靶向Bak1的shRNA表达腺病毒构建及其对人骨肉瘤细胞的影响
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摘要
目的构建沉默人Bcl-2拮抗1基因(Bak1)的重组腺病毒,感染人成骨肉瘤细胞MG63并检测其对细胞活性的影响。方法设计特异性针对Bak1的sh RNA序列及无关对照序列scr,分别构建重组腺病毒表达载体,感染人成骨肉瘤细胞MG63,利用q PCR和Western blot检测Bak1基因沉默后的MG63细胞Bak1 m RNA及蛋白的表达,筛选出能高效沉默目标基因的重组腺病毒表达载体,并对其进行包装扩繁,荧光显微镜观察计算病毒滴度,利用MTT法检测高效沉默的重组腺病毒感染MG63细胞后的细胞活性。结果 (1)测序列结果表明,sh RNA序列已成功克隆入到p Ad-GFP载体上;(2)sh RNA-3的沉默效率最高,扩增纯化后腺病毒滴度达到7.7×108GFU/ml;(3)MTT检测显示与NC对照组相比,sh RNA-3组MG63细胞活性增强(P<0.5)。结论成功构建了沉默Bak1的重组腺病毒,为进一步研究Bak1的功能奠定了基础。
Objective To construct recombinant BCL2-antagonist/killer 1(Bak1)-silenced adenovirus,and to establish a cell model by human osteosarcoma MG63 cells,and to study MG63 cells viability after being infected by the recombinant adenovirus.Methods The specific sequences of Bak1 and controlled sequence(scr) were designed and synthesized and then used to construct recombinant adenovirus. The best recombinant adenovirus were selected after infecting MG63 cells by q PCR and Western blot,and then were packaged and amplified and calculated virus titer with fluorescence microscopy. The viability of infected MG63 cells was determined by MTT. Results It was proved by gene sequencing that the sh RNA sequences were successfully cloned into the adenoviral vectors p Ad-GFP. The sh RNA-3 was the best recombinant adenovirus(P<0.5),and its titer was 7.7×108 GFU/ml after amplified and purification. MTT showed that Bak1 gene silencing could weaken the viability of MG63 cells(P<0.5). Conclusion The recombinant adenovirus which could silence the expression of Bak1 gene after infecting MG63 cells was successfully constructed,which lying a foundation for further research on the function of Bak1.
Objective To construct recombinant BCL2-antagonist/killer 1(Bak1)-silenced adenovirus,and to establish a cell model by human osteosarcoma MG63 cells,and to study MG63 cells viability after being infected by the recombinant adenovirus.Methods The specific sequences of Bak1 and controlled sequence(scr) were designed and synthesized and then used to construct recombinant adenovirus. The best recombinant adenovirus were selected after infecting MG63 cells by q PCR and Western blot,and then were packaged and amplified and calculated virus titer with fluorescence microscopy. The viability of infected MG63 cells was determined by MTT. Results It was proved by gene sequencing that the sh RNA sequences were successfully cloned into the adenoviral vectors p Ad-GFP. The sh RNA-3 was the best recombinant adenovirus(P<0.5),and its titer was 7.7×108 GFU/ml after amplified and purification. MTT showed that Bak1 gene silencing could weaken the viability of MG63 cells(P<0.5). Conclusion The recombinant adenovirus which could silence the expression of Bak1 gene after infecting MG63 cells was successfully constructed,which lying a foundation for further research on the function of Bak1.
引文
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[2]Qi H,Aguiar DJ,Williams SM,et al.Identification of genes responsible for osteoblast differentiation from human mesodermal progenitor cells[J].Proc Natl Acad Sci USA,2003,100(6):3305-3310.
[3]Weinstein RS,Manolagas SC.Apoptosis and osteoporosis[J].Am J Med,2000,108(2):153-164.
[4]Gunaratnam K,Vidal C,Boadle R,et al.Mechanisms of palmitateinduced cell death in human osteoblasts[J].Biol Open,2013,2(12):1382-1389.
[5]Hock JM,Krishnan V,Onyia JE,et al.Osteoblast apoptosis and bone turnover[J].J Bone Miner Res,2001,16(6):975-984.
[6]Youle RJ,Strasser A.The BCL-2 protein family:opposing activities that mediate cell death[J].Nat Rev Mol Cell Biol,2008,9(1):47-59.
[7]曾国勇.Bcl-2、Bak与骨质疏松症及其中医证型的相关性研究[D].广州:广州中医药大学,2016:20-21.
[8]王晨秀,霍亚南.骨健康影响因素的研究进展[J].江西医药,2013,48(3):266-271.
[9]胡瑛,秦淑兰,江芳芳,等.江西省进贤县4515例居民骨密度流行病学调查分析[J].江西医药,2016,51(10):1013-1015.
[10]Farrow SN,White JH,Martinou I,et al.Cloning of a bcl-2homologue by interaction with adenovirus E1B 19K[J].Nature,1995,374(6524):731-733.
[11]Chittenden T,Harrington E,O′Connor R,et al.Induction of apoptosis by the Bcl-2 homologue Bak[J].Nature,1995,374(6524):733-736.
[12]Kiefer MC,Brauer MJ,Powers VC,et al.Modulation of apoptosis by the widely distributed Bcl-2 homolog Bak[J].Nature,1995,374(6524):736-739.
[13]Bleicken S,Classen M,Padmavathi PV,et al.Molecular details of Bax activation,oligomerization,and membrane insertion[J].J Biol Chem,2010,285(9):6636-6647.
[14]Siddiqui WA,Ahad A,Ahsan H.The mystery of BCL2 family:Bcl-2 proteins and apoptosis:an update[J].Arch Toxicol,2015,89(3):289-317.
[15]王静,杨猛,陈曦,等.新型双核铂(Ⅱ)配合物通过p53-Bak途径诱导人乳腺癌MCF-7细胞凋亡[J].中国生物化学与分子生物学报,2017,33(2):160-168.
[16]陈晓,王启之,郦启之,等.NF-ΚB介导的促凋亡蛋白Bak表达在溃疡性结肠炎发病中的作用研究[J].中国免疫学杂志,2016,32(9):1286-1290.
[17]张森旺,卢晓芬,何晓虹,等.半边旗二萜类成分PAG通过ROS诱导的线粒体凋亡克服肝癌多药耐药[J].广东医学院学报,2016,34(4):365-369.
[18]李颖,白波,吴伙燕,等.骨骼肌线粒体通透转换孔在骨质疏松症中的变化[J].中华实验外科杂志,2011,28(7):1071-1073.
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[21]Crystal RG.Adenovirus:the first effective in vivo gene delivery vector[J].Hum Gene Ther,2014,25(1):3-11.