目的 构建靶向CXCR7基因的CRISPR/Cas9基因编辑系统,并应用于HEK 293T细胞系。方法 设计两对靶向CXCR7基因的sgRNAs,分别插入PX458载体中,并转化DH5α大肠埃希菌。经菌液PCR和测序验证,挑选序列正确的sgRNA-CXCR7-PX458质粒,转染HEK 293T细胞,用流式分选转染阳性细胞,提取其DNA,PCR扩增后测序验证。结果 经测序验证,成功构建了靶向CXCR7基因的CRISPR/Cas9系统,转染HEK 293T细胞后,测序鉴定发现成功编辑CXCR7基因。结论 成功构建了靶向CXCR7的sgRNA-CXCR7-PX458质粒,可在HEK 293T上成功编辑CXCR7基因,为进一步的功能研究奠定基础。

Objective To construct the CRISPR/Cas9 gene editing system targeting C-X-C chemokine receptor 7 (CXCR7) gene and to edit CXCR7 gene in 293T cell line. Methods Two pairs of small guide RNAs (sgRNAs) targeting CXCR7 gene were designed and inserted into PX458 vector, which were transformed into host bacterium Escherichia coliDH5α. The correct sgRNA-CXCR7-PX458 plasmids were selected by PCR and further Sanger sequencing verification. HEK 293T cell line was transfected by DNA of sgRNA-CXCR7-PX458 plasmid. After 72 hours,GFP-positive cells were sorted by flow cytometry. We did DNA extraction of the GFP-positive cells and amplified the CXCR7 gene corresponding fragment by PCR and investigated the CXCR7 gene editing results by Sanger sequencing. Results The CRISPR/Cas9 system targeting CXCR7 gene was successfully constructed. After 293T cells were transfected, the CXCR7 gene was edited in HEK 293T cells successfully. Conclusion The sgRNA-CXCR7-PX458 plasmid targeting CXCR7 gene was successfully constructed. The CRISPR/Cas9 gene editing system targeting CXCR7 gene were used on the HEK 293T cell line, which lays a foundation for further study of BCOR function.

目的 研究雷公藤红素对人阿霉素耐药MCF-7/ADR乳腺癌细胞生长的作用。方法 采用MTT试验检测MCF-7/ADR细胞对阿霉素的耐药情况以及雷公藤红素对MCF-7/ADR细胞生长的影响;采用Annexin V-FITC/PI双染试验分析雷公藤红素对MCF-7/ADR耐药细胞凋亡的诱导作用;应用流式细胞周期分析检测雷公藤红素对MCF-7/ADR耐药细胞周期的影响。结果 MCF-7/ADR细胞对阿霉素耐药指数达14.54;而雷公藤红素能有效抑制阿霉素耐药细胞MCF-7/ADR的生长,并呈现浓度依赖性,作用48 h的IC50是1.04 μmol/L,MCF-7/ADR对雷公藤红素的耐药指数仅为0.87。2 μmol/L雷公藤红素作用8 h后,Annexin V-FITC染色阳性的MCF-7/ADR细胞比例较对照显著升高,差异有统计学意义(P＜0.05)。在1 μmol/L雷公藤红素作用24 h后,G1期细胞比例由对照(59.22±3.78)%升高至(77.44±4.21)%,而S期细胞比例由对照(37.51±2.91)%降至(19.65±2.25)%,差异有统计学意义(P＜0.05)。结论 雷公藤红素能激活MCF-7/ADR细胞凋亡的发生,并诱导MCF-7/ADR发生 G1/S细胞周期阻滞,从而对阿霉素耐药MCF-7/ADR细胞的生长发挥高效抑制作用。

Objective To investigate the effect of celastrol on the growth of adriamycin-resistant MCF-7/ADR breast cancer cells. Methods The resistance situation of MCF-7/ADR cells to adriamycin and the effect of celastrol on the growth of adriamycin-resistant MCF-7/ADR cells were evaluated by MTT assay. The effect of celastrol on apoptosis in MCF-7/ADR breast cancer cells was determined by annexin V-FITC/PI double staining. The effect of celastrol on cell cycle progression was determined by flow cytometry analysis. Results The resistance index of MCF-7/ADR cells to adriamycin was 14.54. After treatment with celastrol for 48 h, MCF-7/ADR cells displayed markedly inhibited growth in a dose-dependent manner, and calculated IC50 was 1.04 μmol/L. Celastrol decreased the resistance index of MCF-7/ADR from 14.54 to 0.87. The numbers of apoptotic MCF-7/ADR cells, as revealed by annexin V binding, significantly increased upon celastrol treatment (P＜0.05). Celastrol treatment caused an increase of cells in G1 phase from (59.22±3.78)% to (77.44±4.21)%, while the percentage of cells in S phase was decreased from(37.51±2.91)% to(19.65±2.25)%(P＜0.05). Conclusion These data demonstrated that celastrol induced apoptosis and G1/S cell cycle arrest in MCF-7/ADR cells and consequently displayed potent cytocidal effect on adriamycin-resistant MCF-7/ADR breast cancer cells.