反义Cyclin B1提高Lewis肺癌细胞对健择化学敏感性的体内外研究

Effect of Full-length Cyclin B1 Antisense cDNA on Chemosensitivity of Lewis Lung Carcinoma Cells to Gemicitabine in vitro and in vivo

目的研究CyclinB1反义全长cDNA(AS-CLB1)对小鼠Lewis肺癌细胞(LL/2)化疗敏感性的影响,为将该方法联合健择化疗用于非小细胞肺癌的治疗提供实验依据。方法通过流式细胞术检测LL/2亲本(LP),LL/2空载体(LV)及LL/2/AS-CLB1(LA)细胞的凋亡及细胞周期分布。用健择(20nmol/L～20μmol/L)处理上述三种细胞,分别于1h及24h后用四甲基偶氮唑盐(MTT)比色法评估健择对各组细胞的体外杀伤作用。将上述三种细胞分别接种C57BL/6小鼠,成瘤后用健择〔25～125mg/(kg·d)〕处理各组动物,3d1次,一共4次,观察细胞在体内的成瘤性及动物的生存时间,并用流式细胞仪检测各组动物肿瘤组织的细胞凋亡。结果LA细胞较对照(LP和LV细胞)出现了明显的G1期阻滞及凋亡;健择对LA细胞的体外杀伤作用显著增强;在LA细胞组,细胞的成瘤性明显下降,该组动物肿瘤组织的细胞凋亡明显增加,动物的生存时间也显著延长。结论AS-CLB1可以明显增强LL/2细胞体内外对健择的化学敏感性,其作用可能与AS-CLB1诱导细胞发生G1期阻滞及凋亡,从而增强了健择的抗肿瘤作用有关。

Objective To evaluate the effect of full-length cyclin B1 antisense cDNA (AS-CLB1) on chemosensitivity of Lewis lung carcinoma cells (LL/2) to gemicitabine (GEM) in vitro and in vivo and hence provide a therapeutic regiment for treating non-small cell lung (NSCL) cancer using AS-CLB1 combined with GEM. Methods Cell cycle phase distribution and apoptosis of LL/2 parent cells, LL/2/vect and LL/2/AS-CLB1 transfectants (LP, LV and LA cells) were determined by flow cytometry. In addition, the three kinds of cells were treated with GEM (20 nmol/L-20 μmol/L) in vitro for 1 h and 24 h respectively, and then cytotoxicity of GEM was measured by MTT assay. After inoculation with the three kinds of cells respectively, the C57BL/6 mice were treated with GEM 〔25 125 mg/(kg·day)〕 once every three days for four times when tumors developed; tumorigenicity and survival were observed and cell apoptosis in tumor tissues was determined by flow cytometry. Results LA cells displayed apparent apoptosis and G_1 arrest compared with LP and LV cells (controls). Additionally, cytotoxicity of GEM to LA cells was more obvious than that to controls. Moreover, tumorigenicity was inhibited, cell apoptosis in tumor tissues was induced, and survival was evidently increased in LA cells group. Conclusion AS-CLB1 slightly increased the sensitivity of LL/2 cells to GEM in vitro and in vivo. The function of AS-CLB1 may be associated with its ability to enhance the anti-tumor activity of GEM by inducing cell apoptosis and G_1 arrest.