舒尼替尼阻断STAT3通路抑制头颈癌细胞增殖的实验研究

发布时间：2019-05-11 06:07

【摘要】：目的:舒尼替尼是一种多靶向酪氨酸激酶抑制剂,因其可抑制与血管形成有关的多种受体酪氨酸激酶的活性,具有较好的抗血管生成作用,被应用于治疗多种肿瘤。近来研究发现,舒尼替尼能够诱导肿瘤细胞凋亡而不依赖于其抑制血管生成作用。信号转导及转录激活因子(signal transduction and activator of transcription,STAT)家族中成员STAT3作为多种酪氨酸激酶信号通路的重要组成部分,其活化后在包括头颈癌等多种恶性肿瘤细胞中表达较高。STAT3异常活化并持续高表达与这些恶性肿瘤凋亡的抑制、肿瘤细胞增殖、肿瘤微环境血管形成及转移关系密切。舒尼替尼能否诱导头颈癌细胞凋亡,STAT3在此过程中是否发挥作用,目前相关报道较少。本实验通过使用舒尼替尼分别处理UM-22B和SCC90两种头颈癌细胞系,观察肿瘤细胞增殖和凋亡情况,以及STAT1和STAT3的变化,从而进一步了解舒尼替尼对头颈癌的作用及机制,为舒尼替尼在临床治疗头颈癌提供理论基础。方法:体外培养的SCC90和UM-22B细胞,经过不同浓度舒尼替尼处理不同时间后,分别采用MTT试验、倒置显微镜观察等方法,检测细胞增殖抑制情况;并且采用流式细胞术检测STAT1和STAT3磷酸化水平。结果:1舒尼替尼对两种细胞增殖的影响:MTT显示,舒尼替尼对SCC90细胞增殖的抑制率最高可达92.89%,24,48,72h的IC50分别是2.37、1.17和0.93μmol/L;对UM-22B细胞抑制率最高可达82.56%,24,48h的IC50分别是6.30和2.31μmol/L。用药组不同浓度不同处理时间其生长抑制率有显著差异,抑制率的时间-浓度依赖性明显;2形态学变化:倒置显微镜下,SCC90、UM-22B细胞均显示:对照组细胞生长较用药组迅速,细胞贴壁生长呈“铺路石”状,细胞呈扁平状多角形,胞质饱满,胞质近中央部位有圆形细胞核;用药组细胞增殖受到抑制,贴壁细胞体积变小而呈圆形,连接松解,核颜色较对照组加深;3舒尼替尼对STAT1和STAT3磷酸化水平的影响:SCC90细胞经过0.25,0.5,1μmol/L的舒尼替尼处理2,8,16,24小时后,各实验组中p-STAT1阳性表达与对照组相比,无统计学差异;而p-STAT3阳性表达与对照组相比明显降低,并且随着浓度增高,阳性率显著下降,差异有统计学意义。UM-22B细胞经过0.5,1,2,4μmol/L的舒尼替尼处理30分钟和2小时后,各实验组中p-STAT3阳性表达与对照组相比降低,并且随着浓度增高,阳性率显著下降。其中,两个时间段中,0.5和1μmol/L组与对照组相比无统计学差异,而2和4μmol/L组有显著性差异。结论:1舒尼替尼可以抑制头颈鳞癌UM-22B和SCC90细胞的增殖。2舒尼替尼可以下调UM-22B和SCC90细胞STAT3磷酸化水平。3 STAT1在舒尼替尼抑制头颈癌细胞增殖过程中磷酸化水平未见明显升高。
[Abstract]:Aim: Schnitinib is a multi-target tyrosine kinase inhibitor. It can inhibit the activity of many receptor tyrosine kinases related to angiogenesis and has a good anti-angiogenesis effect. It has been used in the treatment of many kinds of tumors. Recently, it has been found that Schnitinib can induce apoptosis of tumor cells, independent of its inhibitory effect on angiogenesis. STAT3, a member of the signal transduction and activator of transcription (signal transduction and activator of transcription,STAT) family, is an important component of many tyrosine kinase signaling pathways. The abnormal activation and persistent high expression of STAT3 in various malignant tumor cells, including head and neck cancer, were closely related to the inhibition of apoptosis, proliferation of tumor cells and vascular formation and metastasis in tumor microenvironment. There are few reports on whether Schnitinib can induce apoptosis of head and neck cancer cells and whether STAT3 plays a role in this process. In order to further understand the effect and mechanism of Schnitinib on head and neck carcinoma, the proliferation and apoptosis of UM-22B and SCC90 cell lines, and the changes of STAT1 and STAT3 were observed by the treatment of shunitinib, respectively, in order to understand the effect and mechanism of Schnitinib on head and neck cancer. It provides a theoretical basis for the clinical treatment of head and neck cancer with sulnitinib. Methods: SCC90 and UM-22B cells cultured in vitro were treated with different concentrations of sulnitinib for different time. MTT test and inverted microscope were used to detect the inhibition of cell proliferation. The phosphorylation levels of STAT1 and STAT3 were detected by flow cytometry. Results: 1 the effect of sulnitinib on the proliferation of two kinds of SCC90 cells: MTT showed that the inhibition rate of Schnitinib on the proliferation of SCC90 cells reached 92.89%, 24,48 and 2.37 渭 mol / L, 1.17 渭 mol / L and 0.93 渭 mol / L, respectively. The highest inhibition rate of UM-22B cells was 82.56%, 24 h and 48 h IC50 were 6.30 and 2.31 渭 mol / L, respectively. In the treatment group, the growth inhibition rate was significantly different in different concentration and treatment time, and the time-concentration dependence of inhibition rate was obvious. 2 morphological changes: under inverted microscope, SCC90,UM-22B cells in the control group grew faster than those in the treatment group, the adherent growth of the cells was "paving stone", the cells were flat and polyangular, and the cytoplasm was full. Round nucleus was found near the center of cytoplasm. The proliferation of the cells in the treatment group was inhibited, the volume of the adherent cells became small and round, the junction was loosened, and the color of the nucleus was deeper than that of the control group. (3) the effect of sulnitinib on the phosphorylation of STAT1 and STAT3: there was no significant difference in the positive expression of p-STAT1 between the SCC90 cells and the control group 24 hours after treatment with 0.25 渭 mol / L, 0.51 渭 mol / L and 1 渭 mol / L sulnitinib for 2, 8, 16, 24 hours, and there was no significant difference in the positive expression of p-STAT1 between the experimental groups and the control group. The positive expression of p-STAT3 was significantly lower than that of the control group, and the positive rate decreased significantly with the increase of concentration. UM-22B cells were treated with 0.5, 1, 2, 4 渭 mol / L sulnitinib for 30 minutes and 2 hours. The positive expression of p-STAT3 in each experimental group was lower than that in the control group, and the positive rate decreased significantly with the increase of concentration. Among them, there was no significant difference between 0.5 渭 mol / L group and 1 渭 mol / L group and 4 渭 mol / L group, but there was significant difference between 2 渭 mol / L group and 4 渭 mol / L group. Conclusion: 1 Surnitinib can inhibit the proliferation of UM-22B and SCC90 cells in head and neck squamous cell carcinoma. 2 Surnitinib can down-regulate the STAT3 phosphorylation level of UM-22B and SCC90 cells. 3 STAT1 can inhibit the proliferation of head and neck cancer cells during the proliferation of head and neck cancer cells. There was no significant increase in acidizing level.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R739.91

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