纳米介导的缺氧诱导因子1α小干扰RNA抑制人食管鳞癌TE-1细胞生长

Inhibitory effect of small interference RNA targeting hypoxia-inducible factor 1 alpha nanospheres on human esophageal squamous carcinoma TE-1 cell growth

背景:纳米微粒作为一种比较理想的非病毒基因递送载体,具有无免疫原性和致瘤性等优越性。利用纳米技术和基因干扰技术阻断缺氧诱导因子1α(hypoxia-inducible factor 1,HIF-1α)在食管鳞癌组织的表达,降低癌细胞对化疗药物的耐药性,理论上成为能够抑制食管癌细胞生长的有效方法。目的:探讨HIF-1α小干扰RNA纳米微粒对人食管鳞癌TE-1细胞生长的抑制作用。设计、时间及地点:以体外培养的食管鳞癌TE-1细胞为对象,基因水平完全随机对照实验,于2007-01/2008-12在中山大学附属第三医院中心实验室完成。材料:由上海生物工程公司合成HIF-1α小干扰RNA,采用超声乳化法制备纳米微粒。人食管鳞癌TE-1细胞株购自中科院上海细胞库。方法:体外培养的人食管鳞癌TE-1细胞分为4组:分别为生理盐水组,不含基因的纳米微粒组,HIF-1α小干扰RNA组,HIF-1α小干扰RNA纳米微粒组。主要观察指标:RT-PCR检测HIF-1α mRNA的表达,Western-blot法检测HIF-1α蛋白的表达;流式细胞仪检测细胞凋亡情况;MTT法检测细胞增殖情况。结果:处理72h后HIF-1α小干扰RNA纳米微粒组细胞的HIF-1α mRNA表达和HIF-1α蛋白表达低于其他3组(P<0.01),细胞凋亡率高于其他3组(P<0.01)。HIF-1α小干扰RNA纳米微粒组细胞增殖受明显抑制,生长缓慢,与其他3组比较,差异有显著性意义(P<0.01)。结论:HIF-1α小干扰RNA纳米微粒能够有效减少食管鳞癌TE-1细胞的HIF-1α mRNA和蛋白的表达,提高肿瘤细胞的凋亡,显著抑制TE-1细胞的生长。

BACKGROUND： Nanosphere, an ideal nonviral gene delivery vector, is not excellence of immunogenicity and oncogenicity. Nanotechnology and gene interference are used to block hypoxia-inducible factor 1 alpha （HIF-1α） expression in esophageal squamous carcinoma tissue and decrease tolerance of malignant cells to chemotherapeutics. Theoretically, they become effective methods to inhibit malignant cell growth of esophageal squamous carcinoma. OBJECTIVE： To study the inhibitory effect of small interference RNA targeting HIF-1α （siRNA-HIF-1α） nanospheres on human esophageal squamous cancer TE-1 cell growth. DESIGN, TIME AND SETTING： Based on in vitro cultured esophageal squamous cancer TE-1 cells, a completely randomized controlled study was performed at the Central Laboratory, the Third Hospital Affiliated to Sun Yat-sen University from January 2007 to December 2008. MATERIALS： siRNA-HIF-1α was synthesized by Shanghai Bioengineering Company; siRNA-HIF-1α nanospheres were prepared using solvent evaporation technique; human esophageal squamous cancer TE cell strain was provided by Shanghai Cell Bank of the Chinese Academy of Sciences. METHODS： TE-1 cells cultured in vitro were assigned into four groups： saline, gene-free nanospheres, siRNA-HIF-1α, and siRNA-HIF-la nanospheres groups. MAIN OUTCOME MEASURES： HIF-1α mRNA expression was detected by RT-PCR; HIF-1α protein expression was detected by Western blot; apoptosis of TE-1 cells was determined by flow cytometry; TE-1 cell growth was examined by MTT. RESULTS： At 72 hours after treatment, both HIF-1α mRNA expression and HIF-la protein expression in the siRNA-HIF-1α nanospheres group were significantly less than other three groups （P 〈 0.01 ）, but apoptotic rate was significantly greater than other three groups （P 〈 0.01）. TE-1 cell growth was remarkably inhibited in the siRNA-HIF-1α nanospheres group, which was significantly different compared with other three groups （P 〈 0.01）. CONCLUSION： siRNA-HIF-1α nanospheres can specifically reduce both HIF-la mRNA and HIF-1α protein expressions in esophageal squamous carcinoma TE-1 cells, significantly increase tumor cell apoptosis, and remarkably inhibit TE-1 cell growth.