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目的探讨趋化因子 CXCL12及其受体 CXCR4对卵巢上皮性癌(卵巢癌)细胞增殖、迁移和侵袭能力的影响。方法采用 RT-PCR 技术和免疫细胞化学染色法检测卵巢癌细胞株CAOV3细胞中 CXCL12、CXCR4 mRNA 和蛋白的表达,以及 CXCL12(100 ng/ml)作用后 CAOV3细胞中整合素β1和血管内皮生长因子 C(VEGF-C)mRNA 的表达。实验分为6组:对照组(未加药),实验组1(加100 ng/ml 的 CXCL12),实验组2(加10 ng/ml 的 CXCL12),实验组3(加100 ng/ml 的 CXCL12和10μg/ml 的 CXCR4抗体),实验组4(加100 ng/ml 的 CXCL12和1μg/ml 的 CXCR4拮抗剂——AMD3100),实验组5(加10μg/ml 的 CXCR4抗体或卵巢癌腹水)。采用四甲基偶氮唑蓝比色法检测CXCL12对 CAOV3细胞增殖的影响。以穿膜小室为模型,采用重组细胞基底膜迁移、侵袭实验检测CXCL12和卵巢癌腹水对 CAOV3细胞迁移、侵袭的影响。结果 CAOV3细胞中,CXCR4蛋白呈棕黄色强阳性表达,CXCR4 mRNA 的表达水平为0.70±0.10,经100 ng/ml 的 CXCL12作用24 h 时 CXCR4mRNA 的表达水平(1.24±0.14)显著增加(t=-7.1088,P=0.0021);CXCL12蛋白和 mRNA 均无表达。100 ng/ml 的 CXCL12作用3 h 时整合素β1 mRNA 表达水平即显著增加(作用前后分别为0.53±0.10、1.53±0.16,P<0.01),24 h 时 VEGF-C mRNA 表达水平显著增加(作用前后分别为0.52±0.09、1.11±0.15,P<0.01)。对 CAOV3细胞的增殖作用,实验组1分别与对照组和实验组2比较均明显增强(3组分别为0.428±0.051、0.325±0.045、0.328±0.039,P<0.05);实验组1分别与实验组3和实验组4比较均明显被抑制(后两组分别为0.356±0.031、0.373±0.029,P<0.05);而实验组5(0.349±0.038)与对照组比较,差异则无统计学意义(P>0.05)。对 CAOV3细胞的迁移和侵袭作用,实验组1分别与对照组和实验组2比较均明显增强[迁移细胞数分别为(523.3±25.2)、(108.0±7.2)、(211.7±24.7)个,侵袭细胞数分别为(39.3±4.0)、(4.0±1.0)、(15.7±3.1)个;P 均<0.01];实验组1分别与实验组3和实验组4比较均明显被抑制(P<0.05);但实验组5[即加入卵巢癌腹水组;迁移细胞数为(706.6±30.6)个,侵袭细胞数为(61.7±7.6)个]显著多于实验组1(P<0.05)。结论 CXCL12可促进卵巢癌细胞增殖、迁移和侵袭,并上调整合素β1和 VEGF-CmRNA 的表达,此作用可被 CXCR4抗体抑制,表明 CXCL12在卵巢癌的生长和转移中发挥一定作用。
Objective To investigate the effects of chemokine CXCL12 and its receptor CXCR4 on the proliferation, migration and invasion of ovarian epithelial carcinoma (ovarian cancer) cells. Methods The expressions of CXCL12 and CXCR4 mRNA and protein in ovarian cancer cell line CAOV3 were detected by RT-PCR and immunocytochemical staining, and the expressions of integrin β1 and vascular endothelial growth factor (VEGF) in CAOV3 cells after CXCL12 (100 ng / ml) C (VEGF-C) mRNA expression. The experiment was divided into 6 groups: control group (no drug addition), experimental group 1 (plus CXCL12 of 100 ng / ml), experimental group 2 (CXCL12 plus 10 ng / ml), experimental group 3 CXCL4 and 10μg / ml CXCR4 antibody), experimental group 4 (CXCL12 plus 100ng / ml and CXCR4 antagonist - AMD3100) at 1μg / ml, experimental group 5 (CXCR4 plus 10μg / ml or ascites of ovarian cancer) . The effect of CXCL12 on the proliferation of CAOV3 cells was detected by MTT assay. Using the transmembrane cell as a model, the effects of CXCL12 and ascites of ovarian cancer on the migration and invasion of CAOV3 cells were detected by using the migration and invasion of recombinant cell basement membrane. Results The expression of CXCR4 protein in CAOV3 cells was strongly brownish yellow and the expression level of CXCR4 mRNA was 0.70 ± 0.10. The CXCR4 mRNA expression level (1.24 ± 0.14) was significantly increased after treated with 100 ng / ml CXCL12 for 24 h (t = - 7.1088, P = 0.0021); CXCL12 protein and mRNA were not expressed. The level of integrin β1 mRNA increased significantly at 3 h following treatment with 100 ng / ml CXCL12 (0.53 ± 0.10, 1.53 ± 0.16, respectively; P <0.01), and the expression of VEGF-C mRNA increased significantly at 24 h Respectively, before and after 0.52 ± 0.09,1.11 ± 0.15, P <0.01). The proliferation of CAOV3 cells in experimental group 1 were significantly increased compared with control group and experimental group 2 (0.428 ± 0.051, 0.325 ± 0.045 and 0.328 ± 0.039, respectively, P <0.05) in experimental group 1 and experimental group 1 Group 3 and group 4 were significantly inhibited (the latter two groups were 0.356 ± 0.031,0.373 ± 0.029, P <0.05); while the experimental group 5 (0.349 ± 0.038) compared with the control group, the difference was not statistically significant (P> 0.05). The migration and invasion of CAOV3 cells in experimental group 1 were significantly enhanced compared with control group and experimental group 2 [523.3 ± 25.2, (108.0 ± 7.2) and (211.7 ± 24.7, respectively, (39.3 ± 4.0), (4.0 ± 1.0) and (15.7 ± 3.1), respectively, P <0.01]. The experimental group 1 was significantly inhibited compared with experimental group 3 and experimental group 4 (P <0.05) ). However, the number of migrating cells in experimental group 5 [ie, ascites group (706.6 ± 30.6) and invasive cell number (61.7 ± 7.6)] was significantly higher than that in experimental group 1 (P <0.05). Conclusion CXCL12 can promote the proliferation, migration and invasion of ovarian cancer cells and upregulate the expression of integrin β1 and VEGF-C mRNA. This effect can be inhibited by CXCR4 antibody, indicating that CXCL12 plays a role in the growth and metastasis of ovarian cancer.