慢病毒RNA干扰E2F-1对人胃癌裸鼠皮下瘤耐药性的影响

Inhibitory effect of E2F-l-silencing lentivirus vector on chemoresistance of subcutaneous human gastric cancer in nude mice

目的观察E2F-1基因沉默的慢病毒载体E2F-1／RNAi—LV对人胃癌裸鼠皮下瘤耐药性的影响。方法将人胃癌SGC7901-顺铂（DDP）耐药细胞接种于裸鼠皮下，建立人胃癌耐药裸鼠皮下瘤模型。将成瘤的36只裸鼠随机分为E2F-1／RNAi—LV组、LV—scrRNAi组和PBS组，每组12只。隔日向3组裸鼠肿瘤内分别注射E2F—1／RNAi—LV、LV—scrRNAi或PBS，并按体重腹腔注射DDP，共9次。注射期间和处死裸鼠后测量各组裸鼠的肿瘤体积。应用TUNEL法检测肿瘤细胞的凋亡指数，应用半定量RT—PCR和Western blot法检测肿瘤组织中E2F-1、c—Myc、survivin、多药耐药基因1（MDRl）和多药耐药相关蛋白（MRP）mRNA和蛋白的表达。结果注射慢病毒后，E2F-1／RNAi—LV组肿瘤的生长速度明显减慢。处死裸鼠后，E2F-1／RNAi—LV组裸鼠的肿瘤体积为（745．13±154．42）mm^3，明显低于LV—scrRNAi组[（1988．80±171．35）mm^3]和PBS组[（1633．48±215．25）mm^3，P〈0．05]。E2F-1／RNAi-LV组肿瘤组织的凋亡指数为（27．5±9．7）％，明显高于LV-scrRNAi组[（7．0±1．1）％]和PBS组[（7．3±1．2）％，P〈0．05]。E2F-1／RNAi—LV组肿瘤组织中E2F-1mRNA和蛋白的表达量分别为0．40±0．32和0．35±0．01，明显低于LV-scrRNAi组和PBS组（P〈0．05）。E2F-1／RNAi—LV组c—Myc、survivin、MDR1和MRPmRNA和蛋白的表达量亦明显低于LV-scrRNAi组和PBS组（均P〈0．05）。结论慢病毒载体E2F-1／RNAi-LV瘤内注射可降低人胃癌SGC7901-DDP耐药细胞对DDP的耐药性,抑制人胃癌耐药裸鼠皮下瘤的生长。

Objective To study the effects of E2F-1-sileneing lentivirus vector on the growth and chemoresistanee of subcutaneous human gastric cancer in nude mice. Methods Thirty-six nude mice were inoculated subcutaneously with chemoresistant SGC-7901/DDP cells to establish subcutaneous tumor models of gastric carcinoma. The mice were randomly divided into E2F-1/RNAi-LV group, LV-scrRNAi group and PBS group （n = 12）. E2F-1/RNAi-LV, LV-scrRNAi or PBS （0.1 ml per time） was injected into the mice, respectively, every two days. The nude mice received an intraperitoneal injection of cisplatin （25 mg/kg） every two days. The tumor volume was measured and histopathologieal changes of the tumors were observed by HE staining. The expressions of E2F-1, c-Mye, survivin, MDR1 and MRP were detected by semiquantitative reverse transcription polymerase chain reaction （RT-PCR） and Western blot. Apoptosis in tumor xenografts was determined by in situ TUNEL labeling technique. Results The mean tumor growth rate of the E2F-1/RNAi-LV group was significantly slower than that of the LV-scrRNAi and control groups （P 〈 0.05）. The tumor volume of the E2F-1/RNAi-IN group was （745.13 ± 154.42）mm3, significantly lower than that of the LV-serRNAi and PBS groups （ P 〈 0.05 ）. Compared with that in the LV-scrRNAi and PBS groups, the expressions of mRNA and protein of E2F-1, c-Myc, survivin, MDR1 and MRP were significantly decreased in the E2F-1/RNAi-LV group （P 〈 0.05 ）. The apoptotic rate in the E2F-1/RNAi- LV treatment group was （ 27.5 ±9.7 ） %, significantly higher than （ 7.0 ± 1.1 ） % in the LV-scrRNAi group and （7.3 ± 1.2） % in the PBS group （P 〈 0.05）. Conclusion Intra-tumoral injection of E2F-1/RNAi-LV shows significantly inhibitory effect on the tumor growth and chemoresistance of subcutaneous human gastric cancer in nude mice.