本文選題：CAF　切入點(diǎn)：LOXL2　出處：《南方醫(yī)科大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：研究背景:慢性髓系白血病(CML)、原發(fā)性血小板增多癥(ET)、真性紅細(xì)胞增多癥(PV)、原發(fā)性骨髓纖維化(PMF)等MPN病程進(jìn)展中常伴隨骨髓纖維化。進(jìn)行性骨髓纖維化亦是影響MPN向白血病、骨髓衰竭演變的高危因素。因此,研究發(fā)生骨髓纖維化的特異性細(xì)胞因子,可能為深入挖掘MPN的發(fā)病機(jī)制、尋找更有效的靶向治療提供新的切入點(diǎn)�；虮磉_(dá)譜發(fā)現(xiàn)骨髓微環(huán)境的動(dòng)態(tài)演化參與了 MPN骨髓纖維化進(jìn)程。骨髓微環(huán)境處于1%氧濃度左右的乏氧狀態(tài),腫瘤中的乏氧微環(huán)境可通過誘導(dǎo)新生血管形成、抑制免疫監(jiān)視和免疫反應(yīng)、孕育腫瘤干細(xì)胞等機(jī)制促進(jìn)腫瘤耐藥、復(fù)發(fā)及進(jìn)展。腫瘤相關(guān)成纖維細(xì)胞(CAF)是腫瘤微環(huán)境中基質(zhì)細(xì)胞的主要組成成分,在腫瘤生物行為學(xué)特征中起著至關(guān)重要的作用,骨髓間充質(zhì)干細(xì)胞(BMMSC)作為骨髓微環(huán)境中的重要成分,是CAF重要來源。新近研究發(fā)現(xiàn),乏氧微環(huán)境中增多的CAF與MPN纖維化進(jìn)程密切相關(guān),但缺乏腫瘤細(xì)胞刺激的乏氧環(huán)境下將導(dǎo)致CAF的失活。表明CAF的活化需要通過與腫瘤細(xì)胞或其分泌因子發(fā)生“互話”。然而,到底是哪些因素活化CAF促進(jìn)MPN骨髓纖維化尚不清楚。賴氨酰氧化酶樣蛋白2(LOXL2)是賴氨酰氧化酶(LOX)家族成員之一,可通過激活FAK/Src、Erk信號(hào)通路、介導(dǎo)上皮間質(zhì)轉(zhuǎn)化等方式參與多種惡性腫瘤侵襲性生物學(xué)特征形成。研究發(fā)現(xiàn),LOXL2的表達(dá)水平與MPN纖維化程度密切相關(guān)。乏氧微環(huán)境下,腫瘤細(xì)胞來源的LOXL2可激活FAK/Src信號(hào)通路促使纖維母細(xì)胞轉(zhuǎn)化為CAF,后者分泌多種細(xì)胞因子不斷促進(jìn)腫瘤細(xì)胞增殖、轉(zhuǎn)移。但兩種因子介導(dǎo)骨髓纖維化機(jī)制目前尚不清楚。結(jié)合研究現(xiàn)狀,我們推測(cè),MPN病程中,微環(huán)境中的BMMSC可能活化為CAF表型促進(jìn)骨髓纖維化;而LOXL2則可能是乏氧微環(huán)境中促進(jìn)BMMSC活化為CAF的關(guān)鍵因子,進(jìn)一步推進(jìn)MPN骨髓纖維化進(jìn)程。研究目的:驗(yàn)證CAF及LOXL-2與MPN及骨髓纖維化進(jìn)展密切相關(guān);證實(shí)MPN來源的BMMSC活化為CAF表型促進(jìn)纖維化進(jìn)程;探索乏氧環(huán)境下LOXL-2是否作為啟動(dòng)因素刺激MSC活化為CAF,這種效應(yīng)是否能夠被LOX抑制劑所阻斷。研究方法:臨床標(biāo)本水平:1.提取正常人與MPN患者骨髓單個(gè)核細(xì)胞,應(yīng)用RT-PCR及Westen-blot方法檢測(cè)CAF標(biāo)志物(α-SMA、FAP)及LOXL2的表達(dá)水平。2.收集正常人與MPN患者骨髓或外周血血清,應(yīng)用ELISA方法檢測(cè)LOXL2表達(dá)差異。3.收集骨髓病理活檢標(biāo)本,應(yīng)用免疫組化檢測(cè)骨髓活檢中網(wǎng)染蛋白、α-SMA、FAP、LOXL2的表達(dá)情況。細(xì)胞水平:1.經(jīng)知情同意,取正常人及MPN患者骨髓分離培養(yǎng)BMMSC,觀察細(xì)胞形態(tài),檢測(cè)MSC生物學(xué)特性:免疫表型、成骨、成脂肪細(xì)胞誘導(dǎo)分化能力。應(yīng)用RT-PCR、Western blot分別檢測(cè)正常人與MPN患者α-SMA、FAP表達(dá)情況。2.分別在正常氧和1%氧濃度條件下培養(yǎng)BMMSC 24h、48h、72h、96h,觀察細(xì)胞形態(tài)變化,檢測(cè)CAF標(biāo)志物α-SMA、FAP及p-FAK、p-SRC、FAK、SRC通路蛋白表達(dá)情況。3.利用重組人LOXL2(rhLOXL2)刺激BMMSC 24h、48h、72h、96h,檢測(cè)CAF標(biāo)志物α-SMA、FAP、通路蛋白表達(dá)變化。4.應(yīng)用LOX抑制劑β-氨基丙腈處理上述組分,檢測(cè)CAF標(biāo)志物α-SMA、FAP及通路蛋白表達(dá)情況。研究結(jié)果:1.臨床標(biāo)本水平檢測(cè)α-SMA、FAP及LOXL2在MPN患者中表達(dá)顯著升高(P0.05),LOXL2在MPN骨髓或外周血血清中表達(dá)顯著高于正常人水平(P0.05)。2.骨髓活檢免疫組化檢測(cè)結(jié)果顯示,a-SMA、FAP及LOXL2在正常人不表達(dá)或較低表達(dá),在MPN患者中呈不同程度的表達(dá),隨著纖維化程度的增加表達(dá)逐漸升高,在PMF嚴(yán)重纖維化(+++)患者中升高最顯著。3.MPN患者BMMSC具有不同于正常人的生物學(xué)特征,其成骨分化能力增強(qiáng)、成脂分化能力減弱;a-SMA、FAP在MPN患者BMMSC中表達(dá)較正常人明顯升高。4.乏氧環(huán)境下單獨(dú)培養(yǎng)BMMSC,CAF表達(dá)逐漸降低,96h降低最明顯(P0.05);應(yīng)用rhLOXL2刺激后,a-SMA、FAP表達(dá)增高,96h升高最顯著(P0.05);進(jìn)一步應(yīng)用LOX抑制劑β-氨基丙腈處理rhLOXL2 + MSC,a-SMA、FAP表達(dá)明顯下降(P0.05);相關(guān)通路蛋白檢測(cè)顯示,p-FAK、p-Src在rhLOXL2刺激下表達(dá)升高,BAPN可削弱該通路蛋白表達(dá)。正常氧情況下培養(yǎng)的BMMSC,對(duì)rhLOXL2刺激不敏感,a-SMA、FAP表達(dá)水平變化不顯著。研究結(jié)論:1.腫瘤相關(guān)成纖維細(xì)胞、LOXL2與MPN及骨髓纖維化進(jìn)程密切相關(guān);2.MPN患者BMMSC具有不同于正常人的生物學(xué)特征,在MPN骨髓微環(huán)境中獲得CAF表型;3.乏氧環(huán)境下LOXL-2可能通過FAK/Src信號(hào)通路刺激BMMSC活化為腫瘤相關(guān)成纖維細(xì)胞促進(jìn)纖維化進(jìn)程,這可能是骨髓纖維化發(fā)生、進(jìn)展的關(guān)鍵機(jī)制;LOX活性抑制劑β-氨基丙腈對(duì)此種效應(yīng)具有明顯抑制作用。
[Abstract]:Background: chronic myeloid leukemia (CML), essential thrombocythemia (ET), polycythemia vera (PV), idiopathic myelofibrosis (PMF) bone marrow fibrosis often associated with progress in the course of MPN. For myelofibrosis is also the effect of MPN to leukemia, risk factors of bone marrow failure evolution. Therefore, specific cytokine of bone marrow fibrosis, may be the pathogenesis of dig MPN, looking for more effective targeted therapy provides a new starting point. The gene expression profile found in dynamic evolution of the bone marrow microenvironment in MPN bone marrow fibrosis. Bone marrow microenvironment in the hypoxic state about 1% oxygen concentration the tumor hypoxia microenvironment formed by inducing angiogenesis, inhibit the immune surveillance and immune response, inoculation of tumor stem cells mechanism to promote tumor drug resistance, recurrence and progression. Tumor associated fibroblasts (CAF) is The main component of stromal cells in the tumor microenvironment, plays a crucial role in the characteristics of tumor biological behavior of bone marrow mesenchymal stem cells (BMMSC) as an important component of the bone marrow microenvironment, is an important source of CAF. Recent studies showed that CAF and MPN fibrosis increased in hypoxia closely related, but the lack of tumor cells stimulated by hypoxic environment will lead to the inactivation of CAF. That CAF activation occurs through "mutual" with tumor cells or their secreted factors. However, what are the factors that promote the activation of CAF MPN bone marrow fibrosis remains unclear. Lysyl oxidase like protein 2 (LOXL2) is lysyl oxidase (LOX) one of the members of the family can activate FAK/Src, Erk signaling pathway, mediated by mass conversion of way to participate in a variety of epithelial malignant tumor invasive biological characteristics. The study found that the expression level of LOXL2 MPN is closely related with the degree of fibrosis. The hypoxic microenvironment, tumor cell derived LOXL2 can activate FAK/Src pathway promote fibroblasts into CAF, which secrete a variety of cytokines to promote tumor cell proliferation and metastasis. But the two factor mediated bone marrow fibrosis mechanism is unclear. Combining with the research status, we that, in the course of MPN, the micro environment of BMMSC may be activated as CAF phenotype of bone marrow fibrosis; while LOXL2 may be hypoxia in promoting BMMSC activation is the key factor of CAF, to further promote the MPN of bone marrow fibrosis. The purpose of the study is to verify the progress of CAF and LOXL-2 and MPN and confirmed that MPN is closely related to bone marrow fibrosis; the source of the activation of BMMSC CAF phenotype promote fibrosis; explore the hypoxic environment LOXL-2 as start factors to stimulate the activation of MSC is CAF, this effect could be LOX inhibitors The block. Methods: clinical specimen level: 1. extraction of normal and MPN patients bone marrow mononuclear cells, using RT-PCR and Westen-blot methods to detect CAF markers (-SMA, FAP) and the level of expression of LOXL2.2. and MPN in patients with normal serum collected from peripheral blood or bone marrow, ELISA method was used to detect differences in the expression of LOXL2 in.3. collect bone marrow biopsy specimens by immunohistochemical detection of bone marrow biopsy in dye protein, alpha -SMA, FAP, LOXL2 expression. The cell level: 1. after informed consent, patients with normal bone marrow and cultured MPN BMMSC, observe the cell morphology and biological characteristics of MSC detection: immunophenotype, osteogenic, adipogenic cell differentiation ability. The application of RT-PCR, Western blot were detected in normal human and patients with MPN alpha -SMA, FAP expression of.2. were cultured in BMMSC 24h, in the normal oxygen and oxygen concentration 1% 48h, 72h, 96h, morphological changes were observed. CAF marker alpha -SMA, FAP and p-FAK, p-SRC, FAK, SRC protein expression of.3. pathway by recombinant human LOXL2 (rhLOXL2) BMMSC 24h 48h, 72h stimulation, 96h, detection of CAF marker alpha -SMA, FAP, aminoproprionitrile processing and the component change of.4. inhibition of LOX beta protein expression pathway detection of CAF markers, -SMA alpha, FAP expression and protein pathway. Results: 1. clinical specimens to detect the level of alpha -SMA, expression of FAP and LOXL2 in MPN patients was significantly increased (P0.05), the expression of LOXL2 MPN in bone marrow or peripheral blood serum was significantly higher than the normal level (P0.05).2. bone marrow biopsy immunohistochemistry the results showed that the a-SMA expression of FAP and LOXL2 in normal expression and low expression was different in patients with MPN, with the increase of the degree of fibrosis the expression gradually increased in PMF (+ + +) in patients with severe fibrosis increased most significantly in patients with.3.MPN BMMSC is different from the normal The biological characteristics, the osteogenic differentiation ability, adipogenic differentiation capacity decreased; a-SMA, FAP expression is normal in patients with MPN BMMSC significantly increased in.4. cultured alone BMMSC hypoxic environment, CAF expression gradually decreased, 96h decreased most significantly (P0.05); application of rhLOXL2 after stimulation with a-SMA, the increased expression of FAP 96h, the most significant increase (P0.05); the further application of LOX inhibitors of beta aminopropionitrile rhLOXL2 + MSC, a-SMA, FAP expression was significantly decreased (P0.05); detection pathway related proteins revealed that p-FAK and p-Src expression under the stimulation of rhLOXL2 increased, BAPN can weaken the expression of the channel protein. Under the condition of normal oxygen culture BMMSC. Not sensitive to stimulation of rhLOXL2, a-SMA, FAP expression levels did not change significantly. Conclusions: 1. tumor associated fibroblasts, LOXL2 and MPN and the process of bone marrow fibrosis is closely related with 2.MPN; BMMSC has biological characteristics different from normal people, in MPN Get the CAF phenotype in bone marrow microenvironment; 3. hypoxic environment LOXL-2 through FAK/Src signaling pathway to stimulate BMMSC activation for tumor associated fibroblasts promote fibrosis, which may be the key mechanism in the development of bone marrow fibrosis, the inhibitor of LOX activity; beta aminopropionitrile has obvious inhibitory effect on this effect.

【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R551.3;R733

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相關(guān)期刊論文 前2條

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本文編號(hào)：1603903