上调Twist基因对SW480细胞株体外增殖及侵袭性影响的研究

发布时间：2018-02-05 16:52

本文关键词： Twist基因结肠癌细胞株细胞转染 RT-PCR Western-blot　出处：《河北医科大学》2011年硕士论文　论文类型：学位论文

【摘要】：目的:Twist基因是存在于人、果蝇、鼠等生物体内的碱性螺旋-环-螺旋(Basichelix loop helix ,bHLH)蛋白,作为一种转录因子,主要表达于胚胎胎盘、中胚层及成人的某些中胚层来源的未分化组织。Twist表达的蛋白在人有202个氨基酸、小鼠有206个氨基酸、蟾蜍有166个氨基酸序列构成,其中人类和小鼠的氨基酸序列具有96%的同源性,在不同的种属中其DNA结合区域有着100%的序列保守,所有Twist基因都能结合E-box的DNA序列:CANNTG。Twist基因在转录水平上调控多种基因的表达[1],最初是在胚胎发育过程中发现它的生物学功能,它可以通过促进上皮-间叶变(epithelial mesenchymal transition , EMT)来调节胚胎的发育。近期研究结果证实[2,3]Twist具有癌基因的特性,能够编码凋亡抑制蛋白,通过多个信号系统参与肿瘤的发生:抑制分化,通过降低P53上游的调控因子ARF的表达阻断P53信号通路,阻碍N-myc信号系统的传递,和抑制NF-κB通路等。它还参与了多种上皮来源的肿瘤细胞发生EMT并促进癌细胞侵袭转移,同时Twsit基因在肿瘤进展中可以诱导一系列间充质标志物的产生。研究表明, Twist基因高表达于多种实体瘤中(包括乳腺癌、前列腺癌、黑色素瘤以及骨肉瘤) ,并能够促进人乳腺癌和黑色素瘤细胞系的凋亡。之前我们已经有研究证实Twist基因在结肠癌细胞株HCT116中呈高表达,而在SW480细胞株中呈低表达,本实验通过将高表达Twist基因的质粒转染入体外培养的人结肠癌SW480细胞株中,观察Twist基因对细胞增殖及侵袭性的影响。方法:本研究将高表达Twist基因的质粒和阴性对照的空载质粒瞬时转染入SW480细胞株中,通过G418筛选后,分别命名为转染组和对照组,经过RT-PCR和Western-blot方法的鉴定,获得稳定转染Twist基因的SW480细胞,再通过MTT法绘制转染组细胞和对照组细胞的生长曲线,然后通过细胞划痕实验和Transwell侵袭实验,观察Twist基因对细胞迁移能力和侵袭力的影响。结果: 1 RT-PCR和Western-blot方法检测结果显示,稳定转染Twist基因的SW480细胞中TwistmRNA和Twist蛋白表达量均显著高于未转染组(P0.05)。 2通过MTT法绘制细胞生长曲线,结果显示,从第4天开始,转染Twist基因的SW480细胞生长速度明显高于对照组(P0.05)。 3通过细胞划痕实验显示,划痕24小时后转染组细胞迁移了(40.06±5.56),而对照组细胞迁移了(25.24±2.65),差异有统计学意义。48小时后,转染组细胞迁移了(75.77±8.06),而对照组迁移了(35.37±6.79),差异有统计学意义(P0.05)。 4体外侵袭实验结果显示,转染Twist基因的SW480细胞发生侵袭的个数为(154±12) ,未转染组细胞侵袭个数为(73±14),差异有统计学意义(P0.05)。结论: 1通过本实验Twist基因成功转入SW480细胞株并且呈稳定表达。 2上调Twist基因表达后,SW480细胞体外增殖率明显上升,说明Twist基因能够增强SW480细胞的体外增殖能力。 3上调Twist基因表达后,SW480细胞体外迁移能力明显增强,说明Twist基因能够增强SW480细胞的迁移能力。 4上调Twist基因表达后,SW480细胞体外侵袭能力明显增强,说明Twist基因能够增强SW480细胞的体外侵袭能力。
[Abstract]:Objective: Twist gene is present in human, Drosophila, mouse basic helix loop helix and other organisms (Basichelix loop, helix, bHLH) protein, as a transcription factor that is expressed in embryonic placenta, some mesoderm mesoderm and adult undifferentiated tissue expression of.Twist protein has 202 amino acids in human mice, 206 amino acids, 166 amino acid sequence of a toad, the amino acid sequence of human and mouse has 96% homology, in different species in the DNA binding domain have 100% conserved sequences, all Twist genes can be combined with the DNA sequence of E-box: the expression of [1] CANNTG.Twist gene regulation at the transcriptional level a variety of genes, originally discovered its biological functions during embryonic development, it can promote epithelial mesenchymal transition (epithelial mesenchymal, transition, EMT) to regulate embryonic development. Nearly Results confirmed that [2,3]Twist has the characteristics of cancer gene, encoding apoptosis proteins through multiple signaling systems involved in tumor suppression and differentiation, blocking P53 pathway by lowering the expression of transcription factor ARF upstream of P53, blocking N-myc signal transmission system, and the inhibition of NF- B pathway. It is involved in the many kinds of epithelial tumor cells EMT and promoting cancer cell invasion and metastasis, while Twsit gene can induce a series of mesenchymal markers in tumor progression. The results show that Twist gene is highly expressed in a variety of solid tumors (including breast cancer, prostate cancer, melanoma and osteosarcoma). Apoptosis and can promote human breast cancer and melanoma cell lines. Before we have proved the high expression of Twist gene in colon cancer cell line HCT116 was a low expression in SW480 cell line, the In order to observe the effect of Twist gene on cell proliferation and invasion, we transfected the plasmid expressing high Twist gene into human colon cancer cell line SW480.
Methods: the study of Twist gene plasmid and negative control plasmid was transiently transfected into SW480 cell line in the high expression by G418 screening, named as transfection group and control group, identified by RT-PCR and Western-blot method, to obtain a stable transfection of Twist gene in SW480 cells, and then through the MTT method to draw the transfected cells the control group and the cell growth curve, followed by cell scratch assay and Transwell invasion assay, to observe the effect of Twist gene on the invasion and migration ability of cells.
Result:
1 RT-PCR and Western-blot detection results showed that the expression level of TwistmRNA and Twist protein in SW480 cells stably transfected with Twist gene was significantly higher than that in the untransfected group (P0.05).
2 the cell growth curve was plotted by MTT method. The results showed that the growth rate of SW480 cells transfected with Twist gene was significantly higher than that of the control group from fourth days (P0.05).
3 by cell scratch test showed that the scratch after 24 hours of transfection group cell migration was (40.06 + 5.56), while the control group, the cell migration (25.24 + 2.65), the difference was statistically significant.48 hours after transfection group cell migration (75.77 + 8.06), while the control group was (35.37 + 6.79) migration. The difference was statistically significant (P0.05).
4 in vitro invasion test showed that the number of SW480 cells transfected with Twist gene was 154 + 12, and the number of invasion in the untransfected group was (73 + 14), the difference was statistically significant (P0.05).
Conclusion:
1 through this experiment, the Twist gene was successfully transferred into the SW480 cell line and showed a stable expression.
2 after the expression of Twist gene was up-regulated, the proliferation rate of SW480 cells increased significantly in vitro, indicating that the Twist gene could enhance the proliferation ability of SW480 cells in vitro.
3 after the expression of Twist gene was up-regulated, the ability of SW480 cells to migrate in vitro was significantly enhanced, indicating that the Twist gene could enhance the migration ability of SW480 cells.
4 after the expression of Twist gene was up-regulated, the invasion ability of SW480 cells in vitro was significantly enhanced, indicating that the Twist gene could enhance the invasiveness of SW480 cells in vitro.

【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R346

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