反义寡核苷酸减弱转染p16基因对包装细胞生长的抑制

目的:应用p16(MTS1)反义寡核苷酸抑制转染p16逆转录病毒载体后外源性p16基因的表达,减弱p16表达对包装细胞的生长抑制作用.方法:合成p16 cDNA起始密码子区的反义寡核苷酸和正义寡核苷酸,加入筛选的转染p16逆转录病毒载体的包装细胞培养液中,记录各孔中出现细胞克隆的时间和数量,测量各形成细胞克隆的病毒上清滴度.Western blot 检测反义寡核苷酸对外源性P16蛋白表达的影响.MTT法检测加入反义寡核苷酸后对转染的包装细胞生长的影响.结果:p16(MTS1)反义寡核苷酸可以抑制外源性p16基因的表达.加入反义寡核苷酸的包装细胞形成细胞克隆的时间提前,克隆数量和滴度都高于对照组.结论:p16(MTS1)反义寡核苷酸可以抑制外源性p16基因的表达,促进p16逆转录病毒载体转染的包装细胞的生长并提高病毒滴度.

The antisense attenuation to the growth inhibition of packaging cells by p16 transfection

Objective: To inhibit the expression of p16 in p16 retroviral vector transfected packaging cells and attenuate the growth inhibition of p16 to the packaging cells. Methods: An antisense oligonucleotide against the start site of p16 cDNA was designed and added to the medium of cultured p16 retroviral plasmid transfected packaging cell line everyday. The sense oligonucleotide was added as the control. The producing time of cell clones and the numbers of clones were recorded. The titer of every clone was detected. The changes of p16 expression of packaging cells were tested after adding p16 antisense oligonucleotide by Western blot. MTT was used to detect the cell growth of the packaging cells. Results: p16 antisense oligonucleotide could inibit the expression of p16 in p16 transfected packaging cells. The time of cloning appearing in antisense oligonucleotide group was shorter than that in the control group and the viral titer in antisense oligonucleotide group was also promoted. Conclusion: p16 antisense oligonucleotide could attenuate the growth inhibition of p16 to p16 transfected packaging cells and promote viral titer.