LMO3逆转录病毒表达载体的构建及其对SK-N-AS细胞增殖的影响  

Construction of Recombinant Retroviral Vector and Its Effection on Proliferation of SK-N-AS Human Neuroblastoma

作　　者：惠玲[1] 王晓辉[1] 王剑锋[1] 张长菊[2] 王美亮[1] 贾庆华[1] 

机构地区：[1]兰州军区兰州总医院医学实验中心,甘肃省干细胞与基因药物重点实验室,兰州市730050 [2]兰州大学第二医院检验中心,兰州市730030

出　　处：《医学分子生物学杂志》2012年第3期166-170,共5页Journal of Medical Molecular Biology

基　　金：国家自然科学基金（No.30400125）,中国博士后科学基金（No.20070410230）,甘肃省自然科学基金（No.1107RJZA106）

摘　　要：目的构建包含LM03（LIM-only3，LM03）全长基因的逆转录病毒表达载体，感染人神经母细胞瘤SK-N-AS，检测LM03对SK-N-AS细胞增殖的影响。方法将质粒pEGFP-Cl-一LM03经EcoRI和BamHI双酶切后亚克隆至逆转录病毒载体pLXSN，构建重组逆转录病毒表达载体pLXSN-LMO3，导人包装细胞pA317，获得逆转录病毒颗粒，感染SK-N-AS细胞，用RT-PCR及Western印迹鉴定，检测LM03感染后细胞的增殖及细胞周期分布情况。结果获得了能正确表达LM03基因的重组逆转录病毒表达载体pLX-SN-LMO3；LM03基因被逆转录病毒成功导入SK-N-AS细胞后，与对照组细胞相比，LM03感染组G1／G1期细胞减少，S期细胞增加，感染48h后，LM03感染组细胞的增殖能力显著高于空载体对照组及SK-N．AS组（P〈0．05）。结论成功构建了LM03基因的逆转录病毒表达载体，LMO3可以通过促进SK-N-AS细胞由G0／G1期进入S期，从而促进细胞的增殖。Objective To establish a recombinant retroviral vector containing LIM-only 3 （LMO3） gene and investigate its role in SK-N-AS human neuroblastoma ceils. Methods The plas- mid pEGFP-Cl-LMO3 was cut with EcoR Ⅰ and BamH Ⅰ , and subcloned to retroviral vector pLX- SN to construct the recombinant plasmid pLXSN-LMO3. The recombinant plasmid was transferred in- to packaging cell line PA317 by using Lipofectamiue2000. Virus was used to infect SK-N-AS ceils. After selection with G418, the mRNA and protein expressions of LMO3 were determined by the reverse transcription-PCR and Western blotting, respectively. The proliferation and cell cycle was detected by MTY or flow cytometry. Results The recombined LMO3 retroviral expression vector was successfully constructed. The recombinant retrovirus efficiently delivered LMO3 gene into SK-N- AS ceils. After transfected with LMO3, the G1/G0 phase ceils were decreased, whereas S phase cells were increased compared with the control groups. Cell proliferation showed signi？ cantly in- creased in LMO3 infected cells after transfected 48 h （P 〈 0.05 ） . Conclusion The recombinant retroviral vectors pLXSN-LMO3 was successfully constructed. Overexpression of LMO3 can promoteSK-N-AS cells proliferation. The results indicated that LMO3-induced cells proliferation might work through promoting the G1/G0 phase cells into S phase.

关 键 词：LMO3 载体构建 逆转录病毒载体 PLXSN 

分 类 号：Q78[生物学—分子生物学]