小鼠酪氨酸酶siRNA的体外有效性实验研究  

An Efficient siRNA Designed to Suppress Tyrosinase of Mice in Vitro

作　　者：贾秀华[1] 黄冰[1] 庄菁[1] 朱翔[2] 廖东江[1] 葛坚[1] 林少春[1] 徐晓平[1] 彭展[1] 

机构地区：[1]中山大学中山眼科中心眼科学国家重点实验室,广州510060 [2]中山大学附属第三医院,广州510630

出　　处：《中国比较医学杂志》2008年第12期10-16,I0003,共8页Chinese Journal of Comparative Medicine

基　　金：国家自然科学基金委员会专项基金/主任基金(No.30640047);广东省科学事业/科研条件计划项目(No.2006B60101058)

摘　　要：目的通过RNAi技术,筛选出针对小鼠酪氨酸酶的有效siRNA,为酪氨酸酶异常等人类疾病的动物模型制作及该类疾病的预防和治疗奠定基础。方法利用针对小鼠酪氨酸酶mRNA的不同干扰片段分别转染小鼠黑色素瘤B16细胞系,同时采用阴性干扰片段转染作为阴性对照,单纯脂质体转染作为空白对照。使用实时荧光定量PCR、酪氨酸酶活性测定和黑色素含量测定3种方法分3个时间点于转染后24、48、72 h对不同组的细胞干扰结果进行检测。结果siNM-011661-001、siNM-011661-002、siNM-011661-003三个干扰片段均能在转染后发挥一定的干扰作用,其中siNM-011661-001在转染后发挥的干扰效果最强,在降低酪氨酸酶基因mRNA表达、酶活性、以及黑素含量三方面分别能起到82.81%、64.73%、52.53%的抑制作用。结论针对小鼠酪氨酸酶的siNM-011661-001序列能在转录后水平发挥高效、稳定的基因沉默作用,是发挥RNAi的优势序列。Objective To screen efficient small interfering RNA （siRNA） fragments that may suppress tyrosinase expression in mice through RNAi technique, and to lay a solid foundation for establishing animal models, preventing and curing human disease of tyrosinase abnormity. Method Chemically synthesized siRNA designed to suppress tyrosinase expression were transfected into B16 cells by Lipofectamine^TM 2000, simultaneously negative siRNA and empty Lipofectamine^TM 2000 as negative and empty control, respectively. The mRNA expression and protein synthesis were analysed by real-time RT-PCR. The tyrosinase activity and melanin concentration were determined at three different time points, 24 hours, 48 hours and 72 hours after transfection. Result All of the fragments （siNM_011661 _ 001, siNM_ 011661_ 002, siNM_ 011661_ 003） exerted suppressive effect to a certain extent. It was noticed that siNM_ 011661 _ 001 was the most powerful fragment after transfection, which could inhibit the expression of tyrosinase gene at mRNA level, tyrosinase activity, melanin concentration up to 82.81% , 64.73 % , and 52.53 % , respectively. Conclusion siNM _011661 _ 001 can induce an efficient and stable gene-specific inhibition of expression at post-transcriptional level. Thus, it is the superior RNAi sequence in this study.

关 键 词：RNAI 酪氨酸酶 B16细胞 

分 类 号：R-33[医药卫生]