TGF-β1与P38MAPK在绒毛膜癌JEG-3细胞发生发展中的相互关系的研究

发布时间：2018-05-25 16:25

本文选题：绒毛膜癌 + TGF-β1　；参考：《承德医学院》2014年硕士论文

【摘要】：绒毛膜癌简称绒癌，是一种高度恶性的滋养细胞肿瘤。它由滋养细胞干细胞(细胞滋养层细胞)发生恶性转化而形成，可继发于流产、异位妊娠、葡萄胎、侵蚀性葡萄胎、甚至是正常妊娠。绒癌的发生发展与滋养细胞的过度侵蚀作用密切相关，早期即可发生血道转移而危及患者生命。绒癌的发生是多种因素协同作用的结果，其中细胞信号转导通路在绒癌的形成过程中发挥了重要的作用。TGF-β信号转导通路是当今肿瘤防治领域探讨与钻研的焦点之一。转化生长因子β1（transforming growth factor beta，TGF-β1）是滋养细胞在侵袭母体的过程当中研究最多的细胞调节因子，它主要是通过TGF-β1信号转导通路在滋养细胞肿瘤恶性侵袭与转移的过程中发挥自身的重要作用。P38丝裂原活化蛋白激酶（P38mitogen-activedprotein kinase，P38MAPK）是存在于人体细胞内的一类蛋白激酶，它属于丝氨酸/苏氨酸蛋白激酶类别。P38MAPK主要参与人体细胞的生长、细胞发育、细胞分裂以及细胞凋亡等生理过程。P38MAPK信号转导通路是细胞内介导细胞外刺激的重要信号系统，在细胞恶变和肿瘤浸润转移过程中发挥关键作用。TGF-β1可以直接激活P38MAPK信号转导通路的上游激酶，从而间接地激活P38MAPK信号转导通路,参与肿瘤的发生与发展。在多种疾病发病机制的研究中，TGF-β1信号转导通路与P38MAPK信号转导通路存在一定的交互作用，但在绒癌发生的分子机制的研究中，两条信号转导通路之间是否存在相互作用的研究目前国内外罕见报道。本实验通过在低剂量TGF-β1刺激的绒癌JEG-3细胞模型上，分别采用TGF-β1受体（I和II）阻滞剂（LY364947）和P38抑制因子（SB203580）阻断两条信号转导通路，比较P38活化后的核转位以及P38、磷酸化P38蛋白水平的表达，从而揭示TGF-β1与P38MAPK信号转导通路在绒癌发生中的相互作用，对绒癌的恶性侵袭及转移的分子机制进行阐明并积累实验依据，为绒癌的临床治疗寻找新的作用靶点。目的用浓度为5ng/ml的TGF-β1预处理绒癌JEG-3细胞，在建立的细胞模型上，采用免疫荧光染色法检测P38活化后的核转位，Western blotting技术检测P38、磷酸化P38蛋白水平的表达，探讨TGF-β1及P38MAPK信号转导通路间的相互作用及作用的关键步骤，揭示绒癌恶性侵袭及转移的分子机制。方法 1.细胞培养及细胞模型的建立：实验的主要研究对象为人绒癌JEG-3细胞系。此细胞系来源于中国协和医科大学基础医学研究所。将绒癌JEG-3细胞用含10%胎牛血清的1640完全培养基置于37℃、5%CO2培养箱中培养。当细胞长满至70%-80%时，，按0.25%胰酶：0.02%EDTA为1:3的比例进行传代。取对数生长期的绒癌JEG-3细胞进行实验。将浓度为5ng/ml的TGF-β1作用于绒癌JEG-3细胞，同期设立空白对照组、1μM、3μM TGF-β1受体阻滞剂（LY364947）2个剂量组及1μM、3μM P38抑制因子（SB203580）2个剂量组。 2.免疫荧光染色法检测P38活化后的核转位过程。 3.免疫蛋白印迹法检测P38、磷酸化P38蛋白的表达水平。 4.应用SPSS15.0统计学软件对实验数据进行统计学分析。结果 1.免疫荧光染色结果 1.1给予TGF-β1受体（I和II）阻滞剂（LY364947）后绒癌JEG-3细胞中P38的活化及核转位的情况免疫荧光染色法实验表明，TGF-β1受体阻滞剂作用于绒癌JEG-3细胞后，磷酸化P38在细胞核内的荧光染色强度减弱，与TGF-β1刺激组比较，差异具有统计学意义（P＜0.05）；磷酸化P38的核染色强度呈现浓度依赖效应，随着TGF-β1受体阻滞剂浓度的增加，磷酸化P38在细胞核内的荧光染色强度逐渐减弱（P＜0.05）。 1.2给予P38MAPK抑制因子（SB203580）后绒癌JEG-3细胞中P38的活化及核转位的情况免疫荧光染色法实验表明，P38抑制因子作用于绒癌JEG-3细胞后，磷酸化P38在细胞核内的荧光染色强度减弱，与TGF-β1刺激组比较，差异具有统计学意义（P＜0.05）；磷酸化P38的核染色强度呈现一定的浓度效应，随着P38抑制因子浓度的增加，磷酸化P38在细胞核内的荧光染色强度逐渐减弱（P＜0.05）。 2. Western blotting检测结果 2.1TGF-β1受体（I和II）阻滞剂（LY364947）对绒癌JEG-3细胞中P38及磷酸化P38蛋白水平表达的影响实验表明，TGF-β1作用于绒癌JEG-3细胞后，P38及磷酸化P38的蛋白表达量显著增高，与空白对照组比较，差异具有统计学意义（P＜0.05）；而加入TGF-β1受体阻滞剂后，绒癌JEG-3细胞中P38及磷酸化P38的蛋白表达量减少，与TGF-β1刺激组比较，差异具有统计学意义（P＜0.05）；且呈现出浓度依赖效应，随TGF-β1受体阻滞剂作用浓度的增加，P38及磷酸化P38的蛋白表达量逐渐减少。 2.2P38MAPK抑制因子（SB203580）对绒癌JEG-3细胞中P38及磷酸化P38蛋白水平表达的影响实验表明，P38抑制因子（SB203580）作用于绒癌JEG-3细胞后，P38及磷酸化P38的蛋白表达量减少，与TGF-β1刺激组比较，差异具有统计学意义（P＜0.05）；且表现出浓度依赖效应，P38及磷酸化P38的蛋白表达量随P38抑制因子作用浓度的增加而逐渐减少。结论： 1.外源性TGF-β1可促进绒癌JEG-3细胞中P38活化后的核转位，并且可提高绒癌JEG-3细胞中P38及磷酸化P38的蛋白水平的表达。 2. TGF-β1受体（I和II）阻滞剂（LY364947）可减弱绒癌JEG-3细胞中P38活化后的核转位，并且降低绒癌JEG-3细胞中P38及磷酸化P38蛋白水平的表达，且呈现良好的浓度依赖效应。 3. P38抑制因子（SB203580）可减弱绒癌JEG-3细胞中P38活化后的核转位，并且降低绒癌JEG-3细胞中P38及磷酸化P38蛋白水平的表达，且表现出良好的浓度依赖效应。 4. TGF-β1信号转导通路与P38MAPK信号转导通路在绒癌JEG-3细胞的恶性侵袭机制中存在着相互作用。
[Abstract]:Choriocarcinoma, called choriocarcinoma, is a highly malignant trophoblast tumor. It is formed by malignant transformation of trophoblast cells (cell trophoblast cells), secondary to abortion, ectopic pregnancy, hydatidiform mole, erosive mole, and even normal pregnancy. The development of choriocarcinoma is closely related to the overerosion of trophoblast. .TGF- beta signal transduction pathway plays an important role in the formation of choriocarcinoma. Cell signal transduction pathway plays an important role in the formation of choriocarcinoma, which is one of the focal points in the field of cancer prevention and treatment. Transforming growth factor beta 1 (transform Ing growth factor beta, TGF- beta 1) is the most important cell regulating factor in the process of trophoblast invasion of the mother body. It plays an important role in the malignant invasion and metastasis of trophoblast tumor through the TGF- beta 1 signal transduction pathway, which is an important role of.P38 mitogen activated protein kinase (P38mitogen-activedprotein kinase,). P38MAPK) is a kind of protein kinase that exists in human cells. It belongs to serine / threonine protein kinase class.P38MAPK, which is mainly involved in human cell growth, cell development, cell division and cell apoptosis, and.P38MAPK signal transduction pathway is an important signal system to mediate extracellular stimulation in cell, and in cell malignant transformation. .TGF- beta 1, which plays a key role in the invasion and metastasis of tumor, can directly activate the upstream kinase of the P38MAPK signal transduction pathway, thus indirectly activates the P38MAPK signal transduction pathway and participates in the development and development of the tumor. In the study of the pathogenesis of various diseases, the TGF- beta 1 signal transduction pathway and P38MAPK signal transduction pathway exist certain. But in the study of the molecular mechanisms of choriocarcinoma, the study of the interaction between two signal transduction pathways is rarely reported at home and abroad.
In this experiment, two signal transduction pathways were blocked by TGF- beta 1 receptor (I and II) blocker (I and II) blocker (LY364947) and P38 inhibitory factor (SB203580) on the choriocarcinoma cell model stimulated by low dose of TGF- beta 1. The expression of nuclear transposition after P38 activation and P38, and the expression of P38 egg white phosphorylation were compared, and the TGF- beta 1 and signal transduction pathway were revealed. The interaction of the road in the occurrence of choriocarcinoma, clarifies the molecular mechanism of the malignant invasion and metastasis of the choriocarcinoma and accumulates the experimental basis, looking for new targets for the clinical treatment of choriocarcinoma.
objective
TGF- beta 1 with concentration of 5ng/ml was used to pretreat JEG-3 cells in choriocarcinoma. On the established cell model, the nuclear transposition after P38 activation was detected by immunofluorescence staining. Western blotting technique was used to detect P38, the expression of phosphorylated P38 protein level, and the key steps of the interaction and role of TGF- beta 1 and P38MAPK signal transduction pathways were discussed. The molecular mechanism of malignant invasion and metastasis of choriocarcinoma.
Method
1. cell culture and the establishment of cell model:
The main object of the study was the JEG-3 cell line of human choriocarcinoma. The cell line was derived from the Institute of basic medicine of Peking Union Medical College. The 1640 complete medium containing 10% fetal bovine serum was placed in the 1640 complete medium containing 10% fetal bovine serum in the 5%CO2 culture box. When the cell was full to 70%-80%, it was transmitted by 0.25% pancreatin: 0.02%EDTA as 1:3. The JEG-3 cell of the choriocarcinoma cell of the logarithmic growth period was tested. The TGF- beta 1, which was 5ng/ml, acted on the JEG-3 cells of choriocarcinoma, and the blank control group was set up in the same period, 1 mu M, 2 doses of 3 mu M TGF- beta 1 receptor blocker (LY364947) and 1 M, 3 mu M P38 inhibitory factor (SB203580) 2 doses group.
2. immunofluorescence staining was used to detect the nuclear translocation process after P38 activation.
3. the expression level of P38 and phosphorylated P38 protein was detected by Western blotting.
4. statistical analysis of experimental data was performed by SPSS15.0 statistical software.
Result
1. results of immunofluorescence staining
1.1 the activation and translocation of P38 in choriocarcinoma JEG-3 cells after TGF- beta 1 receptor (I and II) blockers (LY364947) were administered.
The immunofluorescence staining experiments showed that after TGF- beta 1 receptor blocker acted on the JEG-3 cells of choriocarcinoma, the fluorescence intensity of phosphorylated P38 in the nucleus was weakened. Compared with the TGF- beta 1 stimulation group, the difference was statistically significant (P < 0.05); the nuclear staining intensity of phosphorylated P38 showed a concentration dependent effect with the concentration of the TGF- beta 1 receptor blocker. The fluorescence staining intensity of phosphorylated P38 in nucleus decreased gradually (P < 0.05).
1.2 the activation and translocation of P38 in choriocarcinoma JEG-3 cells after giving P38MAPK inhibitor (SB203580).
The immunofluorescence staining experiments showed that the fluorescence staining intensity of phosphorylated P38 in the nucleus of choriocarcinoma JEG-3 cells weakened after the action of P38 inhibitory factor. Compared with the TGF- beta 1 stimulation group, the difference was statistically significant (P < 0.05); the nuclear staining intensity of phosphorylated P38 showed a certain concentration effect, with the increase of the concentration of P38 inhibitory factor, phosphorus The intensity of fluorescence staining of acidified P38 in nuclei decreased gradually (P < 0.05).
2. Western blotting detection results
Effect of 2.1TGF- beta 1 receptor (I and II) blocker (LY364947) on the expression of P38 and phosphorylated P38 protein in choriocarcinoma JEG-3 cells
The experiment showed that the protein expression of P38 and phosphorylated P38 increased significantly after the effect of TGF- beta 1 on the JEG-3 cells of choriocarcinoma. Compared with the blank control group, the difference was statistically significant (P < 0.05). After adding TGF- beta 1 receptor blockers, the protein expression of P38 and phosphorylated P38 in choriocarcinoma JEG-3 cells decreased, and the difference was compared with the TGF- beta 1 stimulation group. It was statistically significant (P < 0.05), and showed a concentration dependence effect. With the increase of the concentration of TGF- beta 1 receptor blockers, the protein expression of P38 and phosphorylated P38 gradually decreased.
Effect of 2.2P38MAPK inhibitor (SB203580) on the expression of P38 and phosphorylated P38 protein in choriocarcinoma JEG-3 cells
The experiment showed that the protein expression of P38 and phosphorylated P38 decreased after the effect of P38 inhibitory factor (SB203580) on the JEG-3 cells of choriocarcinoma. Compared with the TGF- beta 1 stimulation group, the difference was statistically significant (P < 0.05), and showed a concentration dependence effect. The protein expression of P38 and phosphorylated P38 gradually decreased with the increase of the action concentration of P38 inhibitory factor.
Conclusion:
1. exogenous TGF- beta 1 can promote the nuclear translocation of P38 in choriocarcinoma JEG-3 cells, and increase the expression of P38 and phosphorylated P38 protein in choriocarcinoma JEG-3 cells.
2. TGF- beta 1 receptor (I and II) blocker (LY364947) reduces the nuclear transposition after P38 activation in choriocarcinoma cell JEG-3 cells and reduces the expression of P38 and phosphorylated P38 protein levels in choriocarcinoma JEG-3 cells, and presents a good concentration dependent effect.
3. P38 inhibitory factor (SB203580) can reduce the nuclear transposition after P38 activation in choriocarcinoma cell JEG-3 cells, and reduce the expression of P38 and phosphorylated P38 protein levels in choriocarcinoma JEG-3 cells, and show a good concentration dependent effect.
4. the interaction between the TGF- beta 1 signal transduction pathway and the P38MAPK signal transduction pathway in the malignant invasion mechanism of choriocarcinoma JEG-3 cells.
【学位授予单位】：承德医学院
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R737.33

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