目的：探讨分析RNA干扰缺氧诱导因子-1α（HIF-1α）对食管鳞癌细胞Eca-109抑制效应的体外及体内的表达情况。方法选取研究用Eca-109细胞，设计RNA干扰片断h-EGFR构建重组质粒pGensil-1-EGFR转染目的基因片段。测定EGFR蛋白/mRNA表达，挑选转染成功的基因修饰细胞株分别植入裸鼠体内，构建移植瘤模型，且随机分为未转染组与实验组。运用免疫组化技术检测各组裸鼠HIF-1α蛋白表达，逆转录聚合酶链反应（RT-PCR）技术检测各组HIF-1αmRNA表达，最后测量裸鼠移植瘤的重量及体积。结果 Eca-109中HIF-1α蛋白伴随氯化钴浓度的升高而升高。并且HIF-1α蛋白水平在缺氧后12小时升高，24小时后降低，且持续减少。转染后EGFR mRNA的阳性表达率为26.55%、9.48%、4.60%，依次降低，P＜0.01。其中EGFR siRNA水平最低。转染后EGFR mRNA阳性表达率为24.30%、34.11%、34.05%，与未转染组（78.27%）相比显著降低（P＜0.01）。经RT-PCR分析，实验组裸鼠EIF-1αmRNA表达较少，与未转染组相比差异无显著性（P＞0.05）。实验组裸鼠HIF-1α蛋白表达的灰度值显著低于未转染组（P＜0.01）。接种1周后裸鼠均出现肿瘤，实验组裸鼠移植瘤体积为（0.20±0.13）cm3，重量为（0.21±0.05）g，均显著少于未转染组。结论 HIF-1α蛋白的表达与氯化钴浓度及缺氧有关，RNA干扰缺氧诱导的HIF-1α只发生在蛋白水平，而非基因水平， RNA干扰技术可以成功抑制食管鳞癌细胞HIF-1α基因，阻碍肿瘤细胞生长、增殖，降低肿瘤恶性程度，值得临床应用与推广。%Objective To study the interference of RNA hypoxia inducible factor-1α(HIF-1α) expression inhibition in vitro and in vivo effect of esophageal squamous cell carcinoma cell line Eca-109. Method Selected in esophageal squamous cancer cells (ESCC) to study the Eca-109, the design of RNA interference fragment h-EGFR to construct recombinant plasmid pGensil-1-EGFR gene transfection purpose. Determine EGFR protein/mRNA expression, pick out the transfection successful nude mice implanted with genetically modiifed cell lines respectively, transplantation tumor model was constructed, and randomly divided into untransfection group and experimental group. Using immunohistochemical techniques to detect alpha groups of HIF-1αprotein expression, reverse transcription polymerase chain reaction (RT-PCR) technology to detect each group HIF-1αmRNA expression, ifnally to measure the weight and volume of the nude mouse transplantation tumor. Result HIF-1αEca-109 cells protein increased along with the increase of the concentration of cobalt chloride. HIF-1αin 12 hours after hypoxia increases, reduced after 24 hours, and had been declining. After transfection EGFR mRNA positive expression rate was 26.55%, 9.48%, 4.60%, in turn (P < 0.01). With EGFR siRNA (small interference RNA) had the lowest levels. After transfection EGFR mRNA positive expression rate was 24.30%, 34.11%, 34.05%, compared with the blank control group (78.27%) (P<0.01). Through RT-PCR analysis, experimental mice HIF-1αmRNA expression was less, there was no signiifcant difference compared with not transfection group (P>0.05). Experimented group HIF-1αprotein expression of grey value signiifcantly lower than untransfection group (P<0.01). Vaccination in 1 week after nude mice tumor, experimental mice transplanted tumors for (0.20±0.13) cm3, weight (0.21±0.05) g, compared with untransfection group, experimental group mice transplanted tumor volume and weight were signiifcantly less than not transfection group. Conclusion HIF-1αprotein expression is associated with cobalt chloride concentration and lack of oxygen, oxygen to RNA interference of HIF-1αonly in protein level, rather than the genetic level, thus a technique called RNA interference can successfully suppress ESCC HIF-1αgene, hinder the growth of tumor cells, value-added, reduce tumor malignant degree, worthy of clinical application and promotion.
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