【摘要】：目的構(gòu)建靶向糖原合成激酶3β(GSK-3β)基因的RNA干擾慢病毒載體,建立其對(duì)GSK-3β基因沉默表達(dá)的人神經(jīng)母細(xì)胞瘤細(xì)胞(SH-SY5Y)的細(xì)胞系模型。方法根據(jù)GSK-3βmRNA序列設(shè)計(jì)合成靶向目的基因的短發(fā)夾狀RNA(shRNA)序列并構(gòu)建慢病毒表達(dá)載體,經(jīng)酶切和測(cè)序鑒定,將慢病毒重組載體與包裝載體共轉(zhuǎn)染293T細(xì)胞,獲得純化濃縮病毒液,侵染SH-SY5Y細(xì)胞,采用綠色熒光示蹤觀察細(xì)胞的轉(zhuǎn)染情況,設(shè)為慢病毒干擾組(實(shí)驗(yàn)組)、陰性對(duì)照組(Lenti-NC組)、空白對(duì)照組(Blank組),利用qRT-PCR和Western blot法檢測(cè)GSK-3βmRNA和蛋白表達(dá)水平。結(jié)果成功構(gòu)建了靶向GSK-3β的shRNA慢病毒,重組慢病毒轉(zhuǎn)染人SH-SY5Y細(xì)胞,72 h后觀察轉(zhuǎn)染效率達(dá)90%以上,與對(duì)照組比較,靶基因變化結(jié)果顯示,實(shí)驗(yàn)組中GSK-3βmRNA和蛋白的表達(dá)均顯著降低(均P0.01)。結(jié)論有效建立慢病毒介導(dǎo)的GSK-3β基因穩(wěn)定沉默表達(dá)的SH-SY5Y細(xì)胞模型,為體外評(píng)價(jià)研究GSK-3β沉默后藥物或行為治療AD提供試驗(yàn)工具細(xì)胞模型。
[Abstract]:Aim to construct RNA interference lentivirus vector targeting glycogen synthesis kinase 3 尾 (GSK-3 尾) gene and establish a cell line model of human neuroblastoma cell line (SH-SY5Y) whose expression of GSK-3 尾 gene is silenced. Methods according to the GSK-3 尾 mRNA sequence, the short hairpin RNA (shRNA) sequence of the target gene was designed and synthesized, and the lentivirus expression vector was constructed. The recombinant lentiviral vector was co-transfected with the packaging vector to 293T cells by restriction endonuclease digestion and sequencing. SH-SY5Y cells were infected with purified and concentrated virion. The transfected cells were divided into lentivirus interference group (experimental group), negative control group (Lenti-NC group) and blank control group (Blank group) by green fluorescence tracer. The expression levels of GSK-3 尾 mRNA and protein were detected by qRT-PCR and Western blot methods. Results shRNA lentivirus targeting GSK-3 尾 was successfully constructed. Recombinant lentivirus was transfected into human SH-SY5Y cells. After 72 hours, the transfection efficiency was more than 90%. Compared with the control group, the target gene changes showed that the recombinant lentivirus was transfected into SH-SY5Y cells. The expression of GSK-3 尾 mRNA and protein in the experimental group was significantly lower than that in the control group (P0.01). Conclusion the lentivirus-mediated stable silencing expression of GSK-3 尾 gene in SH-SY5Y cell model can be established effectively, which provides an experimental tool for evaluating the drug or behavioral therapy of AD after GSK-3 尾 silencing in vitro.
【作者單位】： 山東大學(xué)附屬濟(jì)南市中心醫(yī)院神經(jīng)內(nèi)科;濟(jì)南市第四人民醫(yī)院內(nèi)分泌科;
【基金】：國(guó)家自然科學(xué)基金資助項(xiàng)目(No:30970989)
【分類(lèi)號(hào)】：R749.16

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