RYBP基因稳定沉默的HL-60细胞系的构建

 【摘要】　目的　利用慢病毒介导的RNA干扰（RNAi）技术，建立RYBP基因稳定沉默的HL-60细胞系。方法　设计针对RYBP基因的5条短发夹状RNA（shRNA）序列，构建RYBP shRNA慢病毒重组载体，分别包装成慢病毒颗粒，直接感染HL-60细胞，同时设空载体和阴性对照shRNA慢病毒对照组。通过观察绿色荧光蛋白表达以监测感染效率，经嘌呤霉素（终质量浓度8 μg／ml）筛选出稳定感染细胞。Western blot实验初步鉴定出蛋白水平最低的RYBP shRNA组，用实时定量聚合酶链反应（PCR）进一步检测该组细胞mRNA表达水平。结果　慢病毒感染的HL-60细胞经嘌呤霉素筛选成功，与对照组比较，5条RYBP shRNA组细胞RYBP表达均有不同程度减低（P＜0.01），其中以shRNA2沉默效果最佳，且shRNA2组的RYBP基因mRNA水平降低了95 %以上（P＜0.05）。结论　慢病毒介导的shRNA2能高效、稳定地沉默RYBP基因的表达，成功构建了RYBP基因稳定沉默的HL-60细胞系。

Establishment of a HL-60 cell line with stable RYBP silencing

Objective To establish stable HL-60 cell line with stable RYBP gene silencing using lentivirus-mediated RNA interference. Methods Five special shRNAs for RYBP gene were cloned to lentivirus vector. Recombinate lentivirus vectors were packed into lentivirus, which were used to infect HL-60 cells, and took empty vector and non-specific shRNA as control groups. Stable infected cells were selected with puromycin in 8 μg/ml concentration. The expression levels of RYBP were analyzed by Western blot, and confirmed the most effective RYBP shRNA. Then the level of mRNA was analyzed by real-time PCR. Results Stable infected cells were selected by puromycin successfully. Comparing to control groups, the expression of RYBP were reduced at different degrees （P 〈 0.01）. And RYBP shRNA2 took the most silencing effect, th＇e RYBP mRNA was decreased by more than 95 % （P 〈 0.05）. Conclusion The shRNA2 targeting RYBP gene can effectively inhibit the expression of RYBP. HL-60 cell line with stable RYBP gene silencing were constructed successfully, which had provided experiment fundament for further studying the function of RYBP.