摘要目的 研究人骨髓间质干细胞(MSCs)诱导肿瘤的机制.方法 取18只SCID裸鼠随机分为对照组和实验组,对照组注射MSCs,实验组注射F6细胞,于第4(F6-4)、6(F6-6)、7(F6-7)周取肿瘤组织(每组3只).4例肺癌患者的肺癌标本(Lc)和癌旁标本为分别取自不同部位的手术病理标本.并用荧光差异显示技术(FDD)寻找差异基因;用PCR扩增、蛋白质印迹(WB)加以验证;实时荧光定量逆转录(FQ-RT)-PCR技术检测诱导致瘤细胞在裸鼠体内致瘤后致瘤组织基因表达水平.用粒-巨噬细胞集落刺激因子(GM-CSF)和白细胞介素-4(IL-4)诱导MSCs致肿瘤细胞(F6细胞).结果 FDD结果显示,细胞周期蛋白(cyclin)Ⅰ基因高表达,与PCR、WB结果一致.FQ-RT-PCR表明,cyclin Ⅰ基因在MSCs、F6及3组F6致瘤组织细胞之间的表达水平差异有统计学意义(F=12.29,P<0.01),F6组cyclin Ⅰ基因表达水平为4.49±0.40,MSCs基因表达水平为0.04±0.02,增高112倍(P<0.01).裸鼠皮下注射致瘤细胞后,第4,6,7周分离肿瘤组织内cyclin Ⅰ基因表达分别为1.82±0.80、3.30±0.43和3.68±1.67,且呈上升趋势(与MSCs对比,P分别为<0.05或<0.01).4例肺癌患者肺癌组织基因表达分别为0.15±0.02、0.58±0.23、4.82±1.12、1.21±0.60,癌旁组织基因表达分别为0.04±0.02、0.09±0.04、0.94±0.74、0.15±0.08,肺癌组织内cyclin Ⅰ基因表达明显高于癌旁组织(F=12.39,P<0.01).WB证明F6细胞cyclin Ⅰ蛋白表达为0.32±0.08,MSCs蛋白表达为0.09±0.06,增高3.6倍(t=3.86,P<0.05).结论 cyclin Ⅰ基因在MSCs诱导突变到肿瘤细胞形成过程中高表达,且在MSCs诱导肿瘤发生过程中可能发挥重要作用.

abstractsObjective To study role of bone marrow mesenehyme stem cells in tumor formation.Methods Nude mice (n = 18) were randomly divided into two groups.Mice in the control group were subcutaneously injected with human embryonic MSCs, and F6 cells were injected into the nine mice of the experimental group.Three mice were sacrificed to remove tumor tissue, which was then prepared for real-time BT-PCR detection at 4 (F6-4), 6 (F6-6) and 7 (F6-7) weeks, respectively.Human lung cancer tissue samples and adjacent non-malignant lung tissue samples were collected from 4 lung cancer patients.The difference between gene expression of F6 cells and MSCs was distinguished by fluorescent differential display (FDD) and verificated by PCR and the western blot analysis. Real-time fluorescent quantitative reverse transcription polymerase chain reaction (FQ-BT-PCR) was used to detect gene expression in tumor tissues after tumorigenesis in nude mice.A new tumor cell line, denominated F6, was established from mutated human embryonic bone marrow mesenchymal stem cells (MSCs) which were induced by the GMCSF and IL-4 in vitro.Results FDD analysis confirmed that cyelin Ⅰ was positively up-regulated in F6 cells compared with MSCs. Similar results were obtained by PCR and western blot.The cyclin Ⅰ gene expression levels in MSCs, F6, F6-4, F6-6 and 176-7 were significantly different(F=12.29 ,P ＜ 0.01).The cyclin Ⅰ gene expression level in F6(4.49±0.40) was 112 folds higher than those of MSCs(0.04±0.02,P＜0.01).Expression levels in F6-4 , F6-6 and F6-7 tissue were 1.82±0.80,3.30±0.43,3.68±1.67 which were significantly higher than that in MSCs (P＜0.05 or ＜0.01).The expression of cyclin Ⅰ increased significantly alone with the accreting volume of tumor.The expression levels of cyelin Ⅰ in human lung cancer tissues in four patients were 0.15±0.02, 0.58±0.23, 4.82±1.12, 1.21±0.60,respectively, and were significantly higher than that in adjacent non-malignant lung tissues (0.04±0.02,0.09±0.04,0.94±0.74,0.15±0.08) (F=12.39,P＜0.01).The protein expression level of cyclin Ⅰ in F6 cells was 0.32±0.08, which was 3.6-fold higher than that in MSCs (0.09±0.06, t=3.86,P＜0.05).Conclusions The expression level of cyclin Ⅰ in tumor tissue is higher during the entire course of tumorigenesis.Cyclin Ⅰ might be one of the factors playing important roles during tumorigenesis,especially in MSCs mutation.