本文選題：戊二酰輔酶A脫氫酶　切入點(diǎn)：細(xì)胞活性　出處：《中國當(dāng)代兒科雜志》2017年09期 　論文類型：期刊論文

【摘要】：目的探討戊二酰輔酶A脫氫酶(GCDH)基因沉默和賴氨酸代謝物蓄積對肝細(xì)胞活性的影響。方法將BRL肝細(xì)胞分為正常對照組、陰性對照組和GCDH沉默組。構(gòu)建含靶向沉默GCDH基因的sh RNA慢病毒載體,分別用該病毒和陰性對照病毒感染GCDH沉默組和陰性對照組BRL肝細(xì)胞。感染后細(xì)胞再用含5 mmol/L賴氨酸培養(yǎng)基培養(yǎng)。免疫熒光技術(shù)檢測慢病毒感染效率;Western blot法檢測GCDH蛋白表達(dá)水平;MTT法檢測細(xì)胞活性,Hoechest 33342染色檢測細(xì)胞凋亡,Western blot法檢測細(xì)胞凋亡的經(jīng)典指標(biāo)Caspase3水平。結(jié)果構(gòu)建的慢病毒可有效沉默肝細(xì)胞GCDH表達(dá)(P0.01)。MTT及Hoechest 33342染色檢測各組間細(xì)胞活性及細(xì)胞凋亡比較差異無統(tǒng)計(jì)學(xué)意義(P0.05)。Caspase3蛋白表達(dá)在各組間比較差異亦無統(tǒng)計(jì)學(xué)意義(P0.05)。結(jié)論 GCDH基因沉默和賴氨酸代謝物蓄積對肝細(xì)胞無明顯損傷作用。
[Abstract]:Objective to investigate the effects of glutaryl coenzyme A dehydrogenase (GCDH) gene silencing and lysine metabolite accumulation on hepatocyte activity. Methods BRL hepatocytes were divided into normal control group. Negative control group and GCDH silencing group. The sh RNA lentivirus vector containing the target silencing GCDH gene was constructed. The hepatocytes of GCDH silencing group and negative control group were infected with the virus and the negative control group respectively. After infection, the cells were cultured in 5 mmol/L lysine medium. The efficiency of lentivirus infection was detected by immunofluorescence technique and GCDH was detected by Western blot method. The level of protein expression was determined by MTT assay and the activity of cells was detected by Hoechest 33342 staining. Western blot assay was used to detect the Caspase3 level of apoptosis. Results the constructed lentivirus could effectively silence the expression of GCDH in hepatocytes and detect the expression of GCDH in hepatocytes by Hoechest 33342 staining. There was no significant difference in cell activity and apoptosis between the two groups. There was no significant difference in the expression of P0.05N. Caspase3 between the two groups. Conclusion GCDH gene silencing and lysine metabolite accumulation have no significant damage to hepatocytes.
【作者單位】： 華中科技大學(xué)同濟(jì)醫(yī)學(xué)院附屬同濟(jì)醫(yī)院兒科;
【分類號】：R725.9

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1 安永;吳春;潘征夏;;大鼠體外循環(huán)后肝細(xì)胞膽鹽轉(zhuǎn)運(yùn)功能的變化及意義[A];中華醫(yī)學(xué)會(huì)第八次全國小兒外科學(xué)術(shù)會(huì)論文集[C];2010年

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