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中华肝脏外科手术学电子杂志 ›› 2022, Vol. 11 ›› Issue (03) : 315 -319. doi: 10.3877/cma.j.issn.2095-3232.2022.03.020

基础研究

小鼠VETC(+)肝癌模型构建及索拉非尼对VETC结构的影响
熊培尧1, 唐雨豪1, 杨子良1, 朱应钦1, 王骏成1, 徐立1,()   
  1. 1. 510060 广州,中山大学肿瘤防治中心肝脏外科
  • 收稿日期:2022-03-08 出版日期:2022-06-10
  • 通信作者: 徐立
  • 基金资助:
    国家自然科学基金(81772589)

Establishment of mouse model with VETC(+) liver cancer and effect of sorafenib on VETC structure

Peiyao Xiong1, Yuhao Tang1, Ziliang Yang1, Yingqin Zhu1, Juncheng Wang1, Li Xu1,()   

  1. 1. Department of Hepatic Surgery, Sun Yat-sen University Cancer Center, Guangzhou 510060, China
  • Received:2022-03-08 Published:2022-06-10
  • Corresponding author: Li Xu
引用本文:

熊培尧, 唐雨豪, 杨子良, 朱应钦, 王骏成, 徐立. 小鼠VETC(+)肝癌模型构建及索拉非尼对VETC结构的影响[J/OL]. 中华肝脏外科手术学电子杂志, 2022, 11(03): 315-319.

Peiyao Xiong, Yuhao Tang, Ziliang Yang, Yingqin Zhu, Juncheng Wang, Li Xu. Establishment of mouse model with VETC(+) liver cancer and effect of sorafenib on VETC structure[J/OL]. Chinese Journal of Hepatic Surgery(Electronic Edition), 2022, 11(03): 315-319.

目的

构建具有肿瘤包绕型血管(VETC)的小鼠肝细胞癌(肝癌)模型,探讨索拉非尼对VETC(+)肝癌的影响。

方法

将49只C57小鼠随机分为预防组(15只)、治疗组(15只)和对照组(19只)。预防组小鼠肝包膜下接种HEP1-6肝癌细胞(5×105/只),2 d后开始连续索拉非尼溶液灌胃(25 mg/kg,每日一次);治疗组于接种后10 d开始连续予索拉非尼溶液灌胃(50 mg/kg,每日一次);对照组以相同体积的DMSO灌胃,于接种后5、10、15、20、23、27 d分批处死小鼠,肿瘤组织石蜡包埋,切片行CD34及HE染色,观察小鼠体重及不良反应,记录成瘤情况、肿瘤最大径等。肿瘤最大径及VETC环数等比较采用Kruskal-Wallis检验。

结果

各组小鼠均顺利成瘤,接种后20 d对照组2只小鼠肝内可见转移灶,接种后23 d对照组1只小鼠死于肿瘤进展。治疗组与预防组均未观察到明显肝内转移,小鼠可见脱屑,无明显厌食、腹泻等不良反应,体重无明显变化,饲养笼干燥,无死亡。预防组接种后5、10、15、20、23 d肿瘤最大径均明显小于对照组(H=-4.355,-3.857,-6.000,-6.250,-6.167;P<0.05);接种后15 d,治疗组肿瘤最大径亦明显小于对照组(H=-5.125,P<0.05)。接种后5 d,对照组小鼠肝肿瘤中均可观察到明显的VETC结构;接种后15 d VETC环数较5 d明显减少(H=-3.857,P<0.05),且肿瘤内部均出现大片坏死灶;接种后23 d VETC环数较15 d明显增多(H=4.582,P<0.05)。预防组接种5~23 d VETC形成明显被抑制;5、10、20 d VETC环数均明显少于对照组(H=-3.971,-4.355,-6.369;P<0.05)。

结论

本研究成功构建小鼠VETC(+)肝癌模型。索拉非尼可有效降低小鼠肝癌的肿瘤负荷,破坏VETC结构并抑制其形成,预防组疗效尤为显著。

Objective

To establish a mouse model of hepatocellular carcinoma (HCC) with vessels encapsulating tumor clusters (VETC) and to evaluate the effect of sorafenib on VETC(+) HCC.

Methods

49 C57 mice were randomly divided into the prevention (n=15), treatment (n=15) and control groups (n=19). In the prevention group, HEP1-6 HCC cells were inoculated under the liver capsule with a dosage of 5×105 cells per mouse, and after 2 d, sorafenib solution was continuously administered by gavage (25 mg/kg, once daily). In the treatment group, sorafenib solution was continuously given by gavage (50 mg/kg, once daily) at 10 d after inoculation. In the control group, an equivalent amount of DMSO was given by gavage. Mice were sacrificed at 5, 10, 15, 20, 23 and 27 d after inoculation, respectively. The tumor tissues were embedded in paraffin and sliced up for CD34 and HE staining. The weight and adverse reactions of mice were observed. The tumor formation and maximum tumor diameter were recorded. The maximum tumor diameter and number of VETC rings were compared by Kruskal-Wallis test.

Results

All the mice in the groups successfully developed tumors. At 20 d after inoculation, 2 mice had intraheptic metastatic lesions in the control group, and 1 mouse died of tumor progression at 23 d after inoculation. No evident intrahepatic metastasis was observed in the treatment and prevention groups. Desquamation was observed in the mice. No obvious adverse reactions, such as anorexia and diarrhea, were seen. No significant change in weight was noted. The mouse cage was dry and no mouse died. In the prevention group, the maximum tumor diameter was significantly smaller than those in the control group at 5, 10, 15, 20 and 23 d after inoculation (H=-4.355, -3.857, -6.000, -6.250, -6.167; P<0.05). At 15 d after inoculation, the maximum tumor diameter in the treatment group was significantly smaller than that in the control group (H=-5.125, P<0.05). At 5 d after inoculation, evident VETC structure could be observed in mouse liver tumors in the control group. At 15 d after inoculation, the number of VETC rings was significantly lower compared with that at 5 d (H=-3.857, P<0.05), and a large quantity of necrotic foci were observed in the tumors. At 23 d after inoculation, the number of VETC rings was significantly larger than that at 15 d (H=4.582, P<0.05). In the prevention group, the formation of VETC was significantly inhibited at 5-23 d after inoculation, the number of VETC rings at 5, 10 and 20 d was significantly less than those in the control group (H=-3.971, -4.355, -6.369; P<0.05).

Conclusions

The mouse model of VETC(+) HCC is successfully established in this study. Sorafenib can effectively reduce the tumor load, destroy the VETC structure and suppress the formation of VETC in HCC mouse models, especially in the prevention group.

图1 小鼠肝癌组织CD34染色观察VETC(免疫组化法 ×200)注:a为肿瘤细胞簇形成完整环形包绕的血管环(箭头所示);b为环形不完整,但内皮细胞包绕肿瘤细胞簇大于180°(箭头所示);VETC为肿瘤包绕型血管
表1 不同组别肝癌细胞接种后各时间点处死小鼠的肿瘤最大径(cm)
表2 不同组别肝癌细胞接种后各时间点处死小鼠肿瘤VETC环数(个)
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