目的探讨移植瘤中作为肿瘤间质细胞的骨髓间充质干细胞(BMSCs)和胶质瘤干细胞SU3.红色荧光蛋白(PEP)的相互作用。方法应用6GyX线辐射Balb/c裸小鼠损毁骨髓,尾静脉移植源于绿色荧光蛋白(GFP)裸小鼠的骨髓细胞,建立骨髓损毁重建模型:将SU3.RFP细胞接种骨髓损毁重建模型小鼠皮下并获取移植瘤,HE染色并用激光共聚焦显微镜观察。原代培养移植瘤,克隆出高增殖力的GFP+细胞,免疫细胞化学染色检测该细胞和BMSCs中CD44、Scal、CD90和CD45的表达。CCK-8法、细胞克隆形成实验、Transwell侵袭实验分别检测高增殖力GFP+细胞、BMSCs、SU3.RFP的增殖能力、克隆形成率和侵袭能力;染色体核型分析该细胞、正常BMSCs有丝分裂相的染色体形态;将高增殖力GFP+细胞接种于Balb/c裸小鼠皮下,收获肿瘤组织并行HE染色。结果SU3-RFP细胞对骨髓损毁重建模型小鼠的致瘤率为100%(7/7)。HE染色移植瘤显示血管丰富,管腔内可见红细胞。激光共聚焦显微镜下显示绿色的外源性移植骨髓细胞与红色的肿瘤干细胞相互作用活跃。免疫荧光染色显示高增殖力GFP+细胞与BMSCs均高表达CD44、弱表达Sca1和CD90、不表达CD45,将其命名为已转化的BMSCs(tBMSCs)。CCK-8法、细胞克隆形成实验、Transwell侵袭实验检测显示SU3-RFP、tBMSCs的吸光度似)值f培养后5、6、7d1、克隆形成率、穿膜细胞数均高于BMSCs,差异有统计学意义(P〈0.05)。染色体核型分析显示tBMSCs分裂相染色体较BMSCs明显增多;tBMSCs对Balb/c裸小鼠致瘤率为100%(5/5)。HE染色可见肿瘤细胞呈浸润性生长,血供丰富。结论胶质瘤干细胞可诱导移植瘤中BMSCs的恶性转化,后者参与肿瘤重构。
Objective Both human glioma stem cell line SU3 transfected with red fluorescent protein (SU3-RFP) and bone marrow cells from green fluorescence protein (GFP) transgenic athymic nude mice were transplanted into bone marrow damage-reconstitution Balb/c nude mice to investigate the mutual interactions between bone marrow mesenchymal stem cells (BMSCs) and SU3-RFP in vivo. Methods After irradiation of bone marrow in Balb/c nude mice, allogeneic transplantation with GFP+ bone marrow cells was performed via caudal vein to establish bone marrow damage-reconstitution models. SU3-RFP cells were transplanted subcutaneously to the models and xenograft tumors were harvested; HE staining was performed and observation under laser scanning confocal microscope was performed. Primary culture of the xenograft tumors was performed to sort and clone the high proliferative GFP+ cells. Immunocytochemical staining was employed to detect the expressions of CD44, Scal, CD90 and CD45 in BMSCs; proliferation, clone formation rate and invasive ability of GFP+ cells, BMSCs and SU3-RFP were detected by CCK-8 method, cell clone formation experiment and Transwell invasion assay, respectively. Karyotype analysis was used to analyze the chromosome morphology of BMSCs. High proliferative GFP+ cells were inoculated into the subcutaneous of Balb/c nude mice, and the tumor tissues were harvested to perform HE staining. Results The tumor formation rate of SU3-RFP cells in the bone marrow reconstitution models was 100% (7/7). HE staining indicated that the tumors were rich in blood vessels and erythrocytes in the lumen. Active interaction was noted between BMSCs and SU3-RFP. The high proliferative GFP+ cells and BMSCs both had strong CD44 expression, Scal and CD90 weakly positive expression, and CD45 negative expression, and these cells were named transformational BMSCs (tBMSCs). The proliferation and clone formation rate and invasive ability of tBMSCs and SU3-RFP were significantly higher than those of BMSCs (P〈0.05?