慢病毒载体介导的CXCR 7-shRNA联合重楼总皂苷对胃癌细胞SGC 7901特异性靶向干预的实验研究  

Specific targeted-intervention effect of CXCR 7-shRNA mediated by low viral carrier plus Rhizoma Paridis total saponin on tumor cells:an experimental study

作　　者：赵东文 李谌[3] 王海龙[3] 陆航[3] 

机构地区：[1]辽宁医学院,辽宁锦州121001 [2]瓦房店市中心医院普外科,辽宁瓦房店116300 [3]辽宁医学院附属第一医院普外科,辽宁锦州121001

出　　处：《中国生化药物杂志》2014年第2期22-25,共4页Chinese Journal of Biochemical Pharmaceutics

基　　金：辽宁省自然科学基金资助项目(201102124)

摘　　要：目的探讨慢病毒介导的CXCR7-shRNA转染人胃癌细胞株SGC7901后重楼总皂苷干预对CXCR7蛋白表达的影响。方法设计并合成CXCR7的3条shRNA序列及1条阴性对照序列,与pSilencerTM 4.1系统合成构建重组慢病毒载体,转染HEK293T细胞包装病毒并检测滴度;将3种重组慢病毒载体及阴性对照分别感染人胃癌细胞SGC7901,RT-PCR检测用药前后CXCR7 mRNA的表达情况,测定沉默效率,筛选沉默效率最高的一组CXCR7-shRNA作为后续实验表达载体;MTT法检测转染CXCR7-shRNA对SGC7901细胞的增殖的影响以及重楼总皂苷干预后的影响;Western blot检测转染CXCR7-shRNA后SGC7901细胞蛋白表达情况以及重楼总皂苷干预的影响。结果测序证实3种慢病毒载体及1组阴性对照载体均包装成功,滴度分别4.9×108pfu/mL、3.6×108pfu/mL、5.2×108pfu/mL和2.0×108pfu/mL;3组慢病毒载体转染SGC7901细胞后,CXCR7 mRNA的表达量均较阴性对照组显著降低(P<0.05),其中CXCR7-shRNA-1组和重楼总皂苷组对CXCR7的抑制率显著高于其他2组(P<0.05);CXCR7-shRNA-1转染SGC7901后,肿瘤细胞的生长增殖下降,与其他组相比有显著性差异(P<0.05);CXCR7-shRNA-1转染SGC7901后,与空病毒载体组、空白组相比,CXCR7蛋白表达量显著降低(P<0.05)。结论成功构建了3种CXCR7-shRNA慢病毒表达载体。转染SGC7901细胞以及采用重楼总皂苷处理均可有效下调CXCR7 mRNA和蛋白的表达水平,并能够抑制肿瘤细胞的增殖与侵袭。因此可认为CXCR7基因在该机制中起到了至关重要的作用,为我们进一步研究以CXCR7/CXCL12生物学轴为靶点的胃癌基因治疗奠定了基础。Objective To investigate the CXCR 7 protein expression when CXCR 7-shRNA transfected into human gastric cancer cell which mediated with lentivirus vector combined with Rhizoma Paridis Total Saponin. Methods Three shRNA sequences of CXCR 7 and one negative control sequence were designed and synthesized, and recombinant lentiviral vectors with pSilencerTM 4.1 system were established. Transfection of HEK 293 T cells and packaging viral were finished and the titers were detected. Transfection of all recombinant lentiviral vectors and negative control vector were finished and expression of CXCR 7 mRNA were detected by RT-PCR method. Silence efficiency in groups were determined and the expression vector with highest silence efficiency was selected for next experiments. To detect the effect of SGC 7901 cell proliferation by CXCR 7-shRNA transfection and combined with Rhizoma Paridis Total Saponin intervention with MTT. To detect the effect of SGC 7901 cell expression of protein by CXCR 7-shRNA transfection and combined with Rhizoma Paridis Total Saponin intervention with Western blot. Results The packaging of three lentiviral vector and negative control sequence are successful which is confirmed by gene sequencing and the titer are 4.9×108 pfu/mL, 3.6×108 pfu/mL, 5.2×108 pfu/mL, 2.0×108 pfu/mL respective. The expression quantity of CXCR 7 mRNA in positive groups are lower than negative control group(P<0.05)and inhibition ratio to CXCR 7 in CXCR 7-shRNA-1 and combined with Rhizoma Paridis Total Saponin intervention group is higher than the other two groups(P<0.05). The proliferation level of tumour cell is significant reduction after CXCR 7-shRNA-1 transfection and have a significant difference comparing to the group without transfection(P<0.05). The expression of CXCR 7 protein is significant reduction after CXCR 7-shRNA-1 transfection comparing to the group without virus vector and negative control group and have a significant difference(P<0.05). Conclusion The construction of three CXCR 7-shRNA lentiviral expressi

关 键 词：胃癌 CXCR 7 CXCL12 慢病毒载体 SHRNA 

分 类 号：R735.2[医药卫生—肿瘤]