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本研究旨在分析腺病毒携带的IL-24基因在体内外对人骨肉瘤细胞生长抑制效应及其分子机制。将Ad-IL-24重组腺病毒感染MG-63细胞,用荧光显微镜、RT-PCR法检测IL-24在MG-63细胞中的转录和表达;MTT法、流式细胞技术和Hoechst染色法检测IL-24基因的表达对MG-63细胞的生长抑制和凋亡效应;半定量RT-PCR法检测IL-24基因的表达对MG-63细胞中的bcl-2、bax、caspase-3相关基因表达的影响。用Ad-IL-24重组腺病毒在MG-63骨肉瘤荷瘤裸鼠的瘤体内进行注射治疗,观察肿瘤生长变化,15d后处死裸鼠,摘除瘤体,称瘤重。并通过免疫组化法检测Bcl-2、Bax、Caspase-3等与细胞凋亡相关因子的表达。结果表明Ad-IL-24重组腺病毒感染MG-63细胞后,能明显抑制MG-63细胞增殖,并能通过上调细胞中bax、caspase-3和下调bcl-2基因表达,诱导细胞凋亡,呈现出典型细胞凋亡形态学变化。Ad-IL-24重组腺病毒瘤内注射MG-63裸鼠荷瘤骨肉瘤移植瘤后,能显著抑制肿瘤生长,瘤重的抑制率可达52%,免疫组化结果显示Ad-IL-24重组腺病毒能明显上调与细胞凋亡相关因子Bax和Caspase-3的表达和下调Bcl-2和CD34的表达。本研究结果显示腺病毒介导的IL-24基因在体内外均可明显抑制MG-63人骨肉瘤细胞的生长,诱导其凋亡,其机制可能与下调bcl-2,上调bax、caspase-3的基因表达水平有关,由此为骨肉瘤的基因治疗提供实验依据。
This study aimed to analyze the inhibitory effect of adenovirus carrying IL-24 gene on human osteosarcoma cell growth in vitro and in vivo and its molecular mechanism. MG-63 cells were infected with Ad-IL-24 recombinant adenovirus. The transcription and expression of IL-24 in MG-63 cells were detected by fluorescence microscope and RT-PCR. MTT assay, flow cytometry and Hoechst staining The effect of IL-24 gene expression on the proliferation and apoptosis of MG-63 cells was detected by semi-quantitative RT-PCR. The expressions of bcl-2, bax and caspase-3 in MG-63 cells The impact of expression. Ad-IL-24 recombinant adenovirus was injected into the tumor of MG-63 osteosarcoma-bearing nude mice to observe the changes of tumor growth. After 15 days, the nude mice were sacrificed and the tumors were excised and weighed. The expression of Bcl-2, Bax, Caspase-3 and other apoptosis-related factors were detected by immunohistochemistry. The results showed that MG-63 cells could significantly inhibit the proliferation of MG-63 cells by inducing the apoptosis of MG-63 cells by up-regulating bax, caspase-3 and down-regulating bcl-2 gene expression, The morphological changes of typical apoptotic cells were observed. Ad-IL-24 recombinant adenovirus injected MG-63 nude mice bearing osteosarcoma xenografts significantly inhibited tumor growth, tumor weight inhibition rate of up to 52%, immunohistochemical results showed that Ad-IL-24 Recombinant adenovirus can significantly up-regulate the expression of Bax and Caspase-3, and down-regulate the expression of Bcl-2 and CD34. The results showed that adenovirus mediated IL-24 gene can significantly inhibit the growth and induce apoptosis of MG-63 human osteosarcoma cell in vitro and in vivo, which may be related to down-regulation of bcl-2, up-regulation of bax and caspase-3 Gene expression level, which provides experimental evidence for the gene therapy of osteosarcoma.