靶向CD74基因RNA干扰慢病毒稳定感染人胰腺癌细胞株的建立与鉴定

Construction and identification of CD74 RNA interference lentiviral vector and its stably infected human pancreatic cancer cell line

目的构建靶向CD74基因的小发夹RNA慢病毒载体,感染胰腺癌Capan-2细胞株,筛选并建立稳定细胞株。方法应用q RT-PCR法检测不同胰腺癌细胞株间CD74的m RNA表达情况;根据Gene Bank提供的CD74 c DNA序列,设计3条靶向CD74的si RNA序列,构建重组干扰质粒p SUPERretro-puro-CD74-si RNA;将重组干扰质粒转染Capan-2细胞,经过q RT-PCR和Western blot法筛选出最佳干扰效果质粒;慢病毒包装质粒并感染Capan-2细胞株,利用嘌呤霉素筛选,建立稳定低表达CD74细胞株;应用q RT-PCR和Western blot法检测CD74基因m RNA和蛋白的表达抑制率。结果 Capan-2细胞株的CD74 m RNA平均ΔCt值为102.9±6.4,在各胰腺癌细胞株中表达水平最高(P<0.05);携带干扰效果最高si RNA的慢病毒感染Capan-2细胞,建立了稳定低表达CD74基因的Capan-2细胞株,CD74基因m RNA抑制率61%,蛋白抑制率90%。结论成功筛选出靶向CD74的sh RNA最佳干扰质粒,构建了CD74-RNAi慢病毒载体,并稳定感染建立了低表达CD74基因的Capan-2细胞株。

Objective To construct the CD74-RNAi lentiviral vector and establish a stably infected human pancreatic cancer Capan-2 cell line. Methods CD74 m RNA expression was detected in different pancreatic cancer cell lines using Quantitative Real-time PCR(q RT-PCR). According to genetic information of CD74 c DNA sequence provided by Gene Bank, 3 si RNA sequence targeting CD74 were designed. The corresponding p SUPERretro-puro recombinant plasmids(p SUPERretro-puro-CD74-si RNA) were constructed. Then recombinant interference plasmid was transfected into Capan-2 cells. q RT-PCR and Western blot(WB) were used to detect the recombinant interference plasmid with best effect of interference. Then this plasmid underwent lentiviral packaging and was infected into Capan-2 cells. After selection with puromycin, a stably low-expression of CD74 cell line was established. q RT-PCR and WB were applied to measure the expression inhibition rate of CD74. Results Capan-2 cell line expressed the highest CD74 m RNA level in pancreatic cancer cell lines, which ΔCt value was 102.9±6.4(P<0.05). Lentiviral with the best gene silencing effect si RNA was infected into Capan-2 cell line, and a Capan-2 cell line, stably low-expressing CD74, was established. And the inhibitive rate of CD74 m RNA and protein were 61% and 90%, respectively. Conclusion CD74 lentiviral vector is successively constructed andpancreatic cancer Capan-2 cell line which can stably low-express CD74 is established.