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骨形态发生蛋白9(bone morphogenetic proteins,BMP9)是骨形态发生蛋白(bone morphogenetic proteins,BMPs)家族的一员,具有多种生物学功能。本实验主要是从体内、外两个层面模拟的脂肪微环境中,研究BMP9对乳腺癌细胞生长的影响。将前脂肪细胞3T3-L1诱导为成熟脂肪细胞后,利用Transwell小室共培养技术,将其分别与MDA-MB-231细胞、感染腺病毒AdGFP的对照MDA-MB-231/AdGFP细胞、感染腺病毒AdBMP9的MDA-MB-231/AdBMP9细胞进行共培养。针对共培养体系中的肿瘤细胞,采用EdU实验检测增殖能力的变化;流式细胞术检测细胞周期变化;Western blot检测cyclin D1、c-myc蛋白水平的变化。将乳腺癌细胞与脂肪细胞以1:5的比例混合后,注射到裸鼠皮下,监测瘤体的生长并绘制生长曲线;取离体肿瘤组织进行免疫组化染色,检测瘤体中ki67、cyclin D1和c-myc的表达情况。结果显示,显微镜下可以观察到诱导后的3T3-L1细胞出现明显脂滴,能被油红O染色,证明成熟脂肪细胞诱导成功;AT-PCR及Western blot证实BMP9过表达成功;BMP9可以抑制共培养体系中肿瘤细胞的增殖(p<0.05),使其周期阻滞在G_2/M期(p<0.05),并降低cyclin D1和c-myc的蛋白水平(p<0.05)。动物移植瘤实验发现,BMP9高表达实验组的瘤体明显小于空白组和载体感染对照组;瘤体免疫组化结果显示,实验组ki67、cyclin D1和c-myc的表达均明显下调。本研究证明在体内、外模拟的脂肪微环境中,BMP9可以抑制乳腺癌细胞的增殖,且这种作用可能是通过下调cyclin D1和c-myc来实现的。
Bone morphogenetic protein 9 (BMP9) is a member of the bone morphogenetic proteins (BMPs) family and has multiple biological functions. This experiment mainly from the body and outside the two levels of simulated fat microenvironment, the study of BMP9 on breast cancer cell growth. After induction of preadipocyte 3T3-L1 into mature adipocytes, Transwell chamber co-culture technique was used to infect MDA-MB-231 cells and MDA-MB-231 / AdGFP cells infected with adenovirus AdGFP, AdBMP9 MDA-MB-231 / AdBMP9 cells were co-cultured. For the tumor cells in the co-culture system, the EdU assay was used to detect the proliferation ability; the cell cycle was detected by flow cytometry; the protein level of cyclin D1 and c-myc was detected by Western blot. Breast cancer cells and adipocytes were mixed in a ratio of 1: 5 and then injected into the skin of nude mice to monitor the growth of the tumor and draw the growth curve. The tumor tissues were removed for immunohistochemical staining to detect the expression of ki67, cyclin D1 and c-myc expression. The results showed that under the microscope, obvious lipid droplets could be observed in 3T3-L1 cells after being induced, which could be stained with oil red O, which proved successful induction of mature adipocytes. BMP9 overexpression was confirmed by AT-PCR and Western blot. (P <0.05). The cell cycle was arrested at G2 / M phase (p <0.05) and the protein levels of cyclin D1 and c-myc were decreased (p <0.05). The experiment of animal xenografts found that the tumor size of experimental group with BMP9 overexpression was significantly smaller than that of blank group and vector-infected control group. Immunohistochemistry showed that the expression of ki67, cyclin D1 and c-myc were significantly down-regulated in experimental group. This study demonstrates that BMP9 can inhibit the proliferation of breast cancer cells in vitro and in vivo under the simulated fat microenvironment, and this effect may be achieved through the downregulation of cyclin D1 and c-myc.