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This experiment is used to test the efficacies of chemo treatments or gene therapy in an in vivo system. In this protocol, the mouse xenograft model is used.

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[Bio101] Murine xenograft model
[Bio101] 小鼠异种移植模型

癌症生物学 > 通用技术 > 动物模型 > 细胞侵袭
作者: FengZhi Liu
FengZhi LiuAffiliation: School of Biomedical Sciences, Thomas Jefferson University, Philadelphia, USA
For correspondence: fengzhi6@yahoo.com
Bio-protocol author page: a51
6/5/2012, 9355 views, 1 Q&A
DOI: https://doi.org/10.21769/BioProtoc.221

[Abstract] This experiment is used to test the efficacies of chemo treatments or gene therapy in an in vivo system. In this protocol, the mouse xenograft model is used.

[Abstract] 这个实验方法通常是用来检测在体内环境下化疗或基因治疗的效果。

Materials and Reagents

  1. Severe combined immunodeficiency (SCID) mice
  2. NaCl
  3. Pluronic acid
  4. Phosphate buffered saline (PBS)
  5. HBSS solution

Equipment

  1. 1 ml syringe with 22-24 gauge of needls
  2. Bioluminescent imaging instrument (in university core facility)

Procedure

  1. For single tumor cell (or any kind of tumor cells)
    1. Six-week-old female in-bred Fox Chase SCID mice were obtained from Charles River Laboratories (Hartford, CT, USA). Animals were handled according to a protocol approved by the Institutional Animal Care and Use Committee at our university.
    2. Mice were allowed to acclimate to animal housing, and xenografts were developed by subcutaneously injecting 5 x 106 cancer cells in murine flank bilaterally. Twice weekly, tumor volume was determined using digital caliper measurements and the formula:
      large diameter2 x small diameter
                         2
    3. After 14 days, all mice had measurable tumours and were sorted into treatment and control groups with equal number of animals (n=5). Treatment group mice received some dose of chemodrugs dissolved in vehicle (0.1 M NaCl, 0.05% pluronic acid in PBS) per treatment while control mice received vehicle only.
    4. All mice received 10 intraperitoneal injections over a 14-day period (cycle: Five treatment days followed by two non-treatment days). After 14 days (2 cycles), mice were killed. So the whole experiment lasts 28 days.
    5. The tumor sample can be used either for extract RNA, protein or immunohistochemistry staining.

  2. For interaction of tumor and Stella cells (for cancers containing big volume of stella cells)
    --using pancreatic cancer model.
    1. Bioluminescent imaging was done weekly to follow the luciferase signal from BxPC3 cells.
    2. Mice were sacrificed and tumors were harvested and measured.
      1. BxPC3-FL alone, either 0.5 x 106 or 1 x 106 per mouse.
      2. BxPC3-FL with immortalized HPSCs (human pancreatic stellate cells), in varying tumor-to-stroma ratios (1:1, 1:1, or 1:5).
      3. Immortalized HPSCs alone (0.5 x 106 or 1 x 106). All cell suspensions, including the mixture of BxPC3 and HPSCs, were injected in a 50 μl volume of HBSS.
    3. Statistical analysis was done using GraphPad Prism (GraphPad). Comparisons were made using two-tailed Student’s t test and significant difference was defined as P < 0.05. Data are shown as mean ±SE.

Acknowledgments

This protocol was adapted from Hwang et al. (2008).

References

  1. Hwang, R. F., Moore, T., Arumugam, T., Ramachandran, V., Amos, K. D., Rivera, A., Ji, B., Evans, D. B. and Logsdon, C. D. (2008). Cancer-associated stromal fibroblasts promote pancreatic tumor progression. Cancer Res 68(3): 918-926.

材料和试剂

 

1.        SCID (严重联合免疫缺陷型Severe Combined Immunodeficiency) 小鼠

2.        NaCl(氯化钠)

3.        Pluronic acid(聚丙二醇与环氧乙烷的加聚酸)

4.        PBS(磷酸盐缓冲溶液)

5.        HBSS溶液(汉克斯平衡的盐溶液)

 

仪器

 

1.        1.1ml的注射器配用22-24规格的针头。

2.        生物发光成像仪器(大学的核心设备)

 

实验步骤

 

1.        用于单个肿瘤细胞(任何一种肿瘤细胞)

1)             Charles River LaboratoriesHartford, CT, USA)获得六周大的、先天严重联合免疫缺陷型的雌鼠。在本大学所进行的动物操作都是获得动物实验管理小组批准的。

2)             先让小鼠适应新的动物房,同时通过在小鼠侧腹两侧皮下注射5x106癌症细胞来获得异种移植物。两周后,用数字卡尺来测量肿瘤的体积,公式如下:

   大直径2x 小直径

                                2

3)            14天后,所有小鼠的肿瘤都已被测量,然后将小鼠分为实验组和对照组,每组各5只。将一定计量的化疗药物溶于缓冲液中(0.1 M NaCl, 0.05% 聚丙二醇与环氧乙烷的加聚酸的磷酸缓冲液),然后处理实验组的小鼠,而对照组的小鼠只用缓冲液处理。

4)            在这14天中,对所有的小鼠都进行10次的腹膜内注射(循环:5天注射2天不注射),14天(2个循环),小鼠被杀死。整个实验持续28天。

5)            这些肿瘤样品可以用来提取RNA、蛋白质或者免疫组织化学染色。

2.        用于研究肿瘤细胞和 Stella细胞的互作(也用于含有大体积Stella细胞的肿瘤)

用于胰腺肿瘤模型

1)             用患有胰腺癌的小鼠作为异种移植的模型,这种胰腺癌的肿瘤细胞是用萤火虫荧光素酶(BxPC3—FL)标记的。

2)             所有的小鼠分成3组,均进行胰内注射,但注射药物不同。如下:

a.       只注射萤火虫荧光素酶(BxPC3—FL),每鼠注射量0.5x106  1x106 

b.      注射萤火虫荧光素酶(BxPC3—FL)和永生的 HPSCs(人类胰腺星状细胞),按变化的肿瘤基质比:1:1, 1:1, or 1:5注射。

c.       只注射永生的 HPSCs0.5x106  1x106)。

所有的细胞悬浮液(包括 BxPC3 HPSCs的混合液)都在50mLHBSS中注射。

3.        注意观察细胞中的荧光素酶信号,每周做一次生物发光成像。

4.        杀死小鼠,获得肿瘤并对其进行测量。统计学分析。用GraphPad Prism进行统计学分析。同时用两个学生的跟踪检测做比较,两个人的检测结果不同的概率P < 0.05.数据显示为平均值±SE(标准误差)

 

试剂配方:

 

1.        0.1 M NaCl

2.        含有0.05%聚丙二醇与环氧乙烷的加聚酸的磷酸缓冲液。

 

参考文献

 

1.         Liles J.S., Arnoletti J.P., Kossenkov A.V., Mikhaylina A., Frost A.R., Kulesza P., Heslin M.J., Frolov A. (2011). Targeting erbB3-mediated stromal-epithelial interactions in pancreatic ductal adenocarcinoma. British Journal of Cancer 105(4): 523-33.

2.         Hwang R.F., Moore T., Arumugam T., Ramachandran V., Amos K.D., Rivera A., Ji B., Evans D.B., Logsdon C.D. (2008). Cancer-associated stromal fibroblasts promote pancreatic tumor progression. Cancer Research 68(3): 918-26. 

 

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How to cite this protocol: Liu, F. (2012). Murine xenograft model. Bio-protocol Bio101: e221. DOI: 10.21769/BioProtoc.221; Full Text



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7/8/2012 12:30:51 PM  

Q1. Which part in mouse body do you expect to see the tumors? Does it depend on types of injected tumor cells or injection location?

Q2. Can you elaborate in more details how you visualize the tumors in step 1-(2)?

7/8/2012 9:24:55 PM  

FengZhi Liu (Author)
School of Biomedical Sciences, Thomas Jefferson University, USA

The mouse usually is SCID mouse. Any tumor can grow if you use enough amount. The side is subcutaneous on the back of neck. You just hod the skin with thumb and index and inject the tumor cells.

The tumors take some time to grow to visible. Some tumors grow fast, and may takes one week, some not, it depends. you just measure the sizes in different groups with a gauze.

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