利用实时细胞分析技术检测前列腺与胰腺癌细胞药物敏感性及Trim46基因低表达对胰腺癌细胞Capan-2的增殖和迁移能力的

发布时间：2018-05-13 09:38

本文选题：前列腺癌 + 胰腺癌　；参考：《北京协和医学院》2017年硕士论文

【摘要】：目的基于实时细胞分析的技术发展,探索一种更加简便稳定的方法评估不同肿瘤细胞系对药物的敏感性,为前列腺癌和胰腺癌的临床治疗选择药物提供参考。方法选取VCaP、DU145、PC-3、PC-3M-2B4和PC-3M-IE8五株人前列腺癌细胞系,和SW1900、Capan-2、PANC-1三株人胰腺癌细胞系,选用多西他赛、卡巴他赛和醋酸阿比特龙三种前列腺癌常用治疗药物,及盐酸吉西他滨和替吉奥胶囊两种胰腺癌常用治疗药物,细胞接种24小时后分别梯度浓度给药,利用RTCA监测给药前后的细胞生长曲线,计算药物对前列腺癌细胞和胰腺癌细胞的半抑制浓度(IC50)。对胰腺癌细胞培养板内给药,利用AO/EB染色和激光共聚焦观察,验证盐酸吉西他滨和替吉奥胶囊所得IC50值。结果多西他赛对 VCaP、DU145、PC-3、PC-3M-2B4、PC-3M-IE8 五种细胞系 24h的 IC50 分别为 8.81 nM、11.61 nM、1.78 nM、1.44 nM、8.69 nM。卡巴他赛对五种细胞系 24 h 的 IC50 分别为 3.73 nM、3.96 nM、10.41 nM、5.43 nM、7.37 nM。醋酸阿比特龙对五种细胞系24小时的IC50分别为8.34μM、8.60 μM、24.20 μM、8.59 μM、13.21 μM。盐酸吉西他滨对SW1900、Cpan-2、PANC-1三种细胞系72h的IC50 分别为 1.69μmol/L、10.05μmol/L、12.74μmol/L。替吉奥胶囊对三种细胞系 72h的 IC50 分别为 180.29μmol/L、765.70μmol/L、95.57μmol/L。AO/EB 染色验证 IC50真实可靠。结论PC-3M-2B4及DU145可作为多西他赛的对照;VCaP及PC-3可作为卡巴他赛与醋酸阿比特龙的对照,SW1990及Capan-2可作为盐酸吉西他滨与替吉奥胶囊的对照,建立细胞模型,进行药物体外敏感性筛选,为临床应用提供参考。目的:为了验证Trim46低表达对胰腺癌细胞Capan-2增殖能力及迁移能力的影响,观察Trim46在Capan-2细胞内表达位置、与肿瘤迁移相关蛋白的定位关系及对其表达水平的影响。方法:利用miRNA沉默建立Trim46低表达Capan-2细胞。使用Western blot及细胞免疫荧光检测Trim46在Capan-2细胞内的表达水平,确认Trim46低表达Capan-2细胞系的建立;利用免疫荧光检测Trim46在Capan-2细胞内与肿瘤转移相关蛋白的共定位情况;利用Western blot检测Trim46低表达对Capan-2细胞中相关肿瘤转移蛋白表达水平的影响;利用RTCA检测Trim46低表达对Capan-2细胞增殖能力的影响;利用愈伤实验检测Trim46低表达对Capan-2细胞迁移能力的影响。结果:建立了 Trim46低表达Capan-2细胞系;Trim46主要在Capan-2的细胞质及细胞膜处表达,呈点状或丝状结构;Trim46与actin、fak、及integrinbeta2、integrin beta6共定位较好;Trim46低表达会影响细胞黏附转移相关蛋白的表达,FAK总蛋白表达下调、phoshph-FAKpTyr861表达上调、Cortactin(皮层肌动蛋白)表达下调、ARP2(肌动蛋白相关蛋白)表达上调、CrKL表达上调;Trim46低表达能抑制Capan-2细胞的增殖及迁移能力。结论:Trim46基因可能与肿瘤增殖与迁移能力有关。
[Abstract]:Objective to explore a more convenient and stable method to evaluate the drug sensitivity of different tumor cell lines based on the development of real-time cell analysis, and to provide a reference for the clinical treatment of prostate cancer and pancreatic cancer. Methods five PC-3M-2B4 and PC-3M-IE8 human prostate cancer cell lines, and three human pancreatic cancer cell lines SW1900 Capan-2PANC-1 were selected. Three commonly used prostatic cancer drugs, docetaxel, carbatin and abbitrone acetate, were used to treat prostate cancer. And gemcitabine hydrochloride and tigeo capsule were commonly used in the treatment of pancreatic cancer. After 24 hours of cell inoculation, the cell growth curve was monitored by RTCA. The semi-inhibitory concentration of the drug on prostate cancer cells and pancreatic cancer cells was calculated. AO/EB staining and laser confocal observation were used to verify the IC50 values of gemcitabine hydrochloride and tigueo capsules for intraplate administration of pancreatic cancer cells. Results the IC50 of docetaxel on five PC-3M-2B4 PC-3M-3M-IE8 cell lines was 8.81nM 11.61nM 1.78nM, 1.44nM, 8.69 nMfor 24 h, respectively. The IC50 of Carbataxel on five cell lines for 24 h was 3.73 nMN 3.96 nMU 10.41 nMN 5.43 nMN 7.37 nM. respectively. The IC50 of the five cell lines was 8.34 渭 M, 8.60 渭 M, 24.20 渭 M, 8.59 渭 M, and 13.21 渭 m, respectively. The IC50 of gemcitabine hydrochloride was 1.69 渭 mol / L ~ 10.05 渭 mol 路L ~ (-1) and 12.74 渭 mol / L 路L ~ (-1) for 72 h, respectively. The IC50 of Tigeo capsule was 180.29 渭 mol / L 765.70 渭 mol / L 95.57 渭 mol/L.AO/EB for 72 h, respectively, to verify the authenticity of IC50. Conclusion PC-3M-2B4 and DU145 can be used as control group of docetaxel, VCaP and PC-3 can be used as control of carbataxel and abbibilone acetate. SW1990 and Capan-2 can be used as control of gemcitabine hydrochloride and tigueo capsule to establish cell model and screen drug sensitivity in vitro. To provide reference for clinical application. Aim: to investigate the effect of low expression of Trim46 on the proliferation and migration of Capan-2 in pancreatic cancer cells, and to observe the location of Trim46 expression in Capan-2 cells and its relationship with the localization of tumor migration-associated protein and its effect on the expression level of Trim46. Methods: Trim46 low expression Capan-2 cells were established by miRNA silencing. Western blot and cellular immunofluorescence were used to detect the expression of Trim46 in Capan-2 cells, to confirm the establishment of Capan-2 cell line with low expression of Trim46, and to detect the co-localization of Trim46 in Capan-2 cells associated with tumor metastasis. Western blot was used to detect the effect of low expression of Trim46 on the expression of tumor metastasis protein in Capan-2 cells, RTCA was used to detect the effect of low expression of Trim46 on the proliferation of Capan-2 cells, and callus assay was used to detect the effect of low expression of Trim46 on the migration ability of Capan-2 cells. Results: trim46, a Capan-2 cell line with low expression of Trim46, was mainly expressed in the cytoplasm and cell membrane of Capan-2. The low expression of trim46 and integrin beta6 may affect the expression of cell adhesion and metastasis related protein. The expression of total FAK protein down-regulates the expression of Cortactin (cortical actin) and down-regulates ARP2( actin phase of actin phase). The low expression of Trim46 could inhibit the proliferation and migration of Capan-2 cells. Conclusion\% Trim46 gene may be related to tumor proliferation and migration ability.
【学位授予单位】：北京协和医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.9;R737.25

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