Ad-IL-24对MG-63人骨肉瘤细胞抑癌效应的体内外实验

Interleukin 24 inhibits growth and induces apoptosis of osteosarcoma cells MG-63 in vitro and in vivo

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。

To study the inhibitory effect and anti-cancer mechanisms of interleukin 24 （IL-24） on human osteosarcoma cell MG-63, we delivered IL-24 into MG-63 cells in vitro and in vivo by adenovirus. The expression level of IL-24 was detected by RT-PCR and fluorescence microscope; the growth inhibition, apoptosis rate and apoptosis body were measured by MTT, Flow cytometry and Hoechst staining respectively. Furthermore, we analyzed the expression of bcl-2, bax, caspase3 genes by RT-PCR after overexpression of IL-24. For in vivo study, we first established the MG-63 tumor model by grafting MG-63 cells in athymic nude mice; and then injected Ad-IL-24 into the tumors. Two weeks after injection, we sacrificed the mice, removed the tumors, weighed and calculated the ratios of tumor-suppression. We also detected the expressions of Bcl-2, Bax, Caspase-3 and CD34 with immumohistochemistry. Our in vitro results indicated that Ad-IL-24 was transcribed and translated in MG-63 osteosarcoma cells. More interestingly, IL-24 inhibited the growth of MG-63 cells and induced apoptosis by up-regulation of bax, caspase-3 and down-regulation of bcl-2. The in vivo data showed that IL-24 suppressed the tumor growth conspicuously through down-regulating the expression of bcl-2, and up-regulating the expression of bax, caspase-3. This study would provide evidence for the gene therapy of IL-24 on osteosarcoma.