封闭Skp2基因表达对大肠癌LS-174T细胞裸鼠皮下移植瘤生长与转移抑制的研究

摘要

目的:建立人大肠癌LS-174T细胞的裸鼠皮下移植瘤模型,应用Skp2基因特异性siRNA重组腺病毒治疗,观察其在体内对肿瘤生长和转移的影响,并探讨其相应的作用机制.方法:将大肠癌LS-174T细胞以5×106个/只接种于雌性裸鼠右侧腋窝皮下,待平均瘤体积长至50～150mm3时筛选成模动物分组给药,分别给予重组腺病毒pAD-Skp2/siRNA 5×108和5× 109pfu/只瘤内注射,阳性组给予今又生5×109pfu/只瘤内注射,另设空病毒对照组.每周给药2次,连续4周,以安乐死时肿瘤体积大小和重量来评价治疗效果.然后用Western Blot检测肿瘤组织中Skp2、p27kipl、c-myc、Bcl-2的表达,用免疫组化法检测肿瘤组织中MMP-2的表达.结果:安乐死时阴性对照组平均肿瘤体积与空病毒对照组生长情况基本一致(P≥0.05)；今又生组平均肿瘤体积明显小于模型组(P≤0.05)；pAD-Skp2/siRNA低、高剂量组平均瘤体积均显著低于模型组(P≤0.01),且有剂量依赖性.今又生组瘤重抑制率为58.16％,pAD-Skp2/siRNA低、高剂量组瘤重抑制率分别为75.59％、86.45％.Skp2蛋白表达在模型组、空病毒对照组和今又生组均有较强表达,而在pAD-Skp2/siRNA低剂量和高剂量组均未见表达；而p27kipl仅在pAD-Skp2/siRNA低剂量和高剂量组有表达,其余三组均未见其表达；c-myc和bcl-2表达趋势一致,均在模型和空病毒对照组有表达,而在今又生组和pAD-Skp2/siRNA两个剂量组均表达明显减弱.模型组和空病毒对照组有棕黄色阳性表达,而今又生组和pAD-Skp2/siRNA两个组均未检测到MMP-2阳性表达.结论:在高转移性大肠癌LS-174T裸鼠移植瘤模型中,封闭Skp2基因可以抑制肿瘤的增殖和转移,其作用机制与细胞周期调控、细胞凋亡中的c-myc和bcl-2以及基质金属蛋白酶MMP-2相关.%Objective: To build nude mice subcutaneous xenograft tumor model of colorectal cancer cell line LS-174T, and block S-phase kinase protein 2 (Skp2) gene by specific siRNA adenovirus in order to investigate the effect on the proliferation and metastasis ability and to discuss the mechanism. Methods: Colorectal cancer cell line LS-174T with the concentration of 5 × 106cells/site was injected into female model animals subcutaneously in the right armpit. Model animals with tumor sized 50～150mm3 were divided into groups and administered recombinant adenovirus pAD-Skp2/siRNA with 5 × 108 and 5 × 10'pfu/head by injecting into tumor while Gendicine was injected in positive control group by 5× 109pfu/head. Besides, there was blank virus control group. All the animals were administered twice a week for four weeks and evaluated by tumor size and weight in the end. Then Western Blot was used to detect Skp2, p27kipl, c-myc, bcl-2 expressions in the tumor tissue and immunohistochemical staining was adopted for MMP-2 detection. Results: Tumor size in negative control group was the same with that in blank virus control group (P≥ 0.05) while that in Gendicine group was smaller than that in the negative control group (PS 0.05). And tumor sizes in low and high dose groups of pAD-Skp2/siRNA were much smaller than that in the negative control (PS 0.01) with dose dependency decrease. The tumor weight inhibition was 58.16%, 75.59%, 86.45%, respectively in Gendicine group, low and high dose groups of pAD-Skp2/siRNA. As Western blot seen, Skp2 protein expression was only detected in negative and. Positive control groups, but p27kipl expression was only in low and high dose groups of pAD-Skp2/siRNA. However, c-myc and bcl-2 were both detected in two negative groups and less in Gendicine group and the two pAD-Skp2/siRNA groups. And the immunohistochemical staining result suggested that there were brownish yellow MMP-2 positive stain in two negative groups, but no positive stains in Gendicine group and the two pAD-Skp2/siRNA groups. Conclusion: Blocking Skp2 gene by recombinant adenovirus pAD-Skp2/siRNA could inhibit the proliferation and metastasis abilities in nude mice subcutaneous xenograft tumor model of colorectal cancer cell line LS-174T, which may be related with c-myc and bcl-2 in cellular cycle regulation and apoptosis as well as matrix metalloproteinases MMP-2.