兔骨髓间充质干细胞在肿瘤微环境诱导下向肌纤维母细胞的分化

背景:骨髓间充质干细胞可以促进肿瘤间质重构,但具体机制尚不明确,而肌纤维母细胞则是肿瘤间质重构过程中发挥重要作用的一类细胞.目的:观察骨髓间充质干细胞在肿瘤微环境诱导下能否分化为肌纤维母细胞.设计、时间及地点:细胞学体外观察及体内移植实验,于2006-06/2007-12在山东大学齐鲁医院低温医学实验室完成.材料:3月龄雄性新西兰大白兔12只,购自山东省农业科学院.方法:穿刺抽取兔骨髓,密度梯度法分离培养骨舒间充质于细胞,传至第2代行DAPI标记,调整细胞密度为5×10~(10) L~(-1),分为2组:实验组加入含体积分数为10%胎牛血清、30% VX2瘤细胞株培养上清的低糖DMEM,对照组加入含体积分数为10%胎牛血清的低糖DMEM,培养14 d.向兔大腿肌肉内注入VX2细胞悬液,2周后触摸到生成肿瘤后,抽取DAPI标记的第2代骨髓间充质干细胞悬液100 μL,从前后左右4个方向均匀回输入肿瘤组织内,3周后取材.主要观察指标:采用免疫荧光法和RT-PCR检测骨髓间充质干细胞中α-SMA和Vimentin的表达,免疫荧光法鉴定骨髓间充质干细胞回输后在肿瘤组织中的分布及分化.结果:第2代骨髓间充质干细胞经VX2培养上清诱导2周后,Cy3免疫荧光结果显示α-SMA和Vimentin呈强阳性表达,对照组呈弱阳性表达.与对照组比较,实验组α-SMA及Vimentin mRNA的表达水平均明显升高(P＜0.01,P＜0.05).回输入 VX2肿瘤组织3周后,骨髓间充质干细胞主要分布于肿瘤间质,其细胞核周围胞浆中α-SMA和Vimentin均呈强阳性表达,表明骨髓间充质干细胞在肿瘤组织中已分化为肌纤维母细胞.结论:在肿瘤微环境的诱导下,骨髓间充质干细胞可以分化为肌纤维母细胞,从而促进肿瘤的生长与发展.

Differentiation of mesenchymal stem cells into myofibroblasts under the induction of tumor microenvironment in rabbits

BACKGROUND:Studies have shown that mesenchymsi stem cells (MSCs) participate in tumor stroma remodeling,but the mechanism is not clear.However,myofibroblast is related with tumor strema remodeling.OBJECTIVE:To investigate whether MSCs can differentiate into myofibroblast under the induction of tumor microenvironment.DESIGN,TIME AND SETING:This in vitro observation and in vivo transplantation cytology experiment was performed at institute of Low Temperature Medicine,Qilu Hospital of Shandong University from June 2006 to December 2007.MATERIALS:Twelve male New Zealand white rabbits,with 3-month-old,were purchased from Shandong Academy of Agricultural Sciences.METHODS:MSCs were obtained through bone marrow aspirates in male New Zealand rabbits and cultured by density gradient method.The 2~(nd) passage of MSCs were labeled with DAPI,and adjusted cell density to 5×10~(10) /L.The cells were assigned into experimental and control groups.For the experimental group,the passage two MSCs were cultured in low-glucose DMEM containing 10% fetal bovine serum and 30% VX2-cultured supernatant.For the control group,passage two MSCs were cultured in low-glucose DMEM containing 10% fetal bovine serum.Also,DAPI labeled self passage 2 MSCs of avery animal were delivered uniformly via direct surgical intratumoral injection to observe the differentiation of MSCs in tumor tissue.MAIN OUTCOME MEASURES:Immunofluorescense and RT-PCR were used to test the expression of α-SMA and Vimentin in MSCs of both groups 14 days later.At 3 weeks after VX2.intratumoral injection,Immunofluorescence was used to observe the distribution and differentiation of MSCs in tumor tissue.RESULTS:After stimulated with 30% VX2-cultured supematant for 2 weeks,Cy3 immunofluorescance showed that the expressions of α-SMA and Vimentin were strongly expressed in the experimental group,which was weakly expressed in the control group.Compared to the control group,the levels of α-SMA and Vimentin mRNA were obviously increased in the experimental group (P ＜ 0.01,P ＜ 0.05).At 3 weeks after MSCs intratumoral injection,these MSCs mainly distributed in tumor stroma and the expression of α-SMA and Vimentin in MSCs were also obviously increased,which demonstrated that the MSCs had differentiated into myofibroblasts.CONCLUSIONS:Under the induction of tumor microenivironment,MSCs can differentiate into myofibroblast,accordingly,accelerate the growth of tumor.