白血病中用基因干扰和转染外源性基因的方法证实,Msi2表达与NOTCH信号途径密切相关。然而,在我们以前的研究中检测了正常人骨髓CD34+分选细胞和外周血单个核细胞(主要为成熟B淋巴细胞)的Msi2和c-Myc及HES1表达水平,发现Msi2与其它两项均呈正相关,但在成人B-ALL中,Msi2与两者的表达均没有明显相关性。此外,有研究显示Msi1过表达可以激活Wnt信号,因Msi1和Msi2有高度同源性,我们推测Msi2在B-ALL的白血病细胞中也可激活Wnt信号。因此,本研究以B-ALL细胞株NALM6为研究对象,观察Msi2沉默对细胞生长、凋亡及Wnt信号中LEF1表达影响,初步探讨Msi2在急性B型淋巴细胞白血病中分子作用机制。方法(1)细胞培养:将NALM6细胞在含10%FBS的1640培养基、37℃、5%CO2、饱和湿度培养箱中培养。(2)Msi2 si RNA慢病毒构建:由上海汉恒生物有限公司合成。(3)病毒感染和筛选:采用慢病毒(MOI=100)转染法转染小干扰片段,5μg/m L浓度的嘌呤霉素筛选细胞。荧光显微镜下观察转染效果,当细胞转染率达到90%以上即可进行细胞样本收集。(4)提取细胞总RNA:各组采集的细胞用Trizol-离心柱法提取RNA,测定其纯度和含量,行逆转录实验做合成c DNA。(5)NALM6细胞Msi2 si RNA干扰验证:采用PCR法检测和westernblot法分别检测对照组和小干扰组的Msi2基因和蛋白表达水平。并采用干扰率60%的一组进行以下试验。(6)细胞生长、凋亡及周期检测:采用CCK-8法、Annexin V/PI及PI法分别观察沉默Msi2基因后对细胞增殖率、细胞凋亡和周期的影响;用Hochest染色观察细胞的凋亡情况;用western-blot法检测细胞凋亡蛋白capase-3,PARP及细胞周期蛋白Cyclin D1,P21的水平。(7)细胞分化检测:用流式细胞仪测定CD38和CD10的阳性率,判断细胞的分化情况。(8)采用RT-PCR和western blot法检测观察LEF1基因表达水平。结果(1)NALM6细胞Msi2小干扰验证:各组荧光率均达到90%以上。Msi2si RNA1组,Msi2 si RNA2组,Msi2 si RNA3组的Msi2 m RNA分别下调了24.48±3.65%,71.73±3.20%,33.03±3.81%。以Msi2 si RNA2组的Msi2基因下降最为明显,其相应的蛋白水平也明显下调,因此,用Msi2 si RNA2组的细胞进行后续的实验。(2)CCK-8法检测Msi2小干扰后活力:Msi2 si RNA抑制NALM6细胞生长结果显示,感染24小时后,Msi2 si RNA2组细胞活力为1.093±0.081,与无关序列对照组无明显区别(1.106±0.074,P=0.872);72小时后,Msi2 si RNA2组细胞活力为1.343±0.064,低于无关序列对照组(1.533±0.097,P=0.048);第七天Msi2 si RNA2组细胞活力为2.380±0.503,明显低于无关序列对照组(3.553±0.628,P=0.038)。(3)Msi2 si RNA诱导NALM6细胞凋亡:Msi2 si RNA2组凋亡率为3.6±0.46%,稍高于无关序列组(凋亡率为0.96±0.21%),但统计学有差异P=0.015。凋亡蛋白剪切caspase 3,剪切Parp蛋白条带有所加深但不明显。(4)Msi2 si RNA阻滞NALM6细胞周期:Msi2 si RNA2组G0/G1期细胞比例为72.53±1.28%,高于对照组(G0/G1期比例为65.44±2.33%,P=0.0073)。并伴随相应的周期蛋白Cyclin D1明显下调和P21明显升高。(5)Msi2 si RNA诱导NALM6细胞分化:Msi2 si RNA2组CD38+CD10-的细胞为62.87±2.10%,CD38-CD10+的细胞为0.67±0.21%;无关序列对照组CD38+CD10-的细胞为0.5±0.1%,CD38-CD10+的细胞为47.43±1.68%。结论:(1)下调Msi2表达水平能减低ALL白血病细胞活力、同时导致细胞周期阻滞并促进细胞分化,但对凋亡的影响不明显。(2)在Msi2-Wnt信号通路中,随着细胞Msi2表达水平的下降,LEF1的表达水平也相应下降,提示LEF1可能参与Msi2分子作用机制。
[Abstract]:Background and objective: acute lymphoblastic leukemia (Acute lymphoblastic, leukemia, ALL) is one of the most common pediatric and adult hematopoietic malignancies. With the continuous development of medical technology and treatment effect of children, ALL increased steadily, the 5 year survival rate was 80% on the left and right; but the current adult cure situation is still not optimistic, such as do not transplant, 5 year survival rate is less than 20%.. Therefore, in order to improve the cure rate of adult ALL, we must further clarify the pathogenesis and to find effective molecular targets to improve the clinical prognosis evaluation system.Musashi-2 (Msi2) gene was first discovered by KHARAS: the original cells in the hematopoietic system in a high level the expression and differentiation of primitive cells with mature and rapid decline, suggesting that Msi2 is a bone marrow stem cell self-renewal and pluripotency remains an important regulatory factor. As the people were in all kinds of white The high expression of Msi2 was found in patients with blood diseases, but its molecular mechanism is still not very clear. The research in hematopoietic stem cells and methods of myeloid leukemia with gene interference and transfection of exogenous gene confirmed that the expression of Msi2 is closely related with the NOTCH pathway. However, in our previous studies in the detection of normal people bone marrow CD34+ sorting cells and peripheral blood mononuclear cells (mainly for mature B lymphocytes) of Msi2 and c-Myc and the expression of HES1, Msi2 and other two were positively correlated, but in adult B-ALL, expression of Msi2 and the two had no significant correlation. In addition, studies have shown that overexpression of Msi1 can activate Wnt for Msi1 signal, and Msi2 is highly homologous, we speculate that Msi2 can activate Wnt signal in B-ALL leukemia cells. Therefore, this research is based on the B-ALL cell line NALM6 as the research object, the observation of Msi2 silencing on cell growth, apoptosis Effect of expression of LEF1 and Wnt in apoptosis signal, preliminary study of Msi2 molecules in acute lymphoblastic leukemia type B mechanism. (1) cell culture: NALM6 cells in culture medium containing 1640 10%FBS, 37 C, 5%CO2, saturated humidity incubator. (2) to construct Msi2 Si RNA: chronic disease virus synthesized by Shanghai Han Heng Biotechnology Co. (3) virus infection and screening by lentivirus (MOI=100) transfected siRNA fragment, puromycin 5 g/m L concentration in screening cells. The effect of transfection was observed under fluorescence microscope, when the transfection rate reached 90% to collect cell samples (. 4) RNA Trizol- was extracted by centrifugal column method for extraction of total RNA: cells were collected, determination of the purity and content of reverse transcriptase experiment synthesis of C (5) DNA. NALM6 Msi2 Si RNA interference verification: cells were detected in control group and the small disturbance was detected by PCR method and Westernblot method The expression level of Msi2 gene and protein group. And a group of the following test interference rate 60%. (6) cell growth, apoptosis and cell cycle detection: using CCK-8 method, the rate of cell proliferation were observed in Msi2 gene silencing Annexin V/PI and PI method, the apoptosis and cell cycle was observed by Hochest staining; apoptosis cell apoptosis; detection of protein capase-3 by Western-blot method, PARP and cyclin Cyclin D1 P21. (7) cell differentiation detection: the positive rate of CD38 and CD10 were measured by flow cytometry, determine the cell differentiation. (8) the level of Western blot was detected by RT-PCR and LEF1 observation the gene expression of NALM6 cells. Results (1) Msi2 small interference verification: each fluorescence rate reached more than 90%.Msi2si RNA1 group, Msi2 Si RNA2 Msi2 m RNA Msi2 Si Group, RNA3 group were reduced by 24.48 + 3.65%, 71.73 + 3.20%, 33.03 + 3.81%. to Msi2 Si R The Msi2 gene of NA2 group decreased significantly, and the corresponding protein levels were significantly reduced, therefore, the following experiments were performed with Msi2 Si Cells in the RNA2 group. (2) detected by CCK-8 Msi2: Msi2 Si small interfering activity showed that NALM6 inhibited the growth of RNA cells after 24 hours of infection, Msi2 Si RNA2 group cell viability was 1.093 + 0.081, and the control group had no significant difference between unrelated sequence (1.106 + 0.074, P=0.872); after 72 hours, Msi2 Si RNA2. The cell viability was 1.343 + 0.064, lower than the control group of unrelated sequence (1.533 + 0.097, P=0.048); the seventh day Msi2 Si RNA2. The cell viability was 2.380 + 0.503. The control group was significantly lower than that of unrelated sequence (3.553 + 0.628, P=0.038). (3) Msi2 Si RNA Msi2 Si induced apoptosis in NALM6 cells: the apoptosis rate of RNA2 group is 3.6 + 0.46%, slightly higher than the unrelated sequence group (apoptosis rate of 0.96 + 0.21%), but there was difference of P=0.015. apoptosis protein caspase 3 Par shear shear. P protein bands increased but not obvious. (4) Msi2 Si RNA block the cell cycle of NALM6 cell ratio of Msi2 Si: RNA2 group G0/G1 was 72.53 + 1.28%, higher than the control group (the proportion of G0/G1 was 65.44 + 2.33%, P=0.0073). With the corresponding D1 down-regulation of cyclin Cyclin and P21 were significantly increased. (5) NALM6 cell differentiation induced by Msi2 Si RNA: Msi2 Si RNA2 group CD38+CD10- cells was 62.87 + 2.10%, CD38-CD10+ cells was 0.67 + 0.21%; unrelated sequence control group CD38+CD10- cells was 0.5 + 0.1%, CD38-CD10+ + 1.68%. cells was 47.43. Conclusion: (1) the expression level Msi2 can reduce the activity of ALL at the same time, leukemia cells, leading to cell cycle arrest and promote cell differentiation, but the influence on apoptosis is not obvious. (2) in the Msi2-Wnt signaling pathway, cell with the expression level of Msi2 decreased, the expression level of LEF1 also decreased, suggesting that LEF1 may be involved in Msi2 molecules Mechanism of action.

【学位授予单位】：宁波大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R733.71

【参考文献】