Liang Jun, Xing Huimin, Wu Xiaohua, Zhang Lei, Zhao Jun
Department of Obstetrics and Gynecology, Third Hospital of Hebei Medical University, Shijiazhuang 050051, China.
Department of Obstetrics and Gynecology, Maternal and Child Health Hospital, Shijiazhuang 050050, China.
Nan Fang Yi Ke Da Xue Xue Bao. 2019 Sep 30;39(9):1065-1070. doi: 10.12122/j.issn.1673-4254.2019.09.10.
To isolate tumor stem-like cells from human epithelial ovarian cancer SKOV3 cells and explore their role in the formation of vascularization mimicry (VM).
SKOV3 cells were passaged to the 7th generation by suspension culture in serum-free medium, and the percentages of CD133- and CD117-positive cells in the 1st, 3rd, 5th and 7th generations were analyzed using flow cytometry. The proliferative activity of the cells sorted from the 7th generation SKOV3 cells was assessed with colony formation assay. A three-dimensional cell culture model was established to compare the ability of VM formation between the sorted cells and the parental SKOV3 cells. The expression levels of matrix metalloproteinases-2 (MMP-2) and MMP-9 in the two groups were detected using real-time PCR and Western blotting.
Some SKOV3 cells formed typical cell spheres with suspension growth in serum-free medium and were passaged to the 7th generation. Flow cytometry revealed that the percentage of CD133-positive cells increased with cell passaging. The cloning efficiency of the sorted cells was significantly higher than that of the parental SKOV3 cells (50.33% 5.33%, < 0.001). The VM formation ability of the sorted cells was stronger than that of the parental SKOV3 cells in the three-dimensional cell culture system. RT-PCR and Western blotting showed that the expression levels of MMP-2 and MMP-9 were significantly higher in the 7th passage cells than in the parental cells ( < 0.05).
The sorted cells from SKOV3 cells cultured in serum-free medium exhibit biological properties of tumor stem cells with strong VM formation ability, suggesting their role in VM formation.
从人上皮性卵巢癌SKOV3细胞中分离肿瘤干细胞样细胞，并探讨其在血管生成拟态（VM）形成中的作用。
将SKOV3细胞在无血清培养基中进行悬浮培养传至第7代，采用流式细胞术分析第1、3、5和7代细胞中CD133和CD117阳性细胞的百分比。用集落形成试验评估从第7代SKOV3细胞中分选的细胞的增殖活性。建立三维细胞培养模型，比较分选细胞与亲代SKOV3细胞形成VM的能力。采用实时荧光定量PCR和蛋白质免疫印迹法检测两组中基质金属蛋白酶-2（MMP-2）和MMP-9的表达水平。
部分SKOV3细胞在无血清培养基中悬浮生长形成典型的细胞球，并传至第7代。流式细胞术显示，CD133阳性细胞的百分比随细胞传代而增加。分选细胞的克隆效率显著高于亲代SKOV3细胞（50.33%±5.33%，P<0.001）。在三维细胞培养系统中，分选细胞形成VM的能力强于亲代SKOV3细胞。RT-PCR和蛋白质免疫印迹法显示，第7代细胞中MMP-2和MMP-9的表达水平显著高于亲代细胞（P<0.05）。
在无血清培养基中培养的SKOV3细胞分选得到的细胞具有肿瘤干细胞的生物学特性，具有较强的VM形成能力，提示其在VM形成中发挥作用。
