【作者】 周亚南；

【导师】 陈子兴；

【作者基本信息】 苏州大学 ， 内科血液学， 2009， 博士

【摘要】 目的和意义Notch信号通路是一个广泛存在于生物进化过程中高度保守的介导细胞与细胞之间通讯机制的信号通路之一,与调控细胞的增殖、分化和凋亡有关,在胚胎发育和决定细胞命运的过程中发挥重要作用。其中Notch1受体与肿瘤的相关性最初于人类具有t(7;9)染色体易位的急性T淋巴细胞白血病中被发现,随后越来越多研究表明恶性肿瘤的发生与异常Notch信号通路有关。有关Notch信号通路与急性白血病发病的已有大量研究主要集中在Notch受体突变与急性T淋巴细胞白血病的发病机理方面,在与急性B淋巴细胞白血病和急性髓性白血病的发病关系中,有Notch受体可能扮演致癌基因与抑癌基因两种截然相反观点。本研究目的:1通过检测Notch信号通路相关分子在急性白血病中的表达,以探讨Notch信号通路在急性白血病中的致病机理。2 Notch受体配基Delta1对正常细胞和白血病细胞的体外作用研究。3 Notch受体配基Delta1对白血病细胞株THP1和SHI-1的作用机理研究。通过上述研究对未来急性白血病的个体化治疗,有可能提供新的理论依据。方法第一部分第一章采用RT-PCR方法,检测受体Notch1和配基Jagged1以及内参照β-肌动蛋白(β2-actin)在经MICM诊断明确的118例初诊急性白血病患者(包括T-ALL21例,B-ALL 32例和AML65例)和11例同期正常供体骨髓单个核细胞中的表达水平,以检测基因=(检测基因表达/β2-actin表达)计算检测基因的表达水平。第二、三、四章建立实时定量RT-PCR方法,采用ABI PRISM 7700 PCR仪检测了初诊T-ALL(15例),B-ALL(16例)以及AML(18例)患者骨髓(原始+幼稚淋巴细胞≥80%)和11例正常骨髓移植供者细胞中Notch1、Notch2、Jagged1、Delta1和Hes1及内参GAPDH的表达水平,以检测基因=(检测基因拷贝数/GAPDH拷贝数)×10~6计算检测基因的表达水平。第二部分第一章将配基Delta1蛋白和对照Human IgG F(c)蛋白分别通过包被固相化在96孔板上,采用MTT法检测,研究Delta1对正常供体骨髓CD34~+细胞、脐带血CD34~+细胞、JMML患者的CD34~+白血病细胞、正常人外周血CD3~+细胞和CD20~-CD56~+细胞是否有促进生长或者抑制增殖作用。第二章采用与第一章相同的方法检测,研究Delta1对细胞株NB4、K562、8266、THP1、SHI-1、MO7E是否有促进生长或者抑制增殖作用。第三部分第一章通过第二部分用MTT法检测发现Delta1对其中的两株细胞株THP1和SHI-1有抑制生长作用后,用MTT法进一步观察采用DAPT(信号通路中γ—分泌酶的阻断剂)能否逆转Delta1对THP1细胞的生长抑制作用。采用细胞计数和台盼兰拒染法判断THP1细胞株经Delta1作用后的细胞增殖能力和细胞存活力,并且通过瑞特染色,在显微镜下观察细胞形态,以及计算NBT还原率。用流式细胞仪(FCM)检测配基Delta1作用后THP1细胞表面抗原CD13、CD14和CD11b的表达,以及用FCM检测凋亡Annexin V的表达,采用实时定量RT-PCRE测Delta1作用THP1细胞前后靶分子Hes1的表达。第二章除了采用与第一章相同方法检测Delta1对细胞株SHI-1作用前后的上述指标外,还采用相同的方法检测加入阻断剂DAPT前后,上述相应指标的变化。结果第一部分采用RT-PCR检测发现在T-ALL、B-ALL、AML以及正常供体骨髓中均有Notch1和Jagged1表达。T-ALL组与对照组比较,Notch1表达水平增高,Jagged1表达水平下降;B-ALL组与对照组比较,Notch1的表达水平没有明显差异,Jagged1的表达水平比对照组低;AML组与对照组比较,同样Notch1的表达水平没有明显差异,Jagged1的表达水平比对照组低。第二、第三和第四章实时定量RT-PCR检测示各白血病组分别与正常对照组比较,在T-ALL中,Notch1表达增高,Jagged1表达降低;Notch2、Delta1和Hes1两组之间三个基因表达水平没有明显差异;在B-ALL中,Jagged1表达降低,Delta1表达增高,其余三个基因在两组之间的表达水平没有明显差异。在AML中,Jagged1和Hes1表达降低,其余三个基因的表达水平在两组之间没有明显差异。第一章中采用RT-PCR方法检测结果与第二、第三章和第四章采用实时定量RT-PCR检测方法所检测的基因表达,其表达趋势一致。第二部分第一章Delta1对正常供体骨髓CD34~+细胞、脐带血CD34~+细胞、JMML患者的CD34~+白血病细胞、正常人外周血CD3~+细胞和CD20~-CD56+细胞没有明显的促进生长或者抑制增殖作用。第二章Delta1对THP1和SHI-1细胞株有抑制生长作用,对NB4、K562、8266和MO7E细胞株没有明显的促进生长或者抑制作用。第三部分第一章Delta1对THP-1细胞株具有抑制生长作用,而γ—分泌酶抑制剂DAPT不能逆转Delta1对THP-1细胞的生长抑制作用。Delta1对THP-1细胞作用72小时后,可以明显抑制THP-1白血病细胞株的增殖和活力。Delta1诱导THP-1细胞分化,Delta1作用THP1细胞48小时,其NBT还原率为(60±8)%比对照组(19±5)%升高,且与对照组比较差异有显著性。流式细胞仪检测细胞表面抗原的改变:Delta1作用于THP1细胞株72小时后,CD11b和CD14有明显升高,提示细胞向单核系方向分化。Delta1作用72小时后,测定THP1细胞Annexin V的表达,由对照组[Human IgG F(c)]组的(10.2±5.4)%下降为(6.6±3.7)%,提示Delta1对THP1没有凋亡作用。Delta1作用THP1 72小时后,靶基因Hes1的表达上调。第二章Delta1能抑制SHI-1细胞的生长,这一抑制作用能部分被DAPT抑制(逆转)。Delta1对SHI-1细胞作用72小时后,可以明显抑制SHI-1白血病细胞株的增殖和活力。Delta1能促进SHI-1细胞凋亡。Delta1作用SHI-1细胞48小时,其NBT还原率为(17.6±5.2)%与对照组(15.2±4.6)%无明显差异。Delta1对SHI-1作用后,SHI-1细胞表面CD13,CD11b和CD14的表达没有明显改变,加入DAPT后实验组和对照组同样没有改变。SHI-1细胞经过Delta1作用72小时后,Annexin V上升为(39.1±6.7)%,明显增高,提示Delta1对SHI-1细胞有凋亡作用。加入DAPT 72小时后,Annexin V下降至(21.0±2.8)%,说明DAPT有逆转Delta1对SHI-1的凋亡作用。SHI-1经Delta1作用72小时后,靶分子Hes1约上调10倍(134.5±23.7 vs1375.1±612.1),加入DAPT后,Hes1又回落(1126.5±402.2 vs 375.1±121.3)。结论(1)在T-ALL,B-ALL,AML中均存在异常Notch信号通路。(2) T-ALL发病可能与Notch信号途径中Notch1表达增高,Jagged1表达降低有关;B-ALL发病可能与Notch信号途径中配基Delta1表达增高,Jagged1表达降低有关;AML的发病可能与配基Jagged1降低,靶分子Hes1的表达降低有关。(3)急性白血病中的异常Notch信号通路中,不仅与受体Notch1有关,配基Jagged1和Delta1在异常信号通路中也可能起重要作用。(4)配基Delta1对白血病细胞株THP1和SHI-1有增殖抑制作用;对细胞株NB4、MO7E、K562和8266等细胞株没有明显的促进或抑制生长以及促进凋亡和诱导分化等作用。对骨髓CD34~+等细胞没有明显促进生长或促进凋亡等作用。(5)配基Delta1对THP1细胞株有抑制增殖和促进分化作用,该作用不能被γ—分泌酶的抑制剂DAPT抑制(逆转)。(6)配基Delta1对SHI-1细胞株有促进凋亡作用,该作用能部分被γ—分泌酶的抑制剂DAPT抑制(逆转)。综上所述:T-ALL、B-ALL和AML中均存在异常Notch信号通路,各白血病类型中,Notch受体、配基和靶基因的表达水平不同,推测Notch信号通路中各相关分子的不同表达水平与各白血病的发病有关。配基Delta1对急性单核细胞白血病细胞株THP1和SHI-1均有抑制生长作用,但是Delta1对此两株细胞株抑制增殖的作用机理并不相同。更多还原

【Abstract】 [Objects]Notch signaling is one of evolutionarily conserved signaling pathway that mediates cell-cell interactions required for a variety of cell-fate decisions during development,it is involved in a variety of cell differentiation,proliferation and apoptosis that affect the development and functions of many organ.Members of the Notch family play critical roles in the determination of cell fates and during embryogenesis they also play crucial developmental roles.Mammalian Notch1 was discovered because of its involvement in(7; 9) chromosomal translocation seen in human T cell acute lymphoblastic leukemia(T-ALL), and the aberrant Notch signaling was subsequently shown that it may contribute to tumorigenesis.During the past years,The majority study of Notch signaling pathway was highlighted in the notch1 mutation in the T cell acute lymphoblastic leukemogenesis.Two absolutely opposite view points regarding the function of Notch as Oncogene or Tumor suppressor in B-cell acute lymphoblastic leukemogenesis and acute myeloid leukemogenesis has emerged.In present study,we aim to detect the expression of Notch and its related molecules in bone marrow cells of acute leukemia patients to investigate the possible role of Notch signaling in human leukemogenesis.2 Examine the effects of the Notch ligand Delta1 on the growth and suppression of normal cells and primary leukemia cells in vitro.3 Study the mechanism of Notch ligand Delta1’s effects on the leukemia cell lines THP1 and SHI-1 in vitro.We hope to gain deeper insight into the role of Notch pathway in leukemogenesis and provide new opportunity of individual treatment strategy for AL patients in the future.[Methods][Section 1]Chapter 1 The expression of Notch1 and Jagged1 were detected in BM of 118 acute leukemia patients at diagnosis confirmed by MICM and 11 healthy donors by the RT-PCR methods,these patients included 21 T-ALLs,32 B-ALLs AND 65 AMLs, Normalized gene expression level such as notch1 was determined as a ratio between notch1 andβ2—actin.Chapter 2,3 and 4 Real-time quantitative reverse transcription polymerase chain reaction(RQ-RT-PCR) was established for detecting notch signaling molecules including Notch1、Notch2、Jagged1、Delta1、Hes1 and GAPDH expression levels in BM including 15 T-ALLs,16 B-ALLs and 18 AMLs at diagnosis(the blast plus prolymphocyte over eighty percent) and 11 donors by ABI PRISM 7700 PCR.Normalized gene expression level such as notch1 was determined as a ratio between notch1 and GAPDH times 10~6.[Section 2]Chapter1 The recombinant Notch ligand Delta1 protein and Human IgG F(c)(as control) were immobilized in 96-well culture plates.We examined the effects of Delta1 on the growth of cells including BM CD34~+ cells of the healthy donors,the cord blood CD34~+ cells of consented donors,the BM CD34~+ leukemia cells of JMML patient, PB CD3~+ cells of the donors and also PB CD20~-CD56~+ cells of the donors by the MTT methods respectively.Chapter 2 the Delta1 protein and Human IgG F(c)(as control) were immobilized as above,We examined the effects of Delta1 on the growth of leukemia cell lines including NB4,K562,THP1,SHI-1 and MO7E by the MTT methods respectively.[Section 3]In chapter 1,in section 2 chapater2,we have found that the growth of two leukemia cell lines THP1 and SHI-1 were suppressed by the Delta1 protein using the MTT method.Subsequently to inhibit of Notch signaling,aγ-secretase inhibitor DAPT was used to examine whether DAPT could rescue the suppression by the Delta1 on the growth of leukemia cell lines THP1 using MTT method.We also performed the following experiment to examine the proliferation and differentiation of the THP-1 cell indued by Delta1 protein. Cell count analysis,cell morphological analysis,NBT reduction,the expression of CD13,CD14 and CD11b,and binding of AnnexinⅤtested by FCM.In the meantime,the expression of Notch signaling targeting gene Hes1 was detected by real -time RT-PCR method.In chapter 2,We performed the same experiments to check the apoptosis and differentiation of the SHI-1 cell by the Delta1 protein.In addition,we also looked at the effect of DAPT on Delta1 induced growth.[Results][Section 1]In chapter 1 The expression of Notch1 and Jagged1 could be detected in all samples of T-ALL,B-ALL,AML and normal donors by the RT-PCR method.The expression levels of Notch1 in T-ALL was significantly higher than that in control group, while the expression levels of Jagged1 in T-ALL was statistically lower than that in control group.The expression levels of Notch1 was no statistical differences between the B-ALL group and the healthy donor group,while the expression levels of Jagged1 in B-ALL was statistically lower than that in control group.No statistical differences coule be found between the AML group and the donors group,wherease the expression levels of Jagged1 in AML was also significantly lower than that in control group.In chapter 2,3 and 4,the results in ALs compared to that in control group determined by the real-time RT-PCR method,The Mean expression levels of Notch1 in T-ALLs at diagnosis were statistically higher accompanied with a lower level of jagged1 in comparesinon with that in control group.However,the Mean expression levels of Notch2,Delta1 and Hes1 were found no differences between the T-ALL group and the control group.The Mean expression levels of Delta1 in B-ALLs at diagnosis were markedly higher than that in control group;While the Mean expression levels of Jagged1 in B-ALLs at diagnosis significantly decreased than those in control group.Again,the Mean expression levels of Notch1,Notch2 and Hes1 were no statistical differences between the B-ALL group and the control group.The Mean expression levels of Jagged1 and Hes1 in AML at diagnosis were statistically decreased than those in control group.While,the Mean expression levels of Notch1,Notch2 and Delta1 were not different between the AML group and the control group.These result concluded by the RT-PCP method in chapter was in concordance with the result concluded by the real-time RT-PCR method in chapter 2,3 and chapter4.[Section 2]In chapter 1,Determined the MTT methods,The Notch ligand Delta1 in the cell culture could not promote or suppress the growth of the hematopoietic cells including BM CD34~+ cells of the donors,the cord blood CD34~+ cells of the donors,the BM CD34~+ leukemic cells of JMML patient,the PB CD3~+ cells of the donors and the PB CD20~-CD56~+ cells from the donors.In chapter 2,On terms of leukemia cell lines failed to Notch ligand Delta1 promote or inhibit the growth of NB4,K562 and MO7E while remarkably suppressed the growth of two monocytic leukemia cell lines,namely THP1, SHI-1 by the MTT methods respectively.[Section 3]In chapter1,Delta1 strongly suppressed the growth of THP1 cells while theγ-secretase inhibitor DAPT could not converse this effect.Delta1 strongly suppressed the proliferation and viability of THP1 cells after treatment with Delta1 for 72 hours. Delta1 partially induced THP1 cells differentiation evidenced by an increased NBT reduction rate to(60±8)%after exposure to Delta1 for 48 hours while the NBT reduction rate in control was(19±5)%.The expression of CD11b and CD14 on the THP1 cells were markedly upregulated after being exposure to Delta1 for 72 hours determined by FCM,suggesting that the THP1 cells were partially induced to differentiation towards monocytic lineage.The apoptoic rate of the THP1 cells treated with Delta1 72 hours late were decreased from(10.2±5.4)%of Human IgG F(c) control group to(6.6±3.7)%of Delta1 group respectively determined by FCM,suggesting that Delta1 did not cause apoptosis of the THP1 cells.The Notch signaling targeting gene Hes1 in the THP1 cells after treatment with Delta1 for 72 hours was upregulated determined by real-time RT-PCR.In chapter 2,Delta1 strongly inhibit the growth of SHI-1 cells and theγ-secretase inhibitor DAPT could partially reverse this effect.Delta1 strongly suppressed the proliferation and viability of SHI-1 cells after treatment of Delta1 for 72 hours.Besides, Delta1 enhanced the apoptosis of the SHI-1.The NBT reduction rates of the SHI-1cell line treated with Delta1 after 48 hours were(17.6±5.2)%while the NBT reduction rates of control group of SHI-1 cells were(15.2±4.6)%,demonstrating no statistical difference between these two groups.The expression of CD13,CD11b and CD14 on the SHI-1cells treated with Delta1 showed no statistical difference to the control group treated with Human IgG F(c),either in preasence or absence of DAPT.The apoptoic rate of the SHI-1 cells treated with Delta1 after 72 hours was markedly upregulated to(39.1±6.7) %,suggesting that Delta1 could exert the apoptotic function on the SHI-1 cells.The treatment of DAPT for 72 hours,decreased the apoptoic rate of the SHI-1 cells to (21.0±2.8)%,implying that DAPT could partially reverse the apoptoic function of Delta1 on the SHI-1 cells.The Notch signaling targeting gene Hes1 in the SHI-1 cells was upregulated for 10 folds(134.5±23.7 vs 1375.1±612.1) by exposure to Delta1 for 72 hours determined by real-time RT-PCR,while this effect was decreased from (1126.5±402.2) to(375.1±121.3) in presence of DAPT.[Conclusion](1) The aberrant Notch signaling was found in BM cells of T-ALL,B-ALL and AML.(2) Notch1 expression levels in T-ALL were strikingly higher than in healthy donors while Jagged1 in T-ALL is evidently lower than in donors,suggesting that abberant Notch signaling may play a role during the acute T cell lymphoblast leukemogenesis;Delta1 expression levels in B-ALL were markedly higher than in donors while Jagged1 in B-ALL is evidently lower than in donors,suggesting that abberant Notch signaling may also play an important role in development of the acute B cell lymphoblast leukemia;While Jagged1 and Hes1 expression levels in AML were strikingly lower than in donors,implying that abberant Notch signaling may be one of the primary cause during the acute myeloid leukemogenesis.(3) The aberrant Notch signaling in the acute leukemogenesis was not only correlated with abnormal expression levels of Notch1 receptor,but also with the abnormal expression levels of ligand Jagged1 and Delta1.(4) Delta1 could suppress the proliferation of the leukemia cell lines THP1 and SHI-1. but was unable to inhibit the growth of the leukemia cell lines NB4,MO7E,K562 and 8266,or to induce apoptosis.Besides Delta1 showed no effect on the BM CD34~+ cells.(5) Delta1 inhibted the proliferation of THP1 cells and induced the differentiation of THP1 cells along monocytic lineage,theγ-secretase inhibitor DAPT could not reverse this effect on the THP1 cells induced by Delta1.(6) Delta1 enhanced the apoptosis of SHI-1 cells,and the apoptotic function of Delta1 on SHI-1 cells could be partially inhibited by DAPT.In summary:The aberrant Notch signaling could be found in T-ALL,B-ALL and AML.The expression pattern of Notch receptors,ligands and target genes were different in various subtypes of acute leukemia,suggesting that the difference expression pattern of molecules related to Notch signaling may confer to subtypes of the acute leukemia in during leukemogenesis.Delta1 suppressed the growth of both monocytic leukemia cell lines THP1 and SHI-1 by either induce monocytic differentiation in THP1 or promoting apoptosis in SHI-1.更多还原