Atg7对小鼠神经干细胞增殖及分化能力的影响

发布时间：2018-06-29 01:30

  本文选题：神经干细胞 + Atg7　； 参考：《山西医科大学》2017年硕士论文

【摘要】：目的:探究自噬相关基因Atg7对NSCs增殖及分化潜能的影响,旨在寻找影响NSCs增殖活力及分化能力的关键靶点,为治疗神经退行性疾病提供新的可行性策略。方法:1.设计合成靶向Atg7的siRNA序列及其对照siRNA,电穿孔法分别转染NSCs,转染48h后提取NSCs总的RNA,72h后提取NSCs全蛋白,运用Real-time PCR及Western blot分别从m RNA水平和蛋白水平测定Atg7基因的敲减效率。2.应用Western blot比较si RNA组与对照组siRNA的LC3II以及P62的表达变化,检测敲减Atg7对NSCs自噬水平的影响。3.测量神经球的数量和平均直径,运用溴脱氧尿嘧啶核苷(BrdU)掺入实验检测Atg7基因沉默对NSCs增殖能力的影响。4.利用Tuj1和GFAP免疫荧光染色分析Atg7基因敲减对NSCs分化能力的影响。5.应用SA-β-gal染色检测siRNA组与对照组si RNA衰老细胞阳性率,qPCR分析P16、P21、P27和P53衰老相关基因的转录情况。结果:1.RT-PCR结果显示siRNA组Atg7 mRNA水平亦较对照组显著降低54%(P0.001),但Atg3,Atg5,Beclin1自噬其他相关基因的mRNA水平无显著变化;Western blot显示,Atg7敲减组的蛋白表达水平较对照组降低35%(P0.01),提示siRNA干扰能够显著降低NSCs Atg7的表达水平。2.Western blot结果显示,siRNA组LC3II蛋白水平较对照组降低27.5%(P0.05),P62蛋白水平为对照组的1.5倍(P0.001),提示敲减Atg7基因后,能够降低NSCs的自噬水平。3.si RNA组神经球的数目较对照组降低20%(P0.01)而且平均直径下降30%(P0.01);BrdU免疫荧光染色实验结果显示si RNA组BrdU阳性率较对照组显著下降29.8%(P0.001),提示Atg7基因敲减可以抑制NSCs的增殖的能力。4.Tuj1和GFAP免疫荧光染色显示,Atg7-siRNA实验组Tuj1阳性细胞率较对照组显著降低38.3%(P0.05),而其GFAP阳性细胞率为对照组的1.8倍(P0.01)。提示Atg7基因沉默可以抑制NSCs向神经元方向分化的能力。5.SA-β-gal染色结果显示siRNA组中衰老阳性细胞数是对照组的2.8倍(P0.001),P16、P21、P53的m RNA表达水平分别为对照组的1.5(P0.05)、1.6(P0.05)、1.6(P0.001)倍,而P27的mRNA表达水平无明显变化,提示下调Atg7表达水平可能激活衰老相关基因转录,导致NSCs活力降低。结论:1.Atg7敲减能够显著抑制NSCs增殖与分化能力。2.Atg7敲减能够下调NSCs自噬水平,降低NSCs活力,提示自噬在维持NSCs功能中发挥重要作用。
[Abstract]:Objective: to explore the effect of autophagy related gene Atg7 on proliferation and differentiation potential of NSCs in order to find out the key targets affecting the proliferation activity and differentiation ability of Atg7 and to provide a new feasible strategy for the treatment of neurodegenerative diseases. Method 1: 1. The siRNAs targeting Atg7 and its control siRNAs were designed and synthesized. NSCs were transfected with Atg7 by electroporation. After 48 hours of transfection, the total RNAs were extracted for 72 h and the whole proteins were extracted. The knockdown efficiency of Atg7 gene was determined by real-time PCR and Western blot, respectively, at the level of mRNA and protein. The expression of LC3II and P62 in siRNA group was compared with that in control group by Western blot, and the effect of knockout Atg7 on autophagy was detected. The number and mean diameter of neurospheres were measured, and the effect of Atg7 gene silencing on proliferation of Atg7 was detected by BrdU incorporation assay. Tuj1 and GFAP immunofluorescence staining were used to analyze the effect of knockout on the differentiation of Atg7. The positive rate of siRNA senescent cells in siRNA group and control group was detected by SA- 尾 -gal staining and the transcription of P16P21, P27 and p53 senescence related genes were analyzed by qPCR. Results 1. RT-PCR results showed that the Atg7 mRNA level in siRNA group was also significantly lower than that in control group by 54% (P0.001), but the mRNA level of other genes related to Atg5 Beclin1 autophagy was not significantly changed. Western blot showed that the protein expression level of ATG7 knockdown group was 35% lower than that of control group (P0.01), suggesting that siRNA-interference could interfere with siRNA. The results of Western blot showed that the level of LC3II protein decreased by 27.5% (P0.05) compared with the control group, and the level of P62 protein was 1.5-fold higher than that of the control group (P0.001), suggesting that after knockout of the Atg7 gene, the expression level of LC3II protein was significantly lower than that of the control group (P0.001). The number of neurospheres decreased by 20% (P0.01) and the mean diameter decreased by 30% (P0.01) in the group of siRNA compared with the control group. The results of immunofluorescence staining showed that the positive rate of BrdU in the group of siRNA was 29.8% (P0.001) lower than that in the control group, suggesting that the knockout of Atg7 gene. The ability of inhibiting the proliferation of NSCs. 4. Tuj1 and GFAP immunofluorescence staining showed that the rate of Tuj1 positive cells in Atg7-siRNA group was significantly lower than that in control group by 38.3% (P0.05), and the GFAP positive cell rate was 1.8 times higher than that in control group (P0.01). The results of SA- 尾 -gal staining showed that the number of senescent positive cells in siRNA group was 2.8 times of that in control group (P0. 001) and the expression level of mRNAs in P16 P21 + p53 was 1. 5 (P0.05) / 1. 6 (P0. 001) times as high as that in control group, respectively, and the expression level of senescence positive cells in siRNA group was 1. 5 (P0.05) (P0. 05) and 1. 6 times (P0. 001). However, there was no significant change in the expression of P27 mRNA, which suggested that down-regulation of Atg7 expression might activate the transcription of senescence related genes and decrease the activity of Atg7. Conclusion: 1. Atg7 knockout can significantly inhibit the proliferation and differentiation of NSCs. 2. Atg7 knockdown can down-regulate the level of autophagy and decrease the activity of NSCs, suggesting that autophagy plays an important role in maintaining the function of NSCs.
【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】：R363

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本文编号：2080225