CAJ | 학술논문

目的：设计以 Rap1b 基因为靶点的短发夹状 RNA（shRNA），构建重组慢病毒表达载体并转染食管鳞癌细胞，观察其在食管鳞癌细胞中的表达。方法：应用重组 DNA 技术，将设计好的4条基因特异性 shRNA 序列插至慢病毒表达载体 pGLV3- GFP 中，构建 pGLV3- GFP - Rap1b - shRNA1/2/3/4。并应用脂质体法转染293T 细胞，进行病毒包装及滴度测定。采用实时荧光定量 PCR（RT - PCR）及 Western blot 分别从 mRNA 和蛋白水平检测转染食管鳞癌细胞株 Eca109后 Rap1b 基因的表达情况。结果：测序证实慢病毒载体构建成功，并测定滴度为1×109 TU/ ml，pGLV3- GFP - Rap1b - shRNA3/4转染后72h 和96h 均可显著抑制 Rap1b 基因mRNA 及蛋白的表达。结论：成功构建 Rap1b 基因的 shRNA 慢病毒载体，所介导的 RNAi 能有效抑制食管鳞癌细胞 Eca109中 Rap1b 基因的表达。
목적：설계이 Rap1b 기인위파점적단발협상 RNA（shRNA），구건중조만병독표체재체병전염식관린암세포，관찰기재식관린암세포중적표체。방법：응용중조 DNA 기술，장설계호적4조기인특이성 shRNA 서렬삽지만병독표체재체 pGLV3- GFP 중，구건 pGLV3- GFP - Rap1b - shRNA1/2/3/4。병응용지질체법전염293T 세포，진행병독포장급적도측정。채용실시형광정량 PCR（RT - PCR）급 Western blot 분별종 mRNA 화단백수평검측전염식관린암세포주 Eca109후 Rap1b 기인적표체정황。결과：측서증실만병독재체구건성공，병측정적도위1×109 TU/ ml，pGLV3- GFP - Rap1b - shRNA3/4전염후72h 화96h 균가현저억제 Rap1b 기인mRNA 급단백적표체。결론：성공구건 Rap1b 기인적 shRNA 만병독재체，소개도적 RNAi 능유효억제식관린암세포 Eca109중 Rap1b 기인적표체。
To construct lentiviral vector targeting Rap1b gene and evaluate its silencing effect on Rap1b gene in esophageal squamous cell carcinoma. Methods:Four short hairpin RNA(shRNA)fragments targeting Rap1b were designed and cloned into lentiviral vector pGLV3 - GFP to construct pGLV3 - GFP - Rap1b - shRNA1 /2 / 3 / 4. Then the silencing effect on Rap1b gene were confirmed by real - time PCR and Western bolt in transfected gene in esophageal squamous cell carcinoma cell line Eca109. Results:The Rap1b shRNA lentiviral vectors were suc-cessfully constructed confirmed by sequencing and the virus reached a titer of 1 × 109 TU/ ml. pGLV3 - GFP - Rap1b- shRNA3 / 4 could significantly inhibit the mRNA and protein expression of Rap1b among four shRNA fragments. Conclusion:shRNA expressing lentiviral recombinants targeting the Rap1b gene were successfully constructed,which had silencing effect on Rap1b gene in esophageal squamous cell carcinoma cell line Eca109.