ATRA耐药相关新基因HA117作用、机制及其临床意义研究
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摘要
第一部分HA117在急性髓系白血病细胞株HL-60、NB4中耐药作用及其机制的体外实验研究
目的：采用重组腺病毒介导的基因高表达技术，评价新基因HA117在两种人急性髓系白血病（AML）细胞株HL-60和NB4中的功能性表达。通过研究HA117对这两种细胞株ATRA及多种化疗药物耐药的影响,研究HA117的功能作用。通过研究HA117对HL-60和NB4细胞周期、增殖、凋亡的影响，并从细胞功能形态学上阐明其可能作用机制；通过检测HA117与作用路径可能相关的关键基因蛋白： PML-RARα、RARα、Bcl-2、Caspase3、MDR1(P-gp)、MRP1及LRP的相互关系，从分子水平上阐明其作用机制。为进一步研究HA117基因在SCID小鼠白血病动物模型体内的耐药作用及机制奠定基础。
方法：(1)采用乒乓交互感染法，以人胚肾细胞株HEK293细胞为包装细胞，收集并浓缩携带绿色荧光蛋白及HA117的重组腺病毒（Ad-GFP-HA117）和携带绿色荧光蛋白的空载体重组腺病毒（Ad-GFP）；(2)采用浓缩病毒转染法，建立HA117基因高表达的实验组细胞，以倒置荧光显微镜观察转染后HL-60和NB4的绿色荧光蛋白（green fluorescence protein，GFP）的表达情况；(3)流式细胞仪技术（flow cytometry, FCM）检测不同病毒滴度（multiplicity ofinfection，MOI）下Ad-GFP-HA117和Ad-GFP对HL-60和NB4细胞的转染效率，胎盘蓝染色法检测不同MOI下对应实验细胞的存活率，筛选出最适MOI；(4) Real time PCR和Western blot技术从基因转录和蛋白表达水平检测外源性HA117在两种细胞株中转染后的表达情况；(5)瑞氏染色观察HA117对ATRA诱导HL-60、NB4细胞向正常粒系成熟细胞分化形态学变化的影响；(6) NBT还原能力实验检测HA117对ATRA诱导HL-60、NB4细胞向正常粒系成熟细胞分化时还原功能的影响；(7) FCM检测HA117对ATRA诱导HL-60、NB4细胞凋亡的影响；(8)药物敏感实验检测HA117转染后的两种白血病细胞对多种临床常用化疗药物的耐受能力的影响；(9) MTT法检测HA117对HL-60和NB4细胞增殖能力的影响；(10)流式细胞仪检测HA117对HL-60和NB4细胞周期的影响；(11) Real time PCR及Western blot法检测HA117对ATRA及其他化疗药物多药耐药作用可能相关m RNA和蛋白：PML-RARα、RARα、Caspase3、Bcl-2、MDR1(P-gp)、MRP1和LRP表达水平的影响。
结果：(1)扩增收集获得足量实验应用的的重组腺病毒液，感染滴度为3.2×109PFU/mL；(2)重组腺病毒Ad-GFP-HA117成功转染入了HL-60和NB4实验细胞内，转染后48h检测不同MOI对应实验细胞的存活率和感染率。当MOI为100时最适腺病毒转染率约为30%-40%，细胞存活率大于80%，细胞转染率高、存活率高、存活状态良好，可继续后期实验；(3) Real time PCR和Western blot法证实Ad-GFP-HA117转染后外源性HA117能在基因和蛋白水平于两种实验白血病细胞株中明显高表达；（4）瑞氏染色法结果显示，HA117阻滞了ATRA诱导HL-60和NB4细胞向正常粒细胞分化的形态学变化；（5）NBT还原实验证实HA117阻滞了ATRA诱导HL-60和NB4细胞向正常粒系白细胞分化后所具有的还原功能；(6) FCM检测细胞凋亡结果显示，HA117阻滞了ATRA诱导HL-60和NB4细胞的凋亡；(7)药物敏感性实验结果证实，转染HA117的两种白血病细胞株较未转染及转染空腺病毒载体组细胞株对临床常用化疗药物的耐受性显著提高，分别增高了2.5~10倍（P<0.05或P<0.01）；(8) MTT法细胞增殖实验结果显示，外源性HA117的转染对HL-60和NB4细胞的增殖能力无影响；(9) FCM检测细胞周期结果显示，外源性HA117的表达对两种白血病细胞株的细胞周期无影响；（10） HA117对PML-RARα、RARα、Caspase3、MDR1(P-gp)、MRP1、LRP基因及蛋白的表达的无明显影响，无统计学意义（P＞0.05）。HA117对Bcl-2有上调作用，具有显著的统计学差异（P<0.01）。
结论：重组腺病毒技术可以有效地将外源性人HA117基因高效稳定的转染入实验细胞HL-60和NB4内。HA117在白血病细胞株HL-60和NB4中具有ATRA耐药性及其他化疗药物耐药的作用。HA117并非通过调控HL-60和NB4细胞增殖、细胞周期、PML-RARα、RARα、Caspase3、MDR1(P-gp)、MRP1、LRP基因及蛋白的表达来产生耐药作用的。HA117作用机制有可能通过调控Bcl-2，启动细胞的抗凋亡途径，进而产生多药耐药性。
第二部分SCID小鼠-人AML模型中HA117耐药作用及其机制的实验研究
目的：建立SCID小鼠-人AML模型，进一步体内实验研究，验证HA117的作用及其作用机制。
方法：(1)建模：在给予SCID小鼠150mg/Kg的环磷酰胺预处理后4天，随机选取小鼠分为三组，实验组A（SCID）:尾静脉注射HL-60细胞；实验组B (SCID+AD):尾静脉注射转染了空载体重组腺病毒的HL-60细胞(HL-60+AD)；实验组C (SCID+HA117):尾静脉注射转染了携带HA117基因的重组腺病毒的HL-60细胞(HL-60+HA117)，建立SCID小鼠-人AML模型。(2)鉴定：小鼠尾静脉血涂片瑞氏染色法检测外周血HL-60细胞的增殖，免疫组织化学检测小鼠骨髓单个核细胞CD33蛋白表达阳性率，组织器官HE染色观察HL-60细胞侵润组织情况共同来鉴定建模是否成功，为后期实验作准备。(3) HA117耐药作用的体内实验研究：用治疗剂量的ATRA和AS2O3分别诱导治疗实验组小鼠，同时与未用药物处理的对照组小鼠比较，观察小鼠一般情况，记录小鼠体重及存活时间，尾静脉血涂片瑞氏染色检测小鼠外周HL-60细胞百分比，小鼠骨髓组织化学染色检测CD33阳性率，分析HA117在白血病小鼠体内是否同样具有ATRA及其他化疗药物的耐药性。(4) HA117耐药作用机制的体内实验研究：将建立的SCID-人AML模型的实验组小鼠饲养10天后，抽取小鼠骨髓，分离单个核细胞，提取总RNA并制作骨髓细胞涂片，Real time PCR法及免疫组织化学法检测HA117、PML-RARα、RARα、Caspase3、Bcl-2、MDR1(P-gp)、MRP1及LRP基因及蛋白的表达变化情况，统计分析HA117在SCID小鼠-人AML模型中对PML-RARα、RARα、Caspase3、Bcl-2、
MDR1(P-gp)、MRP1及LRP的影响。结果：(1)环磷酰胺预处理SCID小鼠后,尾静脉注射HL-60、HL-60/AD和HL-60/HA117细胞，尾静脉血涂片瑞氏染色可见白血病细胞逐渐增殖，肝、脾、肾、及肺HE染色显示有大量HL-60白血病细胞浸润，其骨髓单个核细胞CD33呈强阳性表达，证实SCID小鼠-人AML模型建立成功。(2) ATRA和AS2O3分别诱导治疗实验组小鼠后观察，实验组A和B的白血病SCID小鼠精神反应及一般状况良好，未处理对照组和实验组C较差，差异显著。化疗后10天，与未处理对照组和实验组C的小鼠对比，实验组A和B的白血病SCID小鼠体重较重，生存时间较长，外周血HL-60细胞百分率较少，具有显著的统计学差异（P<0.05）（3）Real time PCR及免疫组织化学法检测小鼠骨髓单个核细胞HA117上调对PML-RARα、RARα、Caspase3、Bcl-2、MDR1(P-gp)、MRP1及LRP基因及蛋白的表达变化影响结果显示，HA117上调继发Bcl-2的上调，具有显著统计学意义，HA117上调前后，RARα、Caspase3、MDR1(P-gp)、MRP1及LRP的表达变
化无统计学差异。结论： HA117在白血病动物模型体内可以产生ATRA及其他化疗药物的耐药性。在白血病动物模型体内,上调HA117可继发Bcl-2的上调，可能为HA117多药耐药的分子机制之一。
第三部分新基因HA117编码蛋白在儿童急性白血病中的表达及其临床意义
目的：通过检测HA117、Bcl-2、P-gp、MRP1及LRP在儿童急性白血病患儿骨髓单个核细胞（bone marrowmononuclear cells，BMMNC）中的表达，探讨新基因HA117编码蛋白在儿童急性白血病耐药中的临床意义及其与Bcl-2、P-gp、MRP1、LRP表达之间的相关性。
方法：收集临床急性白血病（AL）患儿的骨髓标本，分为初诊组、完全缓解组和难治复发组。免疫性血小板减少性紫癜（Immunethrombocytopenic purpura，ITP）患儿骨髓标本作为对照组。免疫组织化学法检测BMMNC中HA117、Bcl-2、P-gp、MRP1及LRP蛋白表达情况。
结果：(1)收集88例AL患儿的骨髓标本，其中有36例HA117编码蛋白呈阳性表达，阳性表达率为40.91%。初诊组阳性表达22例，阳性表达率为51.16%（22/43）；完全缓解组阳性表达7例，阳性表达率18.42%（7/38）；难治复发组7例均呈阳性表达；对照组呈阴性表达。HA117编码蛋白的阳性表达率在完全缓解组、初诊组、难治复发组依次递增，难治复发组显著高于诊组和完全缓解组（P<0.05，P<0.01），初诊组显著高于完全缓解组（χ2=9.409，P<0.01）。（2）初诊组患儿中，HA117蛋白表达阳性22例，17例接受治疗，首次缓解（CR1）8例；HA117阴性表达21例，16例接受治疗，首次缓解CR114例。HA117编码蛋白阳性组CR1率（47.06%）低于阴性组CR1率（87.5%），两者比较其差异有统计学意义（P<0.05）(3) HA117编码蛋白在儿童AML和ALL中的阳性表达率分别为40.98%和40.74%，无统计学差异(P＞0.05)。（4）HA117及其耐药可能相关关键蛋白在儿童急性白血病患儿骨髓单个核细胞（BMMNC）中的表达结果显示，HA117在白血病中表达与Bcl-2成显著正相关（χ~2=37.079，P=0.0001，r=0.649），与MRP1具有一定的正相关性（χ2=6.335，
P=0.012，r=0.268），与P-gp、及LRP无相关性。结论：临床白血病骨髓标本检测结果显示，在儿童急性白血病中HA117编码蛋白的表达与临床分期密切相关，是预后的不利因素，其表达与Bcl-2正相关，可能为HA117在白血病中多药耐药的重要机制之一。
PART ONE EFFECTS AND MECHANISMS OF HA117INTREATING ACUTE MYELOIDLEUKEMIA CELL LINEHL-60AND NB4, AND THE RESEARCH ON IN VIRTOLABORATORY EXPERIMENTS OF MECHANISMS
Objective: To evaluate the expression of HA117in human acutemyeloid leukemia cell line HL-60and NB4by using the recombinantadenovirus-mediated gene high expression and interfere technique.Research the functions of HA117through studying effects of HA117tothe two cell lines ATRA and drug-resistance; Demonstrate the possiblemechanisms in morphology through researching effects of HA117to theproliferation, cell cycle, apoptosis in both cell lines; Clarify the actionmechanisms on a molecular level through detecting the relationshipbetween HA117and the potential key gene proteins MDR1(P-gp)、MRP1、LRP、PML-RARα、RARα、Bcl-2and Caspase-3that may associate withthe acting path. Lay a foundation for furthermore research on drug-resistance mechanisms of HA117gene in SCID mouse leukemiamodel.
Methods:(1) Recombinated adenoviral supernatants with greenfluorescent protein and HA117or not (Ad-GFP-HA117and Ad-GFP)were harvested and concentrated by infection each other like Ping-Pongwith package cell HEK293.(2) Concentrated virus transfection methodwas used to establish the HA117gene high expression group, usingFluorescence microscope to observe the transfected HL-60and NB4which express green fluorescence protein (GFP).(3)Flow cytometry (FCM)and trypan-blue-stain was used to detect the transduction efficiency andsurvival rates of Ad-GFP-HA117and Ad-GFP, screening optimummultiplicity of infection(MOI).(4) The transcription and proteinexpression of ectogenous HA117was detected in the two cell lines byReal time PCR and Western blot.(5) Observing effects on morphologicalchange of HA117to ATRA inducing HL-60、NB4towards normal maturecell by Wright's staining;(6) Detecting effects of restore function ofHA117to ATRA inducing HL-60、NB4towards normal mature cell byNBT test;(7) FCM was used to detect apoptosis of the both cell lines;(8)Drug sensitive test were made to detect the effects of HA117gene-transfected leukemia to the drug-resistance capability of manycommon clinical chemotherapeutics.(9) Mthy1-Thiazoly1-Tetrazolium(MTT) assay was used to assess the cell proliferation of HA117to HL-60 and NB4;(10) FCM was used to detect the cell cycle of HL-60and NB4;(11) The effects of HA117to potential key gene proteins MDR1(P-gp)、MRP1、LRP、PML-RARα、RARα、Bcl-2and Caspase-3expression wasdetected by Real time PCR and Western Blot.
Results:(1) The viral titers of Ad-GFP-HA117was3.5×109plaqueforming units (PFU)/ml.(2) Ad-GFP-HA117was successfully transfectedinto HL-60and NB4, after transduction for48h, detecting the transfectionrate and survival rate in different MOI. The optimal MOI was100,transfection rate was30%-40%, survival rate larger than80%. Thetransfection rate and survival rate was high and status well, could be usedto the continual experiments;(3) Real time PCR and Western Blotdemonstrate that after Ad-GFP-HA117infection, the HA117get a hightranscription and protein expression in both cell lines.(4) Wright'sstaining revealed HA117retardant the cell morphology change ofleukemia cell line inducing normal cell line.(5) NBT test clarify thatHA117prevented restore function of ATRA inducing HL-60and NB4towards normal leukocyte differentiation.(6) The results of FCM revealedthat HA117retardant apoptosis of HA117inducing HL-60and NB4.(7)Drug sensitive test clarify the sensitivity of two leukemia cell line afterinfection improved significantly;(8) MTT assay showed HA117had noeffects on the capability of proliferation;(9) The results of FCM revealedthat the transfection and expression of Ad-GFP-HA117did not influence the cell cycle;(10) HA117had no significantly effects on the gene andprotein expression of MDR1(P-gp), MRP1, LRP, PML-RARα, Caspase-3,and RARα(P>0.05), up-regulation of Bcl-2significantly(P<0.01).
Conclusions: Recombinated adenoviral supernatants could transfectectogenic HA117into HL-60and NB4stablely and effectively. HA117was drug-resistance in HL-60and NB4. The drug-resistance of HA117was not effective by regulating the cell proliferation, apoptosis, cell cycle,expression of MDR1(P-gp), MRP1, LRP, PML-RARα, Caspase-3, andRARα. Starting anti-apoptosis path by regulating Bcl-2, then takedrug-resistance may be the mechanism of HA117.
PART TWO THE IN VIVO STUDY ON EFFECTS ANDMECHANISMS OF HA117IN SCID LEUKEMIA ANIMALMODEL
Objective: To establish SCID mice model suffering from humanAML, promote the in vivo experiments, verify effects and mechanisms ofHA117.
Methods:(1) Modeling:150mg/Kg cyclophosphamidepreconditioned SCID mice for4days, divide mice into three groupsrandomly: Group A (SCID): tail vein injection HL-60; Group B(SCID+AD): tail vein injection HL-60+AD; Group C (SCID+HA117): tailvein injection HL-60+HA117, establish the SCID mice model sufferingfrom human AML.(2) Determination: Detecting the proliferation ofperipheral blood by Wrights' Staining in mice tail vein blood smear,positive expression of CD33in bone marrow mononuclear cells detectedby immunohistochemistry and the infiltrated organization observed by HEstaining were used to identify whether the model is successfullyestablished.(3) Experiments in vivo: Using therapeutic dose ATRA andAS2O3to induce mice in treatment group respectively, compared with themice in control group, observing the normal situation of mice, record miceweight and survival time, detecting cell percentage of peripheral blood byWrights' Staining in mice tail vein blood smear, positive rate of CD33in bone marrow mononuclear cells detected by immunohistochemistry,analysis whether there was drug-resistance in AML mice;(4) Feeding theSCID mice suffering from human AML for ten days, extracting mice bonemarrow, separating mononuclear cells, extracting total RNA and makingbone marrow smear, protein expression change of HA117, MDR1(P-gp),MRP1, LRP, PML-RARα, RARα, Bcl-2and Caspase-3was detected byimmunohistochemistry and Real time PCR, analyse the effects of HA117to MDR1(P-gp), MRP1, LRP, PML-RARα, RARα, Bcl-2and Caspase-3by statistical analysis in SCID mice model suffering human AML.
Results:(1) After cyclophosphamide preconditioned SCID mice, tailvain injected HL-60, HL-60/Ad, HL-60/HA117, leukemia cellproliferating increasingly by wrights' Staining, extracted bone marrowmononuclear cells CD33was significantly positive, liver, spleen, kidney,bone marrow and metastatic tumors revealed a heavy AML inflammatoryinfiltrate by HE Staining. It was demonstrated that SCID mice modelsuffering from human AML was established successfully.(2) Observingmice induced by ATRA and AS2O3in treatment group, SCID mice inGroup A and B gave a good response, but the control group and Group Cbad significantly. After chemotherapy for ten days, compared with controlgroup and Group C, SCID mice in Group A and B had a higher weight,more survival time, but HL-60cell percentage in peripheral blooddecreasing significantly (P <0.05). Results of immunohistochemistry and Real time PCR revealed that Bcl-2up-regulate significantly by HA117up-regulation, MDR1(P-gp), MRP1, LRP, PML-RARα, RARαand
Caspase-3had no marked difference.Conclusions: HA117could be ATRA and other drugs resistance inSCID mice model.The Bcl-2up-regulation followed HA117up-regulatingmay be one of molecular mechanism of HA117drug-resistance.
PART THREE CORRELATIVE STUDY ON THEEXPRESSION OF HA117、Bcl-2、P-gp、MRP1AND LRPIN MASS OF AML CLINICAL SAMPLES
Objective: To explore the clinical significance of HA117encodingprotein in child AML and relationship among HA117and Bcl-2、P-gp、MRP1、LRP by detecting expression of HA117、 Bcl-2、P-gp、MRP1and LRP in bone marrow mononuclear cells.
Methods: Clinical AL patiens was collected and divided into threegroups: newly diagnosed group, complete remission group, and refractoryor relapsed group, immune thrombocytopenic purpura as control group.The expression of HA117、Bcl-2、P-gp、MRP1and LRP in BMMNCwas detected by immunohistochemistry.
Results:(1) Eighty-eight AL child patiens was collected, includingpositive expression of HA117encoding protein samples thirty-six,positive rate40.91%. Number of positive expression samples in newlydiagnosed group was22, positive rate51.16%(22/43); Completeremission group7, positive rate18.42%(7/38); all samples in refractory orrelapsed group expressed positively, but negatively in control group.Positive rate of HA117encoding protein in complete remission group,newly diagnosed group and refractory or relapsed group increasedsequentially, and that was higher significantly refractory or relapsed group compared with complete remission group(P <0.05, P<0.01), newlydiagnosed group higher than complete remission group (χ~2=9.409， P <0.01).(2) In newly diagnosed group, number of positive expression ofHA117encoding protein was22,17under-treatment included, CR18;negative expression samples number was21, under-treatment16, CR114;CR1rate in positive group was lower significantly than that in negativelygroup (P <0.05).(3) Positive rate of HA117encoding protein in childAML and ALL was40.98%and40.74%, which had no marked difference(P>0.05).(4) immunohistochemistry results revealed expression ofHA117in AML was positively related to Bcl-2significantly (χ2=37.079，P=0.0001， r=0.649), and MRP1positively (χ~2=6.335， P=0.012，r=0.268), but had no relationship with P-gp and LRP.
Conclusions: Clinical cases detection results revealed thatoverexpression of HA117and its protein-encoding was closely related toclinical drug-resistance, and it was a unfavorable factor for child ALprognosis. The mechanisms of drug-resistance and Bcl-2may havedependence. That could be one of important mechanisms of HA117drug-resistance, but not unique.
目的：采用重组腺病毒介导的基因高表达技术，评价新基因HA117在两种人急性髓系白血病（AML）细胞株HL-60和NB4中的功能性表达。通过研究HA117对这两种细胞株ATRA及多种化疗药物耐药的影响,研究HA117的功能作用。通过研究HA117对HL-60和NB4细胞周期、增殖、凋亡的影响，并从细胞功能形态学上阐明其可能作用机制；通过检测HA117与作用路径可能相关的关键基因蛋白： PML-RARα、RARα、Bcl-2、Caspase3、MDR1(P-gp)、MRP1及LRP的相互关系，从分子水平上阐明其作用机制。为进一步研究HA117基因在SCID小鼠白血病动物模型体内的耐药作用及机制奠定基础。
方法：(1)采用乒乓交互感染法，以人胚肾细胞株HEK293细胞为包装细胞，收集并浓缩携带绿色荧光蛋白及HA117的重组腺病毒（Ad-GFP-HA117）和携带绿色荧光蛋白的空载体重组腺病毒（Ad-GFP）；(2)采用浓缩病毒转染法，建立HA117基因高表达的实验组细胞，以倒置荧光显微镜观察转染后HL-60和NB4的绿色荧光蛋白（green fluorescence protein，GFP）的表达情况；(3)流式细胞仪技术（flow cytometry, FCM）检测不同病毒滴度（multiplicity ofinfection，MOI）下Ad-GFP-HA117和Ad-GFP对HL-60和NB4细胞的转染效率，胎盘蓝染色法检测不同MOI下对应实验细胞的存活率，筛选出最适MOI；(4) Real time PCR和Western blot技术从基因转录和蛋白表达水平检测外源性HA117在两种细胞株中转染后的表达情况；(5)瑞氏染色观察HA117对ATRA诱导HL-60、NB4细胞向正常粒系成熟细胞分化形态学变化的影响；(6) NBT还原能力实验检测HA117对ATRA诱导HL-60、NB4细胞向正常粒系成熟细胞分化时还原功能的影响；(7) FCM检测HA117对ATRA诱导HL-60、NB4细胞凋亡的影响；(8)药物敏感实验检测HA117转染后的两种白血病细胞对多种临床常用化疗药物的耐受能力的影响；(9) MTT法检测HA117对HL-60和NB4细胞增殖能力的影响；(10)流式细胞仪检测HA117对HL-60和NB4细胞周期的影响；(11) Real time PCR及Western blot法检测HA117对ATRA及其他化疗药物多药耐药作用可能相关m RNA和蛋白：PML-RARα、RARα、Caspase3、Bcl-2、MDR1(P-gp)、MRP1和LRP表达水平的影响。
结果：(1)扩增收集获得足量实验应用的的重组腺病毒液，感染滴度为3.2×109PFU/mL；(2)重组腺病毒Ad-GFP-HA117成功转染入了HL-60和NB4实验细胞内，转染后48h检测不同MOI对应实验细胞的存活率和感染率。当MOI为100时最适腺病毒转染率约为30%-40%，细胞存活率大于80%，细胞转染率高、存活率高、存活状态良好，可继续后期实验；(3) Real time PCR和Western blot法证实Ad-GFP-HA117转染后外源性HA117能在基因和蛋白水平于两种实验白血病细胞株中明显高表达；（4）瑞氏染色法结果显示，HA117阻滞了ATRA诱导HL-60和NB4细胞向正常粒细胞分化的形态学变化；（5）NBT还原实验证实HA117阻滞了ATRA诱导HL-60和NB4细胞向正常粒系白细胞分化后所具有的还原功能；(6) FCM检测细胞凋亡结果显示，HA117阻滞了ATRA诱导HL-60和NB4细胞的凋亡；(7)药物敏感性实验结果证实，转染HA117的两种白血病细胞株较未转染及转染空腺病毒载体组细胞株对临床常用化疗药物的耐受性显著提高，分别增高了2.5~10倍（P<0.05或P<0.01）；(8) MTT法细胞增殖实验结果显示，外源性HA117的转染对HL-60和NB4细胞的增殖能力无影响；(9) FCM检测细胞周期结果显示，外源性HA117的表达对两种白血病细胞株的细胞周期无影响；（10） HA117对PML-RARα、RARα、Caspase3、MDR1(P-gp)、MRP1、LRP基因及蛋白的表达的无明显影响，无统计学意义（P＞0.05）。HA117对Bcl-2有上调作用，具有显著的统计学差异（P<0.01）。
结论：重组腺病毒技术可以有效地将外源性人HA117基因高效稳定的转染入实验细胞HL-60和NB4内。HA117在白血病细胞株HL-60和NB4中具有ATRA耐药性及其他化疗药物耐药的作用。HA117并非通过调控HL-60和NB4细胞增殖、细胞周期、PML-RARα、RARα、Caspase3、MDR1(P-gp)、MRP1、LRP基因及蛋白的表达来产生耐药作用的。HA117作用机制有可能通过调控Bcl-2，启动细胞的抗凋亡途径，进而产生多药耐药性。
第二部分SCID小鼠-人AML模型中HA117耐药作用及其机制的实验研究
目的：建立SCID小鼠-人AML模型，进一步体内实验研究，验证HA117的作用及其作用机制。
方法：(1)建模：在给予SCID小鼠150mg/Kg的环磷酰胺预处理后4天，随机选取小鼠分为三组，实验组A（SCID）:尾静脉注射HL-60细胞；实验组B (SCID+AD):尾静脉注射转染了空载体重组腺病毒的HL-60细胞(HL-60+AD)；实验组C (SCID+HA117):尾静脉注射转染了携带HA117基因的重组腺病毒的HL-60细胞(HL-60+HA117)，建立SCID小鼠-人AML模型。(2)鉴定：小鼠尾静脉血涂片瑞氏染色法检测外周血HL-60细胞的增殖，免疫组织化学检测小鼠骨髓单个核细胞CD33蛋白表达阳性率，组织器官HE染色观察HL-60细胞侵润组织情况共同来鉴定建模是否成功，为后期实验作准备。(3) HA117耐药作用的体内实验研究：用治疗剂量的ATRA和AS2O3分别诱导治疗实验组小鼠，同时与未用药物处理的对照组小鼠比较，观察小鼠一般情况，记录小鼠体重及存活时间，尾静脉血涂片瑞氏染色检测小鼠外周HL-60细胞百分比，小鼠骨髓组织化学染色检测CD33阳性率，分析HA117在白血病小鼠体内是否同样具有ATRA及其他化疗药物的耐药性。(4) HA117耐药作用机制的体内实验研究：将建立的SCID-人AML模型的实验组小鼠饲养10天后，抽取小鼠骨髓，分离单个核细胞，提取总RNA并制作骨髓细胞涂片，Real time PCR法及免疫组织化学法检测HA117、PML-RARα、RARα、Caspase3、Bcl-2、MDR1(P-gp)、MRP1及LRP基因及蛋白的表达变化情况，统计分析HA117在SCID小鼠-人AML模型中对PML-RARα、RARα、Caspase3、Bcl-2、
MDR1(P-gp)、MRP1及LRP的影响。结果：(1)环磷酰胺预处理SCID小鼠后,尾静脉注射HL-60、HL-60/AD和HL-60/HA117细胞，尾静脉血涂片瑞氏染色可见白血病细胞逐渐增殖，肝、脾、肾、及肺HE染色显示有大量HL-60白血病细胞浸润，其骨髓单个核细胞CD33呈强阳性表达，证实SCID小鼠-人AML模型建立成功。(2) ATRA和AS2O3分别诱导治疗实验组小鼠后观察，实验组A和B的白血病SCID小鼠精神反应及一般状况良好，未处理对照组和实验组C较差，差异显著。化疗后10天，与未处理对照组和实验组C的小鼠对比，实验组A和B的白血病SCID小鼠体重较重，生存时间较长，外周血HL-60细胞百分率较少，具有显著的统计学差异（P<0.05）（3）Real time PCR及免疫组织化学法检测小鼠骨髓单个核细胞HA117上调对PML-RARα、RARα、Caspase3、Bcl-2、MDR1(P-gp)、MRP1及LRP基因及蛋白的表达变化影响结果显示，HA117上调继发Bcl-2的上调，具有显著统计学意义，HA117上调前后，RARα、Caspase3、MDR1(P-gp)、MRP1及LRP的表达变
化无统计学差异。结论： HA117在白血病动物模型体内可以产生ATRA及其他化疗药物的耐药性。在白血病动物模型体内,上调HA117可继发Bcl-2的上调，可能为HA117多药耐药的分子机制之一。
第三部分新基因HA117编码蛋白在儿童急性白血病中的表达及其临床意义
目的：通过检测HA117、Bcl-2、P-gp、MRP1及LRP在儿童急性白血病患儿骨髓单个核细胞（bone marrowmononuclear cells，BMMNC）中的表达，探讨新基因HA117编码蛋白在儿童急性白血病耐药中的临床意义及其与Bcl-2、P-gp、MRP1、LRP表达之间的相关性。
方法：收集临床急性白血病（AL）患儿的骨髓标本，分为初诊组、完全缓解组和难治复发组。免疫性血小板减少性紫癜（Immunethrombocytopenic purpura，ITP）患儿骨髓标本作为对照组。免疫组织化学法检测BMMNC中HA117、Bcl-2、P-gp、MRP1及LRP蛋白表达情况。
结果：(1)收集88例AL患儿的骨髓标本，其中有36例HA117编码蛋白呈阳性表达，阳性表达率为40.91%。初诊组阳性表达22例，阳性表达率为51.16%（22/43）；完全缓解组阳性表达7例，阳性表达率18.42%（7/38）；难治复发组7例均呈阳性表达；对照组呈阴性表达。HA117编码蛋白的阳性表达率在完全缓解组、初诊组、难治复发组依次递增，难治复发组显著高于诊组和完全缓解组（P<0.05，P<0.01），初诊组显著高于完全缓解组（χ2=9.409，P<0.01）。（2）初诊组患儿中，HA117蛋白表达阳性22例，17例接受治疗，首次缓解（CR1）8例；HA117阴性表达21例，16例接受治疗，首次缓解CR114例。HA117编码蛋白阳性组CR1率（47.06%）低于阴性组CR1率（87.5%），两者比较其差异有统计学意义（P<0.05）(3) HA117编码蛋白在儿童AML和ALL中的阳性表达率分别为40.98%和40.74%，无统计学差异(P＞0.05)。（4）HA117及其耐药可能相关关键蛋白在儿童急性白血病患儿骨髓单个核细胞（BMMNC）中的表达结果显示，HA117在白血病中表达与Bcl-2成显著正相关（χ~2=37.079，P=0.0001，r=0.649），与MRP1具有一定的正相关性（χ2=6.335，
P=0.012，r=0.268），与P-gp、及LRP无相关性。结论：临床白血病骨髓标本检测结果显示，在儿童急性白血病中HA117编码蛋白的表达与临床分期密切相关，是预后的不利因素，其表达与Bcl-2正相关，可能为HA117在白血病中多药耐药的重要机制之一。
PART ONE EFFECTS AND MECHANISMS OF HA117INTREATING ACUTE MYELOIDLEUKEMIA CELL LINEHL-60AND NB4, AND THE RESEARCH ON IN VIRTOLABORATORY EXPERIMENTS OF MECHANISMS
Objective: To evaluate the expression of HA117in human acutemyeloid leukemia cell line HL-60and NB4by using the recombinantadenovirus-mediated gene high expression and interfere technique.Research the functions of HA117through studying effects of HA117tothe two cell lines ATRA and drug-resistance; Demonstrate the possiblemechanisms in morphology through researching effects of HA117to theproliferation, cell cycle, apoptosis in both cell lines; Clarify the actionmechanisms on a molecular level through detecting the relationshipbetween HA117and the potential key gene proteins MDR1(P-gp)、MRP1、LRP、PML-RARα、RARα、Bcl-2and Caspase-3that may associate withthe acting path. Lay a foundation for furthermore research on drug-resistance mechanisms of HA117gene in SCID mouse leukemiamodel.
Methods:(1) Recombinated adenoviral supernatants with greenfluorescent protein and HA117or not (Ad-GFP-HA117and Ad-GFP)were harvested and concentrated by infection each other like Ping-Pongwith package cell HEK293.(2) Concentrated virus transfection methodwas used to establish the HA117gene high expression group, usingFluorescence microscope to observe the transfected HL-60and NB4which express green fluorescence protein (GFP).(3)Flow cytometry (FCM)and trypan-blue-stain was used to detect the transduction efficiency andsurvival rates of Ad-GFP-HA117and Ad-GFP, screening optimummultiplicity of infection(MOI).(4) The transcription and proteinexpression of ectogenous HA117was detected in the two cell lines byReal time PCR and Western blot.(5) Observing effects on morphologicalchange of HA117to ATRA inducing HL-60、NB4towards normal maturecell by Wright's staining;(6) Detecting effects of restore function ofHA117to ATRA inducing HL-60、NB4towards normal mature cell byNBT test;(7) FCM was used to detect apoptosis of the both cell lines;(8)Drug sensitive test were made to detect the effects of HA117gene-transfected leukemia to the drug-resistance capability of manycommon clinical chemotherapeutics.(9) Mthy1-Thiazoly1-Tetrazolium(MTT) assay was used to assess the cell proliferation of HA117to HL-60 and NB4;(10) FCM was used to detect the cell cycle of HL-60and NB4;(11) The effects of HA117to potential key gene proteins MDR1(P-gp)、MRP1、LRP、PML-RARα、RARα、Bcl-2and Caspase-3expression wasdetected by Real time PCR and Western Blot.
Results:(1) The viral titers of Ad-GFP-HA117was3.5×109plaqueforming units (PFU)/ml.(2) Ad-GFP-HA117was successfully transfectedinto HL-60and NB4, after transduction for48h, detecting the transfectionrate and survival rate in different MOI. The optimal MOI was100,transfection rate was30%-40%, survival rate larger than80%. Thetransfection rate and survival rate was high and status well, could be usedto the continual experiments;(3) Real time PCR and Western Blotdemonstrate that after Ad-GFP-HA117infection, the HA117get a hightranscription and protein expression in both cell lines.(4) Wright'sstaining revealed HA117retardant the cell morphology change ofleukemia cell line inducing normal cell line.(5) NBT test clarify thatHA117prevented restore function of ATRA inducing HL-60and NB4towards normal leukocyte differentiation.(6) The results of FCM revealedthat HA117retardant apoptosis of HA117inducing HL-60and NB4.(7)Drug sensitive test clarify the sensitivity of two leukemia cell line afterinfection improved significantly;(8) MTT assay showed HA117had noeffects on the capability of proliferation;(9) The results of FCM revealedthat the transfection and expression of Ad-GFP-HA117did not influence the cell cycle;(10) HA117had no significantly effects on the gene andprotein expression of MDR1(P-gp), MRP1, LRP, PML-RARα, Caspase-3,and RARα(P>0.05), up-regulation of Bcl-2significantly(P<0.01).
Conclusions: Recombinated adenoviral supernatants could transfectectogenic HA117into HL-60and NB4stablely and effectively. HA117was drug-resistance in HL-60and NB4. The drug-resistance of HA117was not effective by regulating the cell proliferation, apoptosis, cell cycle,expression of MDR1(P-gp), MRP1, LRP, PML-RARα, Caspase-3, andRARα. Starting anti-apoptosis path by regulating Bcl-2, then takedrug-resistance may be the mechanism of HA117.
PART TWO THE IN VIVO STUDY ON EFFECTS ANDMECHANISMS OF HA117IN SCID LEUKEMIA ANIMALMODEL
Objective: To establish SCID mice model suffering from humanAML, promote the in vivo experiments, verify effects and mechanisms ofHA117.
Methods:(1) Modeling:150mg/Kg cyclophosphamidepreconditioned SCID mice for4days, divide mice into three groupsrandomly: Group A (SCID): tail vein injection HL-60; Group B(SCID+AD): tail vein injection HL-60+AD; Group C (SCID+HA117): tailvein injection HL-60+HA117, establish the SCID mice model sufferingfrom human AML.(2) Determination: Detecting the proliferation ofperipheral blood by Wrights' Staining in mice tail vein blood smear,positive expression of CD33in bone marrow mononuclear cells detectedby immunohistochemistry and the infiltrated organization observed by HEstaining were used to identify whether the model is successfullyestablished.(3) Experiments in vivo: Using therapeutic dose ATRA andAS2O3to induce mice in treatment group respectively, compared with themice in control group, observing the normal situation of mice, record miceweight and survival time, detecting cell percentage of peripheral blood byWrights' Staining in mice tail vein blood smear, positive rate of CD33in bone marrow mononuclear cells detected by immunohistochemistry,analysis whether there was drug-resistance in AML mice;(4) Feeding theSCID mice suffering from human AML for ten days, extracting mice bonemarrow, separating mononuclear cells, extracting total RNA and makingbone marrow smear, protein expression change of HA117, MDR1(P-gp),MRP1, LRP, PML-RARα, RARα, Bcl-2and Caspase-3was detected byimmunohistochemistry and Real time PCR, analyse the effects of HA117to MDR1(P-gp), MRP1, LRP, PML-RARα, RARα, Bcl-2and Caspase-3by statistical analysis in SCID mice model suffering human AML.
Results:(1) After cyclophosphamide preconditioned SCID mice, tailvain injected HL-60, HL-60/Ad, HL-60/HA117, leukemia cellproliferating increasingly by wrights' Staining, extracted bone marrowmononuclear cells CD33was significantly positive, liver, spleen, kidney,bone marrow and metastatic tumors revealed a heavy AML inflammatoryinfiltrate by HE Staining. It was demonstrated that SCID mice modelsuffering from human AML was established successfully.(2) Observingmice induced by ATRA and AS2O3in treatment group, SCID mice inGroup A and B gave a good response, but the control group and Group Cbad significantly. After chemotherapy for ten days, compared with controlgroup and Group C, SCID mice in Group A and B had a higher weight,more survival time, but HL-60cell percentage in peripheral blooddecreasing significantly (P <0.05). Results of immunohistochemistry and Real time PCR revealed that Bcl-2up-regulate significantly by HA117up-regulation, MDR1(P-gp), MRP1, LRP, PML-RARα, RARαand
Caspase-3had no marked difference.Conclusions: HA117could be ATRA and other drugs resistance inSCID mice model.The Bcl-2up-regulation followed HA117up-regulatingmay be one of molecular mechanism of HA117drug-resistance.
PART THREE CORRELATIVE STUDY ON THEEXPRESSION OF HA117、Bcl-2、P-gp、MRP1AND LRPIN MASS OF AML CLINICAL SAMPLES
Objective: To explore the clinical significance of HA117encodingprotein in child AML and relationship among HA117and Bcl-2、P-gp、MRP1、LRP by detecting expression of HA117、 Bcl-2、P-gp、MRP1and LRP in bone marrow mononuclear cells.
Methods: Clinical AL patiens was collected and divided into threegroups: newly diagnosed group, complete remission group, and refractoryor relapsed group, immune thrombocytopenic purpura as control group.The expression of HA117、Bcl-2、P-gp、MRP1and LRP in BMMNCwas detected by immunohistochemistry.
Results:(1) Eighty-eight AL child patiens was collected, includingpositive expression of HA117encoding protein samples thirty-six,positive rate40.91%. Number of positive expression samples in newlydiagnosed group was22, positive rate51.16%(22/43); Completeremission group7, positive rate18.42%(7/38); all samples in refractory orrelapsed group expressed positively, but negatively in control group.Positive rate of HA117encoding protein in complete remission group,newly diagnosed group and refractory or relapsed group increasedsequentially, and that was higher significantly refractory or relapsed group compared with complete remission group(P <0.05, P<0.01), newlydiagnosed group higher than complete remission group (χ~2=9.409， P <0.01).(2) In newly diagnosed group, number of positive expression ofHA117encoding protein was22,17under-treatment included, CR18;negative expression samples number was21, under-treatment16, CR114;CR1rate in positive group was lower significantly than that in negativelygroup (P <0.05).(3) Positive rate of HA117encoding protein in childAML and ALL was40.98%and40.74%, which had no marked difference(P>0.05).(4) immunohistochemistry results revealed expression ofHA117in AML was positively related to Bcl-2significantly (χ2=37.079，P=0.0001， r=0.649), and MRP1positively (χ~2=6.335， P=0.012，r=0.268), but had no relationship with P-gp and LRP.
Conclusions: Clinical cases detection results revealed thatoverexpression of HA117and its protein-encoding was closely related toclinical drug-resistance, and it was a unfavorable factor for child ALprognosis. The mechanisms of drug-resistance and Bcl-2may havedependence. That could be one of important mechanisms of HA117drug-resistance, but not unique.
引文
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