shRNA沉默Survivin基因对人骨肉瘤MG-63细胞成瘤能力的影响

目的:研究Survivin-shRNA对骨肉瘤MG-63细胞成瘤能力的影响,并探讨其可能的机制.方法:采用皮下异种移植法构建裸鼠骨肉瘤模型,骨肉瘤MG-63细胞培养成功后,重组腺病毒Survivin-shRNA转染.细胞以1:5比例进行传代,挑选稳定转染的MG-63细胞并扩增,Survivin-shRNA组为Survivin-shRNA转染的细胞,GFP组和CON组为阴性对照组.每3天用卡尺测定肿瘤体积,绘制肿瘤生长曲线.颈椎脱臼法处死裸鼠,取骨肉瘤标本称重.Western blot法检测SUV、VEGF、PCNA及CAS-3蛋白的表达.结果:腺病毒介导的RNA干扰构建Survivin-shRNA载体,Survivin-shRNA组转染细胞的胞质及胞核可见绿色荧光,并伴有少量小颗粒物质.骨肉瘤生长曲线显示,与CON和GFP组相比,Survivin-shRNA组的肿瘤增长缓慢,表明Survivin-shRNA在体内能抑制骨肉瘤的生长.肿瘤的重量结果显示,Survivin-shRNA在体内能抑制骨肉瘤重量的增加.Western blot结果显示,SUV、VEGF的表达在Survivin-shRNA组中明显低于对照组,而CAS-3表达相反.结论:Survivin-shRNA可抑制骨肉瘤细胞增殖及成瘤能力,其机制可能是通过调节SUV、VEGF、CAS-3的表达及抑制肿瘤新生血管形成等来实现的.

Effects of silencing of Survivin gene expression by shRNA on tumorigenicity of MG-63 cell

Objective:To explore the effects and molecular mechanisms of baicalin on tumorigenicity of MG-63 cell.Methods:Subcutaneous xenograft tumor model was established.MG-63 cells were cultured in DMEM medium and recombinant adenovirus vector Survivin-shRNA and negative control rAd5-GFP were transfected into MG-63 cells.Cells were subcultured at a 1: 5 dilution.Positive stable transfectants were selected and expanded for further study.The clone in which the rAd5-Survivin-shRNA virus vectors transfected was named as Survivin-shRNA group,the negative control vectors transfected was named as GFP group and MG-63 cells were named as CON group.The tumor volume every three days was measured with a caliper,using the formula volume =(length × width)2/ 2.Western blot assay was used as determine the protein expression level of SUV,VEGF,PCNA and CAS-3.Results:After Survivin-shRNA was transfected into MG-63 cells,it was found that knockdown of SUV could significantly re-duce the proliferative activities of MG-63 cells compared with GFP group and CON group,cell nuclear fragmenta-tion,apoptotic bodies and DNA ladder turned up in group Survivin-shRNA compared with the group GFP.The tumor growth activity was measured.Tumors in therapy groups grew substantially slow compared with the PBS and rAd5-GFP group.When the tumors were harvested,the average weight of tumors in therapy groups was significantly lower than PBS and rAd5-SUV group.Compared with the GFP group and CON group,the protein expression level of SUV, VEGF and PCNA was decreased,while CAS-3 expression was increased in therapy groups.Conclusion:Survivin-shRNA effectively decreases the tumorigenicity of MG-63 cell.The molecular mechanisms may through VEGF-me-diated regulation or regulate the expression of SUV and CAS-3.