通路抑制剂LY294002、PD98059对SDF-1介导的卵巢癌细胞增殖和侵袭能力的影响

发布时间：2018-04-19 23:08

本文选题：卵巢癌细胞 + 基质细胞衍生因子1通路抑制剂　；参考：《郑州大学》2014年硕士论文

【摘要】：背景与目的基质衍生因子-1（stromalcell-derived factorl,SDF-1）是由骨髓基质细胞及其他相关的间皮细胞和上皮细胞分泌的一种趋化蛋白，系统命名为CXCL12。趋化因子受体4(chemokine receptor4,CXCR4)是SDF-1的主要受体。SDF-1与CXCR4特异性结合后形成的SDF-1/CXCR4生物轴广泛参与体内多种病理生理过程，研究证实SDF-1/CXCR4与肿瘤的增殖、浸润和转移密切相关。SDF-1与CXCR4的结构和相互作用机制是发挥其病理、生理功能的基础。尽管有报道称SDF-1、CXCR4在卵巢癌组织及细胞中呈高表达，SDF-1可促进卵巢癌细胞的增殖和侵袭、迁移能力，但关于SDF-1/CXCR4对卵巢癌增殖和侵袭能力影响的作用机制尚不清楚。信号通路的激活是癌发生的早期事件，磷脂酰肌醇3激酶/蛋白激酶B(PI3K/Akt)信号途径是细胞内重要的信号传导通路；分裂原活化抑制剂/蛋白激酶(MEK/ERK)信号途径是重要的细胞外信号传导通路。本实验主要研究SDF-1对卵巢癌细胞的增殖、侵袭能力的促进作用是否能被MEK抑制剂PD98059、Akt抑制剂LY294002所阻断，从而探讨SDF-1/CXCR4生物轴与信号通路MEK/ERK及PI3K/AKT信号通路的关系。方法 1.细胞培养卵巢癌细胞系SKOV3，应用含12%胎牛血清的RPMI-1640培养基培养，置于5%CO2、37℃、饱和湿度的细胞培养箱中孵育。 2.采用细胞免疫化学检测SDF-1作用不同时间以及不同作用浓度，卵巢癌细胞表达磷酸化AKT（P-AKT）和ERK(P-ERK)水平的变化； 3.采用四甲基亚唑蓝（MTT）法检测卵巢癌SKOV3细胞在100ng/ml SDF-1、10μg/ml CXCR4中和抗体、1μg/ml CXCR4抑制剂AMD3100、50μmol/L LY294002、50μmol/L PD98059作用下细胞增殖能力的变化。 4.采用Transwell细胞侵袭实验法检测卵巢癌SKOV3细胞在100ng/ml SDF-1、10μg/ml CXCR4中和抗体、1μg/ml CXCR4抑制剂AMD3100、50μmol/L LY294002、50μmol/L PD98059作用下细胞侵袭能力的变化。 5.采用SPSS16.0统计软件处理数据，定性资料采用等级资料秩和检验；定量资料以x±s表示，方差齐性检验，多组间的比较采用方差分析，多个样本均数的两两比较采用LSD-t检验。检验水准=0.05，以P0.05为差异具有统计学意义。结果 1.磷酸化的ERK蛋白主要表达于SKOV3细胞的胞质和胞核中，以胞核表达为主，磷酸化的Akt主要表达于卵巢癌细胞的胞质中，镜下可见棕黄色颗粒。根据阳性细胞数及细胞染色程度表示蛋白表达水平，随着100ng/mlSDF-1作用时间的延长,棕黄色颗粒由浅至深，差异具有统计学意义（P-ERK:2=24.011,P=0.000；P-AKT:2=16.408,P=0.003）；30分钟组与60分钟组相互比较，细胞内棕黄色颗粒的染色程度基本一致，无明显差异（P-AKT:2=4.835，P=0.089；P-ERK:2=1.725，P=0.422)。可见100ng/mlSDF-1的最佳作用时间为30分钟；当作用时间为30分钟时，随SDF-1浓度的增加，P-ERK、P-Akt蛋白的染色程度也随之加重，差异具有统计学意义（P-ERK:2=8.467,P=0.015；P-AKT:2=8.345,P=0.015）。 2.与无血清对照相比，SDF-1组的吸光度值（OD492）明显较高（d=-0.12378，P=0.007）；但同时加入10μg/mlCXCR4中和抗体(d=0.12956,P=0.005)、1μg/mlCXCR4抑制剂AMD3100后（d=0.13900,P=0.003），与SDF-1组相比，吸光度值（OD492）则无明显升高；此外，在SDF-1中同时加入50μmol/L PD98059（d=0.11594,P=0.014）、50μmol/L LY294002(d=0.10978,P=0.025)后，与SDF-1组相比，吸光度值（OD492）亦无明显升高。 3.与无血清对照相比，SDF-1组的迁移细胞数（平均70.2±9.22316个）明显多于无血清对照组（平均23.1±1.64007个），差异具有统计学意义（d=-47.1,P=0.000）；但同时加入10μg/mlCXCR4中和抗体(平均27.7±4.73873个)、1μg/mlCXCR4抑制剂AMD3100（平均30.3±6.25478个）后，细胞穿透数目无明显增多，与SDF-1组相比差异具有统计学意义；此外，同时加入50μmol/L PD98059（平均20.7±4.34741个）、50μmol/L LY294002（平均24.3±3.59166个）后，，细胞穿透数目亦无明显增多，与SDF-1组相比差异具有统计学意义。结论 SDF-1可促进卵巢癌细胞的增殖和侵袭能力，但这种作用可被MEK抑制剂PD98059、Akt抑制剂LY294002所阻断和拮抗，因此SDF-1/CXCR4可能通过MEK/ERK信号转导通路、PI3K/Akt信号通路发挥作用。
[Abstract]:Background and purpose
The matrix derived factor -1 (stromalcell-derived factorl, SDF-1) is a chemotactic protein secreted by bone marrow stromal cells and other related mesothelial cells and epithelial cells. The system is named CXCL12. chemokine receptor 4 (chemokine receptor4, CXCR4), which is a SDF-1/CXCR4 Sheng formed by the main body.SDF-1 and CXCR4 specificity of SDF-1. The material axis is widely involved in a variety of pathophysiological processes in the body. It is confirmed that SDF-1/CXCR4 is closely related to the proliferation, invasion and metastasis of tumor, and the structure and interaction mechanism of.SDF-1 and CXCR4 is the basis of its pathological and physiological functions. Although it is reported that SDF-1, CXCR4 is highly expressed in the ovarian cancer group and cells, and SDF-1 can promote ovarian cancer. Cell proliferation and invasion, migration ability, but the mechanism of the effect of SDF-1/CXCR4 on the proliferation and invasion of ovarian cancer is not clear. Signal pathway activation is an early event of cancer, phosphatidylinositol 3 kinase / protein kinase B (PI3K/Akt) signal pathway is an important signal transduction pathway in the cell; The protein kinase (MEK/ERK) signal pathway is an important extracellular signal transduction pathway. This experiment mainly studies the proliferation of SDF-1 to ovarian cancer cells. Whether the promoting effect of invasion ability can be blocked by MEK inhibitor PD98059 and Akt inhibitor LY294002, thus exploring the relationship between the SDF-1/CXCR4 biological axis and the signal pathway MEK/ERK and PI3K/AKT signaling pathway. Department.
Method
1. ovarian cancer cell line SKOV3 was cultured in RPMI-1640 medium containing 12% fetal bovine serum and incubated in a 5%CO2,37 cell incubator with a saturated humidity.
2. the changes in the expression of phosphorylated AKT (P-AKT) and ERK (P-ERK) in ovarian cancer cells were detected by cellular immunocytochemistry at different time and different concentrations of SDF-1.
3. using four methazolium blue (MTT) method, the changes of cell proliferation ability of ovarian cancer SKOV3 cells were detected in 100ng/ml SDF-1,10 mu g/ml CXCR4, and 1 mu g/ml CXCR4 inhibitor AMD3100,50 micron mol/L LY294002,50 micron mol/L.
4. the Transwell cell invasion assay was used to detect the SKOV3 cells of ovarian cancer in 100ng/ml SDF-1,10 mu g/ml CXCR4 neutralization antibody, and the change of cell invasiveness under the action of AMD3100,50 micron mol/L LY294002,50 micron mol/L, 1 g/ml CXCR4 inhibitor.
5. SPSS16.0 statistical software was used to deal with data. Qualitative data were tested by rank data and rank and test. Quantitative data were expressed in X + s, variance homogeneity test, analysis of variance among multiple groups, and 22 comparison of multiple samples by LSD-t test. The level =0.05 was tested with P0.05 as a statistical difference.
Result
The ERK protein of 1. phosphorylation is mainly expressed in the cytoplasm and nucleus of SKOV3 cells, mainly expressed in the cytoplasm of the cell nucleus. The phosphorylated Akt is mainly expressed in the cytoplasm of the ovarian cancer cells. The brown yellow granules are visible under the microscope. The protein expression level is expressed according to the number of positive cells and the degree of cell dyed, with the prolongation of the time of the 100ng/mlSDF-1 action, the brown yellow granules From shallow to deep, the difference was statistically significant (P-ERK:2=24.011, P=0.000; P-AKT:2=16.408, P=0.003). The 30 minute group was compared with the 60 minute group, and the staining degree of the brown yellow granules in the cells was basically the same, no significant difference (P-AKT:2=4.835, P=0.089; P-ERK:2= 1.725, P=0.422). The best time for 100ng/mlSDF-1 was found to be 30 minutes. When the time of action was 30 minutes, the staining degree of P-ERK and P-Akt protein increased with the increase of SDF-1 concentration, and the difference was statistically significant (P-ERK:2=8.467, P=0.015; P-AKT:2=8.345, P=0.015).
2. compared with the serum-free control, the absorbance value (OD492) of the SDF-1 group was significantly higher (d=-0.12378, P=0.007), but at the same time, 10 mu g/mlCXCR4 neutralization antibody (d=0.12956, P=0.005) and AMD3100 (d=0.13900, P=0.003) were added to the 1 u g/mlCXCR4 inhibitor (d=0.13900, P=0.003). Compared with the SDF-1 group, the absorbance value was not significantly increased; in addition, 50 micron was added simultaneously. After L PD98059 (d=0.11594, P=0.014), 50 LY294002 mol/L (d=0.10978, P=0.025), the absorbance value (OD492) did not increase significantly compared with the SDF-1 group.
3. compared with the serum-free control, the number of migratory cells in the SDF-1 group (average 70.2 + 9.22316) was significantly more than that in the non serum-free control group (23.1 + 1.64007). The difference was statistically significant (d=-47.1, P=0.000), but at the same time, it was added to the 10 g/mlCXCR4 neutralizing antibody (average 27.7 + 4.73873) and 1 micron g/mlCXCR4 inhibitor AMD3100 (average 30.3 + 6.25478). The number of cell penetration did not increase significantly, and the difference was statistically significant compared with the SDF-1 group. In addition, the number of cell penetration was not significantly increased after adding 50 mu mol/L PD98059 (average 20.7 + 4.34741) and 50 mu mol/L LY294002 (average 24.3 + 3.59166). The difference was statistically significant compared with that of the SDF-1 group.
conclusion
SDF-1 can promote the proliferation and invasion of ovarian cancer cells, but this effect can be blocked and antagonized by the MEK inhibitor PD98059, Akt inhibitor LY294002, so SDF-1/CXCR4 may play a role in the PI3K/Akt signaling pathway through the MEK/ERK signal transduction pathway.

【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R737.31

【参考文献】