miRNAs在骨髓基质细胞介导的AML细胞耐药中的作用
发布时间：2019-05-14 11:34
【摘要】：目的:骨髓微环境中的间充质干细胞(MSC)可以诱导肿瘤细胞生长和存活,并形成适合肿瘤繁殖及耐药的微环境。急性髓系白血病(AML)复发和耐药的关键在于白血病细胞与MSC之间的相互作用。白血病干细胞(LSCs)与MSC的粘附及相互作用,可以直接促进细胞的自我更新、增殖、分化停滞,并“保护”白血病细胞免受化疗药物的毒性作用。micro RNA(微小RNA,mi RNA)作为一种非编码的小RNA分子,能够在转录后水平调节基因的表达。mi RNA在生物发育过程中发挥着重要作用,如细胞分化及凋亡、肿瘤生成等。研究mi RNA的表达及功能,对防治肿瘤性疾病具有重要意义。有关mi RNA在AML发病中的研究近几年刚刚起步,多从表达量高低的角度阐述,与发病机制相关的功能性研究较少,mi RNA在AML中MSC诱导的耐药中作用的研究尚未见报道。我们早期工作发现,AML细胞系KG1a与MSC粘附后,对化疗药物的反应下降,细胞凋亡减少。RT-PCR和Western结果证实与细胞周期有关的基因C-Myc表达上调。本研究拟在将AML细胞系与人骨髓MSC共培养的基础上通过mi RNA芯片寻找调控C-Myc的mi RNAs,并将筛选出的mi RNAs进行表达量验证。为后续功能验证做准备。方法:1.培养AML细胞系KG1a,准备15例健康供者的骨髓原代细胞。2.分离和培养人骨髓MSC。3.建立共培养体系,与MSC共培养48h后CD33阳选出KG1a(样品编号4D,5E,6F),以AML细胞系KG1a单独培养(样品编号1,2,3)作为对照。4.将上述样品分装6管加Trizol处理,送公司进行样本质量检测并制成基因芯片,根据全基因组芯片结果寻找调控C-Myc的mi RNAs。5.重复步骤1,2,3;RT-PCR法检测两种培养条件下筛选出的mi RNAs表达量变化情况。结果:1.基因芯片样本检验合格。2.根据芯片结果初步寻找调控C-Myc的mi RNAs:let-7a,mi R-17-3p,mi R-34c,mi R-135a,mi R-494;U6作为内参。3.RT-PCR法检测结果:let-7a表达上升3.39倍,mi R-34c表达上升621.67倍,mi R-135a表达上升13.00倍,mi R-494表达上升17.51倍,mi R-17-3p表达下降3.70倍。4.mi R-17-3p可能参与调控C-Myc表达。结论:AML细胞粘附于MSC后,通过一系列mi RNA的变化引起C-Myc表达的上调,而mi R-17-3p可能参与调控C-Myc的表达。
[Abstract]:Aim: mesenchymal stem cells (MSC) in bone marrow microenvironment can induce the growth and survival of tumor cells and form a microenvironment suitable for tumor reproduction and drug resistance. The key to the recurrence and drug resistance of (AML) in acute myeloid leukemia lies in the interaction between leukemia cells and MSC. The adhesion and interaction between (LSCs) and MSC can directly promote the self-renewal, proliferation and differentiation stagnation of leukemic cells, and "protect" leukemic cells from the toxicity of chemotherapeutic drugs. Micro RNA (micro RNA,) Mi RNA), as a small non-coding RNA molecule, can regulate the expression of genes at the post-transcriptional level. Mi RNA plays an important role in biological development, such as cell differentiation and apoptosis, tumorigenesis and so on. It is of great significance to study the expression and function of mi RNA in the prevention and treatment of tumor diseases. The study of mi RNA in the pathogenesis of AML has just started in recent years. Most of them are expounded from the point of view of expression level. Few functional studies related to the pathogenesis of, mi RNA have not been reported on the role of, mi RNA in MSC-induced drug resistance in AML. Our early work found that after the adhesion of AML cell line KG1a to MSC, the response to chemotherapeutic drugs decreased and apoptosis decreased. The results of RT-PCR and Western confirmed that the expression of C-Myc, which is related to cell cycle, was up-regulated. In this study, on the basis of co-culture of AML cell line and human bone marrow MSC, the mi RNAs, regulating C-Myc was found by mi RNA chip and the expression of mi RNAs was verified. Prepare for subsequent functional verification. Methods: 1. Primary bone marrow cells from 15 healthy donors were prepared by culturing AML cell line KG1a,. 2. Isolation and culture of human bone marrow MSC.3. The co-culture system was established. After co-culture with MSC for 48 h, KG1a (sample number 4D, 5E, 6F) was selected. AML cell line KG1a was cultured alone (sample number 1, 2, 3) as control. 4. The above samples were divided into 6 tubes and treated with Trizol, and sent to the company for sample quality test and made into gene chip. According to the results of the whole genome chip, the mi RNAs.5. regulating C-Myc was found. Repeat step 1, 2, 3 RT PCR was used to detect the expression of mi RNAs screened under the two culture conditions. Result: 1. The sample of gene chip is up to standard. 2. According to the results of the chip, the mi RNAs:let-7a,mi R 鈮，
本文编号：2476679
[Abstract]:Aim: mesenchymal stem cells (MSC) in bone marrow microenvironment can induce the growth and survival of tumor cells and form a microenvironment suitable for tumor reproduction and drug resistance. The key to the recurrence and drug resistance of (AML) in acute myeloid leukemia lies in the interaction between leukemia cells and MSC. The adhesion and interaction between (LSCs) and MSC can directly promote the self-renewal, proliferation and differentiation stagnation of leukemic cells, and "protect" leukemic cells from the toxicity of chemotherapeutic drugs. Micro RNA (micro RNA,) Mi RNA), as a small non-coding RNA molecule, can regulate the expression of genes at the post-transcriptional level. Mi RNA plays an important role in biological development, such as cell differentiation and apoptosis, tumorigenesis and so on. It is of great significance to study the expression and function of mi RNA in the prevention and treatment of tumor diseases. The study of mi RNA in the pathogenesis of AML has just started in recent years. Most of them are expounded from the point of view of expression level. Few functional studies related to the pathogenesis of, mi RNA have not been reported on the role of, mi RNA in MSC-induced drug resistance in AML. Our early work found that after the adhesion of AML cell line KG1a to MSC, the response to chemotherapeutic drugs decreased and apoptosis decreased. The results of RT-PCR and Western confirmed that the expression of C-Myc, which is related to cell cycle, was up-regulated. In this study, on the basis of co-culture of AML cell line and human bone marrow MSC, the mi RNAs, regulating C-Myc was found by mi RNA chip and the expression of mi RNAs was verified. Prepare for subsequent functional verification. Methods: 1. Primary bone marrow cells from 15 healthy donors were prepared by culturing AML cell line KG1a,. 2. Isolation and culture of human bone marrow MSC.3. The co-culture system was established. After co-culture with MSC for 48 h, KG1a (sample number 4D, 5E, 6F) was selected. AML cell line KG1a was cultured alone (sample number 1, 2, 3) as control. 4. The above samples were divided into 6 tubes and treated with Trizol, and sent to the company for sample quality test and made into gene chip. According to the results of the whole genome chip, the mi RNAs.5. regulating C-Myc was found. Repeat step 1, 2, 3 RT PCR was used to detect the expression of mi RNAs screened under the two culture conditions. Result: 1. The sample of gene chip is up to standard. 2. According to the results of the chip, the mi RNAs:let-7a,mi R 鈮，
本文编号：2476679
本文链接：https://www.wllwen.com/yixuelunwen/zlx/2476679.html
