用小鼠乳腺肿瘤细胞教生物学的核心概念：一个简单的实验模块
Summary
A feasible laboratory module for biology undergraduates that explores advanced cellular and molecular concepts using animal cell culture is described. Students grow, characterize and manipulate a breast cancer cell model by exposure to chemotherapy agents. Cell viability is assayed through cell counting using both a standard and novel method.
Abstract
Undergraduate biology students are required to learn, understand and apply a variety of cellular and molecular biology concepts and techniques in preparation for biomedical, graduate and professional programs or careers in science. To address this, a simple laboratory module was devised to teach the concepts of cell division, cellular communication and cancer through the application of animal cell culture techniques. Here the mouse mammary tumor (MMT) cell line is used to model for breast cancer. Students learn to grow and characterize these animal cells in culture and test the effects of traditional and non-traditional chemotherapy agents on cell proliferation. Specifically, students determine the optimal cell concentration for plating and growing cells, learn how to prepare and dilute drug solutions, identify the best dosage and treatment time course of the antiproliferative agents, and ascertain the rate of cell death in response to various treatments. The module employs both a standard cell counting technique using a hemocytometer and a novel cell counting method using microscopy software. The experimental procedure lends to open-ended inquiry as students can modify critical steps of the protocol, including testing homeopathic agents and over-the-counter drugs. In short, this lab module requires students to use the scientific process to apply their knowledge of the cell cycle, cellular signaling pathways, cancer and modes of treatment, all while developing an array of laboratory skills including cell culture and analysis of experimental data not routinely taught in the undergraduate classroom.
Introduction
往往在普通本科生物学课程，细胞周期调控和癌症的主题上眼，但没有详细探讨，因为内容在这些课程的广度留下一点时间深入。此外，本科学生的生物通常不会暴露在与动物细胞培养相关的先进技术。为了帮助学生养成对这些概念有了更深的了解，同时应用和分析所学，实验室活动是作为研究的沃尔特·里德陆军研究所 （WRAIR）扩展实验活动1的修改。实验室模块使用逐步的，实验策略，其包括生长和表征的癌细胞模型，开发和执行细胞计数的方法，确立最佳时间进程和剂量与抗增殖剂处理的细胞，并确定异常细胞信号传导途径。实验还允许开-ended询问。
大多数需要这种活动的技术可以实现在一个典型的生物学教学实验室。活动开始与学生表征的小鼠乳腺肿瘤（MMT）细胞系的形态和生长速率，模型为人类乳腺癌2。乳腺癌的选择，因为它的人口患病率，其熟悉大学年龄的学生，和现有的广泛的数据作为模型癌症。在MMT细胞系特别选择，因为它是容易获得的，良好表征的，具有短倍增时间，容易成长。此外，MMT细胞是雌激素依赖这与大多数女性乳癌一致。学生然后通过与化疗药物，其作用机理是很好的药物和的处理长度的确立。浓度改变允许学生处理细胞识别所述MMT细胞异常细胞信号传导途径评价这些变量对细胞分裂的速率的影响。重点分析了本次活动是细胞活力，它只是需要细胞计数，使用以下两种方法之一的决心。每种方法依赖于强大的显微镜技术。学生确定细胞存活率通过使用标准的，血球的方法和一种新的显微照相方法和建议。根据他们的研究结果，他们可以提出并测试修改的活性。然后学生表示他们的数据和解释结果，以完善他们的假设，并制定新的实验策略。
该实验室的活动适合于大一大二或水平的学生主修生物科学。它被冷凝成可在第一年完成，一般生物学或第二年，蜂窝/分子生物学过程的一周实验室模块。需要正确完成活动的技能包括基本的算术和代数，熟悉的C数组矿石实验室技能（ 例如 ，移液，溶液制作，无菌技术），数据分析，基本光镜和时间管理，以及细胞培养和电子表格软件的讲师知识。所需试剂包括的动物细胞系模型对癌症（ 例如 ，小鼠乳腺肿瘤细胞，MMT 2），化疗剂（ 例如 ，他莫昔芬，姜黄素，二甲双胍，和阿司匹林），台盼蓝和细胞培养基（ 例如，Eagle氏最低必需培养基 ; EMEM）用适当的补充剂（ 例如 ，供体马和牛胎儿血清）。所需仪器包括一个倒置光学显微镜用数码相机连接，计算机100毫米和24孔组织培养板，CO 2培养箱（或等效物），生物安全柜（BSC; II级），血球和数字显微镜软件。
也有特定的实验室活动依赖于动物细胞培养TEAC很好的例子^ h本科生约在细胞生物学3概念。然而，许多需要物资或技术是不容易（ 例如 ，放射性同位素，活的动物组织，先进的影像设备1,4,5），描述是相当先进（ 例如 ，适合400级课程6）协议，或需要多周或学期长的项目6,7。这里描述的实验活动很简单，可与常见的实验室设备一个星期进行。
综上所述，本实验模块有效地引入或增强细胞周期，细胞信号通路与肿瘤的概念，而教学的基本和高级技能实验室，实验数据分析，动物细胞培养的方法和科学的过程。该实验室模块简单，经济方便和开放式查询提供了灵活性和机会。该活动鼓励学生的创造力通过提供模板实验策略，作为一个指导，但不是一个配方。最重要的是，该活动满足了花开分类8所有学习领域，因为它需要记忆，理解，应用，分析，评估和创建通过参与学生在过程中，拉他们走出课本，走进科研的世界。
Protocol
Representative Results
Discussion
实验室模块提出，旨在通过动物细胞培养的先进技术传授细胞生物学的各种话题。模块通过分析若干抗增殖化学品对细胞模拟人类乳腺癌的复制的效果达到这一点。主要检测依赖于细胞计数的基本技术，并引入了一种新的方法来计算使用显微镜的软件单元。包括该模块的活动可以与仪器和设备，在大多数生物学程序可用来进行。该模块可在5天的日程与用品是廉价且容易地得到实现。虽然数码显微…
Disclosures
The authors have nothing to disclose.
Acknowledgements
This work is supported by the Joseph Alexander Foundation, the ASBMB Undergraduate Research Award, 2013-2014, and a Science Award Grant, Marymount Manhattan College, 2012-2013.
Materials
| Tissue Culture Hood | ESCO Labculture Reliant | Class II Type A2 Biological Safety Cabinet | |
| Waterjactor CO2 Incubator | CEDCO | Model 1510 | |
| Bright-line Hemocytometer | American Optical | with two separate grids | |
| Motic Images Plus | Mac OSX Verison 2.0 or higher | ||
| Gilson Pipetman | Rainin instrument co. inc | P-20D, P-200D, P-1000D | |
| CK30/CK40 Culture Microscope | Olympus | 4 objective inverted light microscope with camera | |
| 200 uL Pipet tips | MidSci | 40200C | |
| 1000 uL Pipet tips | MidSci | AVR4 | |
| 10 mL Seriological Pipets | TPP | TP94010 | |
| 24-well plates | CoStar- Tissue Culture Cluster | 3524 | 24 wells, 16 mm well diameter, Radiation sterilized |
| Trypan Blue Solution 0.4% | Sigma | T8154 | 100 mL, cell culture tested non-haz |
| Bright-line Hemacytometer replacement coverslip, non-haz | Sigma | Z375357 | |
| Mouse Mammary Tumor(MMT) cells | ATCC | CCL-51 | |
| Eagle Minimum Essentail Medium (EMEM) | ATCC | 30-2003 | 500 mL |
| Fetal Bovine Serum | Sigma | F0926 | 500 mL |
| Meformin Hydrochloride | Sigma | PHR1084 | 500 mg |
| Tamoxifen | Sigma | T5648 | white or white-yellow powder |
| Curmumin | Sigma | C1386 | yellow-orange powder |
| Aspirin | Sigma | A2093 | meets USP testing specifications |
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