正常免疫小鼠新型人源肝癌异种移植模型的构建

唐慧昕; 李珊珊; 洪丰; 毕研贞; 王全义; 张小蓓; 程姝敏; 段钟平; 舒振锋; 陈煜

doi:10.3969/j.issn.1001-5256.2021.11.019

正常免疫小鼠新型人源肝癌异种移植模型的构建

DOI: 10.3969/j.issn.1001-5256.2021.11.019

唐慧昕¹,
李珊珊¹,
洪丰²,
毕研贞³,
王全义⁴,
张小蓓⁴,
程姝敏⁵,
段钟平¹,
舒振锋²,
陈煜^{1, 6, ,}

1.
首都医科大学附属北京佑安医院肝病中心四科，北京 100069
2.
上海美峰生物技术有限公司，上海 201203
3.
青岛市立医院感染科，山东青岛 266011
4.
济宁医学院附属医院肝病研究所，山东济宁 272000
5.
济宁医学院临床学院，山东济宁 272000
6.
肝衰竭与人工肝治疗研究北京市重点实验室，北京 100069

基金项目:

国家自然科学基金 (81170395);

国家自然科学基金 (81570556);

中国肝炎防治基金会-天晴肝病研究基金资助课题 (TQGB20210013);

国家重点研发计划资助 (2017YFA0103000);

国家科技重大专项“艾滋病和病毒性肝炎等重大传染病防治” (2017ZX10203201-005);

北京市医院管理局临床医学发展专项经费资助 (ZYLX201806)

详细信息

通信作者:
陈煜，chybeyond1071@ccmu.edu.cn

唐慧昕和李珊珊对本文贡献相同，同为第一作者

中图分类号: R735.7
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出版历程
- 收稿日期: 2021-04-13
- 录用日期: 2021-05-24
- 出版日期: 2021-11-20

Construction of a new patient-derived xenograft model of human liver cancer in mice with normal immunity

1.
The Fourth Liver Disease Center, Beijing YouAn Hospital, Capital Medical University, Beijing 100069, China
2.
Shanghai Meifeng Biotechnology Co., Ltd., Shanghai 201203, China
3.
Department of Infectious Diseases, Qingdao Municipal Hospital, Qingdao, Shandong 266011, China
4.
Institute of Liver Diseases, Affiliated Hospital of Jining Medical University, Jining, Shandong 272000, China
5.
Clinical College of Jining Medical University, Jining, Shandong 272000, China
6.
Beijing Municipal Key Laboratory of Liver Failure and Artificial Liver Treatment Research, Beijing 100069, China

Research funding:

National Natural Science Foundation of China (81170395);

National Natural Science Foundation of China (81570556);

Chinese foundation for hepatitis prevention and control—TianQing liver disease research fund subject (TQGB20210013);

National Key R&D Program of China (2017YFA0103000);

National Science and Technology Key Project on "Major Infectious Diseases such as HIV/AIDS, Viral Hepatitis Preventon and Treatment" (2017ZX10203201-005);

Beijing Municipal Administration of Hospitals Clinical Medicine Development of Special Funding Support (ZYLX201806)

摘要

摘要: 目的基于新型微载体microcarrier 6复合人原代肝癌细胞接种于具有正常免疫功能的小鼠，以期建立新型肝癌人源肿瘤异种移植(PDX)模型。方法从5例新鲜的人源肝癌组织中分离提取出原代肝癌细胞，分别与微载体microcarrier 6共培养，体外构建三维肿瘤细胞培养模型。将75只雄性C57BL/6小鼠按移植物不同随机分为3组：细胞对照组、微载体对照组和实验组(每例患者对应3组，共15组，每组5只)。采用皮下接种法将肝癌细胞-微载体复合体植入小鼠体内，观察小鼠成瘤时间、成瘤率、病理组织学表现等。计数资料两组间比较采用Fisher精确检验。结果 5例患者的肝癌细胞接种小鼠后有3例对应的小鼠出现成瘤；此3例实验中，对照组均没有小鼠成瘤，仅实验组出现成瘤，实验组15只小鼠中有12只成功成瘤，成瘤率高达80%，与细胞对照组、微载体对照组相比差异均有统计学意义(P值均＜0.05)，其成瘤时间为5~7 d，移植瘤生长速度快，HE染色可见排列呈巢状或片状、异形性明显的细胞，可见病理性核分裂象，免疫组化染色CK8/18、Hep、Gpc-3均为阳性，符合人源肝癌细胞特点。结论本实验成功基于microcarrier 6复合人原代肝癌细胞在正常免疫小鼠体内建立了新型人源肝癌PDX模型，此模型可以更好地研究肝癌在免疫功能正常机体中发生、发展机制，同时也为肝癌精准医疗提供了较以往更有价值的新型动物模型。
- 肝肿瘤 /
- 遗传载体 /
- 移植, 异种 /
- 模型, 动物
Abstract: Objective To establish a new patient-derived xenograft (PDX) model of human liver cancer by inoculating the complex of human primary liver cancer cells and a novel microcarrier (microcarrier 6) into mice with normal immune function. Methods Primary liver cancer cells were isolated and extracted from the fresh human liver cancer tissue of five patients and were then co-cultured with microcarrier 6 to construct a three-dimensional tumor cell culture model in vitro. According to the type of graft, 75 male C57BL/6 mice were divided into cell control group, microcarrier control group, and experimental group (each sample corresponded to three groups, with 15 groups in total and 5 mice in each group). The liver cancer cell-microcarrier complex was implanted into the mice by subcutaneous inoculation, and tumor formation time, tumor formation rate, and histopathological manifestations were observed. The Fisher's exact test was used for comparison of categorical data between two groups. Results As for the liver cancer cells from the five patients, tumor formation was observed in the mice corresponding to three patients. In these three experiments, tumor formation was not observed in the control groups and was only observed in the experimental groups, and 12 of the 15 mice in the experimental groups had successful tumor formation, with a tumor formation rate as high as 80%, which was significantly different from that in the cell control groups and the microcarrier control groups (all P < 0.05). The tumor formation time was 5-7 days; the xenograft tumor grew rapidly, and HE staining showed nested or flaky cells with obvious heteromorphism, with the presence of pathological mitosis; immunohistochemical staining showed positive CK8/18, Hep, and Gpc-3, which was in accordance with the characteristics of human liver cancer cells. Conclusion This experiment successfully establishes a new PDX model of human liver cancer based on the complex of microcarrier 6 and human primary liver cancer cells in mice with normal immunity. This model can be used to better elucidate the mechanism of the development and progression of liver cancer in the body with normal immunity, and besides, it also provides a new animal model with higher value for the precise treatment of liver cancer.

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图 1 基于microcarrier 6构建的原代肝癌细胞三维培养体系(×200)

注：a，原代肝癌细胞；b，微载体microcarrier 6；c，原代肝癌细胞与微载体microcarrier 6共培养。

下载: 全尺寸图片幻灯片

图 2 移植瘤生长曲线

下载: 全尺寸图片幻灯片

图 3 肉眼观察荷瘤小鼠体内移植瘤

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图 4 原代肝癌和移植瘤组织HE染色结果(×200)

注：a，原代肿瘤；b，移植瘤。黑色箭头，异形细胞；红色箭头，未被清除的微载体；蓝色箭头，血管。

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图 5 移植瘤免疫组化染色(×200)

注：a，CK8/18；b，Hep；c，Gpc-3。

下载: 全尺寸图片幻灯片

表 1 肝癌相关PDX模型的病例信息

编号	类型	性别	年龄(岁)	肿瘤大小(cm)	AFP(μg/L)	分化程度	肝炎类型	有无肝硬化
LC-1	肝细胞癌	女	74	4.5×3.5×3	14.81	中	HBV	无
LC-2	胆管细胞癌	男	54	3×3×2.5	19.20	低	HBV	有
LC-3	肝细胞癌	男	44	4.5×4×3.5	56.07	中-低	HBV	有
LC-4	肝细胞癌	男	46	13.5×12×8	58 233.14	中	HBV	有
LC-5	肝细胞癌	男	60	13×12×9	6424.04	中	HBV	有

下载: 导出CSV

表 2 3例成功成瘤实验中小鼠成瘤时间和成瘤率

组别	LC-1	LC-2	LC-3	成瘤时间(d)	成瘤率(%)
细胞对照组	0/5	0/5	0/5	-	0¹⁾
微载体对照组	0/5	0/5	0/5	-	0¹⁾
实验组	4/5	5/5	3/5	5~7	80
注：与实验组比较，1)P＜0.05。

下载: 导出CSV

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