中华临床医师杂志（电子版）
中華臨床醫師雜誌（電子版）
중화림상의사잡지（전자판）
CHINESE JOURNAL OF CLINICIANS(ELECTRONIC VERSION)
2013年
15期
7033-7038
,共6页
郭斌%刘晓娟%王莉%范钦华
郭斌%劉曉娟%王莉%範欽華
곽빈%류효연%왕리%범흠화
RNA 干扰%色素上皮,眼%增生%细胞运动%玻璃体视网膜病,增生性%ski基因
RNA 榦擾%色素上皮,眼%增生%細胞運動%玻璃體視網膜病,增生性%ski基因
RNA 간우%색소상피,안%증생%세포운동%파리체시망막병,증생성%ski기인
RNA interference%Retinal pigment epithelium,eye%Proliferation%Migration%Vitroretinopathy,proliferative%ski gene
目的：设计并化学合成针对ski的siRNA分子片段，转染人视网膜色素上皮（hRPE）细胞抑制ski表达，观察其对hRPE细胞增生和迁移等生物学功能方面的影响。方法设计并化学合成3对针对人ski mRNA(NM_003036)中910、1912和389靶位的siRNAs，以脂质体方法转染hRPE细胞，运用RT-PCR法和Western印迹法检测细胞中ski基因在mRNA水平和蛋白水平的变化。然后利用MTT法和Transwell模型检测转染ski-siRNA的hRPE细胞增生活力和迁移能力等生物学功能方面指标的变化。结果 RT-PCR和Western印迹检测结果证实3条ski-siRNA转染48 h后，均不同程度降低了hRPE细胞中ski基因的mRNA和蛋白表达水平(P＜0.05)，其中siRNA-C的干扰效果最强，与阴性对照相比抑制率达到45%左右，被用于后续生物学功能实验的转染。转染ski-siRNA后2～6 d hRPE细胞的增生能力明显受到抑制(P＜0.05)。与空白对照组和NC组相比，在培养12 h和24 h时，siRNA-C转染hRPE细胞迁移数量明显升高(P＜0.01)。结论 siRNA-C (389)是可以高效、特异地沉默hRPE细胞中ski基因。靶向ski基因的siRNA转染可以有效地抑制hRPE细胞增生，但可能促进细胞迁移。ski对hRPE增生和迁移调控可能是PVR治疗的一个新的方向。
目的：設計併化學閤成針對ski的siRNA分子片段，轉染人視網膜色素上皮（hRPE）細胞抑製ski錶達，觀察其對hRPE細胞增生和遷移等生物學功能方麵的影響。方法設計併化學閤成3對針對人ski mRNA(NM_003036)中910、1912和389靶位的siRNAs，以脂質體方法轉染hRPE細胞，運用RT-PCR法和Western印跡法檢測細胞中ski基因在mRNA水平和蛋白水平的變化。然後利用MTT法和Transwell模型檢測轉染ski-siRNA的hRPE細胞增生活力和遷移能力等生物學功能方麵指標的變化。結果 RT-PCR和Western印跡檢測結果證實3條ski-siRNA轉染48 h後，均不同程度降低瞭hRPE細胞中ski基因的mRNA和蛋白錶達水平(P＜0.05)，其中siRNA-C的榦擾效果最彊，與陰性對照相比抑製率達到45%左右，被用于後續生物學功能實驗的轉染。轉染ski-siRNA後2～6 d hRPE細胞的增生能力明顯受到抑製(P＜0.05)。與空白對照組和NC組相比，在培養12 h和24 h時，siRNA-C轉染hRPE細胞遷移數量明顯升高(P＜0.01)。結論 siRNA-C (389)是可以高效、特異地沉默hRPE細胞中ski基因。靶嚮ski基因的siRNA轉染可以有效地抑製hRPE細胞增生，但可能促進細胞遷移。ski對hRPE增生和遷移調控可能是PVR治療的一箇新的方嚮。
목적：설계병화학합성침대ski적siRNA분자편단，전염인시망막색소상피（hRPE）세포억제ski표체，관찰기대hRPE세포증생화천이등생물학공능방면적영향。방법설계병화학합성3대침대인ski mRNA(NM_003036)중910、1912화389파위적siRNAs，이지질체방법전염hRPE세포，운용RT-PCR법화Western인적법검측세포중ski기인재mRNA수평화단백수평적변화。연후이용MTT법화Transwell모형검측전염ski-siRNA적hRPE세포증생활력화천이능력등생물학공능방면지표적변화。결과 RT-PCR화Western인적검측결과증실3조ski-siRNA전염48 h후，균불동정도강저료hRPE세포중ski기인적mRNA화단백표체수평(P＜0.05)，기중siRNA-C적간우효과최강，여음성대조상비억제솔체도45%좌우，피용우후속생물학공능실험적전염。전염ski-siRNA후2～6 d hRPE세포적증생능력명현수도억제(P＜0.05)。여공백대조조화NC조상비，재배양12 h화24 h시，siRNA-C전염hRPE세포천이수량명현승고(P＜0.01)。결론 siRNA-C (389)시가이고효、특이지침묵hRPE세포중ski기인。파향ski기인적siRNA전염가이유효지억제hRPE세포증생，단가능촉진세포천이。ski대hRPE증생화천이조공가능시PVR치료적일개신적방향。
Objective To design and prepare siRNAs targeting ski gene and to observe its effects on the biological characteristics of human retinal pigment (hRPE) cells, such as proliferation and migration. Methods Three pairs of siRNAs targeting ski mRNA 910, 1912 and 389 targets were designed and synthesized by utilizing RNA design software. The most effective siRNA was chosen to transfect into hRPE cells with cathodolyte liposome transfection method. Real-time PCR and Western blotting were used to measure ski expression at mRNA and protein levels. The cell proliferation was assessed by MTT assay and recorded by growth curve. The number of cells which migrate through micropores and stay on the outer bottom side of Transwell systems were observed and calculated under invert microscopy. Results All the 3 specific ski-siRNAs (A, B, C) effectively inhibited the expression of ski gene and protein, with ski-siRNA-C having the highest inhibition rate (45%) compared with the negative control group. Furthermore, the ski-siRNA-C was chose in following transfection studies. The proliferation of hRPE cells was markedly inhibited by ski-siRNA-C after 2 to 6 days(P<0.05), while the migration number of hRPE cells was increased by ski-siRNA after 12 h and 24 h treatment(P<0.01). Conclusions The siRNA-C(389)could effectively knockdown the gene and protein expression of ski in hRPE cells.Silencing ski by siRNA significantly inhibited the proliferation of hRPE cells but may induce migrating of these cells in early stage. The ski might be an efficient target to treat PVR due to its regulation in the proliferation and migration of hRPE cells.
