5HRE增强子和hTERT启动子联合调控CDX2基因对裸鼠移植瘤的抑制作用

Effects of regulation of CDX2 gene by 5HRE enhancer and hTERT promoter on growth of colorectal transplanted tumors in nude mice

目的探讨5HRE增强子和hTERT启动子联合调控CDX2基因对结肠癌LoVo细胞裸鼠皮下移植瘤的抑制作用,为结肠癌生物治疗提供实验依据。方法将复苏前期筛选的稳定表达pLVX-hTERTp-CDX2-3FLAG(hC)的LoVo细胞(hC/LoVo)、空LoVo细胞、稳定表达pLVX-5HRE-hTERTp-3FLAG(5Hh)的LoVo细胞(5Hh/LoVo)及转染pLVX-5HRE-hTERTp-CDX2-3FLAG(5HhC)的LoVo细胞(5HhC/LoVo),接种各组细胞制备裸鼠皮下移植瘤模型,观察构建的载体对LoVo细胞移植瘤生长的影响;免疫组化法观察移植瘤CDX2和Ki-67的表达情况。结果成功复苏转染5HhC的LoVo细胞及各组对照LoVo细胞。各组裸鼠皮下接种肿瘤细胞后均成功致瘤,成瘤率为100%;5HhC组种植瘤生长速度显著低于其他组(P<0.05);终点时间第18天时,5HhC组裸鼠皮下种植瘤的体积明显小于其他各组(P<0.05);5HhC组种植瘤质量为(0.20±0.16)g,明显低于hC/LoVo组(0.34±0.21)g,空LoVo细胞组(0.61±0.19)g及5Hh/LoVo组(0.55±0.27)g(P<0.05);5HhC组种植瘤中CDX2表达为(87±7)%,明显低于hC/LoVo组(55±4)%,空LoVo细胞组(15±3)%及5Hh/LoVo组(17±5)%(P<0.05);5HhC组种植瘤中Ki-67表达为(7±7)%,明显低于hC/LoVo组(13±2)%,空LoVo细胞组(38±5)%及5Hh/LoVo组(85±9)%(P<0.05)。结论构建的双靶向调控载体5HhC可以抑制结肠癌LoVo细胞裸鼠移植瘤的生长。

Objective To verify the effect of pLVX-5HRE-hTERTp-CDX2-3FLAG（5HhC）containing the tumor suppressor gene CDX2 regulated by the hypoxia-induced enhancer（HRE） and the hTERT promoter on growth of colorectal transplanted tumors in mude mice. Methods The LoVo cells with stable expression of pLVX-hTERTp-CDX2-3FLAG （hC）, pLVX-SHRE-hTERTp-3FLAG （5Hh）and pLVX-SHRE-hTERTp-CDX2-3FLAG（5HhC）were obtained by transfection technique, named as hC/LoVo,5Hh/LoVo and 5HhC/Lo- Vo. A xenograft tumor model was established and tumor growth was observed. The expression of CDX2 and Ki-67 protein in tumor tissues were detected by imm^nohistochemistry. Results The xenograft tumor models of human colorectal carcinoma cells in hC/LoVo, LoVo ,5Hh/LoVo and 5HhC/LoVo groups were established successfully. The tumor volume in 5HhC/EoVo group was significantly smal- ler than in the other three groups at every time point （P 〈 0.05 ）. At the termination of observation, the tumor weight in 5HhC/Lovo group was significantly smaller than in hC/LoVo, LoVo and 5 Hh/LoVo groups respectively [ （0.20± 0.16 ） g vs （ 0. 34± 0.21 ） g, （0. 61 ±0. 19）g, （0. 55 ± 0. 27 ）g, P 〈 0.05 ]. The expression of CDX2 protein by immunohistochemistry in tumor tissues in 5HhC/LoVo group [ （ 87 ±7 ） % ] were higher than that of hC/LoVo [ （ 55 ±4 ） % ], LoVo [ （ 15± 3 ） % ] and 5Hh/LoVo [ （ 17 ± 5 ） % ], respectively （P 〈 0. 05 ）, while the expression of Ki-67 protein in tumor tissues in 5 HhC/LoVo group was lower than in the other three groups [ （7 ±7）%vs （13 ±2）%,LoVo（38 ±5）% and 5Hh/LoVo（85 ±9）% ,P 〈0.05]. Conclusion The gene therapy vector 5HhC may in- duce the growth of colorectal transplanted tumors in mude mice.