目的：构建靶向人大肠癌细胞端粒酶逆转录酶（human telomerase reverse transcriptase，hTERT）基因的RNAi载体，探讨其对人大肠癌SW480细胞增殖的影响。方法：设计3条靶向hTERT的shRNA序列和阴性对照序列，分别克隆入pGPU6/GFP/Neo载体，构建RNAi载体pGPU6-hTERT-1、2、3和阴性对照载体pGPU6-hTERT-NC，转染SW480 细胞。RT-PCR检测各组载体对hTERT mRNA表达的影响，MTT法检测下调hTERT mRNA表达对SW480细胞增殖的影响。结果：成功构建3个携带hTERT mRNA序列的重组载体，3种RNAi载体均能明显抑制SW480 细胞hTERT mRNA的表达，pGPU6-hTERT-3组SW480细胞hTERT mRNA 表达水平较空白对照组下调最为显著（0.347±0.028 vs 0.513±0.032，P<0.01）。转染3种RNAi载体均能明显抑制SW480 细胞增殖，pGPU6-hTERT-3 组细胞增殖抑制率较空白对照组、脂质体对照组和pGPU6-hTERT-NC组升高最为显著\[（50.08±0.43)% vs (4.11±0.39)%、(3.88±0.35)%、(3.38±0.35)%，P<0.05\]。结论：转染RNAi载体pGPU6-hTERT-3能够抑制SW480细胞的增殖，其机制可能与降低hTERT基因的表达从而抑制端粒酶活性有关。

Objective: To construct an optimized human telomerase reverse transcriptase (hTERT) gene-specific RNAi and to evaluate its effect on human colon cancer cell proliferation in vitro. Method: Three hTERT-specific RNAi sequences and a negative control (NC) or scrambled sequence were cloned, respectively, into a pGPU6/GFP/Neo vector to generate pGPU6-GFP-hTERT-1, pGPU6-GFP-hTERT-2, pGPU6-GFP-hTERt-3 and pGPU6-GFP-NC. Human colon cancer SW480 cells were transfected with these vectors respectively. At 24, 48 and 72 h after transfection, hTERT mRNA abundance was assessed by RT-PCR and cell viability by MTT assay. Results: The 3 hTERT-specific RNAi vectors constructed were all effective to silence the hTERT gene; hTERT mRNA abundance in SW480 cells transfected with pGPU6-GFP-hTERT-3 was significantly lower than that in SW480 cells transfected with pGPU6-GFP-NC (0.347±0.028 vs 0513 ± 0.032,P<0.01). All the three hTERT sequence-specific RNAi vectors were effective to inhibit the proliferation of SW480 cells; cellular proliferation inhibition rate in SW480 cells of pGPU6-GFP-hTERT-3 group was significantly increased than that of blank contro, liposomal and NC group (\[50.08±0.43\]% vs \[4.11±0.39\]%, \[3.88±035\]% and \[3.38±0.35\]%; P<0.05). Conclusion: RNAi-mediated hTERT gene silencing results in colon cancer cell growth inhibition and may offer a novel therapy for colon cancer.

山东万杰医学院校级课题（No. X10ZK02）。