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

【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)、真性紅細胞增多癥(PV)、原發(fā)性骨髓纖維化(PMF)等MPN病程進展中常伴隨骨髓纖維化。進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胞因子,可能為深入挖掘MPN的發(fā)病機制、尋找更有效的靶向治療提供新的切入點�；虮磉_譜發(fā)現骨髓微環(huán)境的動態(tài)演化參與了 MPN骨髓纖維化進程。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)境可通過誘導新生血管形成、抑制免疫監(jiān)視和免疫反應、孕育腫瘤干細胞等機制促進腫瘤耐藥、復發(fā)及進展。腫瘤相關成纖維細胞(CAF)是腫瘤微環(huán)境中基質細胞的主要組成成分,在腫瘤生物行為學特征中起著至關重要的作用,骨髓間充質干細胞(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,乏氧微環(huán)境中增多的CAF與MPN纖維化進程密切相關,但缺乏腫瘤細胞刺激的乏氧環(huán)境下將導致CAF的失活。表明CAF的活化需要通過與腫瘤細胞或其分泌因子發(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信號通路、介導上皮間質轉化等方式參與多種惡性腫瘤侵襲性生物學特征形成。研究發(fā)現,LOXL2的表達水平與MPN纖維化程度密切相關。乏氧微環(huán)境下,腫瘤細胞來源的LOXL2可激活FAK/Src信號通路促使纖維母細胞轉化為CAF,后者分泌多種細胞因子不斷促進腫瘤細胞增殖、轉移。但兩種因子介導骨髓纖維化機制目前尚不清楚。結合研究現狀,我們推測,MPN病程中,微環(huán)境中的BMMSC可能活化為CAF表型促進骨髓纖維化;而LOXL2則可能是乏氧微環(huán)境中促進BMMSC活化為CAF的關鍵因子,進一步推進MPN骨髓纖維化進程。研究目的:驗證CAF及LOXL-2與MPN及骨髓纖維化進展密切相關;證實MPN來源的BMMSC活化為CAF表型促進纖維化進程;探索乏氧環(huán)境下LOXL-2是否作為啟動因素刺激MSC活化為CAF,這種效應是否能夠被LOX抑制劑所阻斷。研究方法:臨床標本水平:1.提取正常人與MPN患者骨髓單個核細胞,應用RT-PCR及Westen-blot方法檢測CAF標志物(α-SMA、FAP)及LOXL2的表達水平。2.收集正常人與MPN患者骨髓或外周血血清,應用ELISA方法檢測LOXL2表達差異。3.收集骨髓病理活檢標本,應用免疫組化檢測骨髓活檢中網染蛋白、α-SMA、FAP、LOXL2的表達情況。細胞水平:1.經知情同意,取正常人及MPN患者骨髓分離培養(yǎng)BMMSC,觀察細胞形態(tài),檢測MSC生物學特性:免疫表型、成骨、成脂肪細胞誘導分化能力。應用RT-PCR、Western blot分別檢測正常人與MPN患者α-SMA、FAP表達情況。2.分別在正常氧和1%氧濃度條件下培養(yǎng)BMMSC 24h、48h、72h、96h,觀察細胞形態(tài)變化,檢測CAF標志物α-SMA、FAP及p-FAK、p-SRC、FAK、SRC通路蛋白表達情況。3.利用重組人LOXL2(rhLOXL2)刺激BMMSC 24h、48h、72h、96h,檢測CAF標志物α-SMA、FAP、通路蛋白表達變化。4.應用LOX抑制劑β-氨基丙腈處理上述組分,檢測CAF標志物α-SMA、FAP及通路蛋白表達情況。研究結果:1.臨床標本水平檢測α-SMA、FAP及LOXL2在MPN患者中表達顯著升高(P0.05),LOXL2在MPN骨髓或外周血血清中表達顯著高于正常人水平(P0.05)。2.骨髓活檢免疫組化檢測結果顯示,a-SMA、FAP及LOXL2在正常人不表達或較低表達,在MPN患者中呈不同程度的表達,隨著纖維化程度的增加表達逐漸升高,在PMF嚴重纖維化(+++)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特征,其成骨分化能力增強、成脂分化能力減弱;a-SMA、FAP在MPN患者BMMSC中表達較正常人明顯升高。4.乏氧環(huán)境下單獨培養(yǎng)BMMSC,CAF表達逐漸降低,96h降低最明顯(P0.05);應用rhLOXL2刺激后,a-SMA、FAP表達增高,96h升高最顯著(P0.05);進一步應用LOX抑制劑β-氨基丙腈處理rhLOXL2 + MSC,a-SMA、FAP表達明顯下降(P0.05);相關通路蛋白檢測顯示,p-FAK、p-Src在rhLOXL2刺激下表達升高,BAPN可削弱該通路蛋白表達。正常氧情況下培養(yǎng)的BMMSC,對rhLOXL2刺激不敏感,a-SMA、FAP表達水平變化不顯著。研究結論:1.腫瘤相關成纖維細胞、LOXL2與MPN及骨髓纖維化進程密切相關;2.MPN患者BMMSC具有不同于正常人的生物學特征,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信號通路刺激BMMSC活化為腫瘤相關成纖維細胞促進纖維化進程,這可能是骨髓纖維化發(fā)生、進展的關鍵機制;LOX活性抑制劑β-氨基丙腈對此種效應具有明顯抑制作用。
[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.

【學位授予單位】：南方醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R551.3;R733

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本文編號：1603903