腺病毒介导的ING4对裸鼠人骨肉瘤移植瘤的生长抑制作用

The growth-inhibition of osteosarcoma xenografts by adenovirus-mediated ING4 gene in nude mice

目的:研究腺病毒介导的人ING4基因(Ad-ING4)对MG-63人骨肉瘤细胞移植瘤的生长抑制作用及其机制。方法:将本室构建好的重组腺病毒表达载体Ad-ING4,经QBI-293A细胞感染多轮扩增后获得高效价重组病毒子以用于肿瘤基因治疗试验,首先建立MG-63人骨肉瘤细胞移植瘤裸鼠模型,然后将15只荷瘤裸鼠随机分为阴性对照组(PBS组)、空载体对照组(Ad-GFP组)、Ad-ING4实验组(Ad-ING4组),3组均使用瘤体内注射干预用药,隔日一次,共5次。分别于用药前和用药后测量皮下瘤的体积,治疗开始后的第15天将裸鼠脱颈处死,摘取瘤体称重,计算抑瘤率。HE染色观察移植瘤细胞形态,免疫组化法检测瘤体中Bcl-2、Bax、Caspase-3、VEGF、CD34细胞因子的表达。结果:获得了高滴度(109pfu/ml)的重组腺病毒Ad-ING4;经瘤体基因治疗后,Ad-ING4组与PBS组及Ad-GFP组相比,可以明显抑制裸鼠MG-63人骨肉瘤细胞移植瘤生长,瘤重抑制率可达59.3%,移植瘤组织中可出现典型的细胞凋亡、坏死现象,其分子机制可能与Ad-ING4明显上调瘤体中促进凋亡的细胞因子Bax、Caspase-3的表达,下调抑制凋亡因子Bcl-2及血管形成细胞因子VEGF、CD34的表达有关。结论:Ad-ING4可以显著抑制MG-63人骨肉瘤细胞荷瘤生长,其机制可能通过激活细胞凋亡和抑制血管形成等途径来发挥抑瘤作用。

Objective：To study the inhibitory effect and anti-cancer mechanisms of adenovims-mediated ING4 gene on the MG-63 esteosareoma xenografts in nude mice. Methods： Ad-ING4 was transfected into QBI-293 cells and harvested. 15 nude mice of the subcutaneous tumor models were established with MG-63 osteosarcoma cells and were randomly divided into PBS, Ad-GFP and Ad-ING4 groups. Then PBS（ 100 μl ）, Ad-GFP（ 100 μl, 10^9pfu/ml） and Ad-ING4 （ 100μl,10^9pfu/ml） for each one were given respectively QOD for 5 times, with intratumor injections. Tumor volume changes were monitored; and the 15 mice were sacrificed 2 weeks after treatment, the tmnors were removed, weighed and ratios of tumor-suppression were calculated. The morphological changes of apoptotic tumor ceils were observed under microscope. Bcl-2, Bax, Caspase-3, VEGF, CD34 expression was tested by i mmumohistochemistry. Results： High titer（10^9pfu/ml） adenoviral vector of ING4 gene were obtained. In nude mice bearing MG-63 osteosarcoma xenografts, the growth of MG-63 tumors treated by intratumoral injecting of Ad-ING4 was significantly suppressed,compared with PBS group and Ad-GFP group. The ratios of tumor weight-suppression of Ad-ING4 group was 59.3% （ P 〈 0.05）. Immumohistochemistry displayed that the expression of Bax, Caspase-3 was up-regulated and the expression of Bcl-2, VEGF, CD34 was down-regulated by Ad-ING4. Conclusion： Ad-ING4 can inhibit the growth of MG-63 osteosareoma xenografts in nude mice, which may be via activating the apoptosis pathway and inhibiting tumor angiogenesis.