Bcl-2反义寡核苷酸对人肺癌裸鼠移植瘤的抑制作用

Inhibitory Effect of Bcl-2 Antisense Oligodeoxynucleotides on Growth of Human Lung Carcinoma Xenograft in Nude Mice

背景与目的:研究表明,靶向bcl-2mRNA的反义寡核苷酸(antisenseoligodeoxynucleotide,ASODN)在体内外可产生抗肿瘤效应。我们已经在bcl-2mRNA蛋白编码区发现了一个新的有效反义作用靶点,同时证实这个靶点的ASODN能增强白血病及肺癌细胞对化疗药物、放射线的敏感性。本研究进一步探讨bcl-2ASODN对裸鼠非小细胞肺癌NCI-H460细胞移植瘤的成瘤能力和移植瘤的抑制作用。方法:将经硫代修饰的bcl-2ASODN直接加入到培养的NCI-H460细胞内,MTT法检测细胞生长抑制率,然后将这种细胞接种于裸鼠皮下,观察肿瘤出现的时间和肿瘤体积的变化,并计算抑瘤率。同时取未经处理的NCI-H460细胞接种于裸鼠背部皮下建立裸鼠人肺癌移植瘤模型,待瘤结节直径≥5mm后,分为bcl-2ASODN实验组、生理盐水对照组和无义寡核苷酸对照组。实验组用15mg/kgASODN两天一次直接瘤内注射,连用3周,测肿瘤大小和组织形态学改变。结果:bcl-2ASODN显著抑制NCI-H460细胞的生长,并呈时间依赖性,与无义寡核苷酸组相比,有显著性差异(P<0.05)。bcl-2ASODN作用后的NCI-H460细胞在裸鼠皮下成瘤能力降低。平均成瘤时间延长为12.6天(P<0.01),最大抑瘤率达87.5%(P<0.01)。在处理NCI-H460细胞裸鼠移植瘤过程中,生理盐水对照组和无义寡核苷酸对照组的瘤体进行性增大,而bcl-2ASODN组的瘤块生长缓慢,ASODN处理组与两个对照组相比均有显著性差异(P<0.05)。处理后第30天剥离瘤块,ASODN处理组分别与两个对照组瘤重相比均有显著性差异(P<0.01)。bcl-2ASODN处理组裸鼠NCI-H460细胞移植瘤的生长受到明显抑制,抑瘤率为71.0%。病理学检查可见,bcl-2ASODN处理组瘤块内有大片的变性坏死灶,结缔组织增生,炎性细胞浸润;而两个对照组瘤体内癌细胞呈弥漫性密集分布。结论:bcl-2ASODN能降低NCI-H460细胞的成瘤能力,抑制移植瘤生长。

BACKGROUND ＆ OBJECTIVE： Antisense oligodeoxynucleotides （ASODN） targeting bcl-2 have in vitro and in vivo antitumor activities. We have identified a novel antisense sequence in the coding region of bcl-2 mRNA that could enhance chemosensitivity and radiosensitivity of leukemia cells and lung carcinoma cells. This study was designed to investigate inhibitory effects of bcl-2 ASODN on development and growth ability of human lung carcinoma NCl-H460 cells xenograft in nude mice. METHODS： bcl-2 ASODN was added to NCl-H460 cells. Inhibitory rate of cell growth was assayed by MTT assay, bcl-2 ASODN-treated NCl-H460 cells were implanted subcutaneously into nude mice to observe tumor growth and calculate tumor inhibitory rate. Seven days after subcutaneous implantation of untreated NCl- H460 cells, the tumor-bearing mice were randomized into 3 groups： control group, ASODN group, and nonsense oligodeoxynucleotides （NSODN） group, bcl-2 ASODN, bcl-2 NSODN, and saline were separately injected into the tumor bodies of mice in relevant groups for 3 week. The tumor weight and volume were measured, and the morphology of tumor cells was observed under microscope. RESULTS： bcl-2 ASODN reduced the growth of NCl-H460 cells in a time-dependent manner. Inhibitory rate of NCl-H460 cells was significantly higher in bcl-2 ASODN group than in bcl-2 NSODN group （P〈0.05）. The tumorigenic ability of NCl-H460 cells was reduced by bcl-2 ASODN, with the maximum tumor growth inhibitory rate of 87.5% （P〈 0.01）. The tumor formation time was prolonged to 12.6 days. Tumor growth was significantly inhibited in bcl-2 ASODN group as compared with that in NSODN group and control group （P 〈0.01）. The tumor weight was significantly lighter in bcl-2 ASODN group than in bcl-2 NSODN group and control group （P〈0.01）. The growth inhibitory rate was 71.0% in bcl-2 ASODN group. Serious necrosis foci and inflammatory cell infiltration were observed in tumor tissues of bcl-2 ASODN group, while cancer cells were diffused in tumor tissues of bcl-2 NSODN group and control group. CONCLUSION： bcl-2 ASODN can suppress tumorigenic ability of NCl-H460 cells and inhibit tumor growth in nude mice.