RNA干扰技术抑制体外培养的人眼球筋膜囊成纤维细胞增殖的初步研究  

Preliminary study of inhibition of human Tenon′s capsule fibroblasts in vitro by RNA interference targeting IKK-β

作　　者：黄圣松[1] 葛坚[1] 王莉娜[1] 尹心宝[2] 魏雁涛[1] 马萍[1] 

机构地区：[1]中山大学中山眼科中心,广州510060 [2]中山大学附属第二医院眼科

出　　处：《中华眼科杂志》2005年第12期1076-1081,共6页Chinese Journal of Ophthalmology

基　　金：卫生部临床重点学科基金资助项目(3030902005);广东省普通高校自然科学研究基金资助项目(04Z001)

摘　　要：目的探讨RNA干扰技术对体外培养的人眼球筋膜囊成纤维细胞增殖的抑制作用。方法采用化学合成的靶向IKKβ的siRNA,转染体外培养的人眼球筋膜囊成纤维细胞,同时以对任何基因均无作用的siRNA作为阴性对照。以逆转录聚合酶链反应(RTPCR)技术检测IKKβmRNA表达水平,免疫印迹法检测IKKβ蛋白表达水平。将siRNA的转染浓度分别设为5、10、25、50、100、200nmol/L,进行转染,于转染后48h采用四甲基偶氮唑盐法检测其转染后对成纤维细胞的抑制作用。结果经转染了靶向IKKβ的siRNA,其成纤维细胞IKKβ的mRNA和蛋白表达水平均受到抑制。各个转染浓度(5、10、25、50、100、200nmol/L)均可抑制成纤维细胞的增殖(P<0.05),抑制率分别为10.72%、23.35%、30.84%、51.25%、50.06%、49.63%,50nmol/L时已达最大抑制率。结论靶向IKKβ的siRNA转染可以降低IKKβ的表达水平,同时对体外培养的人眼球筋膜囊成纤维细胞的增殖具有抑制作用。抗IKKβ的RNA干扰技术可能是抑制抗青光眼术后滤过道瘢痕化的新手段。Objective To determine whether small interference RNA （siRNA） targeting the inhibitor of κB kinase-β （IKK-β） could be used to suppress the IKK-β expression, and inhibit the proliferation of human Tenon＇s capsule fibrolasts in vitro. Methods IKK-β specific siRNA designed from the human gene sequence was transfected into the cultured human Tenon＇s capsule fibroblasts, and a nontargeted siRNA was transfected as a negative control. The mRNA of IKK-β was assessed by reverse transcription-polymerase chain reaction （RT-PCR） , and the protein expression was determined by Western blotting. Cell viability of the cultured human Tenon＇s capsule fibroblasts with series of RNA interference at 5, 10, 25, 50, 100, and 200 nmol/L was evaluated by MTT assay 48 hours after transfection. Results The expression of IKK-β was significantly （ P 〈 0.05 ） suppressed at both mRNA and protein levels after transfeetion. The proliferation of the cultured human Tenon＇s capsule fibroblasts was inhibited at all the transfected concentrations at different rates （ 10. 72% ,23.35% ,30. 84% ,51.25% ,50. 06% and 49.63% respectively）. The highest level of inhibition was observed at 50 umol/L of siRNA concentration. Conclusions IKK-β specific siRNA is effective in suppressing the IKK-β expression and inhibiting the proliferation of the cultured human Tenon＇s capsule fibrolasts. RNA interference may offer a new alternative to post-operational management of scar tissue formation.

关 键 词：RNA干扰 蛋白质丝氨酸苏氨酸激酶 筋膜 成纤维细胞 细胞分裂 青光眼 

分 类 号：R77[医药卫生—眼科]