利用细胞膜片技术构建新型前列腺癌皮下移植瘤动物模型

周术奎; 张东亮; 王翔; 刘磊; 李曾; 杨盛柯; 廖洪

doi:10.19401/j.cnki.1007-3639.2022.03.002

您当前的位置：

首页 >

文章列表页 >

利用细胞膜片技术构建新型前列腺癌皮下移植瘤动物模型

论著 | 更新时间：2025-12-31

- 利用细胞膜片技术构建新型前列腺癌皮下移植瘤动物模型
- Developing a new animal model of subcutaneous transplanted prostate cancer with cell sheet technology
- 中国癌症杂志 2022年32卷第3期页码：200-206
- 作者机构：
  
  1. 四川省肿瘤医院·研究所,四川省癌症防治中心,电子科技大学医学院泌尿外科,四川成都 610041
  2. 上海交通大学附属第一人民医院泌尿外科,上海 200080
- 作者简介：
  
  廖洪 E-mail: liaohong131@163.com
- 基金信息：
  
  四川省卫生和计划生育委员会重点研究项目(19ZD015)
- DOI：10.19401/j.cnki.1007-3639.2022.03.002
  中图分类号： R737.25
- 收稿：2021-12-28，
  
  纸质出版：2022-03-30
- 稿件说明：
移动端阅览
周术奎, 张东亮, 王翔, 等. 利用细胞膜片技术构建新型前列腺癌皮下移植瘤动物模型[J]. 中国癌症杂志, 2022,32(3):200-206.

Shukui ZHOU, Dongliang ZHANG, Xiang WANG, et al. Developing a new animal model of subcutaneous transplanted prostate cancer with cell sheet technology[J]. China Oncology, 2022, 32(3): 200-206.
周术奎, 张东亮, 王翔, 等. 利用细胞膜片技术构建新型前列腺癌皮下移植瘤动物模型[J]. 中国癌症杂志, 2022,32(3):200-206. DOI： 10.19401/j.cnki.1007-3639.2022.03.002.

Shukui ZHOU, Dongliang ZHANG, Xiang WANG, et al. Developing a new animal model of subcutaneous transplanted prostate cancer with cell sheet technology[J]. China Oncology, 2022, 32(3): 200-206. DOI： 10.19401/j.cnki.1007-3639.2022.03.002.

摘要

背景与目的：

传统前列腺癌皮下移植瘤模型的建立采取细胞悬液注射方式

但该方式存在较大局限性。探索利用细胞膜片技术构建新型前列腺癌皮下移植瘤动物模型的可行性。

方法：

将人前列腺癌细胞系DU145接种至35 mm温度敏感性细胞培养皿中

连续培养制备前列腺癌细胞膜片

并采用H-E染色和免疫组织化学方法鉴定。根据研究目的

设置细胞悬液组和细胞膜片组

细胞悬液组在BALB/c裸小鼠皮下注射DU145细胞悬液

细胞膜片组在裸小鼠皮下注射DU145细胞膜片。移植后4周处死裸小鼠

剥离两组移植瘤作进一步组织学分析

包括胶原纤维含量、局部浸润及血管生成情况。同时

解剖前列腺癌常见的转移脏器

如骨、肺和肝脏等

评估两组的全身转移情况。

结果：

培养1周后可制备前列腺癌DU145细胞膜片

大体观察呈现半透明胶质薄片

具备较好的组织学强度。组织学染色见膜片状结构

平均厚度约为（32.6

#x000b1;7.5）

#x003bc;m。DU145细胞标志物vimentin高表达

E-cadherin低表达

胶原纤维含量丰富

在膜片中高表达。皮下移植4周后

H-E染色显示

细胞膜片组肿瘤结构致密

有明显周围肌肉浸润

而细胞悬液组肿瘤存在较多空泡

与周围组织边界清楚。皮下移植4周后

细胞膜片组移植瘤体积明显大于细胞悬液组

两组测量肿瘤体积分别为（0.967

#x000b1;0.129）和（0.4

#x000b1;0.054）cm

（

= 3.774

= 0.019 5）。马松染色显示

细胞膜片组蓝色胶原纤维含量明显高于细胞悬液组

两组吸光度（

）值分别为0.023 0

#x000b1;0.001 1和0.014 0

#x000b1;0.000 7（

= 7.022

= 0.000 1）。CD31免疫组织化学染色表明

在200倍显微镜视野下细胞膜片组和细胞悬液组移植瘤组织中微血管密度分别为53.20

#x000b1;3.56和32.40

#x000b1;4.98（

= 3.392

= 0.009 5）。皮下移植4周后

两组通过大体解剖和H-E染色均未见到明显的全身转移病灶。

结论：

利用细胞膜片技术构建前列腺癌皮下移植瘤动物模型具备可行性和一定优势

与传统细胞悬液注射方式相比

成瘤体积更大

与周围组织浸润程度更深

新生微血管密度更高

胶原含量丰富

更有助于揭示前列腺癌真实的生物学特征

有望应用于前列腺癌分子遗传学研究、肿瘤耐药机制及新药研发等领域。

Abstract

Background and purpose:

Traditional preparation of subcutaneous transplanted tumor of prostate cancer relies on cell suspension injection. However

there are great limitations. The purpose of this study was to explore the feasibility of developing a new animal model of subcutaneous transplanted prostate cancer with cell sheet technology.

Methods:

Human prostate cancer DU145 cells were inoculated into a 35 mm temperature sensitive cell culture dish

and the prostate cancer cell sheet was prepared by continuous culture and identified by H-E staining and immunohistochemistry. According to the research purpose

cell suspension group and cell membrane group were set up. DU145 cell suspension was subcutaneously injected into nude mice in cell suspension group

and DU145 cell sheet was subcutaneously injected into BALB/c nude mice in cell sheet group. The BALB/c nude mice were sacrificed 4 weeks after transplantation

and the grafted tumors in both groups were dissected for further histological analysis

including collagen fiber content

local infiltration and angiogenesis. Meanwhile

the common organs of prostate cancer metastasis

such as bone

lung and liver

were dissected to evaluate the systemic metastasis in both groups.

Results:

DU145 prostate cancer cell sheet could be prepared at 1 week afte

r continuous culture. Histological staining revealed a membrane structure with an average thickness of (32.6

#x000b1;7.5)

#x003bc;m. The DU145 cell sheet was rich in collagen fibrin with high vimentin expression and low E-cadherin expression. H-E staining showed that the tumor structure was dense with obvious infiltration of surrounding muscles in the cell sheet group

while the tumor had more vacuoles and a clear boundary with surrounding tissues in the cell suspension group 4 weeks after subcutaneous transplantation. Moreover

the transplanted tumor volume was significantly higher in the cell sheet group than in the cell suspension group

and the measured tumor volume in the two groups were (0.967

#x000b1;0.129) and (0.437

#x000b1;0.054) cm

(

= 3.774

= 0.019 5)

respectively. Masson staining showed that the content of blue collagen fiber was significantly higher in the cell sheet group than in the cell suspension group

and the optical densities (

) value of the two groups were 0.023 0

#x000b1;0.001 1 and 0.014 0

#x000b1;0.000 7 (

= 7.022

= 0.000 1)

respectively. Immunohistochemical staining of CD31 showed that the microvascular density per

#x000D7;200 field in the cell sheet group and the cell suspension group were 53.20

#x000b1;3.56 and 32.40

#x000b1;4.98 (

= 3.392

= 0.009 5)

respectively. Gross anatomy and H-E staining showed no obvious systemic metastasis in the two groups 4 weeks after subcutaneous transplantation.

Conclusion:

It is feasible and advantageous to develop the animal model of subcutaneous transplanted prostate cancer using cell sheet technology. Compared with traditional cell suspension injection method

the tumor volum was larger

the infiltration degree was deeper with surrounding tissues

the density of new microvessels was higher

and the collagen content was richer

which is more helpful to revea

l the real biological characteristics of prostate cancer. It is expected to be applied in the research of molecular genetics of prostate cancer

drug resistance mechanism and drug discovery.

关键词

Keywords

references

SUNG H , FERLAY J , SIEGEL R L , et al . Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries [J ] . CA Cancer J Clin , 2021 , 71 ( 3 ): 209 - 249 . DOI: 10.3322/caac.v71.3 http://doi.org/10.3322/caac.v71.3 https://onlinelibrary.wiley.com/toc/15424863/71/3 https://onlinelibrary.wiley.com/toc/15424863/71/3

CULP M B , SOERJOMATARAM I , EFSTATHIOU J A , et al . Recent global patterns in prostate cancer incidence and mortality rates [J ] . Eur Urol , 2020 , 77 ( 1 ): 38 - 52 . DOI: 10.1016/j.eururo.2019.08.005 http://doi.org/10.1016/j.eururo.2019.08.005 https://linkinghub.elsevier.com/retrieve/pii/S0302283819306190 https://linkinghub.elsevier.com/retrieve/pii/S0302283819306190

中国抗癌协会泌尿男生殖系统肿瘤专业委员会前列腺癌学组 . 前列腺癌筛查中国专家共识(2021年版) [J ] . 中国癌症杂志 , 2021 , 31 ( 5 ): 435 - 440 .

Prostate Cancer Group, Urogenital Tumor Professional Committee, Chinese Anti-cancer Association . Urogenital Tumor Professional Committee, Chinese Anti-cancer Association. Chinese expert consensus on prostate cancer screening (2021 version) [J ] . China Oncol , 2021 , 31 ( 5 ): 435 - 440 .

MOLLICA V , RIZZO A , ROSELLINI M , et al . Bone targeting agents in patients with metastatic prostate cancer: State of the art [J ] . Cancers (Basel) , 2021 , 13 ( 3 ): 546 . DOI: 10.3390/cancers13030546 http://doi.org/10.3390/cancers13030546 https://www.mdpi.com/2072-6694/13/3/546 https://www.mdpi.com/2072-6694/13/3/546

PANG K , XIE C Y , YANG Z R , et al . Monitoring circulating prostate cancer cells by in vivo flow cytometry assesses androgen deprivation therapy on metastasis [J ] . Cytometry A , 2018 , 93 ( 5 ): 517 - 524 . DOI: 10.1002/cyto.a.v93.5 http://doi.org/10.1002/cyto.a.v93.5 https://onlinelibrary.wiley.com/toc/15524930/93/5 https://onlinelibrary.wiley.com/toc/15524930/93/5

ZHANG Y , TONERI M , MA H Y , et al . Real-time GFP intravital imaging of the differences in cellular and angiogenic behavior of subcutaneous and orthotopic nude-mouse models of human PC-3 prostate cancer [J ] . J Cell Biochem , 2016 , 117 ( 11 ): 2546 - 2551 . DOI: 10.1002/jcb.v117.11 http://doi.org/10.1002/jcb.v117.11 http://doi.wiley.com/10.1002/jcb.v117.11 http://doi.wiley.com/10.1002/jcb.v117.11

IMASHIRO C , SHIMIZU T . Fundamental technologies and recent advances of cell-sheet-based tissue engineering [J ] . Int J Mol Sci , 2021 , 22 ( 1 ): E425 .

THUMMARATI P , KINO-OKA M . Effect of co-culturing fibroblasts in human skeletal muscle cell sheet on angiogenic cytokine balance and angiogenesis [J ] . Front Bioeng Biotechnol , 2020 , 8 : 578140 . DOI: 10.3389/fbioe.2020.578140 http://doi.org/10.3389/fbioe.2020.578140 https://www.frontiersin.org/article/10.3389/fbioe.2020.578140/full https://www.frontiersin.org/article/10.3389/fbioe.2020.578140/full

WANG Y , ZHOU S K , YANG R X , et al . Bioengineered bladder patches constructed from multilayered adipose-derived stem cell sheets for bladder regeneration [J ] . Acta Biomater , 2019 , 85 : 131 - 141 . DOI: 10.1016/j.actbio.2018.12.016 http://doi.org/10.1016/j.actbio.2018.12.016 https://linkinghub.elsevier.com/retrieve/pii/S1742706118307414 https://linkinghub.elsevier.com/retrieve/pii/S1742706118307414

ZHOU S K , YANG R X , ZOU Q S , et al . Fabrication of tissue-engineered bionic urethra using cell sheet technology and labeling by ultrasmall superparamagnetic iron oxide for full-thickness urethral reconstruction [J ] . Theranostics , 2017 , 7 ( 9 ): 2509 - 2523 . DOI: 10.7150/thno.18833 http://doi.org/10.7150/thno.18833 http://www.thno.org/v07p2509.htm http://www.thno.org/v07p2509.htm

AKIMOTO J , NAKAYAMA M , TAKAGI S , et al . Efficient intrahepatic tumor generation by cell sheet transplantation to fabricate orthotopic hepatocarcinoma-bearing model mice for drug testing [J ] . J Biomed Mater Res A , 2019 , 107 ( 5 ): 1071 - 1079 . DOI: 10.1002/jbm.a.v107.5 http://doi.org/10.1002/jbm.a.v107.5 https://onlinelibrary.wiley.com/toc/15524965/107/5 https://onlinelibrary.wiley.com/toc/15524965/107/5

ALSHAREEDA A T , ALSOWAYAN B , ALMUBARAK A , et al . Exploring the potential of mesenchymal stem cell sheet on the development of hepatocellular carcinoma in vivo [J ] . J Vis Exp , 2018 , ( 139 ): 57805 .

AKIMOTO J , NAKAYAMA M , TAKAGI S , et al . Improved in vivo subcutaneous tumor generation by cancer cell sheet transplantation [J ] . Anticancer Res , 2018 , 38 ( 2 ): 671 - 676 .

BAL E , PARK H S , BELAID-CHOUCAIR Z , et al . Mutations in ACTRT1 and its enhancer RNA elements lead to aberrant activation of Hedgehog signaling in inherited and sporadic basal cell carcinomas [J ] . Nat Med , 2017 , 23 ( 10 ): 1226 - 1233 . DOI: 10.1038/nm.4368 http://doi.org/10.1038/nm.4368 http://www.nature.com/articles/nm.4368 http://www.nature.com/articles/nm.4368

LÓPEZ DE ANDRÉS J , GRIÑÁN-LISÓN C , JIMÉNEZ G , et al . Cancer stem cell secretome in the tumor microenvironment: a key point for an effective personalized cancer treatment [J ] . J Hematol Oncol , 2020 , 13 ( 1 ): 136 . DOI: 10.1186/s13045-020-00966-3 http://doi.org/10.1186/s13045-020-00966-3 https://doi.org/10.1186/s13045-020-00966-3 https://doi.org/10.1186/s13045-020-00966-3

NAMEKAWA T , IKEDA K , HORIE-INOUE K , et al . Application of prostate cancer models for preclinical study: advantages and limitations of cell lines, patient-derived xenografts, and three-dimensional culture of patient-derived cells [J ] . Cells , 2019 , 8 ( 1 ): E74 .

SAKAI Y , YAMANOUCHI K , OHASHI K , et al . Vascularized subcutaneous human liver tissue from engineered hepatocyte/fibroblast sheets in mice [J ] . Biomaterials , 2015 , 65 : 66 - 75 . DOI: 10.1016/j.biomaterials.2015.06.046 http://doi.org/10.1016/j.biomaterials.2015.06.046 https://linkinghub.elsevier.com/retrieve/pii/S0142961215005682 https://linkinghub.elsevier.com/retrieve/pii/S0142961215005682

WANG Z F , HAN L , SUN T Y , et al . Osteogenic and angiogenic lineage differentiated adipose-derived stem cells for bone regeneration of calvarial defects in rabbits [J ] . J Biomed Mater Res A , 2021 , 109 ( 4 ): 538 - 550 . DOI: 10.1002/jbm.a.v109.4 http://doi.org/10.1002/jbm.a.v109.4 https://onlinelibrary.wiley.com/toc/15524965/109/4 https://onlinelibrary.wiley.com/toc/15524965/109/4

PAVLOU M , SHAH M , GIKAS P , et al . Osteomimetic matrix components alter cell migration and drug response in a 3D tumour-engineered osteosarcoma model [J ] . Acta Biomater , 2019 , 96 : 247 - 257 . DOI: 10.1016/j.actbio.2019.07.011 http://doi.org/10.1016/j.actbio.2019.07.011 https://linkinghub.elsevier.com/retrieve/pii/S174270611930491X https://linkinghub.elsevier.com/retrieve/pii/S174270611930491X

WU H W , HE Z X , LI X N , et al . Efficient and consistent orthotopic osteosarcoma model by cell sheet transplantation in the nude mice for drug testing [J ] . Front Bioeng Biotechnol , 2021 , 9 : 690409 . DOI: 10.3389/fbioe.2021.690409 http://doi.org/10.3389/fbioe.2021.690409 https://www.frontiersin.org/articles/10.3389/fbioe.2021.690409/full https://www.frontiersin.org/articles/10.3389/fbioe.2021.690409/full

CHENG Y Q , WANG S B , LIU J H , et al . Modifying the tumour microenvironment and reverting tumour cells: new strategies for treating malignant tumours [J ] . Cell Prolif , 2020 , 53 ( 8 ): e12865 .

RIBATTI D , PEZZELLA F . Overview on the different patterns of tumor vascularization [J ] . Cells , 2021 , 10 ( 3 ): 639 . DOI: 10.3390/cells10030639 http://doi.org/10.3390/cells10030639 https://www.mdpi.com/2073-4409/10/3/639 https://www.mdpi.com/2073-4409/10/3/639

DE PALMA M , BIZIATO D , PETROVA T V . Microenvironmental regulation of tumour angiogenesis [J ] . Nat Rev Cancer , 2017 , 17 ( 8 ): 457 - 474 . DOI: 10.1038/nrc.2017.51 http://doi.org/10.1038/nrc.2017.51 http://www.nature.com/articles/nrc.2017.51 http://www.nature.com/articles/nrc.2017.51

浏览量

1511

下载量

CSCD

文章被引用时，请邮件提醒。

提交

工具集

关联资源

MYC通过靶向CD47促进前列腺癌增殖和转移的机制研究

没食子酸增强CAR-T细胞抗实体瘤作用的研究

循环肿瘤细胞在胆管癌侵袭转移中的作用机制研究进展及展望

生物3D打印在肿瘤研究及组织工程中的应用

普列克底物蛋白2/miR-196a信号轴介导肿瘤微环境中肺癌细胞的通讯机制研究