分泌性表达载体V-pLNCX-s-hri对小鼠B16黑色素瘤生长的抑制作用  

Secretory expression vector V-pLNCX-s-hri inhibits the growth of mouse B16 melanoma

作　　者：盛甫秀[1] 樊建慧[1] 王冬梅[2] 赵宝昌[1] 崔秀云[1] 田余祥[1] 

机构地区：[1]大连医科大学生物化学与分子生物学教研室,辽宁大连116044 [2]大连医科大学机能学实验室,辽宁大连116044

出　　处：《癌症》2009年第3期236-243,共8页Chinese Journal of Cancer

摘　　要：背景与目的:已有研究表明从人胎盘中提取纯化的人核糖核酸酶抑制因子(human ribonuclease inhibitor,hRI)对小鼠某些实体肿瘤的生长具有明显的抑制作用。本研究的目的是构建分泌性表达载体V-pLNCX-s-hri,并观察其对小鼠B16黑色素瘤生长的抑制作用。方法:将合成的小鼠IgG信号肽碱基序列与hRI基因序列连接后,重组到逆转录病毒载体V-pLNCX上构建分泌性表达载体V-pLNCX-s-hri。将PA137细胞用于病毒包装,NIH3T3细胞用于测定病毒滴度。采用RT-PCR和Western blot法检测hRI基因的表达。构建小鼠荷B16黑色素瘤模型,注射V-pLNCX-s-hri,同时用生理盐水、V-pLNCX和V-pLNCX-hri作为对照,用肿瘤组织重和微血管密度来评价V-pLNCX-s-hri对小鼠B16黑素瘤生长的抑制作用。采用ELISA方法测定B16细胞培养上清和小鼠血清中RI含量。结果:V-pLNCX-s-hri对培养的B16细胞的感染效率为38.5%。在感染V-pLNCX-s-hri后的B16细胞中检测到RI mRNA和蛋白的表达。感染后的B16细胞的培养上清中hRI的含量为0.228μg/mL。V-pLNCX-s-hri组小鼠外周血中RI的含量为0.249μg/mL,明显高于生理盐水组、V-pLNCX组和V-pLNCX-hri组的0.035μg/mL、0.028μg/mL和0.169μg/mL(P值均<0.01)。生理盐水组、V-pLNCX组和V-pLNCX-hri组小鼠的瘤组织重分别为(1.90±1.12)g、(1.77±0.21)g和(1.10±0.46)g,显微镜下每10个视野的微血管平均数分别为89±6、87±7和41±8;而V-pLNCX-s-hri组的瘤组织重为(0.82±0.34)g,血管平均数为34±4。V-pLNCX-s-hri组与各对照组相比,其瘤组织重和血管数量的差异都有统计学意义(P值均<0.01)。结论:构建的分泌性表达载体V-pLNCX-s-hri能对B16细胞进行有效的感染,且在感染的B16细胞中分泌性高表达。V-pLNCX-s-hri对小鼠B16黑色素瘤的生长有明显的抑制作用,且效果优于V-pLNCX-hri。Background and Objective： Human ribonuclease inhibitor （hRI） extracted and purified from human placenta has been shown to remarkably inhibit some solid tumors in mice. This study was to construct V-pLNCX-s- hri, a secretory expression vector, and explore its inhibition effects on the growth of mouse B16 melanoma cells. Methods： The hRI gene sequence conjugated with the synthesized signal peptide of mouse IgG was cloned into the retroviral vector V-pLNCX to construct V-pLNCX-s-hri. The PA317 cells were used for viral package and NIH3T3 cells were employed to determine the viral titer. The expression of hRI gene was detected by RT-PCR and Western blot. The content of RI was determined by enzyme-linked immunoabsorption assay （ELISA）. The model of B16 melanoma-carrying mouse was established and received different treatments. The tumor weight and microvessle density （MVD） were assessed. Normal saline （NS）, V- pLNCX, and V-pLNCX-hri were used as controls. Results： The infection efficiency oi V-pLNCX-s-hri on cultured B16 cells reached 38.5%. mRNA and protein levels of hRI were detected in B16 cells infected by V-pLNCX-s-hri. The hRI content in the supernatant of infected B16 cells reached 0.228 μg/ mL. The hRI content in the peripheral blood of experimental mice was significantly higher in the V-pLNCX-s-hri group （0.249 μg/mL） than in the NS group （0.035 μg/mL）, V-pLNCX group （0.028 μg/mL） and V-pLNCX-hri group （0.169 μg/mL） （P〈0.01）. The tumor weight and MVD were significantly lower in the V-pLNCX-s-hri group compared with those in the NS, V-pLNCX and V-pLNCX-hri groups （P〉0.01）. Conclusions： V-pLNCX-s- hri can effectively infect B16 cells and induce high expression of hRI. V- pLNCX-s-hri is superior to V-pLNCX-hri in inhibiting the growth of B16 cells.

关 键 词：核糖核酸酶 抑制因子 信号肽 逆转录病毒 载体 黑色素瘤 基因治疗 小鼠 

分 类 号：R739.5[医药卫生—肿瘤]