论文部分内容阅读
目的:探讨细胞外基质蛋白1(extracellular matrix protein 1,ECM1)对乳腺癌细胞株MCF-7和人脐静脉内皮细胞(human umbilical vein endothelial cell,HUVEC)增殖的影响。方法:构建ECM1-pEGFP-N2真核表达载体,采用PCR方法,扩增出ECM1基因,用BglⅡ和Kpn Ⅰ双酶切ECM1基因和pEGFP-N2载体,连接酶切目的片段,转化大肠杆菌DH5α,筛选阳性克隆酶切、测序鉴定;利用脂质体介导的转染技术转染MCF-7细胞,药物G418筛选稳定转染细胞株,荧光显微镜检测报告基因表达产物EGFP,免疫组化检测ECM1蛋白表达。用MTT比色法分析ECM1对MCF-7和HUVEC增殖的影响。结果:成功构建了ECM1-pEGFP-N2真核表达载体,并在MCF-7中稳定表达;MTT比色法检测MCF-7增殖结果显示未转染组、空载体转染组和ECM1转染组的D570值分别为0.95±0.07、0.97±0.09和1.03±0.12,三者无明显差异;MTT比色法检测HUVEC增殖结果示培养液组、空载体转染上清组、ECM1转染上清组HUVEC D570值分别为0.89±0.06、0.92±0.09和1.39±0.10,差异具有显著性意义(P<0.01)。结论:ECM1对乳腺癌细胞株MCF-7的增殖无影响,但能显著促进血管内皮细胞体外增殖。
Objective: To investigate the effect of extracellular matrix protein 1 (ECM1) on the proliferation of human breast cancer cell line MCF-7 and human umbilical vein endothelial cells (HUVECs). Methods: The ECM1-pEGFP-N2 eukaryotic expression vector was constructed and the ECM1 gene was amplified by PCR. The ECM1 gene and the pEGFP-N2 vector were double-digested with BglII and Kpn Ⅰ, ligated and digested with the target fragment, and then transformed into E. coli DH5α, The positive clones were screened and identified by sequencing. MCF-7 cells were transfected by lipofectamine mediated transfection. The stable transfected cell lines were screened by G418. EGFP was detected by fluorescence microscopy. The expression of ECM1 protein was detected by immunohistochemistry expression. The effect of ECM1 on the proliferation of MCF-7 and HUVEC was analyzed by MTT colorimetry. Results: The eukaryotic expression vector of ECM1-pEGFP-N2 was successfully constructed and stably expressed in MCF-7. The proliferation of MCF-7 was detected by MTT colorimetric assay. The results of non-transfected group, empty vector transfected group and ECM1 transfected group The D570 values were 0.95 ± 0.07, 0.97 ± 0.09 and 1.03 ± 0.12, respectively. There was no significant difference among the three groups. The proliferation of HUVECs was detected by MTT colorimetric assay. The culture medium group, empty vector transfected supernatant group, ECM1 transfection supernatant group HUVEC D570 values were 0.89 ± 0.06,0.92 ± 0.09 and 1.39 ± 0.10, the difference was significant (P <0.01). Conclusion: ECM1 has no effect on the proliferation of breast cancer cell line MCF-7, but can significantly promote the proliferation of vascular endothelial cells in vitro.