本文選題：絨毛膜癌 + TGF-β1��； 參考：《承德醫(yī)學院》2014年碩士論文

【摘要】：絨毛膜癌簡稱絨癌，是一種高度惡性的滋養(yǎng)細胞腫瘤。它由滋養(yǎng)細胞干細胞(細胞滋養(yǎng)層細胞)發(fā)生惡性轉化而形成，可繼發(fā)于流產、異位妊娠、葡萄胎、侵蝕性葡萄胎、甚至是正常妊娠。絨癌的發(fā)生發(fā)展與滋養(yǎng)細胞的過度侵蝕作用密切相關，早期即可發(fā)生血道轉移而危及患者生命。絨癌的發(fā)生是多種因素協同作用的結果，其中細胞信號轉導通路在絨癌的形成過程中發(fā)揮了重要的作用。TGF-β信號轉導通路是當今腫瘤防治領域探討與鉆研的焦點之一。轉化生長因子β1（transforming growth factor beta，TGF-β1）是滋養(yǎng)細胞在侵襲母體的過程當中研究最多的細胞調節(jié)因子，它主要是通過TGF-β1信號轉導通路在滋養(yǎng)細胞腫瘤惡性侵襲與轉移的過程中發(fā)揮自身的重要作用。P38絲裂原活化蛋白激酶（P38mitogen-activedprotein kinase，P38MAPK）是存在于人體細胞內的一類蛋白激酶，它屬于絲氨酸/蘇氨酸蛋白激酶類別。P38MAPK主要參與人體細胞的生長、細胞發(fā)育、細胞分裂以及細胞凋亡等生理過程。P38MAPK信號轉導通路是細胞內介導細胞外刺激的重要信號系統，在細胞惡變和腫瘤浸潤轉移過程中發(fā)揮關鍵作用。TGF-β1可以直接激活P38MAPK信號轉導通路的上游激酶，從而間接地激活P38MAPK信號轉導通路,參與腫瘤的發(fā)生與發(fā)展。在多種疾病發(fā)病機制的研究中，TGF-β1信號轉導通路與P38MAPK信號轉導通路存在一定的交互作用，但在絨癌發(fā)生的分子機制的研究中，兩條信號轉導通路之間是否存在相互作用的研究目前國內外罕見報道。 本實驗通過在低劑量TGF-β1刺激的絨癌JEG-3細胞模型上，分別采用TGF-β1受體（I和II）阻滯劑（LY364947）和P38抑制因子（SB203580）阻斷兩條信號轉導通路，比較P38活化后的核轉位以及P38、磷酸化P38蛋白水平的表達，從而揭示TGF-β1與P38MAPK信號轉導通路在絨癌發(fā)生中的相互作用，對絨癌的惡性侵襲及轉移的分子機制進行闡明并積累實驗依據，為絨癌的臨床治療尋找新的作用靶點。 目的 用濃度為5ng/ml的TGF-β1預處理絨癌JEG-3細胞，在建立的細胞模型上，采用免疫熒光染色法檢測P38活化后的核轉位，Western blotting技術檢測P38、磷酸化P38蛋白水平的表達，探討TGF-β1及P38MAPK信號轉導通路間的相互作用及作用的關鍵步驟，揭示絨癌惡性侵襲及轉移的分子機制。 方法 1.細胞培養(yǎng)及細胞模型的建立： 實驗的主要研究對象為人絨癌JEG-3細胞系。此細胞系來源于中國協和醫(yī)科大學基礎醫(yī)學研究所。將絨癌JEG-3細胞用含10%胎牛血清的1640完全培養(yǎng)基置于37℃、5%CO2培養(yǎng)箱中培養(yǎng)。當細胞長滿至70%-80%時，，按0.25%胰酶：0.02%EDTA為1:3的比例進行傳代。取對數生長期的絨癌JEG-3細胞進行實驗。將濃度為5ng/ml的TGF-β1作用于絨癌JEG-3細胞，同期設立空白對照組、1μM、3μM TGF-β1受體阻滯劑（LY364947）2個劑量組及1μM、3μM P38抑制因子（SB203580）2個劑量組。 2.免疫熒光染色法檢測P38活化后的核轉位過程。 3.免疫蛋白印跡法檢測P38、磷酸化P38蛋白的表達水平。 4.應用SPSS15.0統計學軟件對實驗數據進行統計學分析。 結果 1.免疫熒光染色結果 1.1給予TGF-β1受體（I和II）阻滯劑（LY364947）后絨癌JEG-3細胞中P38的活化及核轉位的情況 免疫熒光染色法實驗表明，TGF-β1受體阻滯劑作用于絨癌JEG-3細胞后，磷酸化P38在細胞核內的熒光染色強度減弱，與TGF-β1刺激組比較，差異具有統計學意義（P＜0.05）；磷酸化P38的核染色強度呈現濃度依賴效應，隨著TGF-β1受體阻滯劑濃度的增加，磷酸化P38在細胞核內的熒光染色強度逐漸減弱（P＜0.05）。 1.2給予P38MAPK抑制因子（SB203580）后絨癌JEG-3細胞中P38的活化及核轉位的情況 免疫熒光染色法實驗表明，P38抑制因子作用于絨癌JEG-3細胞后，磷酸化P38在細胞核內的熒光染色強度減弱，與TGF-β1刺激組比較，差異具有統計學意義（P＜0.05）；磷酸化P38的核染色強度呈現一定的濃度效應，隨著P38抑制因子濃度的增加，磷酸化P38在細胞核內的熒光染色強度逐漸減弱（P＜0.05）。 2. Western blotting檢測結果 2.1TGF-β1受體（I和II）阻滯劑（LY364947）對絨癌JEG-3細胞中P38及磷酸化P38蛋白水平表達的影響 實驗表明，TGF-β1作用于絨癌JEG-3細胞后，P38及磷酸化P38的蛋白表達量顯著增高，與空白對照組比較，差異具有統計學意義（P＜0.05）；而加入TGF-β1受體阻滯劑后，絨癌JEG-3細胞中P38及磷酸化P38的蛋白表達量減少，與TGF-β1刺激組比較，差異具有統計學意義（P＜0.05）；且呈現出濃度依賴效應，隨TGF-β1受體阻滯劑作用濃度的增加，P38及磷酸化P38的蛋白表達量逐漸減少。 2.2P38MAPK抑制因子（SB203580）對絨癌JEG-3細胞中P38及磷酸化P38蛋白水平表達的影響 實驗表明，P38抑制因子（SB203580）作用于絨癌JEG-3細胞后，P38及磷酸化P38的蛋白表達量減少，與TGF-β1刺激組比較，差異具有統計學意義（P＜0.05）；且表現出濃度依賴效應，P38及磷酸化P38的蛋白表達量隨P38抑制因子作用濃度的增加而逐漸減少。 結論： 1.外源性TGF-β1可促進絨癌JEG-3細胞中P38活化后的核轉位，并且可提高絨癌JEG-3細胞中P38及磷酸化P38的蛋白水平的表達。 2. TGF-β1受體（I和II）阻滯劑（LY364947）可減弱絨癌JEG-3細胞中P38活化后的核轉位，并且降低絨癌JEG-3細胞中P38及磷酸化P38蛋白水平的表達，且呈現良好的濃度依賴效應。 3. P38抑制因子（SB203580）可減弱絨癌JEG-3細胞中P38活化后的核轉位，并且降低絨癌JEG-3細胞中P38及磷酸化P38蛋白水平的表達，且表現出良好的濃度依賴效應。 4. TGF-β1信號轉導通路與P38MAPK信號轉導通路在絨癌JEG-3細胞的惡性侵襲機制中存在著相互作用。
[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.
【學位授予單位】：承德醫(yī)學院
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R737.33

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