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Tumorigenicity Assay in Nude Mice
裸鼠中的致瘤性试验   

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Abstract

Tumorigenicity refers to the ability of cultured cells to develop viable tumors in immune-deficient animals. The goal of this protocol is to illustrate tumorigenicity assay by subcutaneous tumor-cell-transplantation in nude mice. Target cells are transplanted to 6-week-old nude mice subcutaneously and the tumor growth is monitored over a period of observation or treatment. When tumor grows to a pre-determined size or by the end of the limited period, the nude mice will be euthanatized and the xenograft will be removed for further examination.

Keywords: Tumorigenicity(致瘤性), Nude mice(裸鼠), Tumor cell transplantation(肿瘤细胞移植), Tumor xenograft model(肿瘤异种移植模型)

Background

With high incidence and mortality, tumor is one of the leading causes of death and is a major public health problem. Extensive studies are conducted every year to explore tumor pathogenesis and anti-tumor therapy. In the process of cancer research, tumorigenicity assay in nude mice is a widely used experiment to monitor tumor growth in vivo (Giovanella et al., 1974). The most commonly used animal system for tumorigenicity assay is athymic nude mouse (Petricciani et al., 1973) where malignant cells can be transplanted either subcutaneously or subrenal-capsularly (van Meir, 1997). For cells with relatively low ability of tumorigenicity, take rate can be improved by irradiation (30-60 Gy) or implantation in Matrigel (Pretlow et al., 1991). Tumorigenicity assay provides a means of generating human cell derived tumor tissues for measurement of tumor cell malignancy and evaluation for anti-tumor drug efficacy.

Materials and Reagents

  1. 0.1-20 ml pipette tips (Eppendorf, catalog number: 22492012 )
  2. 5-200 ml pipette tips (Eppendorf, catalog number: 22492039 )
  3. 50-1,000 ml pipette tips (Eppendorf, catalog number: 22492055 )
  4. Cell culture disc (75-cm2) (Corning, catalog number: 430641 )
  5. Falcon 15 ml conical centrifuge tubes (Corning, catalog number: 430791 )
  6. Counting slides (Bio-Rad Laboratories, catalog number: 1450011 )
  7. Eppendorf tubes (1.5 ml) (Eppendorf, catalog number: 0030120086 )
  8. Tuberculin syringe (1 ml) (BD, catalog number: 300841 )
  9. BD 1 ml syringe (BD, catalog number: 309628 )
  10. BD PrecisionGlide needle (25 G, 0.5 x 40 mm) (BD, catalog number: 301808 )
  11. Female athymic nude mice (6-8 week old, Fourth Military Medical University, Experimental Animal Center, BALB/c)
  12. Phosphate buffered saline (PBS) pH 7.4 (Thermo Fisher Scientific, GibcoTM, catalog number: C10010500BT )
  13. Trypsin-EDTA (0.25%) (Thermo Fisher Scientific, GibcoTM, catalog number: 25200072 )
  14. Ethanol (Tianjin Fuyu Fine Chemical, catalog number: 20160916 )
  15. RPMI 1640 medium (Thermo Fisher Scientific, GibcoTM, catalog number: C11875500BT )
  16. Fetal bovine serum (FBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 10099141 )
  17. Penicillin-streptomycin (5,000 U/ml) (Thermo Fisher Scientific, GibcoTM, catalog number: 15070063 )
  18. L-glutamine (Thermo Fisher Scientific, GibcoTM, catalog number: 25030081 )
  19. Complete 1640 medium (see Recipes)

Equipment

  1. 2-20 μl pipettes (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 4641060N )
  2. 20-200 μl (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 4641080N )
  3. 100-1,000 μl (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 4641100N )
  4. Water-Jacketed CO2 incubators (Thermo Fisher Scientific, model: Model 3100 Series , catalog number: 3131)
  5. Clean bench (Thermo Fisher Scientific, model: HeraguardTM ECO )
  6. Centrifuge (Eppendorf, model: 5424 R )
  7. Automated cell counter (Bio-Rad Laboratories, model: T20TM )
  8. Microscope (Olympus, models: CX23 and BX53 )
  9. Caliper (Mitutoyo, catalog number: 530-118 )

Procedure

  1. Preparing cells for transplantation
    1. Cells were maintained in complete medium at 37 °C in 5% CO2.
    2. Trypsinize cells at 80-90% confluence (in exponential growth phase) and harvest cells with complete medium to a sterile centrifuge tube.
      Note: Cells should be harvested in the exponential growth stage, which can greatly affect the viability of cell line transplantation. If the tumor viability is poor with a cell line xenograft, Matrigel can be used as injection mixture to provide an enriched stromal environment for engraftment.
    3. Pellet cells at 60 x g for 5 min, remove supernatant and resuspend cells homogeneously in PBS.
      Note: Cells should be resuspended into monoplast suspension for cell counting.
    4. Measure and adjust cell concentration to 1-5 x 107/ml
      Note: Ensure that cells suspension is mixed uniformly for accurate cell counting.
    5. Transfer 5 x 106 cells and spin at 60 x g for 5 min for cell collection.
    6. Resuspend the pelleted cells in 0.2 ml PBS and move them into a sterile Eppendorf tube (1.5 ml) for injection.

  2. Subcutaneous injection of cells
    1. Fill injector (1 ml) with appropriate volume of cell suspension (0.2 ml), avoiding any bubbles to enter the syringe.
    2. Restrain the animal in an upright position by grasping the skin over the shoulders with the thumb and index finger, keeping the fore legs from pushing the syringe away (Figure 1).
    3. Disinfect the dorsal skin gently with 70%-ethanol-saturated cotton balls for 2-3 times (Figure 1).


      Figure 1. Disinfection of the nude mice dorsal skin. The nude mice were restrained by grasping the skin over the shoulders with left thumb and index finger. Disinfect the dorsal skin gently with 70%-ethanol-saturated cotton balls around the injection site for 2-3 times.

    4. Gently insert the needle into the skin on the dorsal flank to reach the subcutaneous pocket (avoid the underlying muscle layer) (Figure 2A).
    5. Gently expel the contents of the syringe. (Figure 2B).
      Note: If you inject accurately into the subcutaneous pocket, there will be no resistance to expel the contents of the syringe.


      Figure 2. Subcutaneous injection of cell suspension into the nude mice. A. Inserting the needle into the skin on the dorsal flank to reach the subcutaneous pocket but not the underlying muscle layer; B. Expelling the contents of the syringe for the subcutaneous implant.

    6. Withdraw the needle and place mice back into the cage.
      Note: After injection, stay for a few seconds before pulling out the needle in case of leakage of cell suspension.
    7. Monitor survival of nude mice and tumor growth 2-3 times a week, measuring the maximum (L) and minimum (W) length of the tumor using a slide caliper.
    8. Tumor volume can be calculated by the volume formula (Tomayko and Reynolds, 1989):

      VT = 1/2(L x W x W)

      where, L: the maximum length of the tumor; W: the minimum length of the tumor.
    9. When tumor grows to a diameter of 150-200 mm3, remove the xenograft from sacrificed nude mice, weight and measure tumor size for tumor volume.
    10. Preserve the xenograft appropriately for histological or biochemical investigation if it is required.

Data analysis

  1. Tumor size and weight of different groups are presented as the mean ± SD., which are adapted for tumor growth curve.
  2. Differences between means were assessed using Student’s t-test. P < 0.05 was considered to be statistically significant.

Notes

It is recommended to perform a small pilot study to determine the tumor take rate before the formal experiments. Additional cells and mice needed if the take rate is less than 100%.

Recipes

  1. Complete 1640 medium
    10% FBS
    1% penicillin-streptomycin
    2 mM glutamine

Acknowledgments

This work was funded by NSFC grants 81602641 to Dr. Xiaodi Zhao.

References

  1. Giovanella, B. C., Stehlin, J. S. and Williams, L. J., Jr. (1974). Heterotransplantation of human malignant tumors in "nude" thymusless mice. II. Malignant tumors induced by injection of cell cultures derived from human solid tumors. J Natl Cancer Inst 52(3): 921-930.
  2. Petricciani, J. C., Kirchstein, R. L., Hines, J. E., Wallace, R. E. and Martin, D. P. (1973). Tumorigenicity assays in nonhuman primates treated with antithymocyte globulin. J Natl Cancer Inst 51(1): 191-196.
  3. Pretlow, T. G., Delmoro, C. M., Dilley, G. G., Spadafora, C. G. and Pretlow, T. P. (1991). Transplantation of human prostatic carcinoma into nude mice in matrigel. Cancer Res 51(14): 3814-3817.
  4. Tomayko, M. M. and Reynolds, C. P. (1989). Determination of subcutaneous tumor size in athymic (nude) mice. Cancer Chemother Pharmacol 24(3): 148-154.
  5. van Meir, E. G. (1997). Identification of nude mice in tumorigenicity assays. Int J Cancer 71(2): 310.

简介

致瘤性是指培养的细胞在免疫缺陷型动物中发展活肿瘤的能力。 该方案的目的是通过裸鼠皮下肿瘤细胞移植来说明致瘤性测定。 将靶细胞皮下移植到6周龄的裸鼠中,并在观察或治疗期间监测肿瘤生长。 当肿瘤生长至预定大小或在有限期结束时,裸鼠将被安乐死,异种移植物将被去除以进一步检查。
【背景】肿瘤发病率高,死亡率高,是导致死亡的主要原因之一,也是一个重大的公共卫生问题。 每年进行广泛的研究,以探索肿瘤发病机制和抗肿瘤治疗。 在癌症研究的过程中,裸鼠中的致瘤性测定是用于监测体内肿瘤生长的广泛使用的实验(Giovanella et al。,1974)。 用于致瘤性测定的最常用的动物系统是无胸腺裸鼠(Petricciani等人,1973),其中恶性细胞可以皮下或肾下 - 荚膜移植(van Meir,1997)。 对于具有相对低致瘤性能力的细胞,通过照射(30-60Gy)或植入Matrigel(Pretlow等人,1991)可以提高摄取率。 致瘤性测定提供了产生人细胞衍生的肿瘤组织用于测量肿瘤细胞恶性肿瘤和评价抗肿瘤药物功效的方法。

关键字:致瘤性, 裸鼠, 肿瘤细胞移植, 肿瘤异种移植模型

材料和试剂

  1. 0.1-20毫升移液器吸头(Eppendorf,目录号:22492012)
  2. 5-200 ml移液器吸头(Eppendorf,目录号:22492039)
  3. 50-1,000毫升移液器吸头(Eppendorf,目录号:22492055)
  4. 细胞培养盘(75厘米 2)(康宁,目录号:430641)
  5. Falcon 15 ml锥形离心管(Corning,目录号:430791)
  6. 计数片(Bio-Rad Laboratories,目录号:1450011)
  7. Eppendorf管(1.5ml)(Eppendorf,目录号:0030120086)
  8. 结核菌素注射器(1 ml)(BD,目录号:300841)
  9. BD 1ml注射器(BD,目录号:309628)
  10. BD PrecisionGlide针(25 G,0.5 x 40 mm)(BD,目录号:301808)
  11. 女性无胸腺裸鼠(6-8周龄,第四军医大学实验动物中心,BALB / c)
  12. 磷酸盐缓冲盐水(PBS)pH 7.4(Thermo Fisher Scientific,Gibco TM,目录号:C10010500BT)
  13. 胰蛋白酶-EDTA(0.25%)(Thermo Fisher Scientific,Gibco TM,目录号:25200072)
  14. 乙醇(天津富宇精细化工,目录号:20160916)
  15. RPMI 1640培养基(Thermo Fisher Scientific,Gibco TM,目录号:C11875500BT)
  16. 胎牛血清(FBS)(Thermo Fisher Scientific,Gibco TM,目录号:10099141)
  17. 青霉素 - 链霉素(5,000U / ml)(Thermo Fisher Scientific,Gibco TM,目录号:15070063)
  18. L-谷氨酰胺(Thermo Fisher Scientific,Gibco TM,目录号:25030081)
  19. 完成1640培养基(见食谱)

设备

  1. 2-20μl移液管(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:4641060N)
  2. 20-200μl(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:4641080N)
  3. 100-1,000μl(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:4641100N)
  4. 水封二氧化碳培养箱(Thermo Fisher Scientific,型号:3100系列,目录号:3131)
  5. 清洁工作台(Thermo Fisher Scientific,型号:Heraguard TM ECO)
  6. 离心机(Eppendorf,型号:5424 R)
  7. 自动细胞计数器(Bio-Rad Laboratories,型号:T20 TM
  8. 显微镜(Olympus,型号:CX23和BX53)
  9. 卡钳(三丰,目录号:530-118)

程序

  1. 准备移植细胞
    1. 将细胞在37℃,5%CO 2维持在完全培养基中
    2. 以80-90%汇合(指数生长期)胰蛋白酶处理细胞,并用完全培养基收获细胞至无菌离心管。
      注意:细胞应在指数生长阶段收获,这可能极大地影响细胞系移植的生存力。如果细胞系异种移植瘤的肿瘤生存力差,Matrigel可用作注射混合物,以提供植入物的富集基质环境。
    3. 以60μg的粒细胞5分钟,除去上清液并将细胞重新悬浮于PBS中。
      注意:细胞应重新悬浮于单质细胞悬液中进行细胞计数。
    4. 测量和调整细胞浓度为1-5 x 10 / ml
      注意:确保细胞悬浮液均匀混合,以确保细胞计数。
    5. 转移5×10 6个细胞并以60×g旋转5分钟用于细胞收集。
    6. 将沉淀的细胞重悬于0.2ml PBS中,并将其移至无菌Eppendorf管(1.5ml)中注射。

  2. 皮下注射细胞
    1. 用适量体积的细胞悬浮液(0.2ml)注射器(1ml),避免任何气泡进入注射器。
    2. 用拇指和食指抓住肩膀上的皮肤,将动物放在直立的位置,保持前腿不要将注射器推开(图1)。
    3. 用70%乙醇饱和的棉球轻轻消毒背部皮肤2-3次(图1)。


      图1.裸鼠背部皮肤的消毒裸鼠通过用左拇指和食指抓住肩膀上的皮肤来约束。用注射部位周围70%乙醇饱和的棉球轻轻消毒背部皮肤2-3次。

    4. 轻轻地将针插入背侧皮肤到达皮下袋(避免下面的肌肉层)(图2A)。
    5. 轻轻排出注射器的内容物。 (图2B)。
      注意:如果将其精确注射到皮下的口袋中,则不会排出注射器内容物的阻力。


      图2.将细胞悬液皮下注射到裸鼠中。 :一种。将针插入背侧皮肤到达皮下袋而不是下面的肌肉层; B.排出注射器的内容物用于皮下植入。

    6. 取出针头,将老鼠放回笼子里。
      注意:注射后,停留几秒钟,然后拔出针头,以防细胞悬浮液渗漏。
    7. 监测裸鼠的生存和肿瘤生长每周2-3次,使用滑动卡尺测量肿瘤的最大(L)和最小(W)长度。
    8. 肿瘤体积可以通过体积公式计算(Tomayko和Reynolds,1989):

      V = 1/2(L×W×W)

      其中,L:肿瘤的最大长度; W:肿瘤的最小长度。
    9. 当肿瘤生长至150-200mm 3的直径时,从处死的裸鼠中取出异种移植物,重量并测量肿瘤体积的肿瘤大小。
    10. 如果需要,可以适当地保留异种移植物进行组织学或生化调查。

数据分析

  1. 不同组的肿瘤大小和体重均以±SD表示,适合肿瘤生长曲线
  2. 手段之间的差异使用Student's 测试进行评估。 0.05被认为具有统计学意义

笔记

建议进行一项小型试点研究,以确定正式实验前的肿瘤采集率。如果摄取率小于100%,则需要额外的细胞和小鼠。

食谱

  1. 完成1640媒体
    10%FBS
    1%青霉素 - 链霉素
    2 mM谷氨酰胺

致谢

这项工作由国家自然科学基金委员会拨款81602641资助给赵小迪博士。

参考

  1. Giovanella,BC,Stehlin,JS和Williams,LJ,Jr.(1974)。&lt; a class =“ke-insertfile”href =“http://www.ncbi.nlm.nih.gov/pubmed/4524119” target =“_ blank”>“裸体”无胸腺小鼠中人类恶性肿瘤的异种移植。 II。通过注射来源于人类实体瘤的细胞培养物引起的恶性肿瘤。 J Natl Cancer Inst 52(3):921-930。
  2. Petricciani,JC,Kirchstein,RL,Hines,JE,Wallace,RE和Martin,DP(1973)。&nbsp; 用抗胸腺细胞球蛋白治疗的非人类灵长类动物中的致瘤性测定。美国国立癌症研究所51(1):191-196。
  3. Pretlow,TG,Delmoro,CM,Dilley,GG,Spadafora,CG和Pretlow,TP(1991)。将人类前列腺癌移植到基质胶中的裸鼠中。癌症研究 51(14):3814-3817。
  4. Tomayko,MM和Reynolds,CP(1989)。&nbsp; 确定(裸鼠)小鼠的皮下肿瘤大小。癌症化学药物24(3):148-154。
  5. van Meir,EG(1997)。裸鼠的鉴定在致瘤性测定中。 Int J Cancer 71(2):310.
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Du, F., Zhao, X. and Fan, D. (2017). Tumorigenicity Assay in Nude Mice. Bio-protocol 7(13): e2364. DOI: 10.21769/BioProtoc.2364.
  2. Zhao, X. D., Lu, Y. Y., Guo, H., Xie, H. H., He, L. J., Shen, G. F., Zhou, J. F., Li, T., Hu, S. J., Zhou, L., Han, Y. N., Liang, S. L., Wang, X., Wu, K. C., Shi, Y. Q., Nie, Y. Z. and Fan, D. M. (2015). MicroRNA-7/NF-kappaB signaling regulatory feedback circuit regulates gastric carcinogenesis. J Cell Biol 210(4): 613-627.
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