靶向VEGF-hTERT基因siRNA表达载体抑骨肉瘤生长的实验研究

Experimental Study on SiRNA Targeting VEGF-hTERT Gene Expression Vector Inhibiting the Growth of Osteosarcoma

目的:观察靶向VEGF-hTERT基因siRNA表达载体对骨肉瘤细胞增殖的影响和对裸鼠骨肉瘤生长的影响。方法:构RNA干扰腺病毒载体rA-hTERT和rAd-VEGF,单独和联合转染人骨肉瘤细胞系MG-63细胞后实时荧光定量PCR检测hTERT和VEGF基因表达情况,流式细胞仪检测各组细胞凋亡率;人骨肉瘤细胞系MG-63细胞于裸鼠成瘤后使用siRNA表达载体治疗,观察肿瘤的生长状况及组织病理学改变。结果:①转染后48h,rAd-hTERT对hTERT基因mRNA的抑制率约84%,对VEGF基因表达无明显抑制(P>0.05);rAd-VEGF对VEGF基因mRNA的抑制率约82%,对hTERT基因表达无明显抑制(P>0.05);rAd-hTERT-VEGF干扰组对hTERT基因表达抑制率约87%,VEGF基因表达抑制率约83%,联合干扰与单基因干扰相比,对hTERT-VEGF基因的表达抑制差异无显著性(P>0.05)。②干扰组转染后l～6d均能促进细胞凋亡,联合干扰细胞凋亡率与单基因干扰细胞凋亡率相比,差异均有显著性(P<0.05)。裸鼠实验中,基因组肿瘤的生长均受到明显抑制,且联合干扰组与单基因组比较有显著性差异(P<0.05)。对骨肉瘤的肺转移联合干扰组有显著的抑制作用。结论:靶向VEGF-hTERT基因siRNA表达载体对骨肉瘤细胞系MG-63细胞和裸鼠骨肉瘤的生长及转移均有明显的抑制作用。

Objective To evaluate siRNA targeting VEGF-hTERT gene expression vector on proliferation of osteosarcoma cells and the growth of osteosarcoma innude mice.Methods Constructed RNA interference structure adenovirus vector rA-hTERT and rAd-VEGF,and transfected to human osteosarcoma cell line MG-63 cells alone or combined.Then detected of hTERT and VEGF gene expression with real-time fluorescence quantitative PCR,the apoptotic rate in each group were detected by flow cytometry apoptosis.Treated with siRNA expression vector after Human osteosarcoma cell line MG-63 tumor cellswere implanted into nude mice,and observed the tumor growth and pathological changes in each group after treatment .Results ① After 48h of transfection,compared with A and B group,the inhibition rate of rAd-hTERT gene on hTERT mRNA was about 84%,it was not significantly inhibited on VEGF gene expression （P 0.05）;The inhibition rate of rAd-VEGF mRNA on VEGF gene was about 82%,it was not significantly inhibited on hTERT gene expression （P0.05）;In the rAd-hTERT-VEGF interference group,the inhibition rate of hTERT gene expression was about 87%,and the inhibition rate of VEGF gene expression was about 83%;The hTERT-VEGF gene expression was no significant difference between united interference and single-gene interference （P0.05）;② The apoptosis were promoted in interference groups after transfected l ~ 6 d;Compared with single-gene interference,the apoptosis rate of the combination interference have significant difference （P 0.05）.In Nude mice experiments,Tumor growth was significantly inhibited in all genetic manipulation groups,and There was significant difference between the combination interference group and single-gene interfere group （P 0.05）.Lung metastasis of osteosarcoma was significantly inhibited in the combination interference group.Conclusion The siRNAtargeting VEGF-hTERT gene expression vector was significantly inhibited to MG-63 cell and the growth of osteosarcoma in nude mice.