CRKL、CRKⅡ与人慢性粒细胞白血病临床相关性及各自调控作用机制及相关性

发布时间：2018-04-28 22:31

本文选题：CRKL + CRKⅡ　；参考：《大连医科大学》2017年硕士论文

【摘要】：背景:慢性粒细胞白血病(chronic myelocytic leukemia,CML)约占所有成人白血病的20%,仅次于急粒和急淋白血病,处于白血病的第三位,一般具有BCR-ABL特征性的Ph染色体,是一种具有浸润特征的恶性克隆性疾病。其能够使细胞过度增长、凋亡受到抑制、迁移侵袭能力发生改变、细胞分化受到干扰等。K562是从CML病人红白细胞中分离得到,具有肿瘤干细胞的特征,不具有分化能力,是研究CML理想的细胞模型。CRK接头蛋白在禽类肉瘤病毒CT10(chicken tumor 10)中被发现,由SH2和SH3结构域构成,是致癌基因v-CRK的产物。CRK蛋白家族包括CRKⅠ、CRKⅡ和CRKL,三者均在各种组织中表达。CRK家族通过酪氨酸激酶和小G蛋白等信号分子来调控细胞的转录、增殖、分化和凋亡等生物学行为。CRKL和CRKⅡ作为CRK家族的成员,结构相似,具有高度同源性,可通过SH2和SH3结构域招募信号分子。CRKL和CRKⅡ的异常表达与肿瘤有密切联系,但两者共同对肿瘤的作用研究较少。本组前期研究发现,CRKL影响K562的增殖、迁移侵袭和巨核分化等生物学功能,CRKL下调可引起CRKⅡ表达增加,但具体机制尚不清楚。本论文通过下调K562中CRKL的表达来研究对K562分化的影响、下调CRKⅡ和双敲降CRKⅡ和CRKL来研究两者对K562分化、增殖、迁移和侵袭能力的影响,进而探讨CRKⅡ和CRKL各自功能、作用机制及相互间关联。目的:1、探讨CRKL对K562红系分化影响及分子机制;2、探讨CRKⅡ下调对K562增殖、迁移侵袭和红系分化的影响;3、CRKⅡ和CRKL功能及机制间联系。方法:1、qRT-PCR检测CRKL和CRKⅡ在14例非恶性(各种非恶性血液疾病)、33例初发、5例配对(初发-缓解CR)患者组和正常人组中的表达;2、Hemin诱导和联苯胺染色检测CRKL在K562红系分化中的作用、基因芯片和蛋白质组学检测CRKL下调对K562红系分化特征分子表达水平的影响;3、WB法检测CRKL下调对Raf/MEK/ERK/Elk-1通路中分子表达水平的影响;4、WB法和qRT-PCR检测CRKⅡ蛋白在K562中的下调表达水平;5、CCK-8法检测CRKⅡ下调对K562体外恶性增殖能力的影响、Transwell法检测K562的迁移和侵袭能力、qRT-PCR检测CRKⅡ下调对K562红系分化影响;6、WB法检测CRKⅡ下调对p130Cas/CRK/Rac1和Raf/MEK/ERK/Elk-1通路中分子表达水平的影响;7、免疫共沉淀分析CRKL和CRKⅡ在K562的相互作用;8、台盼蓝计数法检测CRKL+CRKⅡ双敲降K562的增殖能力、Transwell法检测K562的迁移和侵袭能力、qRT-PCR检测K562红系分化影响;9、WB法检测CRKL+CRKⅡ双敲降后,ERK、p-ERK和Rac1的表达。结果:1、qRT-PCR结果显示CRKL在CML初发患者骨髓样本中显著高表达,比正常组高出6.2倍(P=0.009),在CR中低表达。CRKL在5例配对样本中,有5例在CML初发患者中高表达,CR时低表达(P=0.0165)。与正常组相比,CR组CRKL水平降低(P=0.0258)。CRKⅡ在初发患者骨髓样本中略高表达,比正常人高出1.8倍(P=0.0855),在CR中低表达。在5例配对样本中,有3例在CML初发患者中高表达,CR时低表达(P=0.1014)。与正常组相比,CR组CRKⅡ无变化(P=0.1051);2、Hemin诱导K562后CRKL表达量降低,在诱导后的1、2 d时蛋白水平分别降低了52.8%(P=0.0007)和54.6%(P=0.0004),CRKⅡ表达变化不明显;与NC阳性细胞率3.4%相比,CRKL下调引起红系阳性细胞率增加10.5%(P=0.0239),标志分子GPA和γ-globin表达水平分别上升了59.4%(P=0.0096)和96.9%(P=0.0006),出现了HBA和HBD等血红蛋白;3、CRKL下调使GATA-1和HMGB2表达水平升高;4、CRKL下调激活Raf/MEK/ERK/Elk-1通路;5、CRKⅡ下调使K562体外迁移和侵袭能力分别降低了54.7%(P=0.0221)和56.1%(P=0.013),对红系分化无明显影响;6、CRKⅡ下调抑制p130Cas/CRK/Rac1通路,对Raf/MEK/ERK/Elk-1通路影响不大;7、CRKL和CRKⅡ在K562中可以结合形成复合物;8、与CRKL下调细胞相比,CRKL+CRKⅡ双敲降抑制K562增殖能力,细胞迁移侵袭能力又降低了35.9%(P=0.0024)和44.8%(P=0.0115),对红系分化影响不大;9、CRKL+CRKⅡ双敲降抑制Rac1的表达,而对ERK和p-ERK影响不大。结论:1、CRKL和CRKⅡ在CML初发患者骨髓样本中高表达,且CRKL表达水平高于CRKⅡ表达水平,在CR组中明显降低。CR组与正常组相比,CRKL表达下降而CRKⅡ无变化;2、CRKL下调通过激活Raf/MEK/ERK/Elk-1通路促进K562向红系分化,而CRKⅡ下调对K562红系分化无影响;3、CRKⅡ下调抑制K562体外增殖、迁移和侵袭能力;4、CRKⅡ通过p130Cas/CRK/Rac1信号通路调控K562的恶性生物学行为;5、CRKL+CRKⅡ双敲降进一步通过抑制Rac1表达抑制K562体外增殖、迁移和侵袭能力;6、CRKL+CRKⅡ通过p130Cas/CRK/Rac1通路调控K562细胞的增殖、迁移和侵袭能力。
[Abstract]:Background: chronic myelocytic leukemia (CML) accounts for about 20% of all adult leukemia, second only to acute and acute lymphoblastic leukemia, which is in the third place of leukemia, generally having a BCR-ABL characteristic Ph chromosome and is a malignant clonogenic disease characterized by infiltration. It can cause excessive growth and apoptosis of cells. Inhibition, migration and invasion ability change, cell differentiation is disturbed,.K562 is isolated from the red white cells of CML patients and has the characteristics of tumor stem cells and does not have the ability to differentiate. It is the CML ideal cell model.CRK joint protein found in avian sarcoma virus CT10 (chicken tumor 10), which is composed of SH2 and SH3 domains. The.CRK protein family, the product of the oncogene v-CRK, includes CRK I, CRK II and CRKL. All of the three groups express the.CRK family through tyrosine kinase and small G protein to regulate cell transcription, proliferation, differentiation and apoptosis as members of the CRK family, which are similar in structure and highly homologous. The abnormal expression of signal molecules.CRKL and CRK II can be recruited through the SH2 and SH3 domains, and the abnormal expression of.CRKL and CRK II is closely related to the tumor. However, there are few studies on the role of the tumor. Earlier studies in this group have found that CRKL affects the biological functions of K562 proliferation, migration and megakaryocyte differentiation, and the downregulation of CRKL can cause the increase in the expression of CRK II, but the specific mechanism is specific. The effect of CRK II and double knock down CRK II and CRKL on the differentiation, proliferation, migration and invasion of K562 were studied by down regulation of the expression of CRKL in K562, and the effects of CRK II and CRKL on the function, mechanism and correlation of CRK II and CRKL were investigated. Objective: 1, to explore CRKL to K562 red system differentiation. Influence and molecular mechanism; 2, to investigate the effect of CRK II on K562 proliferation, migration and invasion and erythroid differentiation; 3, CRK II and CRKL functions and mechanisms. Methods: 1, qRT-PCR detection CRKL and CRK II in 14 cases of non malignant (various non malignant blood diseases), 33 cases of primary hair, 5 cases of paired (initial remission CR) and normal human groups; 2, Hemin The effect of CRKL in the differentiation of K562 red system induced by induction and diphenyl amine staining. The effect of CRKL down regulation on the expression level of K562 erythroid differentiation molecular expression was detected by gene chip and proteomics; 3, WB method was used to detect the effect of CRKL down regulation on the molecular expression level in Raf/MEK/ERK/Elk-1 pathway; 4, WB and qRT-PCR detected the down regulation of CRK II protein in K562 Expression level; 5, CCK-8 method was used to detect the effect of CRK II on the proliferation of K562 in vitro. Transwell assay was used to detect the migration and invasion of K562. QRT-PCR detected the effect of CRK II on the differentiation of K562 red system; and 6, WB method was used to detect the effect of CRK II on the level of molecular expression in p130Cas/CRK/Rac1 and Raf/MEK/ERK/ pathways; 7, immunoprecipitation The interaction between CRKL and CRK II in K562 was analyzed. 8, trypan blue counting method was used to detect the proliferation ability of CRKL+CRK II double knock down K562, Transwell method was used to detect the migration and invasion ability of K562, qRT-PCR was used to detect the effect of K562 red system differentiation. 9, WB method was used to detect CRKL+CRK II double knockdown. The patient's bone marrow samples were highly expressed, 6.2 times higher than that of the normal group (P=0.009), and the low expression of.CRKL in the CR was found in 5 cases of paired samples. There were 5 cases of high expression in the early CML patients and low expression of CR (P=0.0165). The decrease of CRKL level in the CR group (P=0.0258).CRK II was slightly higher in the bone marrow samples of the primary patients compared with the normal group, which was 1. higher than that of the normal group. 8 times (P=0.0855) and low expression in CR. In 5 cases of paired samples, 3 were expressed in the early CML and low expression at CR (P=0.1014). The CRK II in the CR group was not changed (P=0.1051) compared with the normal group; 2, Hemin induced K562 CRKL expression decreased, and the protein level decreased by 52.8% and 54.6%, respectively, after induction of 1,2. The expression changes were not obvious. Compared with the positive cell rate of NC, the rate of CRKL positive cells increased by 10.5% (P=0.0239), the expression level of the marker molecules GPA and gamma -globin increased by 59.4% (P=0.0096) and 96.9% (P=0.0006) respectively, and HBA and HBD and other hemoglobin appeared. 3, CRKL reduced the GATA-1 and HMGB2 expression levels; 4 The living Raf/MEK/ERK/Elk-1 pathway, 5, CRK II downregulation reduced the migration and invasion ability of K562 in vitro by 54.7% (P=0.0221) and 56.1% (P=0.013), and had no obvious effect on the erythroid differentiation; 6, CRK II down regulated the p130Cas/CRK/Rac1 pathway, and had little effect on Raf/MEK/ERK/Elk-1 pathway; 7, CRKL and CRK II could be combined to form complex in K562; 8, and CRKL. CRKL+CRK II double knockdown inhibited K562 proliferation, and cell migration and invasiveness decreased by 35.9% (P=0.0024) and 44.8% (P=0.0115). 9, CRKL+CRK II double knockdown inhibited Rac1 expression but had little effect on ERK and p-ERK. Conclusion: 1, CRKL and CRK II were highly expressed in the bone marrow samples of CML patients. And the expression level of CRKL was higher than that of CRK II. In group CR, the expression of CRKL decreased and CRK II had no change compared with the normal group. 2, CRKL decreased by activating Raf/MEK/ERK/Elk-1 pathway to promote the differentiation of K562 to the red system, while CRK II downregulation had no effect on the differentiation of K562 red system. 3, CRK II inhibited the proliferation, migration and invasion in vitro. Force; 4, CRK II regulates the malignant biological behavior of K562 through the p130Cas/CRK/Rac1 signaling pathway; 5, CRKL+CRK II double knockdown further inhibits the proliferation, migration and invasion of K562 by inhibiting Rac1 expression; 6, CRKL+CRK II regulates the proliferation, migration and invasion of K562 cells through p130Cas/CRK/Rac1 pathway.

【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R733.72

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