摘要
目的探索应用聚凝胺（polybrene）试剂增强Ror2重组腺病毒对K562细胞转染效率的技术。方法在K562细胞中分别加入0～12μl Ror2重组腺病毒,摸索最佳重组腺病毒用量;同时加入0～5μg/ml polybrene和最佳重组腺病毒用量,培养1～3d后,在荧光显微镜下计数,对比加入polybrene前后被感染的细胞数。台盼蓝染色观察重组腺病毒引起K562细胞死亡的情况。采用RT-PCR和Western blotting检测Ror2基因的表达情况。结果 Polybrene浓度为3μg/ml和重组腺病毒8μl时,感染效果最佳,对细胞生长影响较小,实验组感染效果是对照组的17倍;加入polybrene后,外源性Ror2m RNA和蛋白表达水平均明显增高。结论 Polybrene能显著增强重组腺病毒感染K562细胞的作用,并能高效表达外源性Ror2基因,该法简便、价廉、有效,是血液疾病分子生物学研究的一个良好策略。
Objective To explore a new technique to enhance the efficiency of Ror2 recombinant adenovirus infecting K562 cell line with polybrene. Methods Recombinant adenovirus Ror2 （AdRor2, 0-12μl） was added to K562 cells to look for the optimal dose of recombinant adenovirus. At the same time, polybrene （0-5μg/ml） and optimal dose of recombinant adenovirus were added to K562 cells. The infected cells were counted under fluorescence microscope after they were cultured 1-3 days, and the comparative study was performed between cells treated and not treated by polybrene. The influence of AdRor2 on the death of cells was investigated by trypan blue staining, and the expression of Rot2 gene was assessed with Western blotting and RT- PCR. Results It was found that the infection effect was optimal when the concentration of polybrene was 3μg/ml and the recombinant adenovirus was 8bcl, and there was little effect on the cell growth, mRNA and protein expression of exogenous Ror2 were significantly elevated. Conclusions Polybrene can significantly enhance the infection capability of recombinant adenovirus on K562 cells, and it can also effectively express the exogenous Ror2 gene. This technique is simple, inexpensive and effective, thus providing a good strategy for molecular biology research in the field of blood diseases.
出处
《解放军医学杂志》
CAS
CSCD
北大核心
2015年第12期976-980,共5页
Medical Journal of Chinese People's Liberation Army
基金
国家自然科学基金面上项目(81370624)~~
