人肝肿瘤细胞的裸小鼠原位癌建模条件优化及评价

doi:10.12300/j.issn.1674-5817.2024.048

摘要/Abstract

摘要：

目的优化通过注射人肝肿瘤细胞株构建原位癌裸小鼠模型的条件，并探索适宜的给药治疗时间。方法选用稳定表达萤光素酶报告基因（LUC）的人肝细胞癌Hep3B与肝母细胞瘤HepG2细胞株，使用小动物活体成像系统分析萤光素酶发光强度与肝肿瘤细胞数量之间的线性相关性，验证人源肝肿瘤细胞的发光效率。在5周龄雌性BALB/c裸小鼠的肝叶原位接种不同浓度（8×10⁶、2.4×10⁷、7.2×10⁷个/mL）、不同重悬介质（PBS、Matrigel）的人肝肿瘤细胞悬液HepG2-LUC和Hep3B-LUC（共12组，每组7只），分别构建人肝肿瘤裸小鼠原位癌模型。每7 d为1个周期记录各组小鼠体重，用小动物活体成像系统定期监测原位肿瘤的生长过程，观察肿瘤生长趋势。接种肿瘤细胞后第35天剖取小鼠肝脏，制备病理切片，进行苏木精-伊红（hematoxylin and eosin，HE）染色观察组织病理学变化。结果两种人肝肿瘤细胞株的发光强度均与细胞数量呈正相关（R²=0.983 1，R²=0.970 5），适宜用于原位癌模型的构建。HepG2-LUC高浓度组，HepG2-LUC+Matrigel低、中、高浓度组，Hep3B-LUC中、高浓度组与Hep3B-LUC+Matrigel低、中、高浓度组均成功造模。HepG2-LUC+Matrigel高浓度组较低浓度与中浓度组小鼠的体重显著下降（P<0.05），Hep3B-LUC+Matrigel高浓度组较低浓度与中浓度组小鼠的体重也显著下降（P<0.05）。成功造模组小鼠的荧光发光强度随时间呈指数型增长（R²>0.950 0），且在移植后14 d发光强度至少可达到1.0×10⁷ p/（s·cm2·sr）。 HepG2-LUC低、中浓度组和Hep3B-LUC低浓度组小鼠肝脏未见明显的病理学变化，其余组肝脏肿瘤和肝细胞病变明显。结论对于HepG2-LUC细胞株，推荐肝叶原位注射2.4×10⁷个/mL（50 μL）且与Matrigel重悬的混合细胞液体造模，并于造模后第7天给药或采取预后措施；而对于Hep3B-LUC细胞株，推荐肝叶原位注射7.2×10⁷个/mL（50 μL）（不与Matrigel重悬混合）造模，并于造模后的第14天给药或采取预后措施。

关键词: 肝癌, 原位移植, HepG2-LUC, Hep3B-LUC, BALB/c裸小鼠

Abstract:

Objective The study aims to optimize the conditions for constructing orthotopic nude mouse models of liver cancer by injecting human liver tumor cell lines and to explore appropriate timings for drug administration. Methods Human hepatocellular carcinoma Hep3B and hepatoblastoma HepG2 cell lines, which stably expressing the luciferase reporter gene (LUC), were selected. The linear correlation between the luciferase luminescence intensity and the number of liver tumor cells was analyzed using a Small Animal In Vivo Imaging system to verify the luminescent efficiency of the human liver tumor cells. Different concentrations (8×10⁶, 2.4×10⁷, 7.2×10⁷ cells/mL) and resuspension media (PBS, Matrigel) of human liver tumor cell suspensions HepG2-LUC and Hep3B-LUC were orthotopically inoculated into the liver lobes of 5-week-old female BALB/c nude mice (12 groups, 7 mice each) to construct human liver tumor nude mouse orthotopic cancer models. Every 7 days, the weights of mice were recorded, and the growth of orthotopic tumors was monitored using the Small Animal In Vivo Imaging system. On day 35 post-cell inoculation, mouse livers were dissected, and pathological slices were prepared for HE staining to observe histopathological changes in liver tissues. Results The luminescence intensity of human liver tumor cell lines was positively correlated with the number of cells (R²=0.983 1, R²=0.970 5), indicating their suitability for orthotopic model construction. Successful modeling was achieved in the high-concentration groups of HepG2-LUC, the low-, medium-, and high-concentration groups of HepG2-LUC+Matrigel, the medium- and high-concentration groups of Hep3B-LUC, and the low-, medium-, and high-concentration groups of Hep3B-LUC+Matrigel. For both HepG2-LUC+Matrigel and Hep3B-LUC+Matrigel groups, mice in the high-concentration groups exhibited significantly reduced body weight compared to the low- and medium-concentration groups (both with P<0.05). The luminescence intensity of successfully modeled mice increased exponentially over time (R²>0.950 0), and reached a minimum of 1.0×10⁷ p/(s·cm2·sr) by day 14 post-transplantation. Mice in the low- and medium-concentration groups of HepG2-LUC and the low-concentration group of Hep3B-LUC showed no significant pathological changes, while the other groups exhibited evident liver tumors and hepatocyte lesions. Conclusion For the HepG2-LUC cell line, the recommended injection volume is 50 μL with a cell density of 2.4×10⁷ cells/mL, resuspended with Matrigel, followed by drug administration or prognostic measures on day 7 post-modeling. For the Hep3B-LUC cell line, the recommended injection volume is 50 μL with a cell density of 7.2×10⁷ cells/mL, not resuspended with Matrigel, with administration or prognostic measures on day 14 post-modeling.

Key words: Liver cancer, Orthotopic transplantation, HepG2-LUC, Hep3B-LUC, BALB/c nude mice

中图分类号:

R73-3

孟雨,梁冬丽,郑琳琳,等. 人肝肿瘤细胞的裸小鼠原位癌建模条件优化及评价[J]. 实验动物与比较医学, 2024, 44(5): 511-522. DOI: 10.12300/j.issn.1674-5817.2024.048.

MENG Yu,LIANG Dongli,ZHENG Linlin,et al. Optimization and Evaluation of Conditions for Orthotopic Nude Mouse Models of Human Liver Tumor Cells[J]. Laboratory Animal and Comparative Medicine, 2024, 44(5): 511-522. DOI: 10.12300/j.issn.1674-5817.2024.048.

图/表 4

图1 人肝肿瘤细胞株的细胞生物发光效率注：A和B分别为萤光素酶报告基因（LUC）标记的人肝母细胞瘤HepG2细胞（ HepG2-LUC）、人肝癌细胞Hep3B（ Hep3B-LUC）细胞与D-荧光素钾底物混合孵育后的生物发光效率，一元线性回归及单因素方差分析显示各细胞数目与平均生物发光强度呈线性正相关关系。96孔板中，从左至右每孔含细胞数量依次为2×103、4×103、8×103、1.6×104、3.2×104、4.8×104和6.4×104个（n=3）。

Figure 1 Cell bioluminescence efficiency of human liver tumor cell linesNote: A, Bioluminescence efficiency of human hepatoblastoma HepG2 cells labeled with luciferase reporter gene (LUC) after incubating with D-luciferin potassium; B, Bioluminescence efficiency of human hepatocellular carcinoma Hep3B cells labeled with LUC after incubating with D-luciferin potassium. Simple linear regression and one-way ANOVA test showed that the number of each cell was positively correlated with the average bioluminescence intensity. In the 96-well plate, the number of cells in each well from left to right was 2×103，4×103， 8×103， 1.6×104， 3.2×104， 4.8×104, and 6.4×104, n=3.

图2 不同接种细胞浓度、不同介质的肝肿瘤模型裸小鼠的体重变化注：A～D依次为HepG2-LUC［萤光素酶报告基因（LUC）标记的用PBS稀释成不同细胞浓度的人肝母细胞瘤HepG2细胞］、HepG2-LUC+Matrigel［LUC标记的与Matrigel基质胶（4 mg/mL）混合成不同细胞浓度的人肝母细胞瘤HepG2细胞］、Hep3B-LUC（LUC标记的用PBS稀释成不同细胞浓度的人肝癌Hep3B细胞）、Hep3B-LUC+Matrigel［LUC标记的与Matrigel基质胶（4 mg/mL）混合成不同细胞浓度的人肝癌Hep3B细胞］原位注射裸小鼠的体重随时间增长趋势。与低浓度（8×106个细胞/mL）组相比，#P<0.05，##P<0.01，###P=0.000 1；与中浓度（2.4×107个细胞/mL）组相比，*P<0.05，**P<0.01，***P=0.000 1；3组之间比较，nsP>0.05。

Figure 2 Body weight changes of nude mouse liver tumor models with different cell concentrations and suspension mediaNote：A-D represent body weight over time in nude mouse orthotopic tumor models of HepG2-LUC [human hepatoblastoma HepG2 cells labeled with luciferase reporter gene (LUC) and diluted with PBS into different cell concentrations], HepG2-LUC+Matrigel (human hepatoblastoma HepG2 cells labeled with LUC and diluted with 4 mg/mL Matrigel into different cell concentrations), Hep3B-LUC (human hepatocellular carcinoma Hep3B cells labeled with LUC and diluted with PBS into different cell concentrations), and Hep3B-LUC+Matrigel (human hepatocellular carcinoma Hep3B cells labeled with LUC and diluted with 4 mg/mL Matrigel into different cell concentrations) successively. Compared with the low-concentration (8×106 cells/mL) group of the same time, #P<0.05, ##P<0.01, ###P=0.000 1; Compared with the medium-concentration (2.4×107 cells/mL) group of the same time, #P<0.05, ##P<0.01, ###P=0.000 1; Compared among the three groups of the same time, nsP>0.05.

图3 不同接种细胞浓度、不同介质的肝肿瘤模型裸小鼠体内生物发光强度注：A～D依次为HepG2-LUC［萤光素酶报告基因（LUC）标记的用PBS稀释成不同细胞浓度的人肝母细胞瘤HepG2细胞］、HepG2-LUC+Matrigel（LUC标记的与Matrigel基质胶（4 mg/mL）混合成不同细胞浓度的人肝母细胞瘤HepG2细胞）、Hep3B-LUC（LUC标记的用PBS稀释成不同细胞浓度的人肝癌Hep3B细胞）、Hep3B-LUC+Matrigel（LUC标记的与Matrigel基质胶（4 mg/mL）混合成不同细胞浓度的人肝癌Hep3B细胞）原位注射裸小鼠的每周成像结果，提示造模后各组小鼠体内发光强度随时间的变化趋势（虚线提示呈指数型增长）。

Figure 3 Bioluminescence monitoring of nude mouse liver tumor models with different cell concentrations and suspension mediaNote：A-D represent the weekly bioluminescence monitoring results of nude mouse orthotopic tumor models of HepG2-LUC [human hepatoblastoma HepG2 cells labeled with luciferase reporter gene (LUC) and diluted with PBS into different cell concentrations], HepG2-LUC+Matrigel (human hepatoblastoma HepG2 cells labeled with LUC and diluted with 4 mg/mL Matrigel into different cell concentrations), Hep3B-LUC (human hepatocellular carcinoma Hep3B cells labeled with LUC and diluted with PBS into different cell concentrations), and Hep3B-LUC+Matrigel (human hepatocellular carcinoma Hep3B cells labeled with LUC and diluted with 4 mg/mL Matrigel into different cell concentrations) successively, which show the change trends of the luminescence intensity over time after in-situ modeling (dotted lines indicate exponential growth).

图4 不同接种细胞浓度、不同介质的肝肿瘤模型裸小鼠的肝脏组织典型图片（A）和HE染色图（B）注：HepG2-LUC，用PBS稀释成不同细胞浓度的萤光素酶报告基因（LUC）标记的人肝母细胞瘤HepG2细胞接种裸小鼠左肝叶后35 d；HepG2-LUC+Matrigel，与Matrigel基质胶（4 mg/mL）以1∶1的比例混合成不同细胞浓度的LUC标记的人肝母细胞瘤HepG2细胞接种裸小鼠左肝叶后35 d；Hep3B-LUC，用PBS稀释成不同细胞浓度的LUC标记的人肝癌细胞Hep3B接种裸小鼠左肝叶后35 d；Hep3B-LUC+Matrigel，与Matrigel基质胶（4 mg/mL）以1∶1的比例混合成不同细胞浓度的LUC标记的人肝癌细胞Hep3B接种裸小鼠左肝叶后35 d。A图中比例尺大小为5 mm，B图中各细胞浓度组内左右两图的比例尺大小分别为50 μm和20 μm。

Figure 4 Gross images (A) and HE staining (B) of liver tissue in nude mouse liver tumor models with different cell concentrations and suspension mediaNote：HepG2-LUC, left liver lobe of nude mice 35 days after in-situ modeling with human hepatoblastoma HepG2 cells labeled with luciferase reporter gene (LUC) and diluted with PBS into different cell concentrations. HepG2-LUC+Matrigel, left liver lobe of nude mice 35 days after in-situ modeling with human hepatoblastoma HepG2 cells labeled with LUC and diluted with 4 mg/mL Matrigel into different cell concentrations. Hep3B-LUC, left liver lobe of nude mice 35 days after in-situ modeling with human hepatocellular carcinoma Hep3B cells labeled with LUC and diluted with PBS into different cell concentrations. Hep3B-LUC+Matrigel, left liver lobe of nude mice 35 days after in-situ modeling with human hepatocellular carcinoma Hep3B cells labeled with LUC and diluted with 4 mg/mL Matrigel into different cell concentrations. The scale size is 5 mm in figure A, and the scale sizes of the left and right graphs in each cell concentration group are 50 μm and 20 μm respectively in figure B.

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