本文選題：環(huán)孢素A + 氧化應(yīng)激��； 參考：《復(fù)旦大學(xué)》2012年博士論文

【摘要】：妊娠初期,滋養(yǎng)細(xì)胞具有獨(dú)特的類似腫瘤細(xì)胞的生物學(xué)行為,即高增殖、低凋亡和高遷移及侵襲力；同時(shí)滋養(yǎng)細(xì)胞的生物學(xué)行為受到蛻膜微環(huán)境的嚴(yán)格調(diào)控,這對(duì)囊胚植入、胚胎發(fā)育和正常妊娠的維持起關(guān)鍵作用。近年來(lái)研究發(fā)現(xiàn),氧化應(yīng)激參與多種病理性妊娠的發(fā)病,與流產(chǎn)、先兆子癇、胎兒生長(zhǎng)受限(FGR)、早產(chǎn)及死胎等妊娠并發(fā)癥密切關(guān)聯(lián)。過(guò)量的活性氧自由基(ROS)可使磷脂中的不飽和脂肪酸生成過(guò)氧化脂質(zhì),損傷生物膜；可以抑制蛋白質(zhì)功能,破壞核酸及染色質(zhì),從而傷害機(jī)體的各種組織和細(xì)胞,包括導(dǎo)致滋養(yǎng)細(xì)胞凋亡,降低細(xì)胞侵襲力。因此,控制氧化損傷對(duì)于成功妊娠和正常妊娠的維持具有重要意義。 環(huán)孢素A(CsA)是具有劃時(shí)代意義的免疫抑制劑,其臨床應(yīng)用可顯著改善實(shí)體器官移植的近期存活率,是器官移植后免疫抑制和抗排斥反應(yīng)的首選藥物。我們課題組首次發(fā)現(xiàn),低濃度CsA可顯著提高滋養(yǎng)細(xì)胞的增殖與侵襲力,抑制低血清培養(yǎng)誘導(dǎo)的滋養(yǎng)細(xì)胞凋亡,并改善小鼠流產(chǎn)模型的妊娠結(jié)局,提示CsA可能為滋養(yǎng)細(xì)胞功能障礙導(dǎo)致的妊娠疾病的潛在治療藥物。與CsA經(jīng)典的細(xì)胞內(nèi)信號(hào)轉(zhuǎn)導(dǎo)通路不同,我們課題組的研究結(jié)果顯示,CsA可通過(guò)活化MAPK/ERK通路提高滋養(yǎng)細(xì)胞的增殖及侵襲力。 本研究在前期工作基礎(chǔ)上,進(jìn)一步研究在細(xì)胞生物學(xué)功能調(diào)控中起重要作用的粘著斑激酶(FAK)信號(hào)通路是否參與CsA調(diào)節(jié)滋養(yǎng)細(xì)胞的遷移與侵襲；并通過(guò)用H202處理人絨毛膜上皮癌細(xì)胞系JEG-3細(xì)胞,建立滋養(yǎng)細(xì)胞氧化損傷的體外模型,在氧化應(yīng)激條件下進(jìn)一步解析CsA減輕滋養(yǎng)細(xì)胞損傷的分子機(jī)制,以及相應(yīng)的細(xì)胞內(nèi)信號(hào)轉(zhuǎn)導(dǎo)通路,為拓展CsA的臨床應(yīng)用提供科學(xué)依據(jù)。 第一部分環(huán)孢素A通過(guò)FAK信號(hào)通路促進(jìn)滋養(yǎng)細(xì)胞遷移與侵襲 目的探討FAK信號(hào)通路是否參與介導(dǎo)CsA促進(jìn)滋養(yǎng)細(xì)胞遷移與侵襲,以及FAK信號(hào)通路與ERK信號(hào)通路間的相互作用。 方法用CsA(1gM)處理滋養(yǎng)細(xì)胞,或用FAK抑制劑Y15、Src抑制劑PP2、MEK抑制劑U0126分別預(yù)處理滋養(yǎng)細(xì)胞后再用CsA處理,采用Transwell遷移試驗(yàn)和Matrigel侵襲試驗(yàn)分析滋養(yǎng)細(xì)胞的遷移及侵襲力,Western blot分析滋養(yǎng)細(xì)胞FAK、Src及ERK的磷酸化水平及E-鈣粘蛋白(E-cadherin)和金屬基質(zhì)蛋白酶MMP2、MMP9的表達(dá)水平,明膠酶譜實(shí)驗(yàn)分析滋養(yǎng)細(xì)胞培養(yǎng)上清MMP2、MMP9的活性。 結(jié)果CsA可顯著提高原代滋養(yǎng)細(xì)胞及JEG-3細(xì)胞FAK及其接頭分子Src的磷酸化水平。Y15和PP2均能阻斷CsA升調(diào)節(jié)滋養(yǎng)細(xì)胞的遷移及侵襲。Y15和PP2可抑制CsA增高的FAK、Src及ERK的磷酸化水平,U0126可抑制CsA增高的ERK磷酸化水平,但對(duì)FAK及Src的磷酸化水平無(wú)顯著影響。Y15、PP2和U0126均可消除CsA對(duì)E-cadherin表達(dá)的下調(diào)作用和對(duì)MMP2、MMP9表達(dá)及活性的上調(diào)作用。 結(jié)論CsA通過(guò)FAK-Src信號(hào)通路促進(jìn)ERK信號(hào)通路活化,下調(diào)E-cadherin表達(dá),上調(diào)MMP2、MMP9的表達(dá)及分泌,進(jìn)而促進(jìn)滋養(yǎng)細(xì)胞的遷移和侵襲。 第二部分氧化應(yīng)激損傷滋養(yǎng)細(xì)胞的生物學(xué)行為 目的分析H202對(duì)JEG-3細(xì)胞生物學(xué)行為的影響,建立滋養(yǎng)細(xì)胞氧化應(yīng)激損傷模型。 方法用不同濃度的H202處理JEG-3細(xì)胞24h,采用MTT法分析滋養(yǎng)細(xì)胞活力,倒置相差顯微鏡及熒光顯微鏡觀察細(xì)胞形態(tài),DHE熒光探針檢測(cè)細(xì)胞內(nèi)ROS水平,JC-1熒光探針檢測(cè)細(xì)胞線粒體膜電位,Annexin V/PI雙標(biāo)記檢測(cè)細(xì)胞早期凋亡,Matrigel侵襲試驗(yàn)檢測(cè)細(xì)胞的侵襲力,以建立滋養(yǎng)細(xì)胞氧化損傷模型。 結(jié)果500μM H2O2處理JEG-3細(xì)胞24h后,細(xì)胞活力顯著下降,形態(tài)發(fā)生明顯改變,細(xì)胞皺縮,間隙增大,體積減小,細(xì)胞核縮小,染色質(zhì)凝集,呈顆粒團(tuán)塊狀分布；細(xì)胞ROS產(chǎn)生增加,線粒體膜電位下降,凋亡比例升高,細(xì)胞侵襲力顯著下降。更高濃度的H202(從7501μM開(kāi)始)則誘發(fā)細(xì)胞大量脫落壞死,侵襲力喪失。 結(jié)論JEG-3細(xì)胞經(jīng)500gM H2O2處理24h呈現(xiàn)明顯的氧化應(yīng)激損傷,用此條件建立氧化應(yīng)激模型,為研究滋養(yǎng)細(xì)胞氧化損傷及抗氧化應(yīng)激藥物的作用機(jī)制奠定了基礎(chǔ)。 第三部分環(huán)孢素A緩解氧化應(yīng)激誘導(dǎo)的滋養(yǎng)細(xì)胞損傷 目的解析CsA對(duì)H2O2誘導(dǎo)滋養(yǎng)細(xì)胞生物學(xué)行為損傷的保護(hù)作用及其分子機(jī)制。 方法用低濃度CsA(1μM)預(yù)處理JEG-3細(xì)胞24h,再經(jīng)H2O2(500μM)刺激誘導(dǎo)氧化損傷,采用MTT比色法分析滋養(yǎng)細(xì)胞活力,倒置相差顯微鏡及熒光顯微鏡觀察細(xì)胞形態(tài),Annexin V/PI雙標(biāo)記檢測(cè)細(xì)胞早期凋亡,Matrigel侵襲試驗(yàn)檢測(cè)細(xì)胞的侵襲力,DHE熒光探針檢測(cè)細(xì)胞ROS水平,化學(xué)比色法檢測(cè)細(xì)胞丙二醛(MDA)的含量、超氧化物歧化酶(SOD)及過(guò)氧化氫酶(CAT)的活性,JC-1熒光探針檢測(cè)細(xì)胞線粒體膜電位,Western blot檢測(cè)凋亡相關(guān)蛋白的表達(dá)水平。 結(jié)果與H202單獨(dú)處理組比較,JEG-3細(xì)胞經(jīng)低濃度CsA干預(yù)后,細(xì)胞活力明顯升高,形態(tài)得以改善；細(xì)胞凋亡比例下降,侵襲力增加；細(xì)胞內(nèi)ROS、MDA含量明顯下降,SOD.CAT的活性顯著升高；線粒體膜電位水平恢復(fù)；p53的表達(dá)和磷酸化水平下降,Bax表達(dá)減少,Bcl-2表達(dá)增加,caspase-3前體增多,裂解的PARP大片段減少。 結(jié)論CsA干預(yù)可明顯改善氧化應(yīng)激狀態(tài)下JEG-3細(xì)胞的生物學(xué)行為；減輕細(xì)胞氧化損傷程度,增強(qiáng)細(xì)胞的抗氧化損傷能力；抑制線粒體相關(guān)的凋亡信號(hào)通路及caspase-3活化,減少滋養(yǎng)細(xì)胞凋亡。 第四部分環(huán)孢素A通過(guò)調(diào)節(jié)FAK-Src和MAPK信號(hào)通路緩解滋養(yǎng)細(xì)胞氧化損傷 目的解析CsA緩解滋養(yǎng)細(xì)胞氧化損傷的信號(hào)轉(zhuǎn)導(dǎo)通路。 方法用低濃度CsA(1μM)處理JEG-3細(xì)胞24h,再經(jīng)H2O2(500μM)刺激誘導(dǎo)氧化損傷,采用Western blot分析CsA對(duì)氧化應(yīng)激的滋養(yǎng)細(xì)胞FAK.Src及MAPKs磷酸化水平的影響；在此基礎(chǔ)上,用FAK抑制劑Y15、Src抑制劑PP2分別預(yù)處理處理JEG-3細(xì)胞,MTT法測(cè)定細(xì)胞活力,DHE熒光探針檢測(cè)細(xì)胞內(nèi)ROS水平,JC-1熒光探針檢測(cè)細(xì)胞線粒體膜電位,Annexin V/PI雙標(biāo)記檢測(cè)細(xì)胞早期凋亡,Matrigel侵襲實(shí)驗(yàn)檢測(cè)細(xì)胞的侵襲力；用MAPKs信號(hào)通路抑制劑(SB203580、SP600125、U0126)分別預(yù)處理JEG-3細(xì)胞,MTT法分析細(xì)胞活力。 結(jié)果H202刺激可引起JEG-3細(xì)胞FAK、Src磷酸化水平下降,p38MAPK、JNK和ERK的磷酸化水平升高。CsA預(yù)處理JEG-3細(xì)胞后再經(jīng)H202刺激,FAK、Src磷酸化水平明顯升高,JNK磷酸化水平明顯下降,ERK磷酸化水平無(wú)顯著性變化。Y15和PP2處理細(xì)胞后,再經(jīng)CsA和H202聯(lián)合處理,與CsA和H202聯(lián)合處理組比較,滋養(yǎng)細(xì)胞活力下降,ROS產(chǎn)生增加,線粒體膜電位下降,凋亡比例升高,侵襲力下降。p38MAPK抑制劑SB203580或JNK抑制劑SP600125處理細(xì)胞后,再經(jīng)H202處理,滋養(yǎng)細(xì)胞活力較H202處理組升高；而MEK抑制劑U0126處理后,細(xì)胞活力進(jìn)一步下降。 結(jié)論低濃度CsA通過(guò)促進(jìn)FAK-Src信號(hào)通路活化,抑制p38MAPK和JNK信號(hào)通路活化,發(fā)揮對(duì)氧化損傷的滋養(yǎng)細(xì)胞的保護(hù)作用。 綜上所述,本研究發(fā)現(xiàn)低濃度CsA對(duì)人滋養(yǎng)細(xì)胞的生物學(xué)行為具有多重調(diào)節(jié)作用：(1)通過(guò)FAK-Src信號(hào)通路促進(jìn)ERK信號(hào)通路活化,下調(diào)E-cadherin表達(dá),上調(diào)MMP2、MMP9的表達(dá)及分泌,促進(jìn)正常滋養(yǎng)細(xì)胞的遷移和侵襲；(2)通過(guò)降調(diào)節(jié)ROS及MDA的生成,升調(diào)節(jié)SOD和CAT的活性,緩解滋養(yǎng)細(xì)胞氧化應(yīng)激損傷；(3)通過(guò)抑制線粒體相關(guān)的凋亡通路,降低caspase-3活化水平,抑制氧化應(yīng)激誘導(dǎo)的滋養(yǎng)細(xì)胞凋亡；(4)通過(guò)促進(jìn)FAK-Src信號(hào)通路活化,抑制p38MAPK和JNK信號(hào)通路活化,發(fā)揮對(duì)氧化損傷的滋養(yǎng)細(xì)胞的保護(hù)作用。這些研究結(jié)果顯示,CsA的藥理作用及其細(xì)胞內(nèi)信號(hào)轉(zhuǎn)導(dǎo)通路遠(yuǎn)超過(guò)人們迄今為止的認(rèn)識(shí),有望成為一種新型保胎制劑,為臨床防治妊娠失敗、子癇前期、胎兒生長(zhǎng)受限等病理妊娠提供了一種新的策略。
[Abstract]:In the early stages of pregnancy, the trophoblast has a unique biological behavior similar to the tumor cell, that is, high proliferation, low apoptosis, high migration and invasion. At the same time, the biological behavior of trophoblast is regulated by the decidual microenvironment, which plays a key role in the implantation of blastocyst, embryo development and the maintenance of normal pregnancy. Stress participation in the pathogenesis of multiple pathological pregnancies is closely related to abortion, preeclampsia, fetal growth restriction (FGR), premature and stillbirth complications. Excessive active oxygen free radicals (ROS) can make the unsaturated fatty acids in phospholipid produce lipid peroxidation, damage the membrane of raw materials, inhibit protein function, destroy nucleic acid and chromatin. Therefore, it is important for the maintenance of successful pregnancy and normal pregnancy to control the apoptosis of the trophoblastic cells and reduce the cell invasiveness.
Cyclosporin A (CsA) is an epoch-making immunosuppressant. Its clinical application can significantly improve the survival rate of solid organ transplantation. It is the first choice for immunosuppression and rejection after organ transplantation. We first found that low concentration of CsA can increase the proliferation and invasiveness of trophoblastic cells and inhibit low serum culture for the first time. The induction of trophoblast apoptosis and the improvement of the pregnancy outcome of the mouse abortion model suggest that CsA may be a potential therapeutic drug for gestational disease caused by trophoblastic dysfunction. Unlike the classic CsA intracellular signal transduction pathway, our research group shows that CsA can increase the increase of trophoblastic cells through the activation of the MAPK/ERK pathway. Colonization and invasiveness.
On the basis of earlier work, this study further studies whether the FAK signaling pathway plays an important role in the regulation of cellular biological function, whether CsA regulates the migration and invasion of trophoblastic cells, and establishes an in vitro model of the oxidative damage of trophoblastic cells by using H202 to treat human chorionic epithelial cancer cell line JEG-3 cells. Under the condition of oxidative stress, the molecular mechanism of CsA to reduce the damage of trophoblast and the corresponding intracellular signal transduction pathway are further analyzed to provide a scientific basis for the development of the clinical application of CsA.
Part 1 cyclosporine A promotes the migration and invasion of trophoblast cells through FAK signaling pathway.
Objective to investigate whether FAK signaling pathway mediates CsA promoting the migration and invasion of trophoblasts, and the interaction between FAK signaling pathway and ERK signaling pathway.
Methods the trophoblastic cells were treated with CsA (1gM), or FAK inhibitor Y15, Src inhibitor PP2, and MEK inhibitor U0126 were pretreated with CsA treatment. The migration and invasiveness of trophoblastic cells were analyzed by Transwell migration test and Matrigel invasion test. The expression level of protein (E-cadherin) and matrix metalloproteinase MMP2 and MMP9, and the activity of MMP2 and MMP9 in trophoblast cell culture supernatant were analyzed by gelatinase zymogram.
Results CsA can significantly increase the phosphorylation level of the primary trophoblastic and JEG-3 cell FAK and its joint molecule Src.Y15 and PP2, which can block the CsA liters to regulate the migration of trophoblastic cells and invade the FAK of.Y15 and PP2 to inhibit the increase of CsA. The level of.Y15, PP2 and U0126 could not eliminate the effect of CsA on the expression of E-cadherin and the up regulation of MMP2, MMP9 expression and activity.
Conclusion CsA promotes the activation of ERK signaling pathway through FAK-Src signaling pathway, down regulation of E-cadherin expression, up regulation of the expression and secretion of MMP2, MMP9, and thus promoting the migration and invasion of trophoblast.
The second part is the biological behavior of trophoblast injured by oxidative stress.
Objective to analyze the effect of H202 on the biological behavior of JEG-3 cells and establish a model of oxidative stress injury in trophoblastic cells.
Methods the JEG-3 cell 24h was treated with different concentration of H202. The cell viability was analyzed by MTT method. The cell morphology was observed by inverted phase contrast microscope and fluorescence microscope. The ROS level of the cell was detected by DHE fluorescence probe. The mitochondrial membrane potential was detected by JC-1 fluorescence probe. The early apoptosis of the cell was detected by the Annexin V/PI double labeling, and the Matrigel invasion test was tested. The invasive ability of cells was detected to establish a model of oxidative damage in trophoblastic cells.
Results after 500 M H2O2 treatment of JEG-3 cell 24h, the cell vitality decreased significantly, the morphology changed obviously, the cell crinkle, the gap enlargement, the volume decrease, the nucleus shrinking, the chromatin agglutination, the granular mass distribution, the cell ROS production increased, the mitochondrial membrane potential decreased, the apoptosis ratio increased, the cell invasiveness decreased significantly. A higher concentration of H 202 (starting from 7501 M) induced a large number of cells to shed and necrotic and lose their invasiveness.
Conclusion the 500gM H2O2 treatment of JEG-3 cells showed obvious oxidative stress damage. The oxidative stress model was established by this condition, which laid the foundation for the study of the oxidative damage of trophoblast and the mechanism of antioxidation stress drugs.
The third part cyclosporine A alleviates oxidative stress induced trophoblastic injury.
Objective to analyze the protective effect and molecular mechanism of CsA on H2O2 induced biological behavior damage in trophoblastic cells.
Methods JEG-3 cell 24h was pretreated with low concentration of CsA (1 mu M), and then induced oxidative damage induced by H2O2 (500 M). MTT colorimetric assay was used to analyze the viability of trophoblast. Cell morphology was observed by inverted phase contrast microscope and fluorescence microscope. Annexin V/PI double labeling was used to detect cell early death. Matrigel invasion test was used to detect cell invasiveness and DHE fluorescence detection. The level of cell ROS, the content of MDA, the activity of superoxide dismutase (SOD) and catalase (CAT) were detected by chemical colorimetry. The mitochondrial membrane potential of cell mitochondria was detected by JC-1 fluorescence probe, and the expression level of apoptosis related protein was detected by Western blot.
Results compared with the H202 alone group, the cell viability was significantly higher in the JEG-3 cells after the low concentration of CsA, the cell viability was significantly increased, the cell apoptosis ratio decreased, the invasion force increased, the intracellular ROS, MDA content decreased significantly, the activity of SOD.CAT increased significantly, the mitochondrial membrane potential level was restored, the expression of p53 and the level of phosphorylation decreased, Bax Decreased expression, increased Bcl-2 expression, increased caspase-3 precursors, and reduced PARP fragmentation.
Conclusion CsA intervention can obviously improve the biological behavior of JEG-3 cells under oxidative stress, reduce the degree of oxidative damage and enhance the ability of cell oxidation damage, inhibit mitochondrial related apoptosis signaling pathway and caspase-3 activation, and reduce the apoptosis of trophoblast.
The fourth part of cyclosporine A alleviates trophoblast oxidative damage by regulating FAK-Src and MAPK signaling pathways.
Objective to analyze the signal transduction pathway of CsA in relieving oxidative damage in trophoblastic cells.
Methods the JEG-3 cell 24h was treated with low concentration of CsA (1 mu M), and the oxidative damage induced by H2O2 (500 M) was induced. Western blot was used to analyze the effect of CsA on the level of FAK.Src and MAPKs phosphorylation of the trophoblast cells of oxidative stress. HE fluorescence probe was used to detect intracellular ROS level, JC-1 fluorescence probe was used to detect cell mitochondrial membrane potential, Annexin V/PI was used to detect cell early apoptosis, Matrigel invasion test was used to detect cell invasiveness, and JEG-3 cells were pretreated with MAPKs signal pathway inhibitor (SB203580, SP600125, U0126), and cell viability was analyzed by MTT method.
Results H202 stimulation could cause JEG-3 cells FAK, Src phosphorylation level decreased, p38MAPK, JNK and ERK phosphorylation level increased after.CsA pretreatment JEG-3 cells, FAK, Src phosphorylation level was significantly increased, phosphorylation level decreased obviously, phosphorylation level without significant changes Compared with the combined treatment group of CsA and H202, the vitality of trophoblast, the increase of ROS production, the decrease of the mitochondrial membrane potential, the increase of apoptosis ratio, the decrease of the invasive power of the.P38MAPK inhibitor, SB203580 or the JNK inhibitor SP600125, were followed by the H202 treatment, and the activity of the trophoblastic cells was higher than that of the H202 treatment group, while MEK inhibitor U0126 treated, Cell viability decreased further.
Conclusion low concentration of CsA can promote the activation of FAK-Src signaling pathway, inhibit the activation of p38MAPK and JNK signaling pathways, and play a protective role in the trophoblast cells with oxidative damage.
To sum up, the present study found that low concentration of CsA has multiple regulatory effects on the biological behavior of human trophoblastic cells: (1) promoting activation of ERK signaling pathway through FAK-Src signaling pathway, down regulation of E-cadherin expression, up regulation of MMP2, MMP9 expression and secretion, promoting the migration and invasion of normal trophoblastic cells, and (2) regulating the formation of ROS and MDA by lowering the expression of ROS and MDA, Increase the activity of SOD and CAT to alleviate oxidative stress injury of trophoblast; (3) by inhibiting mitochondrial related apoptosis pathway, reducing caspase-3 activation level and inhibiting the apoptosis of trophoblast induced by oxidative stress; (4) activating the FAK-Src signaling pathway, inhibiting the activation of p38MAPK and JNK signaling pathways and giving full play to the nourishment of oxidative damage The results of these studies show that the pharmacological action of CsA and its intracellular signal transduction pathway are far more than people's understanding so far. It is expected to be a new type of fetal preparation, which provides a new strategy for the clinical prevention and treatment of pregnancy failure, preeclampsia, fetal growth restriction and so on.

【學(xué)位授予單位】：復(fù)旦大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2012
【分類號(hào)】：R321

【參考文獻(xiàn)】

相關(guān)期刊論文 前3條

1 蘇玉慧;漆正堂;丁樹(shù)哲;;p53與線粒體的關(guān)系[J];生命的化學(xué);2010年03期

2 吳霞,李大金,袁敏敏,王明雁;人早孕期絨毛和絨毛外細(xì)胞滋養(yǎng)細(xì)胞的分離、培養(yǎng)及鑒定[J];生殖與避孕;2004年02期

3 張斌;夏作理;趙曉民;張和平;;氧化應(yīng)激模型的建立及其評(píng)價(jià)[J];中國(guó)臨床康復(fù);2006年44期

，

本文編號(hào)：1839523