无需缝合外科嵌入式小鼠原位肝癌肿瘤模型的复制及应用价值

Establishment and application value of mouse model of hepatocellular carcinoma via sutureless surgical orthotopic implantation

摘要:

目的 探讨无需缝合的外科嵌入式方法在复制小鼠原位肝癌（HCC）肿瘤模型中的应用，并评估该模型在肿瘤生物学研究中的价值。方法 通过体外培养Hepa1-6细胞至适宜浓度，并采用小动物一体式麻醉系统对C57 BL/6J小鼠实施麻醉。该研究采用细胞法、挂线法和嵌入法复制原位肝癌移植瘤模型，并对手术时长、小鼠复苏时间及存活率进行比较。此外，通过MRI技术动态监测肿瘤的形成过程，并对比3种方法的肿瘤成瘤率、单瘤率及腹壁肿瘤种植率。通过病理学检测评估肿瘤组织的结构和病理特征。结果 细胞法组手术时间较挂线法组短（P <0.05），嵌入法组手术时间较挂线法组短（P <0.05），细胞法组与嵌入法组手术时间比较，差异无统计学意义（P >0.05）。细胞法组复苏时间较挂线法组短（P <0.05），嵌入法组复苏时间较挂线法组短（P <0.05），细胞法与嵌入法复苏时间比较，差异无统计学意义（P >0.05）。MRI结果显示，挂线法组肿瘤呈块状生长，可见分隔及类圆形伪影，局部观察受限。细胞法组肿瘤呈片状不规则生长，可见多发肿瘤。嵌入法组肿瘤呈团块状均匀生长，边缘较清。细胞法组模型复制后第7天平均肿瘤体积较挂线法组大（P <0.05），嵌入法组模型复制后第7天平均肿瘤体积较挂线法组小（P <0.05），细胞法组与嵌入法组模型复制后第7天平均肿瘤体积比较，差异无统计学意义（P >0.05）。各组模型复制后第21天平均肿瘤体积比较，差异无统计学意义（P >0.05）。各组平均肿瘤重量比较，差异无统计学意义（P >0.05）。各组单瘤率、腹壁肿瘤种植率比较，差异有统计学意义（P <0.05），嵌入法组单瘤率较细胞法组高（P <0.05）。各组成瘤率比较，差异无统计学意义（P >0.05）。HE染色结果示，各组肿瘤细胞呈巢状分布，排列紧密，核深染呈分裂象，肿瘤浸润肝组织，符合肝癌病理学特点。结论 采用嵌入法复制的原位移植瘤小鼠模型操作简单，手术时间短，可重复性高，为肝癌的相关基础实验研究及在体动态监测提供了一种重要的实验工具。

Abstract:

Objective To explore the application of sutureless surgical orthotopic implantation in the establishment of mouse model of hepatocellular carcinoma (HCC) and to assess the value of this model in tumor biology researches.Methods Hepa 1-6 cells were cultured in vitro to an appropriate density, and C57 mice were subjected to anesthesia using a small animal all-in-one anesthesia system. In this study, three techniques, including the cellular method, the hanging method and the implantation method, were used to replicate the orthotopic transplantation hepatocellular carcinoma model. The operative duration, and the recovery time and the survival rate of mice were compared. In addition, the process of tumor formation was dynamically monitored by magnetic resonance imaging (MRI), and the tumor formation rate, the single tumor rate, and the abdominal wall tumor implantation rate were compared among the three methods. The structural and pathological features of the tumor tissues were evaluated by pathological examinations.Results The operative duration of the cellular method was shorter than that of the hanging method (P < 0.05), and that of the implantation method was shorter than that of the hanging method (P < 0.05). There was no difference in the operative duration between the cellular method and the implantation method (P > 0.05). The recovery time of mice in the cellular method group was shorter than that in the hanging method group (P < 0.05), and that in the implantation method group was shorter than that in the hanging method group (P < 0.05). There was no difference in the recovery time of mice between the cellular method group and the implantation method group (P > 0.05). The MRI exhibited that the tumors in the hanging method group grew in a nodular pattern, with septa and roundish artifacts, and that parts of the tumors were not well visualized. The tumors in the cellular method group grew irregularly and in a lamellar pattern, with multiple lesions observed. The tumors in the implantation method group grew in a lump-like, uniform manner with relatively clear margins. The mean tumor volume 7 days after modeling in the cellular method group was larger than that in the hanging method group (P < 0.05), and that in the implantation method group was smaller than that in the hanging method group (P < 0.05). There was no difference in the mean tumor volume 7 days after modeling between the cellular method group and the implantation method group (P > 0.05). The mean tumor volume 21 days after modelling was not different among the three groups (P > 0.05). The mean tumor weight was also not different across the groups (P > 0.05). There were differences in the single tumor rate and the abdominal wall tumor implantation rate among the groups (P < 0.05), and the single tumor rate in the implantation method group was higher than that in the cellular method group (P < 0.05). There was no difference in the tumor formation rate among the groups (P > 0.05). The HE staining demonstrated that tumor cells in all groups were distributed in a nest-like pattern and arranged closely with deeply stained nuclei showing mitotic figures, and that the tumors infiltrated liver tissues, consistent with the pathological characteristics of hepatocellular carcinoma.Conclusions The mouse model of hepatocellular carcinoma via orthotopic implantation is easy to operate, and has a short operative duration and high reproducibility. It provides an important experimental tool for basic researches and in vivo dynamic monitoring of hepatocellular carcinoma.