Survivin启动子调控腺病毒介导富含亮氨酸重复和免疫球蛋白样结构域1基因治疗膀胱癌的实验研究

Gene therapy study on bladder cancer with recombinant adenoviral vector carrying LRIG1 gene driven by Survivin promoter

目的探讨Survivin启动子驱动的重组腺病毒Ad—Surp—LRIG1对膀胱癌的治疗作用及机制。方法用Ad—Surp—LRIG1和Ad—LRIG1分别感染人膀胱癌细胞BIU87及人膀胱上皮永生化细胞SV—HUC-1，根据报告基因表皮生长因子受体（EGFR）表达的不同计算病毒的细胞转染率。MTF法评估Ad—Surp—LRIG1和Ad—LRIG1对BIU87细胞生长的抑制作用。建立膀胱癌裸鼠移植瘤模型，将成瘤裸鼠随机分为3组，Ad—Surp—LRIGI组（尾静脉注射Ad—Surp—LRIG1上清液）、AD-LRIG1组（尾静脉注射Ad—LRIG1上清液）、PBS组（尾静脉注射PBS），观察各组裸鼠移植瘤生长情况并绘制肿瘤生长曲线；RT—PCR检测各组肿瘤组织中LRIG1和EGFRmRNA的表达。结果当感染复数（MOI）=25时，Ad—Surp—LRIG1在BIU87细胞中的转染效率为74．56％，在SV—HUC-1细胞中转染率为0，差异有统计学意义（f=58．640，P=0．ooo）；Ad—LRIGl在BIU87细胞中的转染效率为68．27％，在SV-HUC-1细胞中转染率为72．52％，差异无统计学意义（x2=0．075，P=0．784）。Ad—Surp—LRIG1和Ad—LRIG1在BIU87细胞中的转染效率相比，差异无统计学意义（X2=0．016，P=0．898）。与PBS液对照组比较，Ad-Surp—LRIG1和Ad-LRIG1均能明显抑制BIU87细胞的生长，转染4d后这一差异有统计学意义（F=15．960，P=0．000），而Ad—Surp—LRIG1组和Ad—LRIG1组细胞生长速度无明显差异。Ad—Surp-LRIG1组肿瘤生长速度明显慢于其他2组，治疗结束后Ad—Surp—LRIG1组肿瘤质量小于其他2组，差异有统计学意义（F=97．860，P=0．000），Ad—LRIG1组与PBS组相比差异无统计学意义（t=1．73，P=0．06）。与其他2组比较，Ad—Surp—LRIG1组LRIG1mRNA表达水平明显升高（F=851．343，P=0．000），而EGFRmRNA表达水平则显著降低（F=591．205，P=0．000）。结论Ad—Surp-LRIG1能选择性转染人膀胱癌细胞BIU87，在体内外均能明显抑制膀胱癌的生长，其机制可能部分与LRIG1能下调EGFR的表达有关。

Objective To investigate the treatment efficiency and mechanism of recombinant adenoviral vector carrying LRIG1 gene driven by Survivin promoter for bladder cancer. Methods Human bladder cancer cell line BIU87 and immortalized human bladder epithelial cells SV-HUC-1 were infected with Ad-Surp-LRIG1 and Ad-LRIG, respectively. The selective infection efficiency of Ad-Surp-LRIG1 and Ad- LRIG were evaluated by checking the expression of epidermal growth factor receptor （ EGFR ）. The MTT method was used to test cell growth inhibition ratio of Ad-Surp-LRIG1 and Ad-LRIG. Heterotransplanted models of human bladder cancer cell line BIU87 cells in nude mice were established. The mice were randomly divided into 3 groups during the experiment： Ad-Surp-LRIG1 group received viral supernatant solution of Ad-Surp-LRIG1 by tail vein injection; Ad-LRIG group received viral supernatant solution of Ad- LRIG by tail vein injection; and PBS group received phosphate buffer solution （PBS）. The growth of tumors were observed and the growth curve was mapped. The expression of LRIG1 and EGFR were examined by reverse transcription PCR （ RT-PCR）. Results When Multiplicity of infection was 25, the transfection efficiency of Ad-Surp-LRIG1 was 74. 56% in BIU87 cells and 0 in SV-HUC-1 cells （ X2 = 58. 640, P = 0. 000）, while the transfection efficiency of Ad-LRIG was 68.27% in BIU87 ceils and 72. 52% in SV-HUC- 1 cells （ X： = 0. 075, P = 0. 784）. The transfection efficiency difference of Ad-Surp-LRIG1 and Ad-LRIG in BIU87 cells wasn＇t statistically significant （ X2 = 0. 016, P = 0. 898 ）. Compared with PBS, Ad-Surp-LRIG1 and Ad-LRIG1 could inhibit BIU87 cell growth, the difference was significant in 4 days after transfection （F = 15. 960, P = 0. 000）. There wasn＇t significant difference in cell growth rate of Ad-Surp-LRIG1 group and Ad-LRIG1 group. The tumor growth rate in Ad-Surp-LRIG1 group was slower than that in the other 2 groups. The tumor quality in Ad-Surp-LRIG1 was lighter than that in the other two groups, the differences were statistically significant （ F = 97. 860, P = 0. 000）, the quality difference in Ad-LRIG1 group and PBS group wasn＇t statistically significant difference （t = 1.73, P = 0. 06）. Compared with Ad-LRIG1 group and PBS group, the mRNA expression of LRIG1 was obviously up-regulated and that of EGFR was down- regulated in Ad-Surp-LRIG1 group （ P 〈 0. 01 ）. Conclusions The recombinant adenoviral vector of Ad- Surp-LRIG1 could selectively transfected BIU87 cells, which could inhibit significantly the growth of bladder cancer in vivo and in vitro, the mechanism may be partly LRIG1 can downgrade the expression of EGFR.