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目的 观察人核糖核酸酶抑制因子(RI)逆转录病毒真核表达载体(p L NCX- ri)对小鼠B16黑素瘤生长的抑制作用。方法 将转染细胞PA317- p L NCX和PA317- p L NCX- ri分别置于DMEM培养基上,经G4 18筛选培养。用Sepharose CL- 4 B凝胶过滤层析纯化病毒颗粒。经G4 18筛选培养,在倒置相差显微镜下计数克隆细胞数。用RT- PCR方法和Western blotting方法检测被感染的NIH3T3细胞及B16黑素瘤组织中RI基因的表达。给接种B16黑素瘤细胞的C5 7BL/ 6 J小鼠分别皮下注射纯化的p L NCX- ri病毒和空载体病毒,观察C5 7BL/ 6 J小鼠肿瘤的生长状况,并对肿瘤组织称重。镜下观察肿瘤组织切片的血管密度。结果 RI在感染了p L NCX- ri病毒的NIH3T3细胞和B16黑素瘤组织中得到有效表达。p L NCX- ri病毒治疗组的瘤组织重量(0 .6 0±0 .2 6 ) g明显低于p L NCX病毒对照组(1.84±1.6 4 ) g(P<0 .0 1)和生理盐水对照组(1.77±1.13) g(P<0 .0 1)。p L NCX病毒对照组(1.84±1.6 4 ) g和生理盐水对照组(1.77±1.13) g无显著性差异(P>0 .0 5 )。与p L NCX病毒对照组和生理盐水对照组相比,p L NCX- ri病毒治疗组肿瘤组织的血管密度明显减少,而两对照组间无明显差异。结论 人核糖核酸酶抑制因子(RI)对小鼠B16黑素瘤的生长有明显的抑制作用
Objective To observe the inhibitory effect of pLNCX-ri on the growth of mouse B16 melanoma by using the ribozyme inhibitor (RI) retrovirus eukaryotic expression vector. Methods The transfected cells PA317-p L NCX and PA317-p L NCX-ri were respectively cultured on DMEM medium and screened by G418. The virus particles were purified by Sepharose CL-4 B gel filtration chromatography. The cells were screened by G418 and the number of cloned cells was counted under inverted phase contrast microscope. The expression of RI gene in infected NIH3T3 cells and B16 melanoma tissues was detected by RT-PCR and Western blotting. The C5 7BL / 6 J mice inoculated with B16 melanoma cells were subcutaneously injected with the purified pL NCX-ri virus and the empty vector virus respectively to observe the growth of the tumor in the C5 7BL / 6 J mice and the tumor tissues were weighed . Microscopically observe the blood vessel density of tumor tissue sections. Results RI was efficiently expressed in NIH3T3 cells infected with pL NCX-ri virus and B16 melanoma tissues. The tumor weight (0.60 ± 0.226) g in pCLNCX-ri-treated group was significantly lower than that in control group (1.84 ± 1.6 4) g (P <0.01) Saline control group (1.77 ± 1.13) g (P <0.01). There was no significant difference (p> 0.05) between pL NCX virus control group (1.84 ± 1.6 4) g and saline control group (1.77 ± 1.13) g. Compared with the pL NCX virus control group and the saline control group, the vascular density of the tumor tissue in the pLNCX-ri virus-treated group was significantly reduced, but there was no significant difference between the two control groups. Conclusion Human ribonuclease inhibitor (RI) has a significant inhibitory effect on the growth of mouse B16 melanoma