兰州大学机构库 >学院待认领
靶向TAK1基因的siRNA干扰前列腺癌DU145细胞的实验研究
Alternative TitleExperiment Research of Interfering Effect On prostate carcinoma DU145 Cells Induced By siRNA Targeting TAK1
吕海迪
Thesis Advisor周逢海
2014-05-14
Degree Grantor兰州大学
Place of Conferral兰州
Degree Name硕士
Keyword转化生长因子-β激活激酶 前列腺癌 侵袭 迁移 凋亡 药物敏感性
Abstract研究目的 研究TAK1siRNA对目标基因表达的干扰作用,检测沉默TAK1表达对前列腺癌DU145细胞增殖、侵袭、迁移、凋亡及药物敏感性的影响及相关蛋白的表达情况,并初步探讨其作用机制。 研究方法 1.应用siRNA靶向沉默前列腺癌DU145细胞的TAK1基因的表达, qRT-PCR和western blot检测TAK1的表达情况,计算TAK1siRNA的基因敲除效率。 2.绘制的生长曲线检测沉默TAK1基因对前列腺癌细胞增殖能力影响。另外利用transwell构建体外骨转移模型,并检测TAK1基因对DU145细胞侵袭、迁移、凋亡及药物敏感性的影响。 3.敲除TAK1基因后通过实时荧光定量PCR和Western blot检测前列腺癌细胞EGFR、Bcl-2、COX-2、β-catenin、MMP-2、9及JNK等基因在转录和翻译水平的变化。 研究结果 1.敲除TAK1基因后,前列腺癌DU145细胞的增殖能力、迁移、迁移能力被抑制,而凋亡率增加。 2.骨髓诱导细胞外基质(BM-ECM)对前列腺癌DU145细胞具有促进生长、侵袭和迁移作用。 3.敲除TAK1基因后前列腺癌DU145对化疗药物的敏感性明显增加。 4.敲除TAK1后相关蛋白表达量均低于对照组。 结论: 沉默DU145细胞TAK1基因的表达可以降低凋亡相关蛋白Bcl-2、JNK,cox-2,EGFR等的表达,从而促进细胞凋亡,抑制细胞生长,增加对化疗药物敏感性;同时可以下调MMP2,MMP9表达可以降低DU145细胞侵袭及迁移能力。TAK1基因可能是前列腺癌细胞信号转导网络中的一个关键性靶点。
Other AbstractObjective Research the interference effect of TAK1siRNA target, and detecting the effect of silence TAK1 expression on cell proliferation, apoptosis, invasion, migration and the influence of the drug sensitivity and related protein expression of prostate cancer DU145, and then to discuss its mechanism of action. Method 1.Using RNAi silencing to influence the TAK1 gene in high expressing cell lines of DU145. Using SqRT-PCR to detect the expression and silencing efficiency of TAK1 mRNA, and calculate the TAK1gene silencing rate. Application of western blot to assay the expression changes of TAK1 protein before and after the transfection. 2.After TAK1 gene expression was successfully silenced, drawing the growth curve to detecting the change of cell proliferation of prostate cancer.Using Transwell Chambers and scratch test to analyze the efffect of TAK1siRNA on the invasion and migration ability on prostate cancer cells.. Using extracellular matrix induced bone marrow (BM-ECM) and transwell Chambers (8.0 μm and 8.0 μm) to build bone metastasis model in vitro, and observe the BM-ECM effect on the promote growth and chemotaxis on prostate cancer cells. Using flow cytometry instrument to test the apoptosis changes before and after the prostate cancer cell was transfected TAK1siRNA.Using MTT method to detect the effect of TAK1siRNA on three kinds of chemotherapy drugs sensitivity docetaxel , L - OHP and 5 - fluorouracil (5 FU)) on prostate cancer DU145 cells. 3.Before and after TAK1siRNAwas transfected,The transcription and expression change of JNK、Bcl-2、COX-2、β-catenin、MMP-2、9、EGFR were detected by qRT-PCR and Western blot. Results 1. Using Lipofectamine 2000 transfect TAK1siRNA can effective knockout TAK1 gene expression, and RT-PCR and western blot analysis suggests that TAK1siRNA can significantly inhibit TAK1 gene transcription and translation, the difference was statistically significance (P<0.05).Cell growth curve suggests that when TAK1 of prostate cancer DU145 was silenced using TAK1siRNA, cell proliferation ability was weakened. The difference was statistically significance Starting from the fourth day (P < 0.05). 2. Transwell and scratch test suggests that the invasion and migration ability of prostate cancer DU145 cell were declined when transfected by TAK1siRNA, the difference was statistically significance (P < 0.05).The apoptosis rate of prostate cancer cells before and after transfected were 4.13±0.11%, and 13.21±1.41%, knoc...
URL查看原文
Language中文
Document Type学位论文
Identifierhttps://ir.lzu.edu.cn/handle/262010/223832
Collection学院待认领
Affiliation临床医学院
Recommended Citation
GB/T 7714
吕海迪. 靶向TAK1基因的siRNA干扰前列腺癌DU145细胞的实验研究[D]. 兰州. 兰州大学,2014.
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