肌浆网-内质网钙离子转运ATP酶干扰慢病毒表达载体构建及稳定转染PC 12细胞株的建立
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摘要
目的构建肌浆网-内质网钙离子转运ATP酶(SERCA)基因的干扰慢病毒表达载体,建立稳定转染的大鼠肾上腺嗜铬细胞瘤(PC 12)细胞系。方法人工设计、合成针对SERCA基因干扰序列,退火后连接到PGLV3干扰载体上,与p Gag/Pol、pRev、p VSV-G共转染293T细胞,包装产生慢病毒颗粒并测定病毒滴度,感染PC 12细胞,建立稳定细胞株;应用RT-PCR和Western blot技术检测PC 12稳定细胞中SERCA基因和蛋白表达情况,并与对照组进行比较。结果成功构建了针对SERCA基因的RNAi慢病毒表达载体,病毒滴度为3×108U·m L-1;建立稳定转染的PC 12细胞株。有效干扰验证显示,sh SERCA能明显降低SERCA的mRNA及蛋白水平(P<0.01)。结论成功构建SERCA基因的sh SERCA慢病毒表达载体,建立稳定干扰SERCA表达的PC 12细胞株。
OBJECTIVE To construct the SERCA gene interference lentivirus expression vector and establish stable transfected PC 12 cell line. METHODS The interference sequence targeting at rat SERCA gene was designed and synthesized. p Gag/Pol,pRev,and p VSV-G were co-transfected into 293 T cells. The lentivirus particles were packaged and generated. The virus titer was detected. PC 12 cells were transfected for establishing the stable cell line; RT-PCR and Western blot were used to detect SERCA gene and protein expression in stable PC 12 cells,and the results were compared with those in the control group. RESULTS The lentivirus expression vector targeted at SERCA was successfully constructed and the virus titer was 3 × 108 U·m L-1. Stable transfected PC 12 cells line was established. The effective interference verification revealed that sh SERCA could significantly reduce the mRNA and protein levels of SERCA( P < 0. 01). CONCLUSION The shRNA lentiviral expression vector of SERCA gene is successfully constructed and the PC 12 cell line stably interfering with SERCA expresion is established.
OBJECTIVE To construct the SERCA gene interference lentivirus expression vector and establish stable transfected PC 12 cell line. METHODS The interference sequence targeting at rat SERCA gene was designed and synthesized. p Gag/Pol,pRev,and p VSV-G were co-transfected into 293 T cells. The lentivirus particles were packaged and generated. The virus titer was detected. PC 12 cells were transfected for establishing the stable cell line; RT-PCR and Western blot were used to detect SERCA gene and protein expression in stable PC 12 cells,and the results were compared with those in the control group. RESULTS The lentivirus expression vector targeted at SERCA was successfully constructed and the virus titer was 3 × 108 U·m L-1. Stable transfected PC 12 cells line was established. The effective interference verification revealed that sh SERCA could significantly reduce the mRNA and protein levels of SERCA( P < 0. 01). CONCLUSION The shRNA lentiviral expression vector of SERCA gene is successfully constructed and the PC 12 cell line stably interfering with SERCA expresion is established.
引文
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[3]CARAFOLI E.Calcium pumps:why so many?[J].Compr Physiol,2012,2(2):1045-1060.
[4]LIU B.Research progress on calcium homeostasis and nerve injury of central nervous system[J].Foreign Med(国际脑血管杂志),2006,33(4):193-198.
[5]SHI Y M,GE J W.Research progress on neuronal calcium channels on cerebral ischemia-reperfusion[J].Med Rev,2014,7(20):2507-2509.
[6]SZYDLOWSKA K,TYMIANSKI M.Calcium,ischemia and excitotoxicity[J].Cell,2010,47(2):122-129.
[7]SHAFER T J,ATCHISON W D.Transmitter,ionchanneland receptor propertiesof pheochromocytoma(PC12)cells:a modelforneurotoxicologicalstudies[J].Neurotoxicology,1991,12(3):473-492.
[8]ABU R S,TREMBOVLER V,SHOHAMI E.A tissuecultureischemic device to study eicosanoid release by pheochromocytoma PC1 2 cultures[J].J Neurosci Methods,1 9 9 3,5 0(2):197-203.
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[10]YUAN W Z,WU X S.Research progress onRNAimechanism[J].Life Sci Res,2003,7(1):8-14.
[11]MENG F R,CHEN C.Research progress on lentiviral vector[J].China Lung Cancer J(中国肺癌杂志),2014,17(12):870-876.
[12]CAMPOS-MELO D,DROPPELMANN C A,HE Z,et al.Altered microRNA expression profile in amyotrophic lateral sclerosis:a role in the regulation of NFL mRNA levels[J].Mol Brain,2013,6(1):26-30.
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[14]LI X M.Research progress onsarcoplasmic reticulum Ca2+-ATPase of cardiac muscle[J].Med Rev,2008,14(17):2570-2573.
[15]SUKOVICH D A,SHABBEER J,PERIASAMY M.Analysis of the rabbit cardiac/slow twitch muscle sarcoplasmicreticulum calcium ATPase(SERCA2)genepromoter[J].Nucleic Acids Res,1993,21(11):2723-2728.