乏氧环境下LOXL-2刺激MSC活化为肿瘤相关成纤维细胞促进MPN骨髓纤维化进程研究

发布时间：2018-03-13 00:05

本文选题：CAF　切入点：LOXL2　出处：《南方医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：研究背景:慢性髓系白血病(CML)、原发性血小板增多症(ET)、真性红细胞增多症(PV)、原发性骨髓纤维化(PMF)等MPN病程进展中常伴随骨髓纤维化。进行性骨髓纤维化亦是影响MPN向白血病、骨髓衰竭演变的高危因素。因此,研究发生骨髓纤维化的特异性细胞因子,可能为深入挖掘MPN的发病机制、寻找更有效的靶向治疗提供新的切入点。基因表达谱发现骨髓微环境的动态演化参与了 MPN骨髓纤维化进程。骨髓微环境处于1%氧浓度左右的乏氧状态,肿瘤中的乏氧微环境可通过诱导新生血管形成、抑制免疫监视和免疫反应、孕育肿瘤干细胞等机制促进肿瘤耐药、复发及进展。肿瘤相关成纤维细胞(CAF)是肿瘤微环境中基质细胞的主要组成成分,在肿瘤生物行为学特征中起着至关重要的作用,骨髓间充质干细胞(BMMSC)作为骨髓微环境中的重要成分,是CAF重要来源。新近研究发现,乏氧微环境中增多的CAF与MPN纤维化进程密切相关,但缺乏肿瘤细胞刺激的乏氧环境下将导致CAF的失活。表明CAF的活化需要通过与肿瘤细胞或其分泌因子发生“互话”。然而,到底是哪些因素活化CAF促进MPN骨髓纤维化尚不清楚。赖氨酰氧化酶样蛋白2(LOXL2)是赖氨酰氧化酶(LOX)家族成员之一,可通过激活FAK/Src、Erk信号通路、介导上皮间质转化等方式参与多种恶性肿瘤侵袭性生物学特征形成。研究发现,LOXL2的表达水平与MPN纤维化程度密切相关。乏氧微环境下,肿瘤细胞来源的LOXL2可激活FAK/Src信号通路促使纤维母细胞转化为CAF,后者分泌多种细胞因子不断促进肿瘤细胞增殖、转移。但两种因子介导骨髓纤维化机制目前尚不清楚。结合研究现状,我们推测,MPN病程中,微环境中的BMMSC可能活化为CAF表型促进骨髓纤维化;而LOXL2则可能是乏氧微环境中促进BMMSC活化为CAF的关键因子,进一步推进MPN骨髓纤维化进程。研究目的:验证CAF及LOXL-2与MPN及骨髓纤维化进展密切相关;证实MPN来源的BMMSC活化为CAF表型促进纤维化进程;探索乏氧环境下LOXL-2是否作为启动因素刺激MSC活化为CAF,这种效应是否能够被LOX抑制剂所阻断。研究方法:临床标本水平:1.提取正常人与MPN患者骨髓单个核细胞,应用RT-PCR及Westen-blot方法检测CAF标志物(α-SMA、FAP)及LOXL2的表达水平。2.收集正常人与MPN患者骨髓或外周血血清,应用ELISA方法检测LOXL2表达差异。3.收集骨髓病理活检标本,应用免疫组化检测骨髓活检中网染蛋白、α-SMA、FAP、LOXL2的表达情况。细胞水平:1.经知情同意,取正常人及MPN患者骨髓分离培养BMMSC,观察细胞形态,检测MSC生物学特性:免疫表型、成骨、成脂肪细胞诱导分化能力。应用RT-PCR、Western blot分别检测正常人与MPN患者α-SMA、FAP表达情况。2.分别在正常氧和1%氧浓度条件下培养BMMSC 24h、48h、72h、96h,观察细胞形态变化,检测CAF标志物α-SMA、FAP及p-FAK、p-SRC、FAK、SRC通路蛋白表达情况。3.利用重组人LOXL2(rhLOXL2)刺激BMMSC 24h、48h、72h、96h,检测CAF标志物α-SMA、FAP、通路蛋白表达变化。4.应用LOX抑制剂β-氨基丙腈处理上述组分,检测CAF标志物α-SMA、FAP及通路蛋白表达情况。研究结果:1.临床标本水平检测α-SMA、FAP及LOXL2在MPN患者中表达显著升高(P0.05),LOXL2在MPN骨髓或外周血血清中表达显著高于正常人水平(P0.05)。2.骨髓活检免疫组化检测结果显示,a-SMA、FAP及LOXL2在正常人不表达或较低表达,在MPN患者中呈不同程度的表达,随着纤维化程度的增加表达逐渐升高,在PMF严重纤维化(+++)患者中升高最显著。3.MPN患者BMMSC具有不同于正常人的生物学特征,其成骨分化能力增强、成脂分化能力减弱;a-SMA、FAP在MPN患者BMMSC中表达较正常人明显升高。4.乏氧环境下单独培养BMMSC,CAF表达逐渐降低,96h降低最明显(P0.05);应用rhLOXL2刺激后,a-SMA、FAP表达增高,96h升高最显著(P0.05);进一步应用LOX抑制剂β-氨基丙腈处理rhLOXL2 + MSC,a-SMA、FAP表达明显下降(P0.05);相关通路蛋白检测显示,p-FAK、p-Src在rhLOXL2刺激下表达升高,BAPN可削弱该通路蛋白表达。正常氧情况下培养的BMMSC,对rhLOXL2刺激不敏感,a-SMA、FAP表达水平变化不显著。研究结论:1.肿瘤相关成纤维细胞、LOXL2与MPN及骨髓纤维化进程密切相关;2.MPN患者BMMSC具有不同于正常人的生物学特征,在MPN骨髓微环境中获得CAF表型;3.乏氧环境下LOXL-2可能通过FAK/Src信号通路刺激BMMSC活化为肿瘤相关成纤维细胞促进纤维化进程,这可能是骨髓纤维化发生、进展的关键机制;LOX活性抑制剂β-氨基丙腈对此种效应具有明显抑制作用。
[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.

【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R551.3;R733

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