不同氧环境下川芎和丹参对肺癌干细胞样细胞血管生成拟态的作用

Effects of Chuanxiong(Chuanxiong Rhizoma)and Danshen(Salviae Miltiorrhizae Radix Et Rhizoma)on Vasculogenic Mimicry of Lung Cancer Stem Cell-like Cells under Different Oxygen Environment

目的观察川芎和丹参以及两药配伍后对不同氧环境下肺癌干细胞样细胞(CSLCs)血管生成拟态(VM)的影响。方法采用无血清成球培养法分离A549CSLCs亚群,建立A549 CSLCs荷瘤小鼠模型,分为常氧组和乏氧组,每组进一步分为对照组、川芎组、丹参组、川芎和丹参(川丹)组以及恩度组,通过CD31/PAS双染法检测各组VM形成。结果(1)单细胞克隆形成实验显示A549球细胞形成的单个克隆灶的细胞数目明显多于其亲代细胞,差异具有显著统计学意义(P<0.05)。划痕实验结果显示,分别在6 h、12 h和24 h A549球细胞均比其原代细胞有更强的细胞迁移能力(P<0.01)。(2)CD31/PAS双染法结果显示,常氧环境和乏氧环境下,川芎组、丹参组、川丹组和恩度组较对照组VM的数量均明显减少(P<0.01),不同氧环境比较,各组之间差异无统计学意义。结论(1)无血清培养法分离富集的球细胞具有干细胞样细胞的干性。(2)常氧和乏氧环境下川芎、丹参以及两者配伍均能抑制VM的形成,然而,无论常氧还是乏氧环境,川芎和丹参配伍对抑制VM的形成协同作用不明显。

Objective To observe the effects of Chuanxiong(Chuanxiong Rhizoma)and Danshen(Salviae Miltiorrhizae Radix Et Rhizoma)and their compatibility on angiogenesis mimicry(VM)of lung cancer stem cell-like cells(CSLCs)under different oxygen environments.Methods A549 CSLCs subpopulation was isolated by serum-free spheroid culture,and A549 CSLCs tumor-bearing mice models were established.They were divided into normoxic and hypoxic groups.Each group was further divided into control group,Chuanxiong(Chuanxiong Rhizoma)group,Danshen(Salviae Miltiorrhizae Radix Et Rhizoma)group,Chuanxiong(Chuanxiong Rhizoma)and Danshen(Salviae Miltiorrhizae Radix Et Rhizoma)(Chuandan)group and Endostar group.Each group was tested for VM formation in by CD31/PAS double staining method.Results(1)The single-cell clone formation experiment showed that the number of cells in a single clonal foci formed by A549 sphere cells was significantly more than that of their primary cells(P<0.05).The scratch test results showed that A549 sphere cells had more obvious cell migration ability than their primary cells at 6 h,12 h and 24 h(P<0.01).(2)The results of the CD31/PAS double staining method showed that both in normoxic and hypoxic environments,the number of VM in the Chuanxiong(Chuanxiong Rhizoma)group,Danshen(Salviae Miltiorrhizae Radix Et Rhizoma)group,Chuandan group and Endostar group were significantly reduced compared with that of the control group(P<0.01).Comparing the different oxygen environments,there was no significant difference among the groups.Conclusion(1)The sphere cells isolated and enriched by serum-free culture method have the stemness of stem cell-like cells.(2)Chuanxiong(Chuanxiong Rhizoma),Danshen(Salviae Miltiorrhizae Radix Et Rhizoma)and their compatibility can inhibit the formation of VM in both normoxic environment and hypoxic environment.However,the synergistic effect of Chuanxiong(Chuanxiong Rhizoma)and Danshen(Salviae Miltiorrhizae Radix Et Rhizoma)on the formation of VM is not obvious in either normoxic environment or hypoxia environment.