本文選題：Msi2基因 + 小干擾RNA　； 參考：《寧波大學》2017年碩士論文

【摘要】：背景與目的:急性淋巴細胞白血病(Acute lymphoblastic leukemia,ALL)是兒童和成人常見的造血系統(tǒng)惡性腫瘤之一。隨著醫(yī)療技術和水平不斷發(fā)展,兒童ALL治療效果穩(wěn)步提高,其5年無病生存率可達80%左右;但目前的成人治愈情況仍不樂觀,如不移植,5年無病生存率不超過20%。因此,為了提高成人ALL的治愈率,必須進一步明確其發(fā)病機制、并且尋找有效分子靶點來完善其臨床預后評價體系。Musashi-2(Msi2)基因首先被KHARAS等發(fā)現(xiàn):其在造血系統(tǒng)內的原始細胞呈高水平表達,并隨原始細胞的分化成熟而快速下降,提示Msi2是骨髓干細胞維持自我更新和保持多潛能性的重要調節(jié)因子。隨后人們陸續(xù)在各類白血病患者中發(fā)現(xiàn)Msi2的高表達,但其分子作用機制仍不是很明確。有研究在造血干細胞和髓系白血病中用基因干擾和轉染外源性基因的方法證實,Msi2表達與NOTCH信號途徑密切相關。然而,在我們以前的研究中檢測了正常人骨髓CD34+分選細胞和外周血單個核細胞(主要為成熟B淋巴細胞)的Msi2和c-Myc及HES1表達水平,發(fā)現(xiàn)Msi2與其它兩項均呈正相關,但在成人B-ALL中,Msi2與兩者的表達均沒有明顯相關性。此外,有研究顯示Msi1過表達可以激活Wnt信號,因Msi1和Msi2有高度同源性,我們推測Msi2在B-ALL的白血病細胞中也可激活Wnt信號。因此,本研究以B-ALL細胞株NALM6為研究對象,觀察Msi2沉默對細胞生長、凋亡及Wnt信號中LEF1表達影響,初步探討Msi2在急性B型淋巴細胞白血病中分子作用機制。方法(1)細胞培養(yǎng):將NALM6細胞在含10%FBS的1640培養(yǎng)基、37℃、5%CO2、飽和濕度培養(yǎng)箱中培養(yǎng)。(2)Msi2 si RNA慢病毒構建:由上海漢恒生物有限公司合成。(3)病毒感染和篩選:采用慢病毒(MOI=100)轉染法轉染小干擾片段,5μg/m L濃度的嘌呤霉素篩選細胞。熒光顯微鏡下觀察轉染效果,當細胞轉染率達到90%以上即可進行細胞樣本收集。(4)提取細胞總RNA:各組采集的細胞用Trizol-離心柱法提取RNA,測定其純度和含量,行逆轉錄實驗做合成c DNA。(5)NALM6細胞Msi2 si RNA干擾驗證:采用PCR法檢測和westernblot法分別檢測對照組和小干擾組的Msi2基因和蛋白表達水平。并采用干擾率60%的一組進行以下試驗。(6)細胞生長、凋亡及周期檢測:采用CCK-8法、Annexin V/PI及PI法分別觀察沉默Msi2基因后對細胞增殖率、細胞凋亡和周期的影響;用Hochest染色觀察細胞的凋亡情況;用western-blot法檢測細胞凋亡蛋白capase-3,PARP及細胞周期蛋白Cyclin D1,P21的水平。(7)細胞分化檢測:用流式細胞儀測定CD38和CD10的陽性率,判斷細胞的分化情況。(8)采用RT-PCR和western blot法檢測觀察LEF1基因表達水平。結果(1)NALM6細胞Msi2小干擾驗證:各組熒光率均達到90%以上。Msi2si RNA1組,Msi2 si RNA2組,Msi2 si RNA3組的Msi2 m RNA分別下調了24.48±3.65%,71.73±3.20%,33.03±3.81%。以Msi2 si RNA2組的Msi2基因下降最為明顯,其相應的蛋白水平也明顯下調,因此,用Msi2 si RNA2組的細胞進行后續(xù)的實驗。(2)CCK-8法檢測Msi2小干擾后活力:Msi2 si RNA抑制NALM6細胞生長結果顯示,感染24小時后,Msi2 si RNA2組細胞活力為1.093±0.081,與無關序列對照組無明顯區(qū)別(1.106±0.074,P=0.872);72小時后,Msi2 si RNA2組細胞活力為1.343±0.064,低于無關序列對照組(1.533±0.097,P=0.048);第七天Msi2 si RNA2組細胞活力為2.380±0.503,明顯低于無關序列對照組(3.553±0.628,P=0.038)。(3)Msi2 si RNA誘導NALM6細胞凋亡:Msi2 si RNA2組凋亡率為3.6±0.46%,稍高于無關序列組(凋亡率為0.96±0.21%),但統(tǒng)計學有差異P=0.015。凋亡蛋白剪切caspase 3,剪切Parp蛋白條帶有所加深但不明顯。(4)Msi2 si RNA阻滯NALM6細胞周期:Msi2 si RNA2組G0/G1期細胞比例為72.53±1.28%,高于對照組(G0/G1期比例為65.44±2.33%,P=0.0073)。并伴隨相應的周期蛋白Cyclin D1明顯下調和P21明顯升高。(5)Msi2 si RNA誘導NALM6細胞分化:Msi2 si RNA2組CD38+CD10-的細胞為62.87±2.10%,CD38-CD10+的細胞為0.67±0.21%;無關序列對照組CD38+CD10-的細胞為0.5±0.1%,CD38-CD10+的細胞為47.43±1.68%。結論:(1)下調Msi2表達水平能減低ALL白血病細胞活力、同時導致細胞周期阻滯并促進細胞分化,但對凋亡的影響不明顯。(2)在Msi2-Wnt信號通路中,隨著細胞Msi2表達水平的下降,LEF1的表達水平也相應下降,提示LEF1可能參與Msi2分子作用機制。
[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.

【學位授予單位】：寧波大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R733.71

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本文編號：1736074