【摘要】 背景与目的:CCL28,又称为黏膜相关上皮趋化因子(mucosa-associated epitheliachemokine,MEC),是趋化因子CC家族中的一员。研究表明,CCL28与肿瘤发生、发展有关,但对其在乳腺癌中的功能还知之甚少。本文旨在探讨CCL28过表达在体外对乳腺癌细胞MDA-MB-231增殖的影响。方法:构建CCL28过表达载体pBabe-CCL28表达质粒,将重组的pBabe-CCL28基因和空载体pBabe转染细胞Phoenix,利用逆转录病毒感染乳腺癌细胞MDA-MB-231,通过嘌呤霉素药物筛选获得稳定表达CCL28的MDA-MB-231/CCL28细胞系和感染空载体的MDA-MB-231/vector细胞系,利用半定量RT-PCR、实时荧光定量PCR及蛋白质印迹法(Western blot)检测CCL28的基因表达和蛋白质水平;利用CCK8、软琼脂克隆培养及流式细胞术检测CCL28过表达后细胞增殖、克隆形成能力及凋亡的变化;利用Western blot检测CCL28过表达后凋亡相关蛋白的变化。结果:成功建立了稳定表达CCL28的细胞系MDA-MB-231/CCL28,与空载体对照组相比,pBabe-CCL28质粒感染入细胞后CCL28基因表达水平明显增高,说明CCL28 cDNA重组质粒构建成功。CCK8及软琼脂克隆实验结果显示,MDA-MB-231/CCL28的增殖能力明显高于MDA-MB-231/vector,差异有统计学意义(P<0.05)。流式细胞术结果显示,MDA-MB-231/CCL28细胞凋亡比例减少,差异有统计学意义(P<0.05)。Western blot结果显示,抗凋亡蛋白Bcl-2的表达增加。结论:CCL28过表达可以显著促进人乳腺癌细胞MDA-MB-231的增殖能力,抑制乳腺癌细胞凋亡,后者可能与Bcl-2上调有关。CCL28有望成为临床治疗乳腺癌的潜在基因靶点。更多还原

【Abstract】 Background and purpose: CCL28,also known as mucosa-associated epithelial chemokine(MEC),is a member of the CC subfamily.Studies have shown that CCL28 is associated with tumor progress.However,little is known about its function in breast cancer.In this study,we construct the expression vector of cDNA encoding CCL28 gene and observe its effects on cell growth ability of human breast cancer cell line MDA-MB-231 in vitro.Methods: The pBabe-CCL28 expression vector was constructed,then transfected into Phoenix cells and used to infect cultured breast cancer cells MDA-MB-231 by using retroviruses infection method.The positive clones were selected with puromycin to establish stable transfectants MDA-MB-231/CCL28.Control cell lines were generated by infection with viruses containing empty vector by following the same protocol.CCL28 mRNA and protein in MDA-MB-231/CCL28 were determined by semi-quantitative RT-PCR,realtime PCR and western blot respectively.Cell proliferation was tested by cell counting kit 8(CCK8).Cell apoptosis percentage was observed by flow cytometry.The ability of colony formation was tested by soft agar.The expression of apoptosis associated protein was measured by Western blot.Results: The cell line MDA-MB-231/CCL28 stabling expresses chemokine CCL28 was successfully constructed.In the MDA-MB-231/CCL28 cells,cell proliferation was increased,and cell apoptosis percentage was decreased.Western blot showed that the expression of anti-apoptotic protein Bcl-2 was decreased.Conclusion: Our data provides strong evidence that overexpression of CCL28 can promote the proliferative ability of MDA-MB-231 and inhibit the cell apoptosis,which may be regulated by Bcl-2.Therefore,CCL28 could be a important target for the treatment of breast cancer.更多还原