【作者】 杨秀鹏；

【导师】 李艳；

【作者基本信息】 中国医科大学 ， 内科学， 2010， 博士

【摘要】 目的急性髓细胞白血病(Acute Myeloid Leukemia, AML)是血液系统常见的恶性肿瘤,以幼稚粒细胞克隆性增生且不伴有明显的分化为特征,临床分为多种亚型,大部分亚型的发病机制仍不十分清楚。类胰蛋白酶(Tryptase)是一种具有多种生物学效应的丝氨酸蛋白酶,目前被作为AML发病和临床监测过程中的一种新酶类而受到了人们的关注,以往被普遍认为主要是由肥大细胞和嗜碱性粒细胞(嗜碱性粒细胞产生少量)产生的。随着对tryptase广泛和深入的研究,发现在AML患者的部分FAB亚型也高表达tryptase,同时,人们发现AML细胞系U937也可表达tryptase。但是,AML患者白血病细胞所分泌的tryptase在AML疾病发展中的作用目前仍不十分明确。我们前期研究证实,AML-M2患者骨髓单个核细胞的VEGF与tryptase的表达均明显增高,且二者之间具有显著相关性。最近研究还发现,tryptase阳性的肥大细胞计数与许多恶性肿瘤和炎症的血管新生程度成正相关,可以促进多种肿瘤细胞和炎症细胞表达VEGF,并可作为判断恶性肿瘤患者预后的指标。这说明tryptase可能在AML血管新生中起着一定的作用,这种作用可能与VEGF有关,目前国内外尚未见相关报道。PAR-2是tryptase的细胞表面受体,被tryptase酶切裂解后可介导跨膜信号转导,将细胞外的信号传递到细胞内。以往对多种细胞的研究表明,PAR-2可通过多种途径介导细胞内信号转导,但近年来研究主要集中在丝裂原活化蛋白激酶MAPK信号通路,其中VEGF的表达则多与细胞外信号调节激酶ERK和p38MAPK通路有关。NF-κB是重要的转录调节因子,可作为ERK和p38MAPK信号通路的下游信号分子,被激活后可引起相应靶基因的转录激活,以往的研究表明VEGF的表达与NF-κB也有着密切的关系。为探讨tryptase在AML血管新生中的作用,我们将研究tryptase作用于AMLBMCs后是否上调VEGF的表达,并进一步探讨tryptase促进AML BMCs表达VEGF的可能作用机制：tryptase与细胞表面的PAR-2作用后,依赖细胞内MAPK(ERK和／或p38MAPK)信号转导通路,激活转录调节因子NF-κB,使其转位到核内,促使VEGF表达增加。方法1、细胞培养及鉴定：骨髓肝素钠混合液5ml,加入10ml淋巴细胞分离液,2000rpm,离心10min,取单核细胞层,接种于25ml培养瓶中,15%胎牛血清,DMEM/F12中,培养箱常规培养。根据细胞形态学和流式细胞术检测细胞表面CD14,CD34,CD45,CD13和CD117表达情况,鉴定所培养的细胞为AML BMSCs。2、重组tryptase与AML BMSCs共同孵育后检测VEGF的表达情况：以0-50ng/ml的tryptase作用于AML BMSCs,应用MTT的方法检测细胞活性,从而挑选tryptase的最佳作用浓度。以不同浓度(0.5,5,10 ng/ml)的tryptase作用于AML BMSCs,分别用ELISA、免疫荧光、real time RT-PCR和Western-blot的方法检测VEGF的表达情况。3、以U937细胞的培养上清培养AML BMSCs,检测VEGF的表达：以DMEM／F12培养基、U937细胞培养上清液、U937细胞培养上清液加tryptase抗体作为培养基。培养AML BMSCs 24 h后,应用real time RT-PCR方法检测AMLBMSCs中VEGF mRNA的表达；培养AML BMSCs48 h,应用ELISA方法检测上清液中VEGF的表达情况。4、免疫细胞化学和流式细胞术检测AML BMSCs表面PAR-2的表达：用胰酶将AML BMSCs从细胞培养瓶上消化下来,制成细胞爬片,待细胞生长至80%时,甲醛固定,以PBS作为阴性对照,免疫细胞化学方法检测PAR-2的表达。取AML BMSCs约106个细胞,甲醛固定,FITC标记的PAR-2冰上孵育1小时,PBS洗2次,以100μl PBS重悬细胞后上流式细胞仪检测。5、Western blot检测tryptase处理前后ERK、p38MAPK蛋白：提取细胞总蛋白,上样量为20μg。转印到PVDF膜上后用5%脱脂奶粉封闭, ERK鼠抗人多克隆抗体(1：200)、磷酸化ERK鼠抗人多克隆抗体(1：200)及p38MAPK羊抗人多克隆抗体(1：200)、磷酸化p38MAPK鼠抗人多克隆抗体(1：200)育过夜,分别用相应的二抗室温孵育2 h,显色5min,于凝胶自动成像分析系统下成像,测定条带的平均积分光密度值。6、EMSA检测tryptase处理前后NF-κB活化情况：AML BMSCs以PBS、tryptase、tryptase加NF-κB的阻断剂PDTC孵育24 h后,提取总的核蛋白,标记探针,加样,转膜,用X射线胶片曝光30 min,显影液5 min,定影液5 min。观察tryptase处理前后NF-κB的表达水平。7、Real time RT-PCR检测使用PAR-2、ERK、p38MAPK和NF-κB特异性阻断剂预处理细胞后,tryptase诱导AML BMSCs表达VEGF mRNA的情况：分别使用PAR-2、ERK、p38MAPK和NF-κB特异性阻断剂：FLLSY-NH2 (200μM)、PD98059 (20μM)、SB230580 (10μM)和PDTC(100μM)孵育细胞30 min,然后tryptase与AML BMSCs共孵育24 h。收集细胞提取总RNA, real time RT-PCR检测VEGF mRNA表达。8、Western-blot检测使用PAR-2、ERK、p38MAPK和NF-κB特异性阻断剂预处理细胞后,tryptase诱导AML BMSCs表达VEGF蛋白的情况：分别使用PAR-2、ERK、p38MAPK和NF-κB特异性阻断剂：FLLSY-NH2 (200μM)、PD98059 (20μM)、SB230580 (10μM)和PDTC(100μM)孵育细胞30 min,然后tryptase与AML BMSCs共孵育48 h。收集细胞提取总蛋白,Western-blot检测VEGF蛋白的表达。9、统计学分析：统计数据以SPSS13.0软件进行分,计量资料用均数±标准差表示,组间比较用单因素方差分析,多个样本均数的两两比较用LSD方法。结果以P<0.05认为差异有统计学意义。结果1、ELISA、免疫荧光、real time RT-PCR和Western-blot结果显示,tryptase作用于AML BMSCs后,与对照组相比VEGF表达明显增加(P<0.05),且在一定浓度范围内,成一定剂量关系。2、ELISA和real time RT-PCR结果显示,以U937细胞培养上清液、U937细胞培养上清液培养AML BMSCs后,与对照组相比／AML BMSCs中VEGF表达明显增加(P<0.05),这种增加在使用了tryptase特异性抗体后明显减低(P<0.05)。3、流式细胞术和免疫细胞化学方法结果显示,AML BMSCs细胞表面有tryptase特异性受体PAR-2的表达。4、Western blot结果显示,tryptase与AML BMSCs共同孵育48 h后,与对照组相比,磷酸化的ERK和p38MAPK蛋白表达明显增加(P<0.05),在使用了磷酸化阻断剂PD98059和SB230580后表达降低(P<0.05)。5、EMSA结果显示,tryptase与AML BMSCs共同孵育48 h后,与对照组NF-κB核转位增加,在使用了NF-κB特异性阻断剂PDTC后表达降低。6、Real time RT-PCR和Western-blot结果显示,分别使用PAR-2、ERK、p38MAPK和NF-κB特异性阻断剂(FLLSY-NH2、PD98059、SB230580和PDTC)处理细胞30 min后,tryptase上调AML BMSCs的VEGF mRNA和蛋白的表达明显减低(P<0.05)。结论1、重组的tryptase可以促进AML BMSCs高表达VEGF,在一定浓度范围内呈剂量依赖关系。2、白血病细胞系U937分泌的tryptase可以促进AML BMSCs高表达VEGF。3、AML BMSCs表面表达PAR-2, PAR-2在tryptase上调AML BMSCs的VEGF表达中起着重要作用。4、ERK、p38MAPK和NF-κB通路参与了tryptase上调AML BMSCs的VEGF表达。更多还原

【Abstract】 ObjectiveAcute myeloid leukemia is a common malignancy in blood Disease, which characteristiced by a clonal proliferation in immature granulocytes and not accompanied with a clear differentiation. AML can be divided into a variety of clinical subtype, part of which is still not very clear. Tryptase is a serine protease, which has a variety of biological effects and currently considered as a new enzyme in AML pathogenesis, clinical monitoring process and received extensive attention. It was widely believed to be primarily secreted by mast cells and basophils. But with the extensive deeply study in tryptase, a high expression of tryptase has been found some in AML patients of part of FAB subtype. Previous research has also confirmed that acute myeloid leukemia cell line U937 can secrete tryptase. However, the role of tryptase which secreted by leukemia cells in AML is not very clear in the development of AML.Our previous findings show that the expression of VEGF and tryptase were significantly increased in AML-M2 patients, and has a significant correlation between VEGF and tryptase. Recent studies have also found that, the counts of mast cell of tryptase-positive has a positive correlation with angiogenesis in a number of malignant tumors and inflammation. Tryptase can promote overexpression of VEGF in a variety of tumor and inflammatory cells, and may serve as indicators for judging the prognosis of patients with malignant tumors. This shows that tryptase may plays a certain role in AML angiogenesis, which may be related to VEGF. PAR-2 is the cell surface receptors of tryptase, activated by tryptase and then mediating transmembrane signal transduction. Previous studies shown that, PAR-2 could transmit intracellular information by a variety of ways. The study in recent years mainly focused on MAPK signaling pathway, and the expression of VEGF was related with extracellular signal-regulated kinase ERK and p38MAPK pathways. NF-κB is an important transcription factor, which can be used as downstream signaling molecules in ERK and p38MAPK signaling pathway. Previous studies have also shown that expression of VEGF has a close relationship with NF-κB.To investigate the role of tryptase in AML angiogenesis, we will reseach that wether tryptase could increaseVEGF expression in bone marrow stromal cells of AML, and wether the mechanism of tryptase promote AML BMCs expression VEGF is that tryptase actives PAR-2, then dependenting on MAPK (ERK and/or p38MAPK) signaling pathway and activing NF-κB, increasing the expression of VEGF.Methods1. Cell culture and identification:Bone marrow and heparin sodium mixture 5ml, lymphocyte separation medium 10ml,2000rpm, 10min, mononuclear cell were obtained, then inoculated in 25ml culture flask.15% fetal bovine serum and DMEM/F12 were used to culture cells. According to cell morphology and the express of CD14, CD34, CD45, CD13 and CD117 in cell surface, which detected by FCM, AML BMSCs was identificated.2. The expression of VEGF in AML BMSCs after incubated with tryptase: MTT was used to detect cell viability after incubated with tryptase (0～50 ng/ml). In order to select the best concentration of tryptase, different concentrations (0.5,5,10 ng /ml) of tryptase were used to effect on AML BMSCs. ELISA, immunofluorescence, real time RT-PCR and Western-blot method were used to detect the expression of VEGF.3. The expression of VEGF in AML BMSCs cultured by the supernatants of U937 cells:DMEM/F12 substratum, U937 cell culture supernatant and U937 cell culture supernatant plus tryptase antibody were used to be substratum, then AML BMSCs were cultured for 24 h and VEGF mRNA were detected by real time RT-PCR method. AML BMSCs were cultured for 48 h cultured AML BMSCs 48h, and VEGF expression were detected by ELISA method.4. The expression of PAR-2 in AML BMSCs was detected by immunocytochemistry and flow cytometry surface:AML BMSCs were fixed with Paraformaldehyde, and PBS as negative contro. Immunocytochemistry was used to detect the expression of PAR-2. AML BMSCs about 10°cells were fixed with Paraformaldehyde, and incubated with FITC labeled PAR-2 for 1 hour on ice, and washed two times with PBS and re-suspended cells with 100μl PBS,-then detected by flow cytometry.5. ERK, p38MAPK protein detected by Western blot in AML BMSCs incubated with tryptase:Total cellular protein was extracted. The sample 20μg was transfered to PVDF membrane, and closed with 5% skim milk powder. The membrane were incubated overnight with ERK mouse anti-human polyclonal antibody, phosphorylated ERK mouse anti-human polyclonal antibody, and p38MAPK goat anti-human polyclonal antibody, phosphorylated ERK mouse anti-human polyclonal antibody, then incubated with respective secondary antibody at room temperature for 2 h and were colored 5min.6. The activation of NF-κB was detected EMSA in AML BMSCs after incubating with tryptase:AML BMSCs were incubated with PBS, tryptase, tryptase plus PDTC for 24h, then total nuclear protein were extracted. EMSA method was used to detect the activation of NF-κB in AML BMSCs.7. PAR-2, ERK, p38MAPK and NF-κB-specific inhibitor respectively: FLLSY-NH2 (200μM), PD98059 (20μM), SB230580 (10μM) and PDTC (100μM) were used to treated AML BMSCs for 30 min, then trypase were used to incubate with AML BMSCs, and real time RT-PCR method were used to detect the expression of VEGF mRNA.8. PAR-2, ERK, p38MAPK and NF-κB-specific inhibitor respectively: FLLSY-NH2 (200μM), PD98059 (20μM), SB230580 (10μM) and PDTC (100μM) were used to treated AML BMSCs for 30 min, then trypase were used to incubate with AML BMSCs, and Western blot method was used to detect the protein expression of VEGF.9. Statistical analysis SPSS 13.0 software was used to analysis Statistical data The measurement data was indicated with mean±standard deviation, and one way ANOVE was used to analysi with the methon of LSD P<0.05 was statistically significant.Results1. The result of ELISA, immunofluorescence, real time RT-PCR and Western-blot showed that, after AML BMSCs incubating with tryptase, compared with the control group, VEGF expression was significantly increased (P<0.05), with a dose-dependent relationship in a certain range.2. The results of ELISA and real time RT-PCR showed that, after AML BMSCs cultured with U937 cell culture supernatant, compared with the control group, VEGF expression in AML BMSCs significantly increased (P<0.05), the increased was significantly reduced by antibody of tryptase (P<0.05).3. The result of flow cytometry and immunocytochemistry showed that, tryptase-specific receptor PAR-2 was expressed on AML BMSCs.4. The results of Western blot showed that, after AML BMSCs incubated with tryptase for 48h, compared with the control group, Phosphorylation of ERK and p38MAPK protein expression were significantly increased (P<0.05), and the increasing was significantly reduced by PD98059 and SB230580 (P<0.05).5. The results of EMSA showed that, after AML BMSCs incubated with tryptase for 48h, compared with the control group, the activation of NF-κB were increased, and the increasing was significantly reduced by PDTC (P<0.05).6. The results of Real time RT-PCR and Western-blot showed that after AML BMSCs incubated with PAR-2、ERK、p38MAPK and NF-κB specificity blocking agent respectively (FLLSY-NH2, PD98059, SB230580 and PDTC) treated cells for 30 min, VEGF mRNA and protein expression which increased by tryptase were reduced (P <0.05).Conclusions1. The reorganized tryptase could promote VEGF overexpression in AML BMSCs, showing a dose-dependent relationship in a certain range.2. Tryptase secreted by leukemia cell line U937 can also promote VEGF highly expressed in AML BMSCs.3. PAR-2 were expressed on BMSCs surface, playing an important role in tryptase increaseing the expression of VEGF in AML BMSCs4. ERK, p38MAPK and NF-κB signaling pathway were involved in the tryptase increasing VEGF expression in AML BMSCs.更多还原