本文選題：CRKL + CRKⅡ　； 參考：《大連醫(yī)科大學(xué)》2017年碩士論文

【摘要】：背景:慢性粒細(xì)胞白血病(chronic myelocytic leukemia,CML)約占所有成人白血病的20%,僅次于急粒和急淋白血病,處于白血病的第三位,一般具有BCR-ABL特征性的Ph染色體,是一種具有浸潤特征的惡性克隆性疾病。其能夠使細(xì)胞過度增長、凋亡受到抑制、遷移侵襲能力發(fā)生改變、細(xì)胞分化受到干擾等。K562是從CML病人紅白細(xì)胞中分離得到,具有腫瘤干細(xì)胞的特征,不具有分化能力,是研究CML理想的細(xì)胞模型。CRK接頭蛋白在禽類肉瘤病毒CT10(chicken tumor 10)中被發(fā)現(xiàn),由SH2和SH3結(jié)構(gòu)域構(gòu)成,是致癌基因v-CRK的產(chǎn)物。CRK蛋白家族包括CRKⅠ、CRKⅡ和CRKL,三者均在各種組織中表達(dá)。CRK家族通過酪氨酸激酶和小G蛋白等信號(hào)分子來調(diào)控細(xì)胞的轉(zhuǎn)錄、增殖、分化和凋亡等生物學(xué)行為。CRKL和CRKⅡ作為CRK家族的成員,結(jié)構(gòu)相似,具有高度同源性,可通過SH2和SH3結(jié)構(gòu)域招募信號(hào)分子。CRKL和CRKⅡ的異常表達(dá)與腫瘤有密切聯(lián)系,但兩者共同對(duì)腫瘤的作用研究較少。本組前期研究發(fā)現(xiàn),CRKL影響K562的增殖、遷移侵襲和巨核分化等生物學(xué)功能,CRKL下調(diào)可引起CRKⅡ表達(dá)增加,但具體機(jī)制尚不清楚。本論文通過下調(diào)K562中CRKL的表達(dá)來研究對(duì)K562分化的影響、下調(diào)CRKⅡ和雙敲降CRKⅡ和CRKL來研究兩者對(duì)K562分化、增殖、遷移和侵襲能力的影響,進(jìn)而探討CRKⅡ和CRKL各自功能、作用機(jī)制及相互間關(guān)聯(lián)。目的:1、探討CRKL對(duì)K562紅系分化影響及分子機(jī)制;2、探討CRKⅡ下調(diào)對(duì)K562增殖、遷移侵襲和紅系分化的影響;3、CRKⅡ和CRKL功能及機(jī)制間聯(lián)系。方法:1、qRT-PCR檢測CRKL和CRKⅡ在14例非惡性(各種非惡性血液疾病)、33例初發(fā)、5例配對(duì)(初發(fā)-緩解CR)患者組和正常人組中的表達(dá);2、Hemin誘導(dǎo)和聯(lián)苯胺染色檢測CRKL在K562紅系分化中的作用、基因芯片和蛋白質(zhì)組學(xué)檢測CRKL下調(diào)對(duì)K562紅系分化特征分子表達(dá)水平的影響;3、WB法檢測CRKL下調(diào)對(duì)Raf/MEK/ERK/Elk-1通路中分子表達(dá)水平的影響;4、WB法和qRT-PCR檢測CRKⅡ蛋白在K562中的下調(diào)表達(dá)水平;5、CCK-8法檢測CRKⅡ下調(diào)對(duì)K562體外惡性增殖能力的影響、Transwell法檢測K562的遷移和侵襲能力、qRT-PCR檢測CRKⅡ下調(diào)對(duì)K562紅系分化影響;6、WB法檢測CRKⅡ下調(diào)對(duì)p130Cas/CRK/Rac1和Raf/MEK/ERK/Elk-1通路中分子表達(dá)水平的影響;7、免疫共沉淀分析CRKL和CRKⅡ在K562的相互作用;8、臺(tái)盼藍(lán)計(jì)數(shù)法檢測CRKL+CRKⅡ雙敲降K562的增殖能力、Transwell法檢測K562的遷移和侵襲能力、qRT-PCR檢測K562紅系分化影響;9、WB法檢測CRKL+CRKⅡ雙敲降后,ERK、p-ERK和Rac1的表達(dá)。結(jié)果:1、qRT-PCR結(jié)果顯示CRKL在CML初發(fā)患者骨髓樣本中顯著高表達(dá),比正常組高出6.2倍(P=0.009),在CR中低表達(dá)。CRKL在5例配對(duì)樣本中,有5例在CML初發(fā)患者中高表達(dá),CR時(shí)低表達(dá)(P=0.0165)。與正常組相比,CR組CRKL水平降低(P=0.0258)。CRKⅡ在初發(fā)患者骨髓樣本中略高表達(dá),比正常人高出1.8倍(P=0.0855),在CR中低表達(dá)。在5例配對(duì)樣本中,有3例在CML初發(fā)患者中高表達(dá),CR時(shí)低表達(dá)(P=0.1014)。與正常組相比,CR組CRKⅡ無變化(P=0.1051);2、Hemin誘導(dǎo)K562后CRKL表達(dá)量降低,在誘導(dǎo)后的1、2 d時(shí)蛋白水平分別降低了52.8%(P=0.0007)和54.6%(P=0.0004),CRKⅡ表達(dá)變化不明顯;與NC陽性細(xì)胞率3.4%相比,CRKL下調(diào)引起紅系陽性細(xì)胞率增加10.5%(P=0.0239),標(biāo)志分子GPA和γ-globin表達(dá)水平分別上升了59.4%(P=0.0096)和96.9%(P=0.0006),出現(xiàn)了HBA和HBD等血紅蛋白;3、CRKL下調(diào)使GATA-1和HMGB2表達(dá)水平升高;4、CRKL下調(diào)激活Raf/MEK/ERK/Elk-1通路;5、CRKⅡ下調(diào)使K562體外遷移和侵襲能力分別降低了54.7%(P=0.0221)和56.1%(P=0.013),對(duì)紅系分化無明顯影響;6、CRKⅡ下調(diào)抑制p130Cas/CRK/Rac1通路,對(duì)Raf/MEK/ERK/Elk-1通路影響不大;7、CRKL和CRKⅡ在K562中可以結(jié)合形成復(fù)合物;8、與CRKL下調(diào)細(xì)胞相比,CRKL+CRKⅡ雙敲降抑制K562增殖能力,細(xì)胞遷移侵襲能力又降低了35.9%(P=0.0024)和44.8%(P=0.0115),對(duì)紅系分化影響不大;9、CRKL+CRKⅡ雙敲降抑制Rac1的表達(dá),而對(duì)ERK和p-ERK影響不大。結(jié)論:1、CRKL和CRKⅡ在CML初發(fā)患者骨髓樣本中高表達(dá),且CRKL表達(dá)水平高于CRKⅡ表達(dá)水平,在CR組中明顯降低。CR組與正常組相比,CRKL表達(dá)下降而CRKⅡ無變化;2、CRKL下調(diào)通過激活Raf/MEK/ERK/Elk-1通路促進(jìn)K562向紅系分化,而CRKⅡ下調(diào)對(duì)K562紅系分化無影響;3、CRKⅡ下調(diào)抑制K562體外增殖、遷移和侵襲能力;4、CRKⅡ通過p130Cas/CRK/Rac1信號(hào)通路調(diào)控K562的惡性生物學(xué)行為;5、CRKL+CRKⅡ雙敲降進(jìn)一步通過抑制Rac1表達(dá)抑制K562體外增殖、遷移和侵襲能力;6、CRKL+CRKⅡ通過p130Cas/CRK/Rac1通路調(diào)控K562細(xì)胞的增殖、遷移和侵襲能力。
[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.

【學(xué)位授予單位】：大連醫(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)】：R733.72

【參考文獻(xiàn)】

相關(guān)期刊論文 前5條

1 沈紹華;馬華;楊憲勇;劉愛華;;CRKL、PgP不同表達(dá)情況與白血病細(xì)胞耐藥關(guān)系的臨床研究[J];中華臨床醫(yī)師雜志(電子版);2013年09期

2 李文燕;高一萌;張瑜;王瑾;車亞玲;令狐華;;接合物蛋白Crk與CrkL在卵巢癌細(xì)胞株MCAS細(xì)胞增殖中的不同作用[J];第三軍醫(yī)大學(xué)學(xué)報(bào);2012年12期

3 張小鷹;曾慧蘭;蔣建偉;陳濤;吳風(fēng)云;何金花;韓新愛;廖曉莉;;Bcr/abl融合基因的小干擾RNA對(duì)K562細(xì)胞增殖和凋亡的影響[J];廣東醫(yī)學(xué);2008年11期

4 沈太敏;趙純?nèi)?趙涌;令狐華;;接合物蛋白Crk和CrkL在上皮性卵巢癌中的表達(dá)及意義[J];中國全科醫(yī)學(xué);2008年11期

5 陰傳敏;姚珍薇;;信號(hào)接頭蛋白c-Crk與惡性腫瘤關(guān)系的研究進(jìn)展[J];重慶醫(yī)學(xué);2008年02期

，

本文編號(hào)：1817190