【作者】 王存邦；

【导师】 陈幸华；

【作者基本信息】 第三军医大学 ， 内科学， 2007， 博士

【摘要】 异体造血干细胞移植(allo-HSCT)因具有移植物抗白血病(GVL)效应,移植后复发率低;但移植物抗宿主病fGVHD)及感染等并发症发生率高,致使移植相关死亡率高,且由于存在HLA配型相合问题,大部分患者无行异体造血干细胞移植治疗的可能,致使临床应用受限。自体造血干细胞移植(auto-HSCT)丌展已近40年。它虽不受配型限制,并发症较轻,但因缺乏GVL效应而复发率较高。复发是自体造血干细胞移植失败的重要原因,减少复发成为提高疗效的关键。我们以往的研究证明,以自体为主,混合HLA半相合异体造血干细胞的干细胞移植(混合造血干细胞移植,mixed-HSCT)可在一定程度上兼有两者的优点,克服其不足,诱导形成混合嵌合体,减轻GVHD,同时保留GVL效应。鉴于此,本课题从动物实验入手,应用含有标记基因的K562细胞和BALB/C小鼠制备白血病小鼠模型;应用基因标记技术进行动物实验,探讨以自体为主,混合H-2半相合异体造血干细胞的干细胞移植(即混合造血干细胞移植)及移植后应用供体淋巴细胞输注联合IL-2治疗急性髓性白血病的疗效和嵌合体形成情况,为临床应用提供依据;并应用于临床实践,观察该方法对成人急性髓性白血病的治疗效果。1.研究内容及方法:(1)K562细胞株的基因转移:培养PA317-GCGPXSN和NIH3T3及K562细胞株;应用PA317。GCGPXSN细胞株制备病毒上清,NIH3T3细胞株测定病毒滴度;应用rhIL-3、rhIL-6及rhSCF刺激K562细胞株后,实施基因转移;应用流式细胞仪测定基因转移效率及PCR方法测定NeoR基因,将完成基因转移的K562细胞株命名为K562+细胞株。(2)BALB/C小鼠模型制备:SPF级BALB/C小鼠按实验分组后分别经直线加速器照射2GY和3GY,24小时后取对数生长期的K562+细胞按实验分组以尾静脉一次性注射K562+细胞。观察小鼠的一般情况及生存时间、骨髓细胞分类及外周血白细胞计数、分类、细胞亚群及相关脏器病理检查、流式细胞仪测定K562+细胞、PCR方法测定组织浸润情况。(3)常规方法培养BALB/CxC57BL F1代小鼠骨髓细胞,并应用流式细胞仪测定T亚群及NK细胞,CD34+细胞。在经过PHA,rhIL-2,rhIL.3、rhIL-6及rhSCF刺激后实施基因转移,应用流式细胞仪测定基因转移效率,PCR方法测定NeoR基因。将完成基因转移的F1代细胞命名为F1+细胞。(4)动物实验:用上述方法制备白血病小鼠模型,按实验分组。7天后做6Gy照射,照射后24小时做自体骨髓移植或混合骨髓移植,混合移植间隔1小时。混合移植组每7天输F1代细胞一次,应用IL-2者隔日腹腔注射一次。观察4周后杀死存活小鼠,做外周血及骨髓细胞形态检查,细胞亚群测定,GFP+细胞测定,NeoR基因测定,肝脏、脾脏病理学检查及组织匀浆细胞的GFP+细胞和NeoR基因的测定。(5)13例急性髓性白血病患者在完全缓解期实施了Mixed-HSCT,于造血恢复后,给予DLI联合IL-2治疗2-7次:另有11例急性髓性白血病患者在同样条件下实施Mixed-HSCT,移植后未予特殊治疗。2.实验结果:(1)K562细胞基因转移效率K562-细胞应用流式细胞仪测定的荧光强度数值为0.56%,K562+细胞荧光强度为77.16%。两者差异显著(P＜0.01)。PCR方法扩增到了NeoR基因的特异性片断,说明含GFP和NeoR基因的逆转录病毒载体质粒已转移并整合进入K562细胞的基因组中。(2)白血病模型制备情况①照射对小鼠免疫功能的影响E组小鼠经2Gy照射后外周血CD3、CD4、CD8、CD16+CD56+细胞呈进行性降低,至第3周开始出现逐渐恢复迹象,4周后恢复正常。F组在3GY照射后CD3、CD4、CD8、CD16+CD56+细胞呈进行性降低,至第4周开始出现逐渐恢复迹象,5周后恢复正常。在同一时间,3GY照射较2GY照射所导致的细胞亚群降低的程度更为明显(P＜0.05)。脾、胸腺在照射后外观较正常小鼠缩小,重量减轻并延续至4周才基本恢复至照射前的水平,肝脏重量无明显变化。②发病情况照射2、3GY后未接种K562+细胞的BALB/C小鼠(E、F组)观察4周未出现自然死亡。照射2、3GY后分别接种K562+细胞2x10~6、5x10~6的BALB/C小鼠(A、B、C、D组)在接种5-7天时发病。A组于30天内全部死亡;C组于24天内全部死亡;B组于23天内全部死亡;D组于17天内全部死亡;各组小鼠生存天数有显著性差异,均显著短于正常对照组(P＜0.01)。体重均较正常对照组显著下降(P＜0.01)。小鼠的白血病发病率为100%,无自发缓解。随着接种白血病细胞数量的增加和照射剂量的增加,小鼠的存活时间逐渐缩短,且外周血及骨髓中人原、早幼粒白血病细胞所占比例逐渐增加。病理学检查证实有肿瘤细胞浸润。流式细胞仪测定结果与此相一致,PCR方法也证实了NeoR基因的存在。(3)F1代细胞基因转移效率F1(-)细胞应用流式细胞仪测定的荧光强度数值为0.63%,F1(+)细胞荧光强度为76.04%。两者差异显著(P＜0.001)。PCR方法扩增到了NeoR基因的特异性片断,说明含GFP和NeoR基因的逆转录病毒载体质粒已转移并整合进入F1(+)细胞的基因组中。(4)小鼠骨髓移植实验①骨髓移植细胞数目测定实验组小鼠移植自体骨髓细胞中,CD34+细胞为1.32%,CD3+细胞26.56%,CD4+细胞13.44%,CD8+细胞3.09%,CD16+CD56+细胞7.53%。移植异体骨髓细胞(F1+)中,CD34+细胞为1.51%,CD3+细胞61.41%,CD4+细胞39.57%,CD8+细胞27.61%,CD16+CD56+细胞9.59%;移植异体骨髓细胞(F1-)中,CD34+细胞为1.49%,CD3+细胞65.86%,CD4+细胞40.13%,CD8+细胞19.16%,CD16+CD56+细胞9.70%;其中F1(+)与F1(-)细胞各亚群之间没有明显差别(P＞0.05),即每只实验鼠所移植的各类细胞数目没有明显差别。②各实验组疗效观察白血病小鼠模型组(A组)在20天内全部死亡,白血病小鼠模型经6GY照射后(F组)在14天内全部死亡,在濒死小鼠骨髓及外周血中均检测到GFP及NeoR基因。A组濒死小鼠外周血及骨髓见11%-64%不等的原、幼稚细胞。F组濒死小鼠外周血及骨髓细胞稀少,主要为淋巴细胞。A组和F组濒死小鼠骨髓及外周血中均检测到GFP及Neo基因的存在。两组濒死小鼠外周血T细胞亚群及NK细咆均极低。其它实验组小鼠均有6-3只不等的小鼠存活时间＞28天,存活的小鼠外周血及骨髓细胞数量及形态同正常小鼠,外周血T细胞亚群及NK细胞已恢复至正常范围,各组之间无明显差别(P＞0.05)。在存活小鼠中输注F1(+)细胞者检测到了GFP及Neo基因。存活小鼠肝、脾检查未发现肿瘤细胞在肝、脾组织中的浸润及充血等现象。俎织匀浆细胞的GFP和NeoR基因的测定中未检测到GFP+细胞,亦未扩增到特异性的Neo基因片段的存在。证明K562+的肿瘤细胞在存活小鼠的肝、脾中消失。(5)临床疗效观察混合造血干细胞移植患者均获得造血重建。无GVHD发生。观察组中有3例M2a患者在接受1、2、2次DLI+IL-2治疗后+3、+4、+7月复发死亡。1例M2a患者接受2次DLI+IL-2治疗后,于+1年时出现MDS,死于脑出血。1例M5患者应用DLI+IL-2治疗1次后出现不明原因高热、抽搐,死于癫痫持续状态。其余8例患者随访1-5年均无病存活。其中1例M4、1例M5患者有嵌合体形成。长期生存率为61.5%(8/13例)。对照组有2例M2a,2例M4,1例M5于移植后+1-+7月复发死亡,1例M3患者于+25天死于脑出血。其余2例M3,2例M4,1例M5随访3-5年均无病存活。其中后3例均有嵌合体形成。长期生存率为45.4%(5/11例)。3.结论:(1)含有GFP基因和NeoR基因的逆转录病毒成功地转移进入K562细胞株和BALB/CxC57BL F1代小鼠骨髓细胞中。(2)BALB/C小鼠经照射后从尾静脉注射K562细胞株可以制备获得白血病小鼠模型。(3)白血病小鼠在经过混合造血干细胞移植后给予DIL联合IL-2治疗,可以提高长期生存率。(4)临床应用证实,应用混合造血干细胞移植治疗,在移植后给予DIL联合IL-2过继免疫治疗,可以提高急性髓性白血病患者的长期生存率。更多还原

【Abstract】 Owing to graft-verus-leukemia(GVL) effects,allogeneic hematopoietic stem cell transplantation(allo-HSCT) has an advantage of low relapse rate,but a high incidence of transplant-related mortality.Due to the complications such as graft-versus-host disease(GVHD),infection and so on;On the other hand,most patients have no chance to receive allo-HSCT because of HLA restriction.Up to now,autogeneic hematopoietic stem cell has been applied nearly for forty years in clinics,it has the advantage of unlimited donor,fewer complications,but it has a high relapse risk for lack of GVL effects.Relapse is the major reason for failure of auto-HSCT, decreasing relapse is the key point to increase therapeutic effect.Our previous research showed:autologous marrow mixed with HLA-haploidentical allogeneic stem cell transplantation(Mixed-HSCT) could exploit the advantage of both sides in a certain degree,overcome their shortcomings,develop mixed chimerism,reduce GVHD,and keep GVL effects.By leukemia rat model using K562 cell line with gene marker and BALB/C mouse,we studied the therapeutic effects and the forming of chimera of Mixed-HSCT and post transplant donor lymph cell infusion combined IL-2 for acute myeloid leukemia,provided evidence for clinical application;Furthermore,we also observed clinical therapeutic efficacy of mixed-HSCT.1.Methods:(1) Gene transfer:Cultured PA317-GCGPXSN,NIH3T3 and K562 cell lines;prepare virus supernatant with PA317-GCGPXSN cell line;Performed the gene transfer after stimulating K562 cells with rhIL-3、rhIL-6 and rhSCF;Evaluate efficiency of gene transfer by flow cytometry and assess the NeoR gene by PCR.The K562 cells having fulfilled gene transfer were defined as K562+ cells. (2) BALB/C mouse model:SPF(specific-pathogen free) BALB/C mice were irradiated 2Gy or 3Gy for 24 hours respectively according to experiment groups,then injected into log phase growth K562+ cells by vena caudalis.observed general state of health,survive time,bone marrow cell classification,and peripheral blood leucocyte count,classifcation, subset,related organ pathology checking;detected K562+ cells by flow cytometry;detected tissue infiltration by PCR.(3) Cultured bone marrow cells of BALB/C×C57BL first filial generation(F1) mice according to routine methods,detected T cell subsets,NK cells,CD34+ cells.After gene transfer,detected the efficiency of gene transfer by flow cytometry,detected NeoR gene by PCR.The F1 ceils having fulfilled gene transfer were defined as F1+ cells.(4) Animal experiment:Seven days later,the leukemia mice prepared as above methods were performed ABMT or MBMT(allogeneic bone marrow cells were reinfused 1h after ABMT),after irradiated 6Gy for 24 hours.The mice of MBMT group were reinfused F1 cells every 7 days,and administered IL-2 by intraperitoneal injection every other day.Killed the surviving mice after 4 week observation,observed peripheral blood and bone marrow cell morphous,detected cell subsets,GFP+ cells,NeoR gene,examined the liver,spleed pathology and detected GFP+ cells and NeoR gene of their tissue homogenate.(5) 13 patients with acute myeloid leukemia(AML),in complete remission(CR),were undergone Mixed-HSCT,after hemopoiesis recovery,administered donor lymphocyte infusion(DLI) for 2～7 times.Another 11 AML patients also in CR period were undergone Mixed-HSCT,administered no special treatment post transplantation.2.Results:(1) The gene transfer efficiency in K562 cells:fluorescence intensity value measured by flow cytometry was 0.56%in K562- cells,and 77.16%in K562+ cells,there is significant difference between them(P＜0.001>.The special segment of NeoR gene was amplificated by PCR,which showed that retroviral vector plasmid contained GFP and NeoR gene had been transferred and integrated into the genome of K562 cells.(2) The preparation of leukemia model①The effects of irradiation on immune function of mice:after the mice of E group were irradiated 2Gy,their CD3+,CD4+,CD8+,CD16+CD56+ cells in peripheral blood decreased progressively,up to third week,begun to recover gradually,four weeks later recovered to normal.In F group,after irradiated 3Gy,the CD3+,CD4+,CD8+,CD16+CD56+ cells also decreased progressively,while up to fourth week,begun to recover gradually,five weeks later recovered to normal.At the same time point,the extent that cell subsets in mice irradiated by 3 GY decreased was more notable than that by 2Gy(P＜0.05).After irradiation, the spleed,thymus diminished compared with normal mice,their weight lightened and four weeks later recovered to the level before irradiation,the weight of liver had no change.②The incidence:the BALB/C mice(E,F group) irradiated by 2 or 3Gy but without K562 cells infused had no natural death after observed for four weeks.The BALB/C mice(A,B,C,D group) irradiated by 2 or 3Gy and infused 2×10~6,5×10~6 K562 cells fell ill 5～7 days later,all of mice in A group died within 30 days,C group within 24 days,B group within 23 days,D group within 17 days;there is significant difference in survival time,which is shorter than that of normal control group(P＜0.01).The weight in four groups decreased significantly than that in normal control group(P＜0.01).The incidence of leukemia in processed mice was 100%,and had no natural relief.With the increase of infused leukemia cell population and the increase of irradiation dose,survive time of mice shorten gradually,Furthermore the percentage of myeloblast or premyelocyte in peripheral blood and bone marrow increased gradually.Tumour cell infiltration was confirmed by pathology-scopy.The results detected by flow cytometry coincided with that,the existence of NeoR gene was verified by PCR.(3) The gene transfer efficiency in F1 cells:Fluorescence intensity value measured by flow cytometry was 0.63%in F1(-) cells,and 76.04%in F1(+) cells,there is significant difference between them(P＜0.001>.The special segments of NeoR gene were amplificated by PCR,which showed that retroviral vector plasmid contained GFP and NeoR gene had been transferred and integrated into the genome of F1(+)cells.(4) Bone marrow transplant on mice①The cell population of bone marrow transplant:In the experiment group,of the transplanted autologous bone marrow cells,the percentage of CD34+ cells is 1.32%;CD3+ cells,26.56%;CD4+ cells,13.44%;CD8+ cells,3.09%;CD16+CD56+ cells,7.53%.Of the transplanted allogeneic bone marrow cell(F1+),the percentage of CD34+ cells is 1.51%;CD3+ cells,61.41%;CD4+ cells,39.57%;CD8+ cells,27.61%;CD16+CD56+ cells,9.59%;Of the transplanted allogeneic bone marrow cell(F1-),the percentage of CD34+ cells is 1.49%;CD3+ cells,65.86%;CD4+ cells,40.13%;CD8+ cells,19.16%;CD16+CD56+ cells,9.70%.There is no significant difference between F1(+)and F1(-)cell subsets(P＞0.05), that means:there is no difference about all kinds of cell population transplanted to each experiment mouse.②Therapeutic effects:All of mice in leukemia model group(A group) died within 20 days;the mice of leukemia model irradiated 6 GY(F group),died within 14 days.There was 11%～64%myeloblast or premyelocyte in peripheral blood or bone marrow of dying mice in A group;while in F group,peripheral blood or bone marrow cells were rare,mainly lymph cells.GFP and NeoR gene were detected in bone marrow and peripheral blood of both A group and F group dying mice.The T cell subsets and NK cells were scarce in dying mice of both groups.There were 3～6 mice surviving above 28 days in other experiment groups,the quantity and morphous of survival mouse’s bone marrow and peripheral blood cells identified with normal mice,T cell subsets and NK cells of peripheral blood had recovered to normal range,there was no significant difference among each group(＞P0.05).GFP and NeoR gene were detected in survival mice infused F1(+) cells.Tumour cell infiltration was not found in liver or spleed of survival mice.GFP and NeoR gene fragments were not detected in tissue homogenate,which demonstrated K562+ tumour cells disappeared in survival mouse liver and spleed.(5) Clinical therapeutic effects:In observation group,all of patients undergone Mixed HSCT had hematopoietic reconstitution,no GVHD occurred.three M2a patients,who received once,twice,twice DLI+IL-2 therapy,relapsed and died at 3,4,7 months post therapy respectively.One M2a patients receiving twice DLI+IL-2 therapy suffered myelodysplastic syndrome 1 year after therapy,and died of cerebral hemorrhage at last.One M5 patient emerged unknown hyperthermy and twitch after receiveing once DLI+IL-2 therapy,and died of statural epilepticus.Other 8 patients had been disease-free survival(DFS) with follow-up 1～5 years.Among them,one M4 patient,one M5 patient were found to have mixed chimera.The long term survival was 61.5%(8/13).In control group,2 cases of M2a,2 cases of M4,1 case of M5 relapsed and died 1～7months post transplantation,one M3 patients died of cerebral hemorrhage at 25 days post transplantation.Other 2 cases of M3,2 cases of M4,1 case of M5 patients had been disease-free survival(DFS) with follow-up 3～5 years.Among them,the later three cases had mixed chimera formed.The long term survival was 45.4%(5/11).3.Conclusion:(1) The retrovirus contained of GFP gene and NeoR gene can be successfully transferred into K562 cell line and bone marrow cells of BALB/C×C57BL F1 mice.(2) The leukemia mouse model can be made by infusing K562 cells by vena caudalis into BALB/C mouse after irradiated.(3) Long term survival can be promoted when the leukemia mice are administered DLI combined with IL-2 therapy post Mixed-HSCT.(4) It shows according to clinical application:long term survival can be promoted when the AML patients are administered DLI combined with IL-2 adoptive immunotherapy post Mixed-HSCT.更多还原