细胞迁移/细胞侵袭/细胞功能学

细胞迁移/细胞侵袭/侵袭小室实验/transwell实验/细胞功能检测/细胞迁移与侵袭/细胞实验

摘要：在A549细胞中过表达human Oct-4基因，通过对Oct-4过表达细胞组、空细胞组与阴性对照组进入下室的细胞数量进行测定，研究Oct-4基因对细胞迁移能力的影响。实验结果显示Oct-4基因可以促进A549细胞的迁移能力。为进一步深入研究Oct-4基因的功能提供实验数据。

一、实验原理

Transwell小室底层的一张有通透性的膜（一般为聚碳酸酯膜），将其放置于孔板中，小室内称上室，培养板内称下室。由于聚碳酸酯膜有通透性，下层培养液中的成分可以影响到上室内的细胞。应用Transwell可研究下层培养液中的成分对细胞生长、运动等的影响。

肿瘤迁移实验：研究肿瘤细胞的迁移能力或特定情况下肿瘤细胞的迁移能力。常用8.0、12.0µm膜，上室种肿瘤细胞，下室加入FBS或某些特定的趋化因子，肿瘤细胞会向营养成分高的下室跑，计数进入下室的细胞量可反映肿瘤细胞的迁移能力。

肿瘤侵袭实验：研究肿瘤细胞的侵袭能力或特定情况下肿瘤细胞的侵袭能力。在聚碳酸酯膜上涂上一层基质胶，模仿细胞外基质，上室种肿瘤细胞，下室加入FBS或某些特定的趋化因子，细胞要把基质消化后才可以从上室迁移到下室，计数进入下室的细胞量测定细胞的侵袭能力。

二、 实验目的

用质粒Oct-4-EGFP转染后的A549细胞与正常细胞组、阴性对照组进行比较分析，考察基因Oct-4对A549细胞的迁移能力产生的影响。

三、 实验步骤

实验共分以下4个主要步骤：

1.质粒转染细胞：

准备A549细胞，转染前一天按照70-80%密度铺在6cm培养皿中。16h后，按照转染试剂说明书转染细胞（8μg质粒，20μl lipofectamine 2000转染试剂） 。

2.Transwell铺板；

4.统计分析。
显微镜下拍照，使用Image-Pro Plus软件进行细胞计数。

四、实验结果

图1. 过表达Oct-4对A549细胞迁移能力的影响

从左开始依次为A549空细胞组，NC对照组（过表达EGFP），实验组（过表达Oct-4-EGFP），每组实验均重复3次

 图2. 过表达Oct-4对A549细胞迁移能力影响的统计分析

**表示p<0.01
结论：从实验结果以及进入小室的细胞的数量统计分析可以发现，A549细胞在过表达Oct-4-GFP 72h以后，迁移能力均高于空细胞组以及对照组（p<0.01）。

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