Objective: To establish a mouse model bearing orthotopic temozolomide (TMZ)-resistant glioma that mimics the development of drug resistance in gliomas in vivo.
Methods: Seventy-eight adult C57BL/6 mice were randomly divided into 6 groups (n=13), including 3 TMZ induced groups with low, medium and high doses (5, 25, and 50 mg/kg, respectively) and 3 control groups. In each group, 5 mice were used for evaluating tumor size, 5 for observing survival, and 3 for collecting tumor tissues for primary cell culture. In low-dose TMZ induced group, 3 mice bearing orthotopic murine glioma GL261 cell xenografts received intraperitoneal injections of 5 mg/kg TMZ for 5 days followed by a 10-day washout period before collecting glioma tissues. Tumor cell suspensions were prepared and injected in the mice in the medium-dose group, which were treated with the same protocol but with an increased TMZ dose, and the tumor cells harvested from 3 mice were injected in the high-dose group. The mice bearing GL261 cell xenografts in the 3 control groups received no treatment or were injected with medium- or high-dose TMZ. Cell colony forming assay was used to assess TMZ resistance of each generation of the tumor cells; CCK8 assay was used to determine drug resistance index of the cells.
Results: The mouse models bearing TMZresistant glioma was successfully established. The cells from the high-dose induced group showed a significantly higher colony-forming rate than those from the high-dose control group (P < 0.05), and had a drug resistance 4.25 times higher than that of the cells from untreated control group. High-dose TMZ significantly reduced the tumor volume in the control group (P < 0.05) but not in the high-dose induced group (P < 0.01). The survival time of the tumor-bearing mice was significantly shortened in the high-dose induced group (P=0.0018).
Conclusions: Progressive increase of TMZ doses in mice bearing orthotopic gliomas can effectively induce TMZ resistance of the gliomas.
目的: 通过颅内原位成瘤小鼠体内连续传代技术，建立更为有效的替莫唑胺（TMZ）耐药胶质瘤模型，评估其耐药性和相关生物学特性。
方法: 取成年C57BL/6小鼠78只，按随机数字表法分为6组（13只/组）：诱导组共3组（小剂量、中剂量和大剂量诱导组），对照组共3组（不给药组、中剂量和大剂量对照组）。每组中5只磁共振评估肿瘤大小，5只观察小鼠生存，3只取肿瘤行原代培养。使用GL261鼠胶质瘤细胞系，对诱导组1和对照1、2、3组小鼠行原位成瘤。诱导组1使用小剂量TMZ（5 mg/kg）诱导5 d后停药10 d，再将诱导组1小鼠取瘤制备单细胞悬液，流式分选得到胶质瘤细胞后继续培养，用于接种诱导组2。中剂量TMZ（25 mg/kg）处理诱导组2荷瘤鼠，同法取瘤分选后培养并接种诱导组3，大剂量TMZ（50 mg/kg）处理诱导组3荷瘤鼠。以此逐代适度递增药物剂量诱导耐药，建立小鼠胶质瘤TMZ耐药模型。对照组1使用不含TMZ的溶剂对照处理，对照组2、3分别使用中、大剂量（25、50 mg/kg）TMZ处理。细胞集落法检测每代小鼠原位肿瘤细胞的TMZ耐药性；CCK8法检测TMZ作用下的细胞耐药指数。
结果: 成功建立了小鼠胶质瘤TMZ耐药模型并确定了其耐药性。大剂量诱导组细胞相对集落率明显高于大剂量对照组（P < 0.05），相比于不给药组产生了4.25倍耐药性。大剂量TMZ能明显减小对照组荷瘤鼠肿瘤体积（P < 0.05），而大剂量诱导组肿瘤体积明显大于大剂量对照组（P < 0.001），且荷瘤鼠生存时间较大剂量对照组明显缩短（P=0.0018）。
结论: 逐代适度递增药物剂量刺激原位成瘤小鼠，能建立更为有效的小鼠胶质瘤TMZ耐药株模型。
Keywords: animal model; glioma; temozolomide resistance.