Effect of different passage number on the biological characteristics of U87 glioblastoma cell line
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摘要:
目的 探索不同传代次数对人胶质瘤U87细胞系生物学特性的影响及其分子机制。 方法 以两种不同传代次数的U87（I）、U87（II）为研究对象，使用MTT细胞增殖实验、Transwell小室迁移实验及Boyden小室侵袭实验分别检测U87细胞的增殖、迁移和侵袭能力；使用Western Blot技术检测U87（Ⅰ）及U87（Ⅱ）的CTHRC1, FOXM1, PLOD2, MMP9, TGF-β, E-cadherin, Slug, Snail, Vimentin, PI3K, p-PI3K, Akt, p-Akt的表达量差异。 结果 U87（Ⅰ）较U87（Ⅱ）更容易形成网状结构，有更强的侵袭能力，但在增殖和迁移能力上二者无明显差异。在EMT相关蛋白表达水平上，U87（Ⅰ）中的Snail、Vimentin的表达量较U87（Ⅱ）的高，而E-Cadherin、Slug则较低；在PI3K/Akt通路蛋白表达水平上，U87（Ⅰ）的p-Akt，Akt的表达量均低于U87（II），而p-PI3K的表达量却高于U87（II），两者在PI3K的表达量上无明显差异；除此之外，U87（I）中PLOD2、CTHRC1及MMP9的表达量也明显高于U87（II），而TGF-β的表达量则低于后者。 结论 随着传代次数的增加，U87细胞在侵袭能力以及多个促癌基因表达上发生了变化，这可能造成分子机制研究的前后结果不一致，降低结果的可信程度，因此研究人员在进行细胞系实验时，应尽可能在短时间内，使用同一批次细胞完成生物学特性、分子机制研究，以减少传代次数对肿瘤细胞系生物学特性以及基因表达的影响。 Abstract:Objective To unravel the differences of biological characteristics and the uderlying molecular mechanisms between different passages of U87 glioblastoma cell line. Methods U87 (I) and U87 (II), with less or more passage number respectively, were established examined separately by MTT assay, transwell chamber assay, boyden chamber assay. Western blot was used to analyze the expression of CTHRC1, FOXM1, PLOD2, MMP9, TGF-β, E-cadherin, Slug, Snail, Vimentin, PI3K, p-PI3K, Akt and p-Akt separately in these two types of cells. Results Compared to U87 (II), U87 (I) was more prone to form tubules and showed more invasiveness, while there were no differences between U87 (I) and U87 (II) in proliferation and migration. The expression of markers of EMT varies in these two types of cells, with more expression of E-cadherin and Slug and less expression of Vimentin and Snail in U87 (II). Moreover, U87 (II) was found to have greater amount of Akt and p-Akt, but have smaller amount of p-PI3k. No significant difference was found on the expression of PI3K between these cells. Additionally, the expression of PLO2, CTHRC1 and MMP9 were higher in U87 (I) than that in U87 (II), while the expression of TGF-β in U87 (I) was lower than that in the latter one. Conclusion As the passage number increased, U87 cell lines exhibited changes in invasiveness as well as some oncogenic genes’ expressions. It may lead to the different results during different periods, making the results inconvincible. Together, our results showed that, to lessen the influence of passage numbers on cells’ biological characteristics, scientists should use cells with same passage numbers and finish the experiments as quickly as they can. -
Key words:
- glioma /
- U87 /
- passage number /
- invasiveness
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