NK细胞在异基因骨髓移植中作用的动物实验研究

本文探讨NK细胞在异基因小鼠骨髓移植GVHD和GVL反应中的作用。受体小鼠以60 Co照射 ,总剂量 8 0Gy;骨髓细胞经尾静脉注入受体小鼠中 ;GVHD反应指标包括 (1)弓背体位、大小便及脱毛等一般表现 ;(2 )嵌合体、体内混合淋巴细胞反应、 30d生存率为检测GVHD反应的定量指标 ;GVL作用指标包括外周血白细胞总数及白血病细胞比例 ,脾脏白血病细胞浸润比例 ,以及死亡率观察 ;病理学检查作为鉴别GVL实验中是否主要死于GVHD的指标。GVHD反应结果表明 ,异基因与后输NK细胞的异基因骨髓移植组均于 15d内死亡。而先输NK细胞组的GVHD反应明显减轻 ,30d死亡率为5 1%。同时输NK细胞组 30d内死于GVHD反应。移植后第 7天与 15d时 ,以先输NK细胞组的嵌合率高 ,第 30天时各组均未检出明显嵌合体存在。GVL作用实验方面 ,异基因、同时输NK细胞组及后输NK细胞的骨髓移植组虽然于 12d内表现出抗白血病作用 ,但在 30d内相继死于GVHD反应。先输NK细胞组于移植后 12d开始观察到较明显GVHD样反应 ,而外周血或脾细胞中L615细胞比例变化结果证实该移植途径具有较好的抗白血病作用。先输注供体NK细胞的异基因骨髓移植方式既能减轻GVHD反应 ,又能保留较好的GVL作用     

异基因骨髓移植小鼠免疫功能缺损机制的探讨

异基因骨髓移植(Allogeneic Bone Marrow Transplantation,ABMT)后,受体的免疫功能长期缺损,是患者术后极易感染死亡的重要原因之一.本文对ABMT小鼠(C57BL/6→BALB/c)免疫功能缺损的机制进行了探讨,发现ABMT小鼠IL-2产生明显受损;其脾细胞与(C57BL/6小鼠脾细胞一起过继转移到致死量照射的BALB/C小鼠体内,能抑制移植物抗宿主病(GVHD)的发生.去除其脾T细胞后,这种抑制作用丧失,ABMT小鼠脾细胞上清中发现一种非特异的抑制因子,能抑制正常小鼠脾细胞产生混合淋巴细胞反应的能力;能抑制正常小鼠的脾细胞产生IL-2;抑制正常小鼠脾细胞毒T淋巴细胞(CTL)的杀伤活性.用抗Thy-1.2单抗和补体去除ABMT小鼠脾T细胞后,其脾细胞培养上(?)的上述抑制活性丧失.这说明ABMT小鼠脾T(?)细胞活性增强是其免疫功能缺损的重要原因之一,它通过释放非特异的抑制因子执行其免疫抑制功能.     

髓腔内骨髓移植诱导异基因小鼠皮肤移植耐受的研究

探讨髓腔内骨髓移植(IBM-BMT)对异基因小鼠皮肤移植耐受的诱导效果。受鼠为雌性C57BL/6(H-2b,B6)小鼠,于第0天接受60Coγ射线全身照射(TBI),4 h内输注雄性BALB/c(H-2d)小鼠来源的骨髓细胞(BMC),2 d后腹腔注射环磷酰胺(CTX)。通过皮肤移植、混合淋巴细胞反应(MLR)检测耐受状态,并通过骨髓染色体分析了解BMC的植入程度。结果显示,接受IBM-BMT的B6小鼠对BALB/c小鼠的皮肤移植物平均存活时间(MST)超过300 d,显著长于其余两组(P<0.001);MLR结果证明,实验组B6小鼠获得供体特异性耐受,在耐受小鼠骨髓中可检测到一定比例的Y染色体存在。以上表明髓腔内骨髓移植可以保证异基因骨髓细胞的植入并形成混合嵌合状态,从而有效地诱导免疫耐受。     

小鼠异基因骨髓移植急性移植物抗宿主病模型的建立

目的：建立稳定可靠的小鼠异基因骨髓移植急性移植物抗宿主病（GVHD）模型，为异基因骨髓移植中GVHD研究提供理想的实验参照。方法：以C57BL/6 (H-2^b) 小鼠为供鼠、BALB/c (H-2^d) 小鼠为受鼠，在移植物中混合不同比例的供鼠外周淋巴细胞以引起不同程度的GVHD。根据临床表现、存活期及病理检查等判断GVHD程度。结果：单纯异基因骨髓组只有部分小鼠出现急性GVHD，20%的小鼠长期存活（超过120 d），无急性GVHD表现；混合不同比例外周淋巴细胞的异基因骨髓移植小鼠出现GVHD的时间和程度均有差异，其中骨髓与外周淋巴细胞1:1混合组小鼠全部在移植后5-14 d出现典型的弓背体位，存活期6-24 d，临床和病理均可在相对集中的时间内观察到典型的急性GVHD改变。结论：以供鼠骨髓与外周淋巴细胞1:1混合进行的异基因骨髓移植，100%引出致死性的急性GVHD。     

NK细胞在小鼠异基因骨髓移植中的作用

目的:研究自然杀伤(NK)细胞在异基因骨髓移植中对移植物抗宿主病(GVHD)、移植排斥、骨髓植入及造血重建的影响。方法:以近交系小鼠C57/6j(H-2^b)为供鼠、BALB/c(H-2^d)为受鼠,在移植物中增加供者的外周T细胞和/或NK细胞进行异基因骨髓移植,用流式细胞仪检测受鼠的CD₃₄^＋细胞计数和H-2K^b＋细胞表达水平,血细胞自动分析仪检测外周血白细胞计数,并结合临床表现和病理检查,比较不同移植组的存活率、GVHD、植入水平及造血重建等。结果:增加NK细胞组的小鼠存活率显著大于不增加NK细胞组,小鼠出现GVHD的数量少、程度轻,外周血白细胞及骨髓CD₃₄^＋细胞恢复快、H-2K^b＋细胞表达水平高。结论:NK细胞抑制小鼠异基因骨髓移植中的GVHD和移植排斥,促进骨髓植入及造血重建。     

不同预处理方案对异基因骨髓移植物抗宿主病的影响

目的　建立大鼠异基因骨髓移植模型 ,应用不同的预处理方案 ,研究它们对移植物抗宿主病 (GVHD)严重程度的影响。方法　Wistar大鼠为供鼠 ,SD大鼠为受鼠 ,受鼠分 4组 ,分别予以不同的预处理方案 ,A、B组受鼠自移植前 4～ 1d ,腹腔注射氟达拉宾 (Fludarabine ,Flu) 1mg kg ,移植当天分别接受 2、6Gy的全身照射 ;C、D组受鼠在移植当天分别接受 2、6Gy的全身照射。制备供鼠骨髓细胞。照光 4h后 ,经尾静脉输注供鼠骨髓细胞 3.6× 10 7。观察各受鼠GVHD反应。结果　GVHD程度依次为 :Flu +2Gy组 <2Gy组 相似文献   

HLA-DR4基因亚型在骨髓移植中的应用研究

骨髓移植供、受体的ＨＬＡ相合程度是决定移植成败的关键因素之一。ＨＬＡＤＲ４亚型在各人种中的频率均较高,且与骨髓移植后ＧＶＨＤ（ｇｒａｆｔｖｅｒｓｕｓｈｏｓｔｄｉｓｅａｓｅ,移植物抗宿主病）的发生有关〔１３〕。为探讨ＨＬＡＤＲ４基因亚型在骨髓移植中的意义,设计合成了ＨＬＡＤＲ４等位基因引物〔４〕,建立巢式ＰＣＲＳＳＰ分型方法,并对７５例临床标本进行了分析。材料与方法研究对象：来我院拟进行骨髓移植的患者及其家属,其中正常无血缘关系个体４１例,患者３４例,包括１例无血缘关系的供、受体。引…     

小鼠同种异基因骨髓腔内骨髓移植促进早期造血功能重建

目的探讨同种异基因骨髓腔内骨髓移植(IBM-BMT)对小鼠早期造血功能重建的影响。方法将BALB/c小鼠骨髓单个核细胞(BMNCs)分别用胫骨骨髓腔内注射(IBMI)和尾静脉注射(IV)两种方法移植入经致死量60Coγ射线辐照后的60只C57BL/6小鼠。受鼠随机分为3组:骨髓腔内注射高和低剂量组(IBM1和IBM2组)、尾静脉注射组(IV组),每组20只。在骨髓移植后1、3、6和9d分别计数各组受鼠胫骨骨髓腔内有核细胞总数,并用流式细胞术检测供体植入水平(供体来源有核细胞总数、供体来源髓系细胞数)。结果于移植后6d,IBM1组和IBM2组注射侧胫骨骨髓腔内有核细胞总数、供体来源有核细胞总数、供体来源髓系细胞总数均明显高于IV组(P<0.05或P<0.01?。结论IBM-BMT较IV-BMT更能促进同种异基因骨髓移植后的早期造血功能重建。     

异基因骨髓移植中PCR-SSP和RFLP对HLA-DRB、DPB1分型的应用

目的:探讨序列特异性引物聚合酶链反应（PCR-SSP）和聚合酶链反应限制性片段长度多态性分析（PCR-RFLP）对HLA-DRB、DPB1分型在异基因骨髓移植中应用的可行性。方法:应用19对不同的DRB抗原所对应等位基因序列特异性的引物,PCR扩增供受者各20个样本DRB,产物直接琼脂糖凝胶电泳分析;同时用另一对引物PCR扩增DPB1第二外显子,产物10种限制性内切酶酶切分析。结果:各DRB带清晰可辨,直接确定为该型;DPB1的多态性片段编码转换后确定其基因型;4例找到供者。结论:它们是异基因骨髓植前快速、精确和可靠的基因分型手段。     

骨髓间充质干细胞对大鼠异基因骨髓移植后移植物抗宿主病的影响

目的：探讨大鼠骨髓间充质干细胞（Mesenchymal stem cells，MSCs）对异基因骨髓移植（allo—BMT）后移植物抗宿主病（GVHD）的影响。方法：从大鼠骨髓中分离培养问充质干细胞，通过形态学观察及流式细胞术检测表面标志以鉴定其纯度。通过混合淋巴细胞培养（MLR）检测MSCs体外对T细胞增殖的影响。建立大鼠GVHD模型（SD—Wistar），对其进行allo—BMT的同时，共移植不同数量供者源性的MSCs，观察受体大鼠的临床表现、绘制Kaplan—Meier生存曲线，并通过病理分析，综合评价MSCs，对allo-BMT后GVHD的影响。结果：在混合淋巴细胞培养体系中加入与反应淋巴细胞同源的MSCs，能够抑制T细胞的增殖，不同MSCs浓度组之问存在显著性差异（P〈0．01）。异基因骨髓移植时，供鼠MSC与T细胞比例为1：5、2：5共输注时，能推迟受鼠GVHD的发生时间，延长生存期，但并未避免GVHD的发生；当比例为1：1时，只有20％的受鼠发生了慢性GVHD反应，1—2周后逐渐缓解，并得到了长期生存，其余80％大鼠均未出现GVHD反应而长期生存。结论：大鼠MSCs不仅在体外能够抑制T细胞的增殖，体内共移植骨髓和高数量的MSCs也可以降低GVHD的发生率和严重程度，并延长受鼠的生存时间。本研究结果为临床GVHD的防治提供了新的思路。     

Reconstructing immunity after allogeneic transplantation

Immunologic Research - A major goal of our research is to reduce the graft-vs-host disease (GVHD) activity of allogeneic donor T cells in bone marrow transplantation (BMT), while preserving...     

The identification and characteristics of IL-22-producing T cells in acute graft-versus-host disease following allogeneic bone marrow transplantation

Graft-versus-host disease (GVHD) remains the major obstacle for allogeneic bone marrow transplantation, in which many proinflammatory cytokines secreted by alloreactive donor T cells are involved. Role of IL-22 as a member of IL-10 family in GVHD is still disputed and the properties of IL-22-producing cells are unclear. We demonstrated here that CD4⁺ T cells but not CD8⁺ T cells involved in GVHD were the main cellular source of donor-derived IL-22. Th1 and Th17 cells were detected not only express classical cytokine IFN-γ or IL-17, but also contributed to IL-22 secretion in GVHD. Th22 cells characterized by the independent secretion of IL-22 were identified and occupied almost half percentage of IL-22-producing CD4⁺ T cells. The frequency of IL-22-producing CD4⁺ T cells showed dynamic changes with the development of GVHD. Finally, we observed that IL-22-producing CD4⁺ T cells in GVHD mouse carried CD62L⁻CD44^high/low surface markers. In conclusion, we illuminate the characteristics of donor-derived IL-22-producing CD4⁺ T cells, which may have potent implication for further study of pathogenesis of GVHD.     

IL‐12/IL‐18‐preactivated donor NK cells enhance GVL effects and mitigate GvHD after allogeneic hematopoietic stem cell transplantation

Adoptive transfer of donor NK cells has the potential of mediating graft‐versus‐leukemia (GVL) effect while suppressing acute graft‐versus‐host‐disease (aGVHD) during allogeneic hematopoietic stem cell transplantation (allo‐HSCT). However, these beneficial effects are limited by the transient function of adoptively transferred NK cells. Previous studies demonstrate that cytokine‐induced memory‐like NK cells that are preactivated by IL‐12, IL‐15, and IL‐18 have enhanced effector functions and long life span in vivo. Here, we investigated the effects of IL‐12/18‐preactivated and IL‐12/15/18‐preactivated donor NK cells on GVL and aGVHD in a murine model of allo‐HSCT. We found that both IL‐12/18‐ and IL‐12/15/18‐preactivated NK cells mediated stronger GVL effect than control NK cells mainly due to their elevated activation/cytotoxicity and sustained proliferative potential. Interestingly, we observed that although both IL‐12/18‐ and IL‐12/15/18‐preactivated NK cells significantly inhibited severe aGVHD, only the IL‐12/18‐preactivated NK cells maintained the beneficial effect of donor NK cells on mild aGVHD. The IL‐12/15/18‐preactivated NK cell infusion accelerated aGVHD in the fully‐mismatched mild aGVHD model. Our results demonstrated that IL‐12/18‐preactivated NK cells displayed sustained and enhanced GVL functions, and could mitigate aGVHD despite the severity of the disease. IL‐12/18‐preactivated donor NK cell infusion may be an effective and safe adoptive therapy after allo‐HSCT.     

Histopathology of bone marrow reconstitution after allogeneic bone marrow transplantation

In order to study haematopoietic reconstitution in allogeneic bone marrow transplantation we investigated bone marrow histology in 61 biopsies of 37 patients, treated with HLA-compatible bone marrow grafts for leukaemia or severe aplastic anaemia. The biopsies were taken from the day of transplantation until 100 d after transplantation. Stromal changes, in particular oedema, fibrosis and granulomas, were found during the whole period of observation. These changes were more prominent in biopsies from leukaemia patients than from patients with aplastic anaemia. The cellularity in the biopsies increased until 28 d after bone marrow transplantation and was stable thereafter. Initially, only clusters of cells belonging to a single cell lineage were seen, suggesting that the first outgrowth of haematopoietic cells is by proliferation of committed precursor cells. Long-lasting abnormalities in localization of haematopoietic cells in the bone marrow space and of the myeloid: erythroid ratio were seen; dyserythropoiesis was common.     

Cytogenetic markers in allogeneic bone marrow transplantation

Engraftment after bone marrow transplantation (BMT) can be monitored with genetic markers. Cytogenetic markers are the ones that can be studied earliest. Of 27 patients studied, 16 had a sex-mismatched donor, and in 8 cases, there were discriminative markers identified with regular Giemsa and Q-banding. In one case, there was a rather slight polymorphism of the C-bands of chromosomes #9. Thus, donors' cells were identifiable in 25 of 27 cases (93%). The markers studied easiest are the sex chromosomes in sex-mismatched BMT. Different fluorescence of the centromeric regions of chromosomes #3 and characteristic satellites of acrocentric chromosomes are rather frequent and also well suited to discriminate donors' from patients' cells. Nonprominent variabilities (fluorescence of the centromeric regions of chromosomes #4 and #6 after Q-banding), rare polymorphisms (pericentric inversions of chromosomes #1 and #9), and difficult to evaluate polymorphisms (heterochromatic regions of chromosomes #1, #9, and #16) are not very practical for the routine monitoring of engraftment after BMT.     

Stress among allogeneic bone marrow transplantation patients

BMT (bone marrow transplantation) is acknowledged as one of the most stressful treatments in modern cancer care. When investigating the impact of BMT on patients it is crucial that the analytic method employed captures direct patient perceptions, allowing the patient to define the domains under investigation. In this study, a multivariate analytic method, concept mapping (CM), was used to identify perceived stressors among 109 allogeneic BMT recipients. CM employs multidimensional scaling and hierarchical cluster analyses to empirically identify the structure underlying the investigated conceptual domain. The analyses of the BMT stress data resulted in an eight-cluster solution. The stress clusters, ranked from the most severe to the least severe, were identified as: Change of life and impact of long-lasting treatment; Side-effects; Distress related to treatment outcome and physiological status; Family-related stress; Fear of death and depressive thoughts; Other concerns; Negative social support; and Stress related to lack of information and the medical staff. A number of stressors generated by the patients were quite novel, and identified important information likely to be useful in clinical settings as well as subsequent research with this high-risk population.