小鼠肠腺瘤类器官培养及其辐射敏感性

目的 建立小鼠肠腺瘤类器官的体外培养方法,观察其对电离辐射的反应。方法 采用氧化偶氮甲烷(azoxymethane,AOM)和葡聚糖硫酸钠(detrain sodium sulfate,DSS)诱导小鼠产生肠腺瘤。体外分离腺瘤类隐窝结构,接种于基质胶。通过培养基筛选,确定肠腺瘤类器官的体外培养条件,采用免疫组化染色检测Ki67和β-catenin表达水平。进一步采用X线照射,观察肠腺瘤类器官损伤情况,比较其与大、小肠类器官的辐射敏感性。 结果 经AOM/DSS诱导,小鼠肠腺瘤成瘤率达95%,肿瘤均位于结肠靠近直肠处,肠腺瘤类器官在改良的小肠类器官中生长良好,Ki67阳性腺瘤细胞比例高且β-catenin入核特征明显。经X线照射,各类器官存活比例随辐射剂量增加而降低。9 Gy照射7天后,腺瘤类器官存活率为11.96%±1.42%,高于同剂量大肠类器官的5.46%±1.22% (t=6.0082,P＜0.01),小肠类器官几乎未见存活。腺瘤类器官剂量存活曲线较大、小肠类器官右移,提示其辐射敏感性低于大肠和小肠。 结论 在AOM/DSS诱导产生的小鼠肠腺瘤中成功分离培养出腺瘤类器官,其辐射敏感性低于大、小肠类器官。

Culture method and radiosensitivity of intestinal adenoma organoids from mice

Objective To establish a culture method of intestinal adenoma organoids in mice and to investigate its radiosensitivity in vitro. Methods The adenomas were induced in the colon epithelia of mice through azoxymethane (AOM)/detrain sodium sulfate (DSS) strategy. The crypts like structures of adenomas were isolated from mouse intestinal adenoma, and embedded in matrigel for tumor organoid development. The culture medium was optimized to support tumor organoid growth and budding. The expression of Ki67 and β-catenin was detected by immunohistochemical staining. The radiosensitivity was evaluated by survival rate of organoids after X rays exposure compared with colon and intestinal organoids. Results Adenomas was developed in colon close to rectum in 95% mice treated with AOM/DSS. The intestinal adenoma organoids grew well in the modified medium of small intestinal organoid. The high rate of Ki67 positive cells and high rate of β-catenin in nuclear were identified. After X rays exposure, a dose-dependent loss of organoids was showed in all kinds of organoids. The survival rate of adenoma organoids was 11.96%±1.42%, which was more than that of colon organoids 5.46%±1.22% (t=6.008 2, P<0.01) 7 days post irradiation of 9 Gy. While the survival organoid was almost disappeared in small intestine group. The dose survival curve of adenoma organoid was right shifted with lower radiosensitivity compared to colon and small intestinal organoids. Conclusions An adenoma organoids was successfully established from induced adenoma in colon of mice, which appeared more radiation tolerance than colon and small intestinal organoids.