缺氧诱导因子1α小干扰RNA联合顺铂抑制人食管鳞癌TE-1细胞裸鼠移植瘤的生长

Hypoxia-inducible factor 1α small interfering RNA plus cisplatin in vivo inhibits growth of human esophageal squamous carcinoma TE-1 cells transplanted in nude mice

目的探讨缺氧诱导因子1α（HIF-1α）小干扰RNA（siRNA）联合顺铂对人食管鳞癌TE-1细胞裸鼠移植瘤生长的影响。方法体外培养人食管鳞癌TE-1细胞，接种于12只裸鼠右上肢外侧皮下，建立裸鼠荷瘤模型开始抑瘤实验。实验分为4组，每组3只，A组用生理盐水瘤内注射；B组用顺铂（20mg／L）瘤内注射；C组用HIF-1α siRNA（20umol/L）瘤内注射；D组用HIF—1α siRNA（20umol/L）和顺铂（20mg／L）协同瘤内注射。各组注射体积均为20ul，每周测量肿瘤体积变化。3周后处死裸鼠，测量各组移植瘤质量；RT-PCR半定量检测各组移植瘤TE-1细胞HIF—1α mRNA的表达；免疫组织化学法检测其HIF—1α蛋白的表达；流式细胞仪Annexin V-FITC／PI双标检测其细胞的凋亡。结果抑瘤实验开始3周后，A、B、C和D组的移植瘤体积分别为（1．477±0．132）cm^3、（1．200±0．114）cm^3、（1．223±0．129）cm^3和（0．890±0．141）cm^3；移植瘤质量分别为（7．38±0．96）g、（6．35±0．73）g、（6．12±0．65）g和（3．51±0．42）g。B、C、D3组的抑瘤率分别为14．0％、17．1％、52．4％，其中D组移植瘤的生长受抑制最明显，与其他3组比较差异有统计学意义（P〈0．05）。C和D组TE-1细胞的HIF-1α mRNA表达下调至0．343±0．080和0．312±0．055，蛋白表达下调至0．196±0．018和0．229±0．035，与A（mRNA1．315±0．179，蛋白0．523±0．102）、B（mRNA1．483±0．460，蛋白0．542±0．174）两组差异有统计学意义（P〈0．01），而C、D两组间差异无统计学意义（P〉0．05）。A、B、C和D组的TE-1细胞凋亡率分别为（6．13±1．38）％、（28．30±4．82）％、（34．10±5．56）％、（52．88±8．77）％，其中D组与其他3组比较，差异有统计学意义（P〈0．01）。结论HIF—1α siRNA能有效下调裸鼠体内食管鳞癌TE-1细胞HIF—1α的表达，提高顺铂的细胞毒性作用，诱导细胞凋亡，显著抑制移植瘤的生长。

Objective To study the effect of hypoxia-inducible factor 1α （HIF-1 α） small interfering RNA （siRNA） combined with cisplatin on growth of human esophageal squamous carcinoma cells transplanted in nude mice. Methods Human esophageal squamous carcinoma TE-1 cells cultured in vitro were transplanted under the lateral skin of right upper limb in 12 nude mice to establish tumor-bearing models. Anti-tumor experiment was carried out. The nude mice were then divided into four groups （n=3 for each） to receive intratumor injection of normal saline （Group A）, 20 mg/L cisplatin （Group B）, 20 umol/L HIF-1α siRNA （Group C） or both 20 mg/L cisplatin and 20 umol/L HIF-1α siRNA （Group D）. The volume of injected agent was 20 ul for each group. Change in tomor size was recorded every week. At week 3, all the nude mice were sacrificed and the transplanted tumor volume and weight were measured. In transplanted tumor of all the mice, the expression of HIF-1α mRNA was detected by semi-quantitative RT-PCR and HIF- 1α protein was detected by immunohistochemistry. Tile apoptosis of TE- 1 cells was determined by flow cytometry with Annexin V-FITC/PI dual staining. Results After anti-tumor experiment for three weeks, the transplanted tumor volumes of groups A, B, C and D were （1.477 ±0.132） cm^3, （1.200±0.114） cm^3, （1.223±0.129） cm^3 and （0.890±0.141） cm^3, and the tumor weight was （7.38±0.96） g, （6.35±0.73） g, （6.12±0.65） g and （3.51±0.42） g respectively. The rates of tumor suppression were 14.0%, 17.1% and 52.4% in groups B, C and D. Group D mice experienced strongest tumor suppression as compared with the other 3 groups （P〈0.05）. The expressions of HIF-1α mRNA and protein were down-regulated to 0.343±0.080 and 0.196±0.018 in Group C, 0.312±0.055 and 0.229±0.035 in Group D with no significant difference between these two groups （P〉0.05）, but were significantly different with those in Group A （mRNA 1.315± 0.179, protein 0.523±0.102） and Group B （mRNA 1.483±0.460, protein 0.542±0.174）（P〈0.01 ）. The rates of TE-1 cell apoptosis in groups A, B, C and D were （6.13±1.38）%, （28.30±4.82）%, （34.10±5.56）% and （52.88 ± 8.77）% respectively, presenting a significant difference when comparing Group D with the other three groups （P〈0.01）. Conclusion HIF-1α siRNA ean down-regulate HIF-1α mRNA and protein expressions in nude mice in vivo, induce cell apoptosis, significantly inhibit tumor growth of esophageal squamous carcinoma TE-1 cells, and improve the cytotoxicity of cisplatin chemotherapy.