本文選題：卵巢癌細(xì)胞 + 基質(zhì)細(xì)胞衍生因子1通路抑制劑��； 參考：《鄭州大學(xué)》2014年碩士論文

【摘要】：背景與目的 基質(zhì)衍生因子-1（stromalcell-derived factorl,SDF-1）是由骨髓基質(zhì)細(xì)胞及其他相關(guān)的間皮細(xì)胞和上皮細(xì)胞分泌的一種趨化蛋白，系統(tǒng)命名為CXCL12。趨化因子受體4(chemokine receptor4,CXCR4)是SDF-1的主要受體。SDF-1與CXCR4特異性結(jié)合后形成的SDF-1/CXCR4生物軸廣泛參與體內(nèi)多種病理生理過程，研究證實(shí)SDF-1/CXCR4與腫瘤的增殖、浸潤和轉(zhuǎn)移密切相關(guān)。SDF-1與CXCR4的結(jié)構(gòu)和相互作用機(jī)制是發(fā)揮其病理、生理功能的基礎(chǔ)。盡管有報(bào)道稱SDF-1、CXCR4在卵巢癌組織及細(xì)胞中呈高表達(dá)，SDF-1可促進(jìn)卵巢癌細(xì)胞的增殖和侵襲、遷移能力，但關(guān)于SDF-1/CXCR4對卵巢癌增殖和侵襲能力影響的作用機(jī)制尚不清楚。信號通路的激活是癌發(fā)生的早期事件，磷脂酰肌醇3激酶/蛋白激酶B(PI3K/Akt)信號途徑是細(xì)胞內(nèi)重要的信號傳導(dǎo)通路；分裂原活化抑制劑/蛋白激酶(MEK/ERK)信號途徑是重要的細(xì)胞外信號傳導(dǎo)通路。本實(shí)驗(yàn)主要研究SDF-1對卵巢癌細(xì)胞的增殖、侵襲能力的促進(jìn)作用是否能被MEK抑制劑PD98059、Akt抑制劑LY294002所阻斷，從而探討SDF-1/CXCR4生物軸與信號通路MEK/ERK及PI3K/AKT信號通路的關(guān)系。 方法 1.細(xì)胞培養(yǎng)卵巢癌細(xì)胞系SKOV3，應(yīng)用含12%胎牛血清的RPMI-1640培養(yǎng)基培養(yǎng)，置于5%CO2、37℃、飽和濕度的細(xì)胞培養(yǎng)箱中孵育。 2.采用細(xì)胞免疫化學(xué)檢測SDF-1作用不同時間以及不同作用濃度，卵巢癌細(xì)胞表達(dá)磷酸化AKT（P-AKT）和ERK(P-ERK)水平的變化； 3.采用四甲基亞唑藍(lán)（MTT）法檢測卵巢癌SKOV3細(xì)胞在100ng/ml SDF-1、10μg/ml CXCR4中和抗體、1μg/ml CXCR4抑制劑AMD3100、50μmol/L LY294002、50μmol/L PD98059作用下細(xì)胞增殖能力的變化。 4.采用Transwell細(xì)胞侵襲實(shí)驗(yàn)法檢測卵巢癌SKOV3細(xì)胞在100ng/ml SDF-1、10μg/ml CXCR4中和抗體、1μg/ml CXCR4抑制劑AMD3100、50μmol/L LY294002、50μmol/L PD98059作用下細(xì)胞侵襲能力的變化。 5.采用SPSS16.0統(tǒng)計(jì)軟件處理數(shù)據(jù)，定性資料采用等級資料秩和檢驗(yàn)；定量資料以x±s表示，方差齊性檢驗(yàn)，多組間的比較采用方差分析，多個樣本均數(shù)的兩兩比較采用LSD-t檢驗(yàn)。檢驗(yàn)水準(zhǔn)=0.05，以P0.05為差異具有統(tǒng)計(jì)學(xué)意義。 結(jié)果 1.磷酸化的ERK蛋白主要表達(dá)于SKOV3細(xì)胞的胞質(zhì)和胞核中，以胞核表達(dá)為主，磷酸化的Akt主要表達(dá)于卵巢癌細(xì)胞的胞質(zhì)中，鏡下可見棕黃色顆粒。根據(jù)陽性細(xì)胞數(shù)及細(xì)胞染色程度表示蛋白表達(dá)水平，隨著100ng/mlSDF-1作用時間的延長,棕黃色顆粒由淺至深，差異具有統(tǒng)計(jì)學(xué)意義（P-ERK:2=24.011,P=0.000；P-AKT:2=16.408,P=0.003）；30分鐘組與60分鐘組相互比較，細(xì)胞內(nèi)棕黃色顆粒的染色程度基本一致，無明顯差異（P-AKT:2=4.835，P=0.089；P-ERK:2=1.725，P=0.422)�？梢�100ng/mlSDF-1的最佳作用時間為30分鐘；當(dāng)作用時間為30分鐘時，隨SDF-1濃度的增加，P-ERK、P-Akt蛋白的染色程度也隨之加重，差異具有統(tǒng)計(jì)學(xué)意義（P-ERK:2=8.467,P=0.015；P-AKT:2=8.345,P=0.015）。 2.與無血清對照相比，SDF-1組的吸光度值（OD492）明顯較高（d=-0.12378，P=0.007）；但同時加入10μg/mlCXCR4中和抗體(d=0.12956,P=0.005)、1μg/mlCXCR4抑制劑AMD3100后（d=0.13900,P=0.003），與SDF-1組相比，吸光度值（OD492）則無明顯升高；此外，在SDF-1中同時加入50μmol/L PD98059（d=0.11594,P=0.014）、50μmol/L LY294002(d=0.10978,P=0.025)后，與SDF-1組相比，吸光度值（OD492）亦無明顯升高。 3.與無血清對照相比，SDF-1組的遷移細(xì)胞數(shù)（平均70.2±9.22316個）明顯多于無血清對照組（平均23.1±1.64007個），差異具有統(tǒng)計(jì)學(xué)意義（d=-47.1,P=0.000）；但同時加入10μg/mlCXCR4中和抗體(平均27.7±4.73873個)、1μg/mlCXCR4抑制劑AMD3100（平均30.3±6.25478個）后，細(xì)胞穿透數(shù)目無明顯增多，與SDF-1組相比差異具有統(tǒng)計(jì)學(xué)意義；此外，同時加入50μmol/L PD98059（平均20.7±4.34741個）、50μmol/L LY294002（平均24.3±3.59166個）后，，細(xì)胞穿透數(shù)目亦無明顯增多，與SDF-1組相比差異具有統(tǒng)計(jì)學(xué)意義。 結(jié)論 SDF-1可促進(jìn)卵巢癌細(xì)胞的增殖和侵襲能力，但這種作用可被MEK抑制劑PD98059、Akt抑制劑LY294002所阻斷和拮抗，因此SDF-1/CXCR4可能通過MEK/ERK信號轉(zhuǎn)導(dǎo)通路、PI3K/Akt信號通路發(fā)揮作用。
[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.

【學(xué)位授予單位】：鄭州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R737.31

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