婆娑双树基因4干扰载体对儿童睾丸卵黄囊瘤的转染及干扰效果评价  

Efficacy of RNA interference after a transfection of SALIA shRNA vector in pediatric testicular yolk sac tumor

作　　者：吕敏[1] 张浩川[1] 同毓龙 林孝坤 陈聪德[1] 陈肖鸣[1] 

机构地区：[1]温州医科大学附属第一医院小儿外科,325000

出　　处：《中华小儿外科杂志》2016年第9期673-676,共4页Chinese Journal of Pediatric Surgery

摘　　要：目的构建婆娑双树基因SALL4的特异性干扰载体并转染儿童睾丸卵黄囊瘤细胞，评价干扰效果，为研究SALL4基因功能提供实验工具。方法针对不同干扰位点，设计4条特异性siRNA和1条阴性对照序列。构建SALL4干扰质粒载体pGPU6／GFP／Neo-SALL4 shRNA，分空白对照组（B1ank组）、阴性对照组（NC组）、单加转染试剂组（MOcK组）、干扰组（SALIA—homo-483、SALL4-homo-1874、SALL4-homo-951、SALL4-homo-3110），分别转染体外培养的儿童睾丸卵黄囊瘤细胞，采用荧光定量PCR和Western bolt技术比较各组细胞SALL4基因mRNA及蛋白表达情况，筛选出最佳干扰序列。结果荧光定量PCR结果显示，Blank组、NC组、MOCK组、干扰组（SALIA—homo-483、SALL4-homo-951、SALIA-homo-1874、SALL4-homo-3110）SALL4基因mRNA的表达水平分别为100％、（118．0±18．0）％、（128．0±15．0）％、（91．0±21．O）％、（25．0±8．10）％、（59．0±11．0）％、（49．0±13．0）％。SALL4-homo-951、SALIA-homo-3110相比Blank组、NC组、MOCK组mRNA表达下降，差异有统计学意义（F=3．63，P〈O．05）。Westernbolt的检测结果显示Blank组、NC组、MOCK组、干扰组（SALIA—homo-483、SALIA—homo-951、SALIA—homo-1874、SALL4-homo-3110）SALIA基因蛋白表达水平分别为1、1．1±0．1、1．3±0．3、1．7±0．4、0．2±0．1、0．9±0．2和0．4±0．1。相对Blank组、NC组、MOCK组，SALIA干扰组中SALIA-homo-951、SALL4-homo-3110均能降低蛋白表达，其中sALL4-homo-951降低最多，差异有统计学意义（F=7．96，P〈0.05）。结论SALL4-homo-951、SALIA-homo-3110均能有效干扰SALL4基因在儿童卵黄囊瘤中的表达，其中SALIA—homo-951干扰效果最佳，可用于下一步实验研究。Objective To construct the special SALL4 shRNA vector for transfeetion into cultured testicular yolk sac tumor cells, evaluate the effect of interference and provide experimental rationales for elucidating the function of SALL4. Methods Four special interference siRNAs and 1 negative siRNA were designed for different interference loci. And pGPU6/GFP/Neo-SALL4 shRNA vector was constructed and transfected into blank, negative（NC）, MOCK and interference groups of testicular yolk sac tumor cells. The expression quantities of SALIA mRNA and protein were measured by reverse transcription-quantitative polymerase chain reaction （RT-qPCR） and Western blot. And the optimal interference sequence was selected. Results The mRNA expression magnitudes of Blank, negative（NC）, MOCK group, SALL4-homo-483, SALL4-homo-951, SALL4-homo-1874 and SALL4- homo-3110 were（100） %, （118. 0 ± 18. 0）%, （128. 0 ± 15.0）%, （91.0 ± 21.0）%, （25.0 ± 8. 0）%, （59.0 ± 11.0）% and（49.0 ± 13.0）% respectively. As compared with Blank, NC and MOCK, SALIA-homo-951 and SALIA-homo-3110 had lower levels of mRNA expression. And the protein expressions of Blank, NC, MOCK group, SALL4-homo-483, SALL4-homo-951, SALIA-homo-1874 and SALL4-homo-3110 were 1, 1.1±0. 1, 1.3±0.3, 1.7±0.4, 0. 2±0. 1, 0.9±0.2 and 0.4±0. 1 respectively. SALL4-homo-951 and SALL4-homo-3110 lowered the SALL4 protein expression. Conclusions SALIA-homo-951 and SALIA-homo-3110 may interfere with the expression of SALL4 intesticular yolk sac tumor cells. Especially the former has the optimal efficacy.

关 键 词：睾丸卵黄囊瘤 基因表达 转染 

分 类 号：R737.21[医药卫生—肿瘤]