Bmil特异性siRNA慢病毒载体的构建及其对神经干细胞增殖的影响

发布时间：2018-12-13 16:49

【摘要】：目的：神经干细胞是修复中枢神经系统损伤具有发展前景的工具细胞，但在体外培养易出现细胞衰老，严重影响移植效果。最新研究表明Bmi1基因是维持干细胞增殖所必须的一种重要基因，在生理情况下能保持特异靶基因处于稳定的被抑制状态，起到维持细胞存活、生长的重要功能。本研究拟构建Bmi1特异性siRNA慢病毒载体并探讨其对人类胚胎纹状体来源神经干细胞增殖的调控作用，为神经干细胞临床应用修复中枢神经系统损伤提供理论依据。 方法：本实验首先从8-12周自然流产胎儿的大脑皮层区中有效分离出神经干细胞并在含有丝分裂原的无血清培养基中进行传代培养，通过特异标记物染色（Nestin）以及将神经干细胞向神经元（Tuj1）和星形胶质细胞（GFAP）分化来鉴定其为神经干细胞，将神经干细胞分成三组进行实验，即Bmi1转染组、GFP空病毒转染组和未经病毒转染组。其次，构建Bmi1的慢病毒载体，进行RNA干扰，检测慢病毒转染效率，并通过荧光实时定量PCR的方法检测Bmi1基因的干扰后的表达情况。再次，在有效干扰Bmi1基因后，通过BrdU掺入率检测细胞的增生能力的改变情况。最后，用β半乳糖苷酶检测细胞的衰老情况。通过以上数据比较三组神经干细胞的增殖能力以及衰老情况。 结果：(1)经Nestin、Tuj1、GFAP染色后证明实验所取细胞为神经干细胞。(2)在转染48h后进行显微镜下观察，发现两病毒组转染组的神经干细胞均带有绿色荧光，并且转染效率都超过了90％，对比转染后0h的Bmi-1基因的相对转录水平(99.6±8．25)％，48、72、168h的Bmi-1基因的相对转录水平均下降，数值分别为：(35.6±9.67)％、(26.4±12.05)％和(35.6±10.42)％，差异有统计学意义。(3)检测不同时间点的Bmi1干扰后细胞的增殖情况，，不同时间点的Bmi1干扰组和GFP对照空病毒干扰组的BrdU掺入率比值分别为1.00±0.27、0．58±0．27、0．54±0.23、0.55±0.25，单因素方差分析后，48h、72h、168h与0h相比BrdU掺入率均下降，差异有统计学意义(P0．01)。(4)Bmi1干扰组和GFP-对照空病毒组神经干细胞的衰老率分别为(59．53±15．38)％、(41．76±12．45)％，两组神经干细胞的衰老率之间差异有统计学意义(P0．01)。经过Bmi1干扰一周后，神经干细胞的增殖能力发生了显著的下降，随之而来的是神经干细胞衰老数量发生显著的增加。 结论：本实验成功分离培养了人胚胎纹状体来源神经干细胞，构建Bmi1的慢病毒干扰载体转染效率高。Bmi1基因是神经干细胞保持增殖活性状态关键调控基因，使神经干细胞长期保持增殖活性从而满足脑修复重建的需要。
[Abstract]:Objective: neural stem cells (NSCs) are promising tool cells for repairing central nervous system injury, but cell senescence is easy to occur in vitro culture, which seriously affects the transplantation effect. New research shows that Bmi1 gene is an important gene necessary to maintain stem cell proliferation. It can keep the specific target gene in a stable and inhibited state under physiological conditions and play an important role in maintaining cell survival and growth. The aim of this study was to construct a Bmi1 specific siRNA lentivirus vector and to investigate its regulatory effect on the proliferation of neural stem cells derived from human embryonic striatum, and to provide a theoretical basis for the clinical application of neural stem cells to repair central nervous system injury. Methods: neural stem cells (NSCs) were isolated from the cortical region of spontaneous abortion fetus at 8-12 weeks and cultured in serum-free medium containing mitogen. Neural stem cells were identified as neural stem cells by staining (Nestin) and differentiating neural stem cells into neurons (Tuj1) and astrocytes (GFAP). Neural stem cells were divided into three groups: Bmi1 transfection group. GFP empty virus transfection group and no virus transfection group. Secondly, the lentivirus vector of Bmi1 was constructed, the RNA interference was carried out, the efficiency of lentivirus transfection was detected, and the expression of Bmi1 gene after interference was detected by real-time quantitative PCR. Thirdly, after effectively interfering with Bmi1 gene, BrdU incorporation rate was used to detect the change of cell proliferation ability. Finally, 尾-galactosidase was used to detect cell senescence. The proliferative ability and senescence of three groups of neural stem cells were compared with the above data. Results: (1) after Nestin,Tuj1,GFAP staining, the cells were identified as neural stem cells. (2) after 48 hours of transfection, the neural stem cells in the two groups were observed under microscope. The relative transcription level of Bmi-1 gene at 0 h after transfection was (99.6 卤8.25)%, and the relative transcription level of Bmi-1 gene at 48 h after transfection was (35.6 卤9.67)%. The difference was statistically significant between (26.4 卤12.05)% and (35.6 卤10.42)%. (3) the proliferation of cells after Bmi1 interference at different time points was detected. The ratios of BrdU incorporation rates in Bmi1 interference group and GFP control group were 1.00 卤0.27 卤0.27 卤0.57 卤0.54 卤0.23 卤0.55 卤0.25, respectively. After univariate ANOVA, the incorporation rate of BrdU decreased at 48h / 72h / 168h compared with 0h respectively. The senescence rates of neural stem cells in Bmi1 interference group and GFP- control group were (59.53 卤15.38)% and (41.76 卤12.45)%, respectively. There was significant difference in senescence rate of neural stem cells between the two groups (P 0.01). After a week of Bmi1 interference, the proliferation of neural stem cells decreased significantly, followed by a significant increase in the number of neural stem cells aging. Conclusion: neural stem cells derived from human embryonic striatum were isolated and cultured successfully in this experiment. The lentivirus interference vector of Bmi1 was constructed with high transfection efficiency. Bmi1 gene is the key regulation gene of neural stem cells to maintain proliferative activity. Neural stem cells to maintain long-term proliferative activity to meet the need for brain repair and reconstruction.
【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R363

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