IGF-IR反义硫代磷酸型寡核苷酸诱导胶质瘤细胞凋亡的病理学研究

Pathologic observation of apoptosis induced by IGF-IR antisense phosphorothioate oligodeoxynucleotides in malignant glioma cell

目的 :探讨IGF IR基因的反义硫代磷酸型寡核苷酸对胶质瘤细胞的形态学影响。方法 :根据IGF IRcDNA序列设计正义、反义寡核苷酸片段 ,并对其部分碱基进行硫代磷酸修饰。体外培养的胶质瘤细胞分别经正义寡核苷酸和反义寡核苷酸处理 ,应用倒置显微镜活细胞观察、HE染色光镜观察、透射电镜及DNA琼脂糖凝胶电泳等方法研究IGF IR反义硫代磷酸型寡核苷酸诱导胶质瘤细胞凋亡作用。结果 :经反义寡核苷酸转染的胶质瘤细胞中 ,呈现典型的凋亡形态学改变。凋亡细胞最早期表现为细胞体积、容量减少 ,染色质凝聚 ,继之染色质集聚于核膜下成新月状或块状 ;细胞核内染色质可完全固缩成团呈“黑洞”样或萎缩的核破裂形成一些较小的膜包绕的球体位于胞质内 ,最后出泡形成凋亡小体。DNA琼脂糖凝胶电泳分析经反义寡核苷酸处理的胶质瘤细胞DNA降解片段 ,可见有明显的小分子量DNA梯状条带 ,而野生型和正义寡核苷酸处理的胶质瘤细胞未见DNA梯状条带。结论 :IGF IR所介导的分泌环路IGF I/IGF IR ,在胶质瘤细胞增殖和维持恶性表型中起重要作用 ;IGF IR反义硫代磷酸型寡核苷酸能诱导胶质瘤细胞凋亡。

Purpose To study the apoptosis inducing effects of IGF IR antisense phosphorothioate oligodexynucleotides(antisense ODNs) on malignant glioma cell. Methods Antisense and sense ODNs were designed according to the sequence of IGF IR mRNA. The apoptosis induced by antisense ODNs was investigated by phase contrast microscopy, ligtht microscopy, transmission electron microscopy, and electrophoresis. Results The data showed that there were apoptosis in antisense ODNs treated C6 cells. The morphological features of apoptotic cell were chromatin condensation and aggregation to the nuclear margin, cytoplasmic condensation, convolution of the nuclear and cellular membranes and nuclear condensation into 'black holes' in early stage, and nuclear fragmentation and apoptotic body formation in latter stage. DNA fragmentation was detected by gel electrophoresis in C6 cell undergoing apoptosis after treatment with antisense ODNs, whereas this phenomena was not detected in C6 cells with untreated or treated with sense ODNs. Conclusions IGF IR mediated autocrine loop may play an important role in glioma cell proliferation through autocrine or paracrine mechanisms. The IGF IR antisense ODNs can inhibit the proliferation and induce apoptosis in malignant glioma cells.