RNAi干扰联合化疗对人喉鳞癌裸鼠移植瘤STAT3信号传导通路的阻滞作用

The influence of RNAi silence combined chemotherapy for STAT3 signaling pathways in nude mice transplantation tumor

目的利用人喉鳞癌裸鼠癌移植瘤,研究RNAi干扰协同化疗对移植瘤中STAT3信号转导通路的影响。方法建立喉鳞癌裸鼠模型:选择Balb/c纯系裸鼠,在裸鼠的背部注射0.2ml 2×106喉癌细胞,移植瘤直径达到9-11mm时,实验分组:Ⅰ重组载体+顺铂组;Ⅱ重组载体组;Ⅲ顺铂组;Ⅳ阴性对照组,3周后处死实验鼠,利用电子显微镜、流式细胞仪,研究移植瘤抑制、Hep-2细胞凋亡及STAT3信号传导通路相关调控蛋白的变化情况。结果实验鼠皮下注射Hep-2细胞后,约1个月移植瘤直径约11mm。成瘤达到97.34%,病理显示:移植瘤(鳞状细胞癌、中分化)。电镜下:重组载体+顺铂组中的喉癌细胞核膜锐利凸起,细胞核中染色质高密度浓集,近C型改变,微量细胞膜圆形肿胀。线粒体水肿,粗面内质网脱颗粒,嵴无规则排列,或者消失。免疫组化结果:细胞浆中见到高表达染色成棕褐色物质的STAT3,细胞核中可见棕褐色点状沉积的p-STAT3。Ⅰ实验组STAT3、p-STAT3蛋白表达明显减弱,其灰度值明显低于Ⅱ、Ⅲ、Ⅳ组(P<0.05)。流式细胞仪显示Ⅰ实验组中STAT3、p-STAT3、Bcl-2、CyclinDl蛋白表达显著减低,大大低于另外三组,(P<0.05)。结论通过对活体裸鼠喉癌移植瘤模型的实验研究,验证了应用RNAi沉默STAT3基因对喉癌化疗的增敏作用。

Objective By using Human laryngeal squamous carcinoma nude mice transplantation tumor model, to study the influence of RNA interference in combined chemotherapy for nude mouse transplantation tumor STAT3 signal transduction pathway. Methods Establish laryngeal squamous carcinoma in nude mice model：choose pure Balb/c nude mice,in the back subcutaneous injection of 0.2 ml 2× 10^6 hep- 2 cells, when the tumor will be 9-11 mm long, study groups： I Restructuring carrier ＋ cisplatin; ]I Restructuring carrier group; Ⅲ The negative control group; IV Cisplatin group,chemotherapy the nice will be treated on execution after 3 weeks, the tumor suppressor , tumor cell apoptosis and changes of STAT3 signaling pathways regulatory proteins will be observed by flow cytometry and electron microscope. Results After injection of laryngeal cancer cells, experimental mice grown to 11 mm size long by nearly one month . ＂rumor rate was 97.34 %, pathologic examination： intermediate differentiation, squamous cell carcinoma. Electron microscopy results ： Restructuring carrier＋cisplatin group of laryngeal cancer cells in the nuclear membrane sharp protuberance,chromatin in the nucleus of high concentration, nearly C type change, trace round cell membrane swelling. Mitochondria edema, rough endoplasmic reticulum degranulation, crest arrangement without rules, or disap- pear. Immunohistochemical method showed that：Meet high expression in cytoplasmic staining into brown substances of STAT3,seen in the nuclei brown dot sedimentary p-STAT3. I STAT3,p-STAT3 protein expression decreased significantly,the grey value significantly below Ⅱ , Ⅲ, Ⅳ group （P〈0.05）. Flow eytometry instrument display I STAT3 ,p - STATg,Bcl - 2 ,CyclinDl protein expression significantly lower in the experimental group,much lower than the other three groups, （P〈0.05）. Conclusion It is demonstrated that applying RNAi to silence STAT3 gene can enhance chemosensitivity by studying therapeutical effect of human laryngeal squamous carcinoma in nude mice.