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2型糖尿病胰腺癌裸鼠模型的建立及活体成像观察

许永宁 黄雪桓 唐芷盼 李若涵 秦雯

引用本文:
Citation:

2型糖尿病胰腺癌裸鼠模型的建立及活体成像观察

DOI: 10.12449/JCH240625
基金项目: 

国家自然科学基金资助项目 (81860508);

广西医疗卫生适宜技术开发与推广应用项目 (S2018040);

广西壮族自治区卫生和计划生育委员会自筹经费科研课题 (Z20180950)

伦理学声明: 本研究方案于2018年3月6日经由广西医科大学第一附属医院伦理委员审批,批号为2018-KY-国基-153,符合实验室动物管理与使用准则。
利益冲突声明:本文不存在任何利益冲突。
作者贡献声明:许永宁负责实验操作、研究过程的实施、起草论文、论文框架、统计学分析及绘制图表;黄雪桓、唐芷盼及李若涵负责细胞培养、科研蜡块制备;秦雯负责课题设计、提供基金资助、拟定写作思路、论文修改和指导撰写文章并最后定稿。
详细信息
    通信作者:

    秦雯, fenglingcao1980@163.com (ORCID: 0009-0002-8282-7388)

Establishment and in vivo imaging observation of a nude mouse model of type 2 diabetes mellitus and pancreatic cancer

Research funding: 

National Natural Science Foundation of China (81860508);

Guangxi Medical and Health Appropriate Technology Development and Promotion Project (S2018040);

Guangxi Health and Family Planning Commission Self-raised Scientific Research Projects (Z20180950)

More Information
  • 摘要:   目的  建立可动态观察成瘤过程并进行体内研究的2型糖尿病(T2DM)胰腺癌裸鼠模型。  方法  首先,通过慢病毒载体GV260转染人胰腺癌细胞(PANC-1细胞)构建能稳定表达萤火虫荧光素酶的胰腺癌细胞株(PANC-1-Luc细胞)。然后,将36只SPF级裸鼠随机分为对照组(n=12,血糖正常的胰腺癌裸鼠)和模型组(n=24,T2DM胰腺癌裸鼠)。对照组:先给予繁殖饲料喂养,之后将PANC-1-Luc细胞异位种植于裸鼠皮下;模型组:先给予高脂饲料喂养联合腹腔注射1% STZ,之后将PANC-1-Luc细胞异位种植于裸鼠皮下。用荧光活体成像系统和人工测量法同步动态监测2组裸鼠胰腺癌生长情况,绘制肿瘤生长曲线、分析荧光值与肿瘤体积的关系。显微镜下观察裸鼠皮下肿瘤及胰岛,验证造模是否成功;同时,通过免疫组化检测肿瘤组织Ki-67的表达来分析高血糖对裸鼠胰腺癌生长的影响。正态分布计量资料组间比较采用成组t检验,非正态分布计量资料组间比较采用Mann-Whitney U检验。  结果  确定PANC-1细胞慢病毒载体稳定转染的最佳病毒滴度为5×107 TU/mL,用嘌呤霉素筛选的最佳浓度为20 μg/mL、最佳筛选时间为9天;PANC-1-Luc细胞的荧光值与细胞数量呈线性正相关,线性方程为y=42.56x-42 504(r=0.977,P=0.004)。T2DM裸鼠模型血糖值为23.05(19.25~26.40)mmol/L,且每只裸鼠的血糖均高于11.1 mmol/L,与对照组裸鼠血糖值[6.15(5.20~7.30)mmol/L]相比,差异有统计学意义(Z=-8.45,P<0.001)。与对照组相比,模型组胰腺组织内胰岛数量减少、体积减小、形状不规则、边界模糊,同时移植瘤病理学检查确认镜下为胰腺癌组织,可判定T2DM裸鼠胰腺癌模型造模成功。模型组皮下肿瘤大小与荧光值呈线性正相关,线性方程为y=232 348 691x-8 258 608(r=0.911,P=0.031);模型组移植瘤Ki-67免疫组化阳性率显著高于对照组[(50.333±7.808)% vs (15.917±4.055)%,t=13.55,P<0.001],说明模型组肿瘤增殖较快。  结论  本研究所构建的T2DM裸鼠胰腺癌模型可模拟T2DM背景下胰腺癌发生、发展的病理过程,动态观察高血糖对体内胰腺癌细胞生长的影响,从而为T2DM背景下胰腺癌发生、发展的体内研究提供新的实验载体。

     

  • 图  1  细胞系筛选实验(×180)

    注: a,第3天;b,第5天;c,第7天;d,第9天。

    Figure  1.  Cell line screening experiment (×180)

    图  2  细胞系筛选实验前后细胞形态对比(×180)

    注: a,未经筛选的细胞;b,经嘌呤霉素筛选后的细胞。

    Figure  2.  Comparison of cell morphology before and after cell line screening experiment (×180)

    图  3  慢病毒载体转染前后细胞形态对比(×180)

    注: a,PANC-1(转染前);b,(转染后)PANC-1-Luc。

    Figure  3.  Comparison of cell morphology before and after lentiviral vector transfection (×180)

    图  4  PANC-1-Luc的荧光值及其与细胞数量的关系

    注: a,实验组和对照组的荧光值比较;b,不同细胞数量PANC-1-Luc荧光值的比较;c,PANC-1-Luc荧光值跟细胞数量呈线性正相关。

    Figure  4.  Fluorescence values of PANC-1-Luc and its relationship with cell number

    图  5  模型组及对照组第5周皮下肿瘤的活体生物发光成像

    注: a,模型组;b,对照组。

    Figure  5.  Vivo bioluminescence imaging of subcutaneous tumors in the model group and the control group at week 5

    图  6  模型组及对照组裸鼠皮下肿瘤大小变化

    Figure  6.  Changes in the size of subcutaneous tumors in nude mice in the model group and the control group

    图  7  模型组及对照组裸鼠皮下肿瘤荧光值动态变化

    Figure  7.  Dynamic changes in subcutaneous tumor fluorescence values in the model group and the control group

    图  8  模型组裸鼠荧光值与肿瘤生长体积动态分析

    Figure  8.  Dynamic analysis of fluorescence values and tumor growth volume in the model group

    图  9  模型组裸鼠荧光值与肿瘤体积相关性分析

    Figure  9.  Correlation analysis between fluorescence values and tumor volume in the model group

    图  10  模型组和对照组裸鼠形成的皮下肿瘤肉眼观察

    注: a,模型组(部分示例);b,对照组(部分示例)。

    Figure  10.  Macroscopic observation of subcutaneous tumors formed in nude mice in the model group and the control group

    图  11  模型组裸鼠皮下肿瘤HE染色

    注: a,×40,白色箭头为胰腺癌形成皮下肿瘤,红色箭头为肌肉组织,黑色箭头为皮肤及皮脂腺;b,×400。

    Figure  11.  The histological observation of subcutaneous tumors in the model group with HE staining under a microscope

    图  12  对照组与模型组裸鼠胰岛组织HE染色(×200)

    注: a,对照组;b,模型组;箭头处为胰岛。

    Figure  12.  HE staining of pancreatic islet tissue in the control group and model group (×200)

    图  13  对照组与模型组裸鼠皮下肿瘤Ki-67免疫组化结果(×200)

    注: a,对照组;b,模型组。

    Figure  13.  Immunohistochemical results of subcutaneous tumor Ki-67 in the control group and model group (×200)

    表  1  模型组及对照组裸鼠皮下肿瘤大小变化(cm3

    Table  1.   Changes in subcutaneous tumor size of nude mice in the model group and the control group (cm3

    时间 对照组(n=12) 模型组(n=24) t P
    第1周 0.013±0.007 0.036±0.021 3.535 0.004
    第2周 0.020±0.012 0.046±0.022 3.490 0.002
    第3周 0.027±0.015 0.051±0.023 3.490 0.002
    第4周 0.035±0.019 0.061±0.034 2.285 0.035
    第5周 0.039±0.019 0.070±0.037 2.549 0.018
    下载: 导出CSV

    表  2  模型组及对照组裸鼠皮下肿瘤荧光值变化(p⋅s-1⋅cm-2⋅sr-1

    Table  2.   Changes in subcutaneous tumor values of nude mice in the model group and the control group (p·s-1·cm-2·sr-1

    时间 对照组(n=12) 模型组(n=24) t P
    第1周 6 460.83±4 200.33 985 416.67±97 565.32 34.726 <0.001
    第2周 28 169.17±20 112.81 2 029 250.00±612 538.25 11.311 <0.001
    第3周 131 601.67±101 868.71 3 251 666.67±981 546.40 10.953 <0.001
    第4周 174 560.00±133 203.77 3 897 500.00±1 248 631.07 10.270 <0.001
    第5周 359 734.17±236 685.20 9 650 833.33±4 384 569.45 7.330 <0.001
    下载: 导出CSV
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