南方医科大学学报

南方医科大学学报 ›› 2020, Vol. 40 ›› Issue (08): 1155-1164.doi: 10.12122/j.issn.1673-4254.2020.08.13

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生酮饮食抑制裸鼠皮下移植人神经母细胞瘤的生长

贺娇娇，吕麟亚，彭俊伟，李长春，孔祥如，章 均，彭 亮   

  • 出版日期:2020-08-20 发布日期:2020-08-20
  • 基金资助:

Inhibitory effect of ketogenic diet on neuroblastoma in BALB/c-nu mouse models

  

  • Online:2020-08-20 Published:2020-08-20

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摘要： 目的 探究生酮饮食（KD）抑制人神经母细胞瘤裸鼠皮下移植瘤生长的作用。方法 选取雌性BALB/c-nu裸鼠建立人神经母细胞瘤细胞株SH-SY-5Y皮下移植瘤模型，当移植瘤体积约250 mm3时，随机分为标准饮食组（SD，标准饲料喂养+PBS 60 mg· kg-1· d-1）、生酮饮食组（KD，生酮饲料喂养+PBS 60 mg· kg-1· d-1 ），环磷酰胺联合生酮饮食组（CP+KD，生酮饲料喂养+环磷酰胺mg· kg-1· d-1），每组8只。测量瘤体长径、短径，计算移植瘤体积。检测裸鼠体质量、血糖、血酮体（β-羟基丁酸）水平和肝脏脂肪变性。Western blot检测凋亡蛋白caspase3、caspase8；免疫组化（IHC）检测增殖蛋白Ki67。透射电镜观察自噬体；IHC和Western blot检测自噬蛋白Beclin1、LC3A/B及P62。结果 种瘤后第28天，KD和CP+KD组裸鼠生存期较SD组显著延长（P< 0.001）；接种第22 天，KD组瘤积小于SD组（P<0.05）；接种第16、19、22 天，CP+KD组瘤积明显小于SD组（P<0.01）。接种第19天，KD组裸鼠体质量较SD组减轻（P<0.05），其余测量时间点上无显著差异（P>0.05）；接种第13天，KD组血糖较SD组下降（P< 0.05），其余测量时间点上的KD和CP+KD组血糖与SD组无显著差异（P>0.05）；KD和CP+KD组血酮全程较高（P<0.05），而血糖/血酮值较低（P<0.05）；KD和CP+KD组裸鼠肝脏脂肪变性评分高于SD组（P<0.05）。Ki67和凋亡蛋白在各组肿瘤组织中均表达。透射电镜下KD组中自噬体较多于SD组；KD组P62蛋白表达明显低于SD组（P<0.01），而Beclin1、LC3A/B蛋白表达增加（P<0.05），与IHC结果较一致。结论 KD能抑制人神经母细胞瘤裸鼠移植瘤的生长，其可能的抗肿瘤机制与细胞自噬相关。

Abstract: Objective To investigate the inhibitory effect of ketogenic diet (KD) on growth of neuroblastoma in mice. Methods BALB/c-nu mouse models bearing neuroblastoma xenografts were established by subcutaneous injection of human neuroblastoma cell line (SH-SY5Y). When the tumor volume reached 250 mm3, the mice were randomized into SD group with standard diet and PBS treatment, KD group with ketogenic diet and PBS treatment, and CP+KD group with ketogenic diet and cyclophosphamide (60 mg· kg-1· day-1 ) treatment, n=8. The tumor volume, body weight, blood glucose, ketone body (β-Hydroxybutyrate) levels, and hepatic steatosis in the mice were assessed. The expressions of caspase-3 and caspase-8 were detected by Western blotting, and Ki67 expresison was detected using immunohistochemistry (IHC). Transmission electron microscopy (TEM) was employed for the autophagosomes, and the autophagic protein Beclin1, LC3A/B and P62 were detected by IHC and Western blotting. Results On day 28 post tumor cell injection, the mice in KD and CP+KD groups could prolong the overall survival rates than that in SD group (P<0.001). On day 22 post the injection, the tumor volume in KD group was smaller than that in SD group (P<0.05); on 16, 19, and 22 day post the injection, the tumor volume in CP+KD group was smaller than that in SD group (P<0.01). The mice in SD group showed greater body weight on day 19 and higher blood glucose level on day 13 post the injection than those in the other two groups (P<0.05). Blood ketone level and hepatic steatosis score were higher and glucose ketone index (GKI) was lower in KD and CP + KD groups than those in SD group (all P<0.05). The expressions of Ki67 and apoptotic proteins were detected in the tumor tissues of all groups. TEM revealed more autophagosomes in the tumor tissues of KD group than that of SD group. P62 expression was lowered (P<0.01) and Beclin1 and LC3A/B expressions were up-regulated in the tumor tissues of KD group (P<0.05), which is consisitent with IHC. Conclusion KD has a strong anti-tumor effect in the xenograft mouse model possibly by regulating cell autophagy.