人脐带间充质干细胞对肝癌细胞增殖性及凋亡的影响

Effects of Human Umbilical Cord Mesenchymal Stem Cell on the Proliferation of Apoptosis of Hepatoma Cells

目的探讨人脐带间充质干细胞(human umbilical cord mesenchyma cell,MSC)对肝细胞性肝癌(hepatocellular carcinoma,HCC)癌细胞增殖和凋亡的影响,为HCC的治疗提供新的思路。方法取50%覆盖率的MSC培养皿,换上新鲜的DMEM/F-12培养基,待其培养至100%的覆盖率后收集培养基备用,即为MSC条件培养基。用新鲜DMEM/F-12培养基加上等量的MSC条件培养基的混合培养基培养Hep G2人类肝癌细胞株24、48和72h,使用MTT法测定Hep G2细胞的增殖活性、通过Hoechest33342和PI双染色后在荧光倒置显微镜下观察计数以测定Hep G2细胞的凋亡、用Transwell侵袭实验和黏附实验测定Hep G2细胞侵袭能力以及通过Western blot法检测凋亡相关信号通路蛋白的表达。结果混合培养基培养Hep G2细胞24h后,对其生长和凋亡及侵袭黏附能力没有显著影响(P>0.05)。但是培养延长到48h和72h后,Hep G2细胞的活性、增殖能力、侵袭能力和黏附能力都受到显著的抑制,这些变化伴随着细胞分裂相关因子Ki-67、PCNA和组蛋白H3磷酸化水平下调,以及细胞凋亡执行者caspase-3的激活和抗凋亡蛋白Bcl-2的抑制。结论体外间接共培养实验表明,人脐带间充质干细胞具有抑制肝脏肿瘤细胞增殖及促进其凋亡的作用。

Objective To examine the effects of human umbilical cord mesenchymal stem cell （ MSC） on the proliferation of apopto-sis of hepatoma cells and to provide novel therapeutic strategy for liver cancer .Methods Culture of MSC with 50% confluence was re-placed with fresh DMEM/F-12 medium.When the confluence reached 100%, all culture used DMEM/F-12 was considered as the conditioning medium.This kind of medium was mixed with fresh DMEM/F-12 at 1：1 to treat human hepatoma cell line HepG 2 for 24, 48 and 72 hours.Cellual viability was measured by MTT assay , apoptosis was quantified by Hoechst 33342/PI co-staining, cell invasion ability and adhesion ability were measured by transwell assay and in vitro adhesion assay , respectively .Change of key signaling compo-nents were studied by Western blot .Results Conditioning medium showed no significant impact on the proliferation , apoptosis and inva-sion of HepG2 after 24-hour incubation （P 〉0.05）.However, when treatment during was extended to 48 and 72 hours, the prolifera-tion and invasion were significantly inhibited by the conditioning medium while cellular apoptosis was invoked .These were accompanied by the down-regulation of Ki-67, PCNA and histone H3 phosphorylation.Cleaved caspase-3 was increased while the expression of anti -apoptotic protein Bcl-2 was inhibited .Conclusion Human umbilical cord mesenchymal stem cell was capable of inhibiting proliferation and invasion , as well as promoting apoptosis of human hepatoma cells in vitro .