检测已构建的缺氧反应元件（hypoxia response element,HRE）和人端粒酶催化亚单位(human telomerase reverse transcriptase，hTERT）启动子调控CDX2基因表达的载体pLVX-5HRE-hTERTp-CDX2-3FLAG(5HhC)对人结肠癌细胞系LoVo增殖的影响。方法：复苏前期筛选的转染pLVX-hTERTp-CDX2-3FLAG(hC)的LoVo细胞（hC/LoVo）、转染pLVX-5HRE-hTERTp-3FLAG(5Hh)的LoVo细胞（5Hh/LoVo）、转染pLVX-5HRE-hTERTp-CDX2-3FLAG(5HhC)的LoVo细胞（5HhC/LoVo）及空白LoVo细胞，各组细胞均在常氧条件和缺氧条件（加入缺氧模拟剂CoCl2）展开实验。MTT检测细胞增殖情况，平板克隆实验观察细胞集落形成，流式细胞术分析细胞周期。结果：成功复苏转染5HhC的LoVo细胞及各组对照LoVo细胞。hC/LoVo、5HhC/LoVo细胞增殖显著低于LoVo及5Hh/LoVo，且缺氧微环境下的5HhC对于LoVo细胞的增殖速度抑制更为明显[细胞增殖率，第5天，常氧 vs 缺氧：(48.62±3.32)% vs (36.81±2.83)%, P<0.05；第7天，常氧 vs 缺氧：(56.44±228)% vs (38.51±3.21)%,P<0.05]。hC/LoVo、5HhC/LoVo组的平板克隆数显著低于LoVo及5Hh/LoVo组，且缺氧微环境下的5HhC对LoVo细胞集落形成能力的抑制较常氧环境下更为显著[集落数：（44.2±3.5）个 vs （90.8±9.3）个,P<0.05]；hC/LoVo、5HhC/LoVo组G1期细胞比例较LoVo细胞及5Hh/LoVo组明显提高[(63.59±0.55)%、(64.82±222)% vs (51.38±070)%、(51.59±0.38)%，P<0.05]，且在缺氧微环境下，5HhC/LoVo组G1期细胞比例提高更加显著[(71.38±3.02 )% vs (64.82±2.22)%，P<0.05]。结论： 治疗载体5HhC可使人结肠癌细胞系LoVo发生G1期阻滞，增殖及集落形成能力受到抑制，且在缺氧诱导下5HhC的抑制能力更为显著。

Homebox transcription factor 2 (CDX2) has been described as a colorectal tumor suppressor. This study aimed to evaluate the effect of overexpression of the hypoxia response element enhancer (HRE) sequence-inserted CDX2 gene under the control of the human telomerase reverse transcriptase (hTERT) promoter on proliferation of human colon adenocarcinoma LoVo cells in vitro. Methods: Stable clones of LoVo cells expressing pLVX-hTERTp-CDX2-3FLAG (hC), pLVX-5HRE-hTERTp-3FLAG (5Hh) and pLVX-5HRE-hTERTp-CDX2-3FLAG (5HhC), respectively, were generated. LoVo cells stably expressing hC, 5Hh, and 5HhC, respectively, were cultured under hypoxia control (CoCl2 200 μmol/L) and normoxia, respectively. At the designated time points of culture, cell viability was assessed by MTT assays, colony-forming capacity by trypan blue staining and cell cycle progression by flow cytometry. Result: Viability was significantly decreased in hC- and 5HhC-expressing LoVo cells as compared with wild-type LoVo cells and 5Hh-expressing LoVo cells. The proliferation rates under normoxia and hypoxiarespectively were (48.62±3.32)% and (36.81±2.83)% at 5 days (P<0.05) and (56.44±2.28)% and (38.51±3.21)% at 7 days (P<0.05) in 5HhC-expressing cells. LoVo cells stably expressing hC and 5HhC, respectively, formed significantly less clones than cells stably expressing 5Hhunder a hypoxic condition (44.2±3.5 vs 90.8±9.3, P<0.05). The proportion of cells at G1 phase arrest was (63.59±0.55)% and (64.82±2.22)% in cells stably expressing hC and 5HhC respectively, significantly higher (P<0.05) than that in wild type LoVo cells and cells stably expression 5Hh (51.38±0.70 and 51.59±0.38) under normoxia. Under a hypoxic condition, as high as 71.38±3.02 5HhC-expressing LoVo cells were arrested at G1 phase. Conclusion: Overexpression of the CDX2 gene carrying the HRE sequence driven by the hTERT promoter may significantly inhibit proliferation and colony formation of human colon adenocarcinoma LoVo cells in vitro, through inducing G1 phase arrest. These effects were more significant under hypoxia.

国家自然科学基金资助项目（No.81101874,No.81172362,No.81172359）；陕西省科学技术研究发展计划项目（No.2011-K12-19）；陕西省科技统筹创新工程计划项目（No.2013KTCQ03-08）