Caspase-3基因过表达抑制LN-18人神经胶质瘤细胞移植瘤生长

Inhibitive role of caspase-3 gene overexpression in growth of human LN-18 neurospongioma cell transplanted tumor

目的观察靶向caspase-3基因过表达对LN-18人神经胶质瘤细胞移植瘤生长的影响并探讨其机制。方法化学合成caspase-3基因序列,通过同源重组,获得caspase-3基因过表达慢病毒。设置转染组、对照组和空白组,其中转染组细胞转染caspase-3基因过表达慢病毒,对照组细胞转染含阴性序列基因重组慢病毒,空白组细胞不加病毒上清液,慢病毒转染LN-18人神经胶质瘤细胞72 h后测定病毒转染效率,蛋白质印迹法评估caspase-3蛋白表达。制备裸鼠神经胶质瘤荷瘤模型,细胞接种后每3天测量肿瘤体积,21 d后处死动物,剥离肿瘤组织,比较各组瘤块大小,TUNEL法观察各组肿瘤组织内细胞凋亡情况。结果 caspase-3基因过表达慢病毒能有效感染LN-18人神经胶质瘤细胞,体外细胞转染率达到83.7%,转染72 h后,转染组caspase-3蛋白表达显著增加;转染组LN-18人神经胶质瘤细胞移植裸鼠皮下,肿瘤生长速度明显小于对照组和空白组,至移植后21 d,转染组、对照组和空白组移植瘤体积分别为(0.52±0.17)cm^3、(2.61±0.22)cm^3和(2.38±0.19)cm^3,F=104.241,P=0.021 89;移植后21 d,与对照组和空白组相比,转染组移植瘤细胞凋亡显著增加(P <0.001)。结论 caspase-3基因过表达慢病毒转染LN-18人神经胶质瘤细胞可明显促进caspase-3蛋白合成,抑制LN-18神经胶质瘤细胞体内移植后生长,显著增加肿瘤细胞凋亡,可作为神经胶质瘤治疗新手段。

Objective To explore the effect of caspase-3 gene overexpression on the growth of human LN-18 neurospongioma cell transplanted tumor in vivo. Methods caspase-3 gene was prepared using chemical synthesis technology,and caspase-3 gene overexpression lentivirus was obtained through homologous recombination. There are three groups in this study. Transfection group was transfected with caspase-3 gene overexpression lentivirus;control group with negative sequence lentivirus,and blank group was untreated. Transfection efficiency was detected and analyzed by fluorescence microscopy and flow cytometry 72 hours after lentivirus transfection ,and the expression of caspase-3 protein was measured by using Western blotting. The neurospongioma cancer model of nude mouse was established and the tumor volume measured every 3 days after injection,then the animals were sacrificed 21 days after the transplantation. The tumor tissue was harvested and the cell apoptosis tested by TUNEL staining. Results The caspase-3 gene overexpression lentivirus could effectively infect human LN-18 neurospongioma cells,and the cell transfection rate was 83.7% in vitro. Caspase-3 gene overexpression lentivirus transfection can significantly increase the caspase-3 protein synthesis. Transfected LN-18 human neurospongioma cells were subcutaneously implanted into nude mice. The tumor growth rate in transfection group was significantly shorter than that in the control group and blank group. After 21 days of transplantation,tumor volume in transfection group,control group and blank group was( 0.52 ± 0.17) cm^3,(2.61 ± 0.22) cm^3 and( 2.61 ± 0.19) cm^3 respectively(F = 104.241, P = 0.021 89). Compared with that in the control group and blank group,the cell apoptosis in transfection group was increased significantly(P < 0.001). Conclusions caspase-3 gene overexpression lentivirus transfection can significantly promote the caspase-3 protein synthesis in LN-18 human neurospongioma cells,obviously inhibit the growth of transplanted neurospongioma tumor and induce the apoptosis of neurospongioma cells. Thus,it might be a potential target for anti-neurospongioma cancer therapy.