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[目的]探讨骨唾液酸蛋白(BSP)与人乳腺癌细胞黏附性的相关性。[方法]构建pCDNA3.1-myc-hisA(+)-BSP真核表达载体,在COS7细胞中表达并纯化BSP全长蛋白。以纤黏连蛋白、胶原和重组BSP蛋白包被96酶标板,细胞黏附实验检测MCF-7和MDA-MB-231在上述基质的黏附性。构建BSP正义和反义表达载体,及同pCDNA3.1(-)空载体稳定转染人乳腺癌细胞MCF-7获得稳定细胞株。细胞黏附实验检测不同BSP表达水平的各组MCF-7稳定细胞株在纤黏连蛋白(FN)和胶原(collagen)上的黏附性。免疫细胞化学检测各组MCF-7稳定细胞株中E-cadherin的表达。[结果]成功表达人骨唾液酸蛋白并证实其具有黏附性。分析不同BSP表达水平的乳腺癌细胞在不同基质上黏附性,结果显示转染正义BSP的MCF-7细胞在不同基质上的OD595值(FN:1.93±0.06;collagen:1.82±0.15)显著高于反义组(FN:0.83±0.19;collagen:0.89±0.08)、空载体转染组(FN:1.49±0.18;collagen:1.44±0.09)和亲本细胞组(FN:1.52±0.27;collagen:1.42±0.12),有统计学差异(P<0.05)。转染正义BSP的MCF-7细胞中E-cadherin的表达与其它组别相比明显降低。[结论]BSP以RGD序列介导细胞黏附,其表达水平与乳腺癌细胞黏附性相关。
[Objective] To investigate the correlation between bone sialoprotein (BSP) and adhesion of human breast cancer cells. [Methods] The pCDNA3.1-myc-hisA(+)-BSP eukaryotic expression vector was constructed, and the full-length BSP protein was expressed and purified in COS7 cells. 96-well plates were coated with fibronectin, collagen, and recombinant BSP proteins. Cell adhesion assays were performed to determine the adhesion of MCF-7 and MDA-MB-231 in the above matrix. BSP sense and antisense expression vectors were constructed and stable cell lines were obtained by stable transfection of human breast cancer MCF-7 cells with pCDNA3.1(-) empty vector. Cell adhesion assays were used to detect the adhesion of various stable MCF-7 cell lines with different BSP expression levels on fibronectin (FN) and collagen. The expression of E-cadherin in MCF-7 stable cell lines was detected by immunocytochemistry. [Results] The human bone sialoprotein was successfully expressed and its adhesion was confirmed. Analysis of different BSP expression levels of breast cancer cells adhered to different matrices. The results showed that the OD595 values (FN: 1.93±0.06;collagen:1.82±0.15) of MCF-7 cells transfected with sense BSP were significantly higher on different matrices. Antisense group (FN: 0.83±0.19; collagen: 0.89±0.08), empty vector transfection group (FN: 1.49±0.18; collagen: 1.44±0.09) and parental cell group (FN: 1.52±0.27; collagen: 1.42± 0.12), statistically significant (P<0.05). The expression of E-cadherin in MCF-7 cells transfected with sense BSP was significantly lower than that of other groups. [Conclusion] BSP mediates cell adhesion by RGD sequence, and its expression level is related to the adhesion of breast cancer cells.