小鼠循环肝癌细胞模型的建立与评价

发布时间：2019-03-27 14:12

【摘要】：目的采用小鼠肝癌Hepa 1-6细胞建立小鼠肝癌循环肿瘤细胞(CTCs)模型。方法采用雄性C57BL/6小鼠108只,按照体重分为3组,每组36只。3个组分别每只尾静脉接种0.2 m L的浓度为1×106、5×106和1×107个/m L的小鼠肝癌Hepa 1-6细胞单细胞悬液。各组分别于接种后第1、5、9、13、17、21天检测采血,采用流式细胞术检测,计算2万个有核细胞中CTCs数目及比例、相对CTCs抑制率,记录动物死亡率。(2)采用雄性C57BL/6小鼠80只,按体重分为2组,分别为模型对照组、甲苯磺酸索拉菲尼组,每组40只。每只尾静脉接种0.2m L的浓度为5×106个/m L的Hepa 1-6细胞单细胞悬液,接种后第2天开始灌胃给予甲苯磺酸索拉菲尼(50 mg/kg),连续21 d,并于给药第3、8、15、21天采血进行CTC检测。结果 (1)接种浓度为1×106个/m L的细胞悬液后,第1、5、9、13、17、21天的动物CTCs比例为:25.1%、18.1%、8.9%、4.4%、2.9%、0.3%,未出现动物死亡;接种浓度为5×106个/m L的细胞悬液后,第1、5、9、13、17、21天的动物CTCs比例为:40.4%、35.4%、15.4%、9.0%、6.6%、4.1%,未出现动物死亡;接种浓度为1×107个/m L的细胞悬液后,第1、5天的动物CTCs比例为:39.1%、33.5%,在接种完毕之后立即出现动物死亡,且所有动物在接种后第7天全部死亡。(2)甲苯磺酸索拉菲尼给药期间D3、D8、D15、D21相对循环肿瘤细胞清除率分别为:-7.5%、4.6%、55.3%、-94.5%,与模型对照组比较差异有显著性(P0.05或P0.01)。结论采用浓度为5×106个/m L小鼠肝癌细胞Hepa 1-6悬液尾静脉注射可建立小鼠肝癌CTC模型,可用于抑制循环肿瘤细胞药物的筛选与评价。
[Abstract]:Objective to establish a mouse model of circulating tumor cells (CTCs) using mouse hepatoma Hepa 1 / 6 cells. Methods one hundred and eight male C57BL/6 mice were divided into three groups according to body weight, 36 mice in each group. 3 groups were inoculated with 1 脳 10 6, 5 脳 10 6 and 1 脳 10 7 cells / mL of single cell suspension of hepatoma Hepa 1 / mL in each of the three groups by intravenous inoculation of 0.2 mL, 5 脳 10 6 and 1 脳 10 7 cells / mL, respectively. Blood samples were collected 1,5,9,13,17,21 days after inoculation in each group. Flow cytometry was used to calculate the number and proportion of CTCs in 20 000 nucleated cells, the relative CTCs inhibition rate and animal mortality rate were recorded. (2) 80 male C57BL/6 mice were used. According to body weight, they were divided into two groups: model control group and sulafinib group with 40 rats in each group. The single cell suspension of Hepa 1 / 6 cell was injected into each tail vein at a concentration of 5 脳 10 6 cells / mL. On the second day after inoculation, the rats were intragastrically administrated with 50 mg/kg toluene sulfonate for 21 days, and then given 3,8, 8, 8, 10 6 cells per tail vein for 21 consecutive days, with a concentration of 5 脳 10 6 cells / mL single cell suspension. Blood samples were collected for CTC test on day 15 and 21. Results (1) at 1,5,9,13,17,21 days after inoculation with 1 脳 10 6 cells / mL of cell suspension, the percentage of CTCs in animals was 25.1%, 18.1%, 8.9%, 4.4%, 2.9%, 0.3%, and no animal death was observed. At 1,5,9,13,17,21 days after inoculation with 5 脳 10 ~ 6 cells / mL of cell suspension, the percentage of CTCs in animals was 40.4%, 35.4%, 15.4%, 9.0%, 6.6%, 4.1%, and no animal death was observed. After inoculation with 1 脳 10 ~ 7 cells / mL cell suspension, the CTCs ratio of animals on day 1 and 5 was 39.1%, 33.5%, and immediately after inoculation, animal death occurred. All animals died on the 7th day after inoculation. (2) the clearance rates of D3, D8, D15 and D21 were-7.5%, 4.6%, 55.3% and 94.5%, respectively, during the administration of sulafinib toluene sulfonate, and the relative circulating tumor cell clearance rates were-7.5%, 4.6%, 55.3%, 94.5%, respectively. Compared with the model control group, there was significant difference (P0.05 or P0.01). Conclusion the mouse hepatoma CTC model can be established by intravenous injection of 5 脳 10 6 / mL of mouse hepatoma cell line Hepa 1 / mL, which can be used to screen and evaluate the drugs for inhibiting circulating tumor cells.
【作者单位】： 湖南省药物安全评价研究中心新药药效与安全性评价湖南省重点实验室;中南大学湘雅医院血液科;
【基金】：湖湘青年创新创业平台专项(2014) 湖南省科技计划重点项目(2012TT1002)
【分类号】：R-332;R735.7
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