【作者】 陈广华；

【导师】 吴德沛；

【作者基本信息】 苏州大学 ， 内科血液学， 2009， 博士

【摘要】 一、小鼠脾NK细胞纯化、体外培养和鉴定目的:探讨小鼠NK细胞分选方法、体外NK细胞培养扩增体系的建立及体外培养对NK细胞杀伤活性的影响。方法:采用流式细胞术检测小鼠脾脏淋巴细胞亚群百分比。采用抗Thy-1.2抗体标记免疫磁珠去除T细胞及抗DX5抗体标记免疫磁珠正选NK细胞。流式细胞仪检测分选NK细胞纯度。采用含IL-2或(和)IL-15的体外培养体系,比较不同培养体系培养扩增效率。采用LDH释放法检测NK细胞杀伤活性。结果:B6成年小鼠脾脏CD3~-DX5~+NK细胞占3.0-5.5%。经磁珠两步法分选后CD3~-DX5~+NK细胞纯度高于95%。含10ng/mL rhlL-2和100ng/mL rhIL-15的DMEM完全培养基培养分选的脾NK细胞第3天、6天、9天、12天、15天时细胞计数倍数为1.27±0.05、3.14±0.18、6.23±0.28、10.81±0.92、23.10±1.44。NK细胞培养后免疫表型为CD3~-DX5~+,CD3~-DX5~+NK细胞占95.0%以上。静息NK细胞杀伤活性弱。效:靶比为10:1时NK细胞毒性为28.7%,而培养激活后效:靶比为10:1时NK细胞毒性为96.3%。结论:采用抗Thy-1.2和DX5免疫磁珠可高纯度分选小鼠NK细胞,体外含IL-2和IL-15的DMEM完全培养基可有效扩增NK细胞,联合应用可提高培养扩增效率,体外培养扩增的NK细胞免疫表型仍为CD3~-DX5~+,扩增后NK细胞杀伤活性比静息NK细胞杀伤活性高。二、建立小鼠异基因造血干细胞移植aGVHD和白血病模型目的:建立B6→BALB/c小鼠异基因造血细胞移植后非致死性aGVHD模型和EL9611白血病异基因造血细胞移植模型。方法:32只SPF级BALB/c小鼠随机分为4组,各组小鼠分别接受6、7、8、9Gy X射线全身照射。C57BL/6(H-2~b,♂)小鼠为供鼠,BALB/c小鼠(H-2~d,♀)为受鼠。32只BALB/c小鼠经预处理后随机分为4组,分别尾静脉输注1×10~6、2.5×10~6、5×10~6、10×10~6个骨髓细胞重建造血。在能重建造血的基础上建立aGVHD模型,输注BMCs基础上输注5×10~6、10×10~6或20×10~6个脾细胞。6-7周龄SPF级BALB/C小鼠尾静脉接种1×10~6个EL9611细胞。接种8天后分组:无TBI对照、TBI对照、同基因BMT对照、异基因BMT组,每组8只。结果:接受6Gy剂量的小鼠全部生存。接受7Gy TBI预处理的小鼠中位生存期为28.0天,33天内全部死亡。8Gy TBI预处理的小鼠中位生存期为12.7天,15天内全部死亡。四组生存时间采用Log-rank检验,x~2=47.24,P＜0.0001。输注2.5×10~6骨髓细胞不能全部重建造血,100天生存率分别为62.5%,其余死于内脏出血。输注5×10~6骨髓细胞可全部重建造血,100天生存率为100%。单纯输注骨髓细胞不能诱发aGVHD。低剂量脾细胞输注小鼠aGVHD疾病程度轻未出现aGVHD相关死亡,中剂量脾细胞输注小鼠25%出现aGVHD相关死亡,高剂量脾细胞输注小鼠41天内100%死于aGVHD。单纯TBI处理组小鼠处理后14天内全部死于造血衰竭。同基因对照小鼠20天内全部死于白血病。异基因BMT组1/8只小鼠长期生存,7/8只小鼠于BMT后45天内死于白血病复发。四组生存时间采用Log-rank检验,x~2=40.22,P＜0.0001。+28d外周血细胞及脾细胞均达到完全供者嵌合体状态。结论:TBI 8Gy对于BALB/c小鼠是清髓性预处理。预处理后输注输注5×10~6个骨髓细胞可全部重建造血。输注5×10~6个脾细胞可诱发轻至中度aGVHD,不出现致死性aGVHD。移植前8天尾静脉接种1×10~6个EL9611白血病细胞可成功建立白血病微小残留病模型。三、供者NK细胞输注及IL-2、IL-15治疗对异基因造血干细胞移植后GVHD、白血病复发和免疫重建的影响目的:探讨体外培养扩增的供者NK细胞输注及IL-2、IL-15治疗对异基因造血干细胞移植后GVHD、白血病复发和淋系免疫重建的影响。方法:采用临床GVHD积分方法评价小鼠GVHD严重程度;定量PCR方法检测脾细胞sjTREC评价胸腺输出功能;RT-PCR方法检测T细胞受体β链谱型;流式细胞仪检测淋系重建;外周血涂片瑞氏染色检测白血病复发。在GVHD模型中40只小鼠随机分为4组。BMT对照组:输注B6小鼠来源BMCs 5×10~6个;GVHD对照组:输注B6小鼠来源BMCs 5×10~6个+SCs 5×10~6个;NK细胞输注组:输注B6小鼠来源BMCs 5×10~6个+SCs 5×10~6个+NK 1×10~7个;NK细胞输注及IL2、IL-15治疗组:输注B6小鼠来源BMCs 5×10~6个+SCs 5×10~6个+NK 1×10~7个,移植后每天腹腔注射IL-2、IL-15共7天。在EL9611白血病模型中BALB/c小鼠移植前.8d尾静脉接种1×10~6个EL9611细胞,70只小鼠分为6组,每组9-12只。TBI对照组:单纯TBI处理;同基因造血细胞移植对照组:输注BALB/c小鼠来源BMCs 5×10~6个+SCs 5×10~6个;异基因造血细胞移植对照组:输注B6小鼠来源BMCs 5×10~6个+SCs 5×10~6个;NK细胞输注组:输注B6小鼠来源BMCs 5×10~6个+SCs 5×10~6个+NK 1×10~7个;NK细胞输注及IL2、IL-15治疗组一:输注B6小鼠来源BMCs 5×10~6个+SCs 5×10~6个+NK1×10~7个,移植后每天腹腔注射IL-2、IL-15共7天;NK细胞输注及IL2、IL-15治疗组二:输注B6小鼠来源BMCs 5×10~6个+SCs 5×10~6个+NK 1×10~7个,移植后每天腹腔注射IL-2、IL-15共14天。结果:骨髓移植对照组BALB/c小鼠不出现GVHD表现。GVHD对照组小鼠移植后7天时出现不同程度的GVHD表现,并出现GVHD相关体重减轻。NK细胞输注组小鼠也出现aGVHD表现,但临床GVHD评分比对照组评分低(P＜0.05)。NK细胞输注及IL2、IL-15治疗组小鼠各观察时间点临床GVHD评分比对照组评分明显低(P＜0.01)。单纯异基因骨髓移植组小鼠的皮肤、肝脏和肠道未见明显的病理改变。GVHD组小鼠出现皮肤、肝脏和肠道可见明显的病理改变。NK细胞输注组小鼠GVHD靶器官病理改变较GVHD对照组轻。同基因造血细胞移植组小鼠20天内全部死于白血病,无长期生存小鼠。异基因造血细胞移植组小鼠中位生存28天,90%(9/10只)于移植后52天内死于白血病复发,10%(1/10只)小鼠长期生存。NK细胞输注组小鼠中44.4%(4/9只)死于白血病复发,55.6%(5/9只)小鼠长期生存,与异基因HCT组比较生存期明显延长(x~2=4.487,P=0.0342)。NK细胞输注及IL2、IL-15治疗组一小鼠20%死于白血病复发,80%(8/10只)长期生存,与NK细胞输注组比较生存期延长(x~2=1.065,p=0.3022)。NK细胞输注及IL2、IL-15治疗组二小鼠8.3%(1/12只)死于白血病复发,91.7%(11/12只)长期生存,与NK细胞输注组比较生存期延长(x~2=4.018,P=0.0450)。NK细胞输注及IL-2、IL-15治疗组、NK细胞输注组和对照组小鼠移植后28天脾细胞计数分别为(4.78±0.51)×10~7、(3.98±0.43)×10~7、(3.45±0.23)×10~7,NK细胞输注及细胞因子治疗小鼠脾细胞总数明显高于对照组小鼠(P＜0.01)。而且NK细胞输注及细胞因子治疗组小鼠T细胞、B细胞、NK细胞均高于对照组小鼠(P＜0.01)。对照组移植小鼠sjTREC拷贝数为136.6±13.7/10~5,NK细胞输注组和NK细胞输注及细胞因子治疗组小鼠SjTREC拷贝数为222.2±11.4/10~7、287.5±10.9/10~5,均明显高于对照组(P＜0.01)。移植后一个月时NK细胞输注组脾脏TRBV重建明显比对照组TRBV重建快。结论:体外培养扩增的供者NK细胞输注及IL-2、IL-15治疗可减轻异基因造血干细胞移植后GVHD严重程度,保护胸腺增强输出功能促进T细胞谱型重建,促进外周T细胞、B细胞、NK细胞等淋系重建,增强移植物抗白血病作用减少白血病复以。四、供者NK细IN输注及IL-2、IL-15治疗对单倍型相合造血干细胞移植后GVHD、白血病复发和免疫重建的影响目的:建立B6→CB6F1单倍型相合造血细胞移植后非致死性aGVHD模型,探讨体外培养扩增的供者NK细胞输注及IL-2、IL-15治疗对单倍型相合造血细胞移植后GVHD、白血病复发和免疫重建的影响。方法:采用临床GVHD积分和病理组织学方法评价小鼠GVHD严重程度;实时定量PCR方法检测SjTREC拷贝数评价移植后胸腺输出功能;RT-PCR结合毛细管电泳检测T细胞受体β链谱型评价TRBV谱型;多参数流式细胞仪检测脾脏淋系重建;外周血涂片瑞氏染色检测白血病复发。在GVHD模型中40只小鼠随机分为4组,每组10只。骨髓移植对照组:输注B6小鼠来源BMCs 5×10~6个;GVHD对照组:输注B6小鼠来源BMCs 5×10~6个+SCs 15×10~6个;NK细胞输注组:输注B6小鼠来源BMCs 5×10~6个+SCs 15×10~6个+NK 1×10~7个;NK细胞输注及IL2、IL-15治疗组:输注B6小鼠来源BMCs 5×10~6个+SCs 15×10~6个+NK 1×10~7个,移植后每天腹腔注射IL-2、IL-15共7天。在EL9611白血病模型中CB6F1小鼠移植时尾静脉接种2×10~6个EL9611白血病细胞,每组10只。单倍型相合造血细胞移植对照组:输注B6小鼠来源BMCs 5×10~6个+SCs 10×10~6个;NK细胞输注组:输注B6小鼠来源BMCs5×10~6个+SCs 10×10~6个+NK 1×10~7个;NK细胞输注及IL2、IL-15治疗组一:输注B6小鼠来源BMCs 5×10~6个+SCs 10×10~6个+NK 1×10~7个,移植后每天腹腔注射IL-2、IL-15共7天;NK细胞输注及IL2、IL-15治疗组二:输注B6小鼠来源BMCs5×10~6个+SCs 10×10~6个+NK 1×10~7个,移植后每天腹腔注射IL-2、IL-15共14天。结果:11.5Gy X线TBI对于CB6F1小鼠为清髓性预处理。GVHD对照组小鼠移植后7天时出现GVHD表现。NK细胞输注组小鼠各时间点临床GVHD评分比对照组评分低(P＜0.05)。NK细胞输注及IL2、IL-15治疗组小鼠各观察时间点临床GVHD评分比对照组评分明显低(P＜0.01)。GVHD对照组小鼠出现皮肤、肝脏和肠道可见明显的病理改变。NK细胞输注组小鼠GVHD靶器官病理改变较GVHD对照组轻。单倍型相合造血细胞移植对照组小鼠100天生存率为20%(2/10只),80%(8/10只)于移植后死于白血病复发。NK细胞输注组小鼠中10%(1/10只)死于白血病复发,90%(9/10只)小鼠长期生存。NK细胞输注IL-2I与L-15治疗组小鼠均长期生存,无死于白血病小鼠。四组生存时间采用Log-rank检验,x~2=30.69,P＜0.0001。NK细胞输注及IL-2、IL-15治疗组、NK细胞输注组和对照组小鼠移植后28天脾细胞计数分别为(5.39±0.59)×10~7、(4.42±0.39)×10~7、(3.68±0.47)×10~7,NK细胞输注及细胞因子治疗小鼠脾细胞总数明显高于对照组小鼠(P＜0.01)。而且NK细胞输注及细胞因子治疗组小鼠T细胞、B细胞、NK细胞、NKT细胞计数均高于对照组小鼠(P＜0.01)。对照组移植小鼠sjTREC拷贝数为155.3±10.7/10~5,NK细胞输注组和NK细胞输注及细胞因子治疗组小鼠sjTREC拷贝数为246.5±29.4/10~5、298.5±16.0/10~5,均明显高于对照组(P＜0.05)。移植后一个月时NK细胞输注组脾脏TRBV重建明显比对照组TRBV重建快。结论:体外培养扩增的供者NK细胞输注及IL-2、IL-15治疗可减轻单倍型相合造血干细胞移植后aGVHD严重程度,保护胸腺增强输出功能促进T细胞谱型重建,促进外周T细胞、B细胞、NK细胞等淋系重建,增强移植物抗白血病作用减少白血病复发。更多还原

【Abstract】 PartⅠ:Isolation,culture and identification of mouse splenic NK cellsObjective:To explore NK cell separation method and NK cell culture system and to study the cytotoxicity activity of expanded NK cells.Methods:Lymphoid cell subset percentages were analysed by flow cytometry (FCM).T cells were depleted by using CD90(Thy1.2) MicroBeads prior to NK cell enrichment by using CD49b(DX5) MicroBeads,an MiniMACS separator and an MS column.The purity of NK cell separation product was assaied by FCM.NK cells were cultured in complete DMEM medium containing IL-2 and/or IL-15.NK cell cytotoxicity was detected by using CytoTox96 kit.Results:CD3~-DX5~+NK accounts for 3.0-5.5%of total splenocytes of adult B6 mice.NK cell separation product had a purity of more than 95%.NK cell count increased by 1.27±0.05,3.14±0.18,6.23±0.28,10.81±0.92,23.10±1.44 folds after 3,6,9,12,15 days culture in complete DMEM medium containing 10ng/mL rhIL-2 and 100ng/mL rhIL-15. NK cell immunotype was CD3~-DX5~+,CD3~-DX5~+NK cells accounts for 95.0%.Resting NK cell had mild cytotoxicity of 28.7%at an E:T ratio of 10:1,while activated NK cell had a cytotoxicity of 96.3%at an E:T ratio of 10:1.Conclusion:NK cell could be highly pure separated from mouse splenocytes by using CD90(Thy1.2) MicroBeads and CD49b(DX5) MicroBeads.NK cell could be efficiently expanded in complete DMEM medium containing IL-2 and IL-15.The immunotype of expanded NK was CD3~-DX5~+.NK cell cytotoxicity was increased after expanded and activated by IL-2 and IL-15.PartⅡEstablishment of a murine nonlethal aGVHD model post allogeneic hematopoietic stem cell transplantation and transplantable leukemia model Objective:To establish a murine nonlethal aGVHD model post allogeneic hematopoietic stem cell transplantation and EL9611 erythroleukemia allogeneic hematopoietic stem cell transplantation model.Methods:Thirty-two SPF grade BALB/c mice were randomly assigned to four groups,mice received 6,7,8 or 9Gy X ray total body irradiation.Adult C57BL/6(H-2~b) males were donor mice and BALB/c(H-2d,♀) were recipient mice.Thirty-two SPF grade BALB/c mice were randomly assigned to four groups post TBI conditioning and were reconstituted with 1×10~6,2.5×10~6,5×10~6 or 10×10~6 BMCs via lateral tail vein injection.GVHD model was established by coinfusion of 5×10~6,10×10~6 or 20×10~6 splenocytes via tail vein.Six- to seven-weeks old SPF grade BALB/c females were inoculated with 1×10~6 EL9611 cells via lateral vein.Leukemia-loaded mice were randomly assigned to 4 groups as no treatment group,TBI group,syngeneic group,allogeneic HCT.Results:All mice survived 6 Gy TBI conditioning.The median survival time of mice conditioned with 7Gy TBI was 28.0d and for 8Gy TBI was 12.7d.The survival time was compare by Log-rank test,with x~2=47.24,P＜0.0001.The survival rate at 100d was 62.5%when mice were injected with 2.5×106BMCs.All mice survived 100d post-HCT when reconstituted with 5×10~6BMCs.No overt aGVHD was observed when mice were reconstituted with BMCs alone.Non-lethal aGVHD was induced when mice were reconstituted with 5×10~6 splenocytes.Lethal aGVHD was induced when mice were reconstituted with 10×10~6 or 20×10~6 splenocytes.Mice received syngeneic HCT died of EL9611 leukemia within 20 days.In the allo-BMT group 1/8 mouse survived 100d free of leukemia.The survival time was compare by Log-rank test,with x~2=40.22,P＜0.0001. Complete donor chimerism was achieved on +28d after allo-HCT.Conclusion:It was myeloablative conditioning when BALB/c mice received 8Gy TBI treatment.Hematopoietic recovery could achieve when BALB/c mice were reconstituted with 5×10~6 BMCs.Nonlethal aGVHD could induced when mice were injected with 5×10~6 splenocytes.Leukemia residual disease could be established when BALB/c mice were inoculated with 1×10~6 EL9611 leukemia cells on -8d.PartⅢDonor-derived NK cell infusion and IL-2,IL-15 treatment efficacy in allogeneic hematopoietic cell transplantationObjective:To explore the effects of ex vivo-expanded donor NK cell infusion and IL-2,IL- 15 treatment on GVHD,leukemia relapse,and immune reconstitution.Methods:Real-time quantitative PCR was used to assay sjTREC in splenocytes. RT-PCR and runoff PCR were used to assay TRBV family spectrum.FCM was used to assay splenic lymphoid immune reconstitution.Forty BALB/c mice were randomly assigned to BMT control group,GVHD control group,NK cell infusion group,and NK cell infusion,cytokines treatment group in the GVHD model.Seventy BALB/c mice were inoculated with 1×10~6 EL9611 cell on -8d and were assigned to six groups.Results:GVHD was observed as early as +7d after allo-HCT in the control mice.The clinical GVHD score of NK cell infusion group was lower than that of GVHD control group.The lesion of skin,liver and ileum of NK cell infusion group was less severe than that of GVHD control group.All syngeneic HCT control recipient mice died of leukemia within 20 days.The median survival time of allogeneic HCT recipient mice was 28 days and 10 percent(1/10) survived +100 d.In the NK cell infusion group,44.4 percent died of leukemia and 55.6 percent survived +100 day,had prolonged survival than allogeneic HCT control group(x~2=4.487,P=0.0342).Eighty percent(8/10) mice of NK cell infusion and IL-2,IL-15 treatment for one week group survived +100 day.Eleven of 12 mice of NK cell infusion and IL-2,IL-15 treatment for two week group survived +100 day.The survival time of this group was longer than that of NK infusion group.The total splenocytes of mice in NK cell infusion and IL-2,IL-15 treatment group were(4.78±0.51)×10~7, (3.98±0.43)×10~7 and(3.45±0.23)×10~7,respectively.The splenic T,B,NK cell counts in NK cell infusion and cytokines treatment group were higher than those of control group. The sjTREC level of control group was 136.6±13.7 per 10~5 cells,while the sjTREC level of NK cell infusion group and combination treatment group were 222.2±11.4 and 287.5±10.9 per 10~5 cells,respectively.TRBV spectrum reconstitution in NK cell infusion group was faster than that of control group.Conclusion:Ex vivo-expanded NK cell infusion and IL-2,IL-15 treatment could mitigate GVHD severity,protect thymus output,promote lymphoid immune reconstitution and enhance graft-versus-leukemia effect in murine allogeneic HCT.PartⅣDonor-derived NK cell infusion and IL-2,IL-15 treatment efficacy in haploidentical hematopoietic cell transplantationObjective:To establish a nonlethal GVHD model post B6→CB6F1 haploidentical hematopoietic cell transplantation(HID) and to explore the effects of ex vivo-expanded donor NK cell infusion and IL-2,IL-15 treatment on aGVHD,leukemia relapse,and lymphoid immune reconstitution.Methods:Clinical GVHD score and histopathological examination were used to evaluate GVHD severity.Real-time quantitative PCR was used to assay sjTREC level post haploidentical HCT.RT-PCR and runoff PCR were used to assay TRBV family spectrum. FCM was used to assay splenic lymphoid immune reconstitution.In the GVHD model, forty CB6F1 mice were randomly assigned to four groups as BMT control group,GVHD control group,NK cell infusion group and NK cell infusion and IL-2,IL-15 treatment group.We established the CB6F1 leukemia model by coinfusion of 2×10~6 EL9611 leukemia cells with stem cell graft per mice at the time of HCT.In the leukemia model, fourty CB6F1 females were randomly assigned to four groups as HCT control group,NK cell infusion group,NK cell infusion and cytokine treatment for 1 week group and NK cell infusion and cytokine treatment for 2 weeks group.Results:Lethal myeloablative conditioning was achieved after CB6F1 mice were treated with 11.5 Gy TBI.GVHD was evident in GVHD control mice on +7d post HID. NK cell infusion group mice had lower clinical GVHD scores than GVHD control group mice(P＜0.05).The clinical GVHD scores of NK cell infusion and cytokines treatment group mice were lower than those of control group mice at several time points(P＜0.01). Overt GVHD lesion could be detected in skin,liver and ileum of GVHD control mice. Twenty percent(2/10) of control group mice survived +100 d post HID and eighty percent of mice died of leukemia.Ten percent of mice died of leukemia and ninety percent of mice survived +100 d post HID in the NK cell infusion group.All mice of NK cell infusion and cytokines treatment group mice survived +100d post HID.The survival time of four group mice were significantly different(x~2=30.69,P＜0.0001).Total splenocyte counts of NK cell infusion and cytokines treatment group,NK cell infusion group,control group were (5.39±0.59)×10~7,(4.42±0.39)×10~7,(3.68±0.47)×10~7 at +28d,respectively.The sjTREC level of control group was 155.3±10.7 per 10~5 cells,while the sjTREC level of NK cell infusion group and combination treatment group were 246.5±29.4 and 298.5±16.0 per 10~5 cells,respectively.TRBV spectrum reconstitution in NK cell infusion group was faster than that of control group.Conclusion:We established a nonlethal GVHD model and a leukemia model post HID.Donor NK cell infusion and IL-2,IL-15 treatment could mitigate GVHD severity, promote lymphoid immune reconstitution and enhance graft-versus-leukemia effect in murine haploidentical HCT.更多还原