發(fā)布時(shí)間：2018-06-29 01:30

  本文選題：神經(jīng)干細(xì)胞 + Atg7�。� 參考：《山西醫(yī)科大學(xué)》2017年碩士論文

【摘要】：目的:探究自噬相關(guān)基因Atg7對(duì)NSCs增殖及分化潛能的影響,旨在尋找影響NSCs增殖活力及分化能力的關(guān)鍵靶點(diǎn),為治療神經(jīng)退行性疾病提供新的可行性策略。方法:1.設(shè)計(jì)合成靶向Atg7的siRNA序列及其對(duì)照siRNA,電穿孔法分別轉(zhuǎn)染NSCs,轉(zhuǎn)染48h后提取NSCs總的RNA,72h后提取NSCs全蛋白,運(yùn)用Real-time PCR及Western blot分別從m RNA水平和蛋白水平測(cè)定Atg7基因的敲減效率。2.應(yīng)用Western blot比較si RNA組與對(duì)照組siRNA的LC3II以及P62的表達(dá)變化,檢測(cè)敲減Atg7對(duì)NSCs自噬水平的影響。3.測(cè)量神經(jīng)球的數(shù)量和平均直徑,運(yùn)用溴脫氧尿嘧啶核苷(BrdU)摻入實(shí)驗(yàn)檢測(cè)Atg7基因沉默對(duì)NSCs增殖能力的影響。4.利用Tuj1和GFAP免疫熒光染色分析Atg7基因敲減對(duì)NSCs分化能力的影響。5.應(yīng)用SA-β-gal染色檢測(cè)siRNA組與對(duì)照組si RNA衰老細(xì)胞陽(yáng)性率,qPCR分析P16、P21、P27和P53衰老相關(guān)基因的轉(zhuǎn)錄情況。結(jié)果:1.RT-PCR結(jié)果顯示siRNA組Atg7 mRNA水平亦較對(duì)照組顯著降低54%(P0.001),但Atg3,Atg5,Beclin1自噬其他相關(guān)基因的mRNA水平無(wú)顯著變化;Western blot顯示,Atg7敲減組的蛋白表達(dá)水平較對(duì)照組降低35%(P0.01),提示siRNA干擾能夠顯著降低NSCs Atg7的表達(dá)水平。2.Western blot結(jié)果顯示,siRNA組LC3II蛋白水平較對(duì)照組降低27.5%(P0.05),P62蛋白水平為對(duì)照組的1.5倍(P0.001),提示敲減Atg7基因后,能夠降低NSCs的自噬水平。3.si RNA組神經(jīng)球的數(shù)目較對(duì)照組降低20%(P0.01)而且平均直徑下降30%(P0.01);BrdU免疫熒光染色實(shí)驗(yàn)結(jié)果顯示si RNA組BrdU陽(yáng)性率較對(duì)照組顯著下降29.8%(P0.001),提示Atg7基因敲減可以抑制NSCs的增殖的能力。4.Tuj1和GFAP免疫熒光染色顯示,Atg7-siRNA實(shí)驗(yàn)組Tuj1陽(yáng)性細(xì)胞率較對(duì)照組顯著降低38.3%(P0.05),而其GFAP陽(yáng)性細(xì)胞率為對(duì)照組的1.8倍(P0.01)。提示Atg7基因沉默可以抑制NSCs向神經(jīng)元方向分化的能力。5.SA-β-gal染色結(jié)果顯示siRNA組中衰老陽(yáng)性細(xì)胞數(shù)是對(duì)照組的2.8倍(P0.001),P16、P21、P53的m RNA表達(dá)水平分別為對(duì)照組的1.5(P0.05)、1.6(P0.05)、1.6(P0.001)倍,而P27的mRNA表達(dá)水平無(wú)明顯變化,提示下調(diào)Atg7表達(dá)水平可能激活衰老相關(guān)基因轉(zhuǎn)錄,導(dǎo)致NSCs活力降低。結(jié)論:1.Atg7敲減能夠顯著抑制NSCs增殖與分化能力。2.Atg7敲減能夠下調(diào)NSCs自噬水平,降低NSCs活力,提示自噬在維持NSCs功能中發(fā)揮重要作用。
[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.
【學(xué)位授予單位】：山西醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R363

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本文編號(hào)：2080225