摘要
目的:探讨慢病毒介导的CXCR7-shRNA转染人结肠癌细胞株SW620后对CXCR7蛋白表达的影响。方法:(1)设计并合成CXCR7的3对shRNA序列及1对阴性对照序列,与pSilencerTM4.1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;(2)将3种重组慢病毒载体及阴性对照分别感染人结肠癌细胞SW620,RT-PCR检测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;(3)MTT法检测转染CXCR7-shRNA对SW620细胞生长增殖的影响;(4)通过细胞划痕实验检测转染CXCR7-shRNA对SW620细胞侵袭迁移能力的影响;(5)Western blot检测转染CXCR7-shRNA对SW620细胞蛋白表达情况。结果:(1)测序证实3对慢病毒载体及1对阴性对照载体均包装成功,滴度分别为3.16×108TU/ml、4.27×108TU/ml、3.93×108TU/ml和2.95×108TU/ml;(2)3组慢病毒载体转染SW620细胞后,CXCR7 mRNA的表达量均较阴性对照组明显降低(P<0.05),其中CXCR7-shRNA-1对CXCR7的抑制率明显高于其他两组(P<0.05);(3)CXCR7-shRNA-1转染SW620后,肿瘤细胞的增殖程度显著减少,与空白组相比有显著性差异(P<0.05);(4)SW620细胞在划痕24h后,空白对照组和实验组的细胞迁移指数(MI)分别为(49.92±6.41)%和(29.13±5.38)%,有统计学意义(P<0.05),划痕48h后,对照组与实验组的MI分别为(96.52±7.44)%和(72.03±8.29)%,有统计学意义(P<0.05);(5)CXCR7-shRNA-1转染SW620细胞后,与空病毒载体组、空白组相比,CXCR7蛋白表达量明显降低,具有统计学意义(P<0.05)。结论:CXCR7-shRNA慢病毒表达载体转染SW620细胞后可有效下调CXCR7 mRNA和蛋白的表达水平并能够抑制肿瘤细胞的增殖与迁移,为下一步研究以CXCR7/CXCL12生物学轴为靶点的结直肠癌慢病毒基因沉默治疗打下了良好的基础,为结直肠癌的基因治疗提供了新方向。
Object： To investigate the change of CXCR7 protein expression after CXCR7-shRNA transfered into human colon cancer cell SW620 which is lentivirus-mediated. Methods： 1. To design and synthesis three shRNA sequence and one negative control sequence of CXCR7. To synthesis and construct recombinant lentiviral vector by pSilencerTM 4.1 system and CXCR7. To transfer the vector into HEK293T cell for packaging viral and to detect titer. 2. To transfer the three different recombinant lentiviral vector and one negative control vector into human colon cancer cell SW620. To detect expression and silence efficiency of CXCR7 mRNA by RT-PCR. The group expressing the best silence efficiency of CXCR7-shRNA is selected as expression vector for next experiment. 3. To detect the change of SW620 cell proliferation after CXCR7-shRNA transfection by MTF. 4. To detect the change of SW620 cell invasion and migration after CXCR7-shRNA transfection by cell scratching experiment. 5. To detect the SW620 protein expression after CXCRT-shRNA transfection by Western blot. Results： 1. Packaging the three different recombinant lentiviral vector and one negative control vector is successful by sequencing and titer is 3.16×108TU/ml, 4.27 ×108TU/ml, 3.93×108TU/ml, 2.95×108TU/ml respectively. 2. Expression of CXCR7 mRNA in three positive group is lower than negative control group after transfection（ P 〈 0.05 ）. The inhibition rate of CXCR7-shRNA-1 to CXCR7 is higher than the another group. 3. The tumor cell proliferation is reduced after CXCRT-shRNA-1 transfection into SW620 cell and there was significant difference comparing to control group （ P 〈 0.05 ）. 4. The migration index of control group and positive group are （49.92± 6.41 ） %, （ 29.13 ± 5.38 ） % respectively 24 hours after cell scratching experiment and it has statistical significance （ P 〈 0.05 ）. The migration index of control group and positive group are （ 96.52 ±7.44） % , （72.03± 8. 29 ）% respectively 48h hours after cell scratching experiment and it has statistical significance （P 〈 0.05 ）. 5. Expression of CXCR7 is reduced after transfection into SW620 cell comparing to empty virus vector group and control group and it has statistical significance （ P 〈 0.05 ）. Conclusion ： Expression of CXCR7 mRNA and CXCR7 protein is down-regulated effectively after CXCRT-shRNA recombinant lentiviral vector transfection into SW620 cell and proliferation and migration of tumor cell is inhibited effectively. It is a good foundation on next study of colon cancer RNAi therapy with lentiviral which target CXCR7/CXCL12 biological axis. And it is a new direction for colon cancer gene therapy.
出处
《中国生物工程杂志》
CAS
CSCD
北大核心
2014年第2期14-20,共7页
China Biotechnology
基金
辽宁省自然科学基金资助项目(201102124)
