成纤维细胞介导白细胞介素6基因疗法对化疗导致的造血损伤的恢复作用

研究了成纤维细胞介导的人白细胞介素６（Ｉｎｔｅｔｒｌｅｕｋｉｎ－６，ＩＬ－６）基因疗法对预先体内注射１５０ｍｇ／ｋｇ５－氟尿嘧啶（５－ＦＵ）所造成的造血损伤小鼠模型的治疗作用。结果发现，外周血血小板数量在移殖高分泌ＩＬ－６成纤维细胞后第１０天开始持续上升，第２２天血小板数量恢复到化疗前水平，中性粒细胞数量也显著增高，但对白细胞恢复没有明显的促进作用。骨髓和脾脏ＣＦＵ－ＧＭ及ＣＦＵ－ＭＫ在体内移殖高分泌ＩＬ－６成纤维细胞后，体外动态观察发现：骨髓和脾脏ＣＦＵ－ＧＭ、ＣＦＵ－ＭＫ在第４～７天左右数量为０，在第１０天左右数量开始上升，于第１６天左右数量显著增高，对ＣＦＵ－ＧＭ、ＣＦＵ－ＭＫ具有明显的恢复作用，ＣＦＵ－Ｓ数量也显著增高。表明成纤维细胞介导的白细胞介素６基因疗法能显著治疗化疗导致的造血损伤，可望为肿瘤放、化疗导致的机体造血损伤提供新的治疗途径。     

成纤维细胞介导白细胞介素6基因疗法对化疗导致的造血损伤的…

研究了成纤维细胞介导的人白细胞介素６（Ｉｎｔｅｔｒｌｅｕｋｉｎ－６，ＩＬ－６）基因疗法对预先体内注射１５０ｍｇ／ｋｇ，５－氟尿嘧啶（５－ＦＵ）所造成的造血损伤小鼠模型的治疗作用，结果发现，外周血血小板数量在移植高分泌ＩＬ－６成纤维细胞后第１０天开始持续上升，第２２天血小板数量恢复到化疗前水平，中性粒细胞数量也显著增高，但对白细胞恢复没有明显的促进作用，骨髓和脾脏ＣＦＵ－ＧＭ及ＣＦＵ－ＭＫ在体内移植     

应用IL—2和IL—3联合基因疗法对骨髓移植后造血及免疫…

以大剂量化疗后骨髓功能严重损伤再给予同基因骨髓移植的小鼠为实验模型，动态观察了联合应用成纤维细胞介导的ＩＬ－２基因疗法和ＩＬ－３基因疗法对同基因骨髓移植后实验小鼠骨髓造血免疫功能重建的影响。结果表明，ＩＬ－２基因疗法对造血重建无明显效果，ＩＬ－３基因疗法对免疫重建无明显作用，但联合应用ＩＬ－２基因疗法和ＩＬ－３基因疗法能显著加快骨髓ＣＦＵ－ＧＭ、ＣＦＵ－ＭＫ及ＣＦＵ－Ｅ恢复过程及提高骨髓细胞ＮＫ、     

IL—3基因疗法对化疗造血损伤的恢复作用

白细胞介素３（ＩＬ－３）是一种具有很强造血调控作用的细胞因子。本实验观察了成纤维细胞介导的ＩＬ－３基因疗法对大剂量环磷酰胺体内注射后造血功能损伤小鼠的恢复作用。结果表明：接爱ＩＬ－３基因疗法的实验小鼠外周血白细胞和血小板数量降低程度减弱，回升速度加快，其脾脏和骨髓ＣＦＵ－ＧＭ、ＣＦＵ－Ｅ、ＣＦＵ－ＭＫ、ＣＦＵ－Ｓ水平显著地高于对照小鼠，可见成纤维细胞介导的ＩＬ－３基因疗法可显著地降低大剂量化疗后造     

应用IL-2和IL-3联合基因疗法对骨髓移植后造血及免疫功能重建的影响

以大剂量化疗后骨髓功能严重损伤再给予同基因骨髓移植的小鼠为实验模型，动态观察了联合应用成纤维细胞介导的ＩＬ－２基因疗法和ＩＬ－３基因疗法对同基因骨髓移植后实验小鼠骨髓造血免疫功能重建的影响。结果表明，ＩＬ－２基因疗法对造血重建无明显效果，ＩＬ－３基因疗法对免疫重建无明显作用，但联合应用ＩＬ－２基因疗法和ＩＬ－３基因疗法能显著加快骨髓ＣＦＵ－ＧＭ、ＣＦＵ－ＭＫ及ＣＦＵ－Ｅ恢复过程及提高骨髓细胞ＮＫ、ＬＡＫ活性和ＣｏｎＡ刺激的骨髓细胞增殖反应。提示联合应用成纤维细胞介导的ＩＬ－２和ＩＬ－３基因疗法能显著加快骨髓移植后造血及免疫重建过程，从而有可能提高骨髓移植成功率。     

IL－3基因疗法对化疗造血损伤的恢复作用

白细胞介素３（ＩＬ－３）是一种具有很强造血调控作用的细胞因子，本实验观察了成纤维细胞介导的ＩＬ－３基因疗法对大剂量环磷酰胺体内注射后造血功能损伤小鼠的恢复作用。结果表明：接受ＩＬ－３基因疗法的实验小鼠外周血白细胞和血小板数量降低程度减弱，回升速度加快，其脾脏和骨髓ＣＦＵ－ＧＭ、ＣＦＵ－Ｅ、ＣＦＵ－ＭＫ、ＣＦＵ－Ｓ水平显著地高于对照小鼠，可见成纤维细胞介导的ＩＬ－３基因疗法可显著地降低大剂量化疗后造血损伤程度，并能显著地促进受损的造血功能尽快恢复，提示若将ＩＬ－３基因疗法与大剂量化疗联合应用将提高肿瘤治疗的效果。     

白细胞介素—11对小鼠骨髓造血的影响

白细胞介素－１１（ＩＬ－１１）是一种新的造血生长因子，于１９９０年基因克隆成功。本文对ＩＬ－１１的造血调节作用进行了初步的研究，探讨了ＩＬ－１１及其与其它造血因子协同对小鼠骨髓造血祖细胞增殖的促进作用。结果表明，ＩＬ－１１单独对骨髓集落的形成无显著的促进作用，但与多系集落刺激因子ＩＬ－３一起则具有很强的协同作用，能明显促进小鼠骨髓Ｍｅｇ－ＣＦＵ和Ｍｉｘ－ＣＦＵ的形成。     

白细胞介素－11对小鼠骨髓造血的影响

白细胞介素－１１（ＩＬ—１１）是一种新的造血生长因子，于１９９０年基因克隆成功．本文对ＩＬ—１１的造血调节作用进行了初步的研究，探讨了ＩＬ—１１及其与其它造血因子协同对小鼠骨髓造血祖细胞增殖的促进作用．结果表明，ＩＬ—１１单独对骨髓集落的形成无显著的促进作用，但与多系集落刺激因子ＩＬ－３一起则具有很强的协同作用，能明显促进小鼠骨髓Ｍｅｇ—ＣＦＵ和Ｍｉｘ－ＣＦＵ的形成．     

“秘灵王”对骨髓受抑模型鼠的免疫调整作用

观察了应用适当浓度“秘灵王”（ＭＬＷ）在体外可诱导小鼠骨髓细胞（ＢＭＣ）增殖，促进其产生ＩＬ－３、ＩＬ－６样活性物质，其中水平与ＢＭＣ增殖能力呈高度正相关，在体内ＭＬＷ亦可促进正常鼠的骨髓细胞活性。在此基础倒ＭＬＷ对骨髓受抑制模型鼠骨髓干细胞增殖、粒－巨细胞集（ＧＭ－ＣＦＵ）形成能力皆有显著调整作用，对ＢＭＣ培养上清中ＩＬ－３、ＩＬ－６样活性物质水平也具有调整作用，其水平的增高与骨髓细胞增殖能力，     

联合应用IL—2基因疗法和IL—3基因疗法对骨髓移植后荷瘤小鼠免 …

以大剂量化疗后基因骨髓移植（ＢＭＴ）的荷瘤小鼠为实验模型。动态观察了联合应用ＩＬ－２基因疗法和ＩＬ－３基因疗法配合ＢＭＴ对荷瘤小鼠抗肿瘤免疫功能的影响。结果表明，联合应用基因疗法能显著提高ＢＭＴ治疗后葆瘤小鼠脾细胞诱导的ＣＴＬ杀伤活性、腹腔巨噬细胞杀伤活性及其所分泌的ＩＬ－１、ＴＮＦ水平，但是对荷瘤小鼠脾脏分泌ＩＮＦ＿γ无协同诱导效应。提示联合应用ＩＬ－２基因疗法和ＩＬ－３基因疗法通过显著地协同促     

Effects of fibroblast-mediated interleukin 3 and interleukin 6 gene therapy on hematopoiesis in mice treated with 5-fluorouracil

Fibroblast-mediated cytokine gene therapy has proven to be a promising strategy for restoring hematopoiesis following repeated chemotherapy. Interleukin 3 (IL-3) and interleukin 6 (IL-6) can synergistically promote the recovery of hematopoiesis following chemotherapy. In this investigation, combined use of fibroblast-mediated IL-3 and IL-6 gene therapy was tested for hematopoietic effects on mice with or without 5-fluorouracil administration. The results demonstrated that combined therapy with IL-3 gene-modified NIH3T3 cell (NIH3T3-IL-3) and IL-6 gene-modified fibroblast NIH3T3 cell (NIH3T3-IL-6) implantation achieves obvious stimulation of hematopoiesis in normal mice and accelerates recovery of hematopoiesis. In normal mice the quantities of platelets, neutrophils, and total white blood cells in peripheral blood increased significantly after the combined implantation of NIH3T3-IL-3 and NIH3T3-IL-6 cells. The numbers of colony-forming unit (CFU) granulocyte/macrophage (CFU-GM) and CFU megakaryocyte (CFU-MK) formed by stem cells in bone marrow was significantly higher after the combined implantation of NIH3T3-IL-3 and NIH3T3-IL-6 cells than after the implantation of NIH3T3-IL-3 alone, NIH3T3-IL-6 alone, or neomycin gene-modified NIH3T3 cells. In hematopoiesis-depressed mice induced by preinjection with 5-fluorouracil at the dose of 150 mg/kg before cell implantation, the platelets, neutrophils, and white blood cells showed accelerated recovery, and the numbers of CFU-GM and CFU-MK formed by bone marrow cells were also markedly higher after the combined implantation of NIH3T3-IL-3 and NIH3T3-IL-6 cells than in control groups. Our data show that combined use of fibroblast-mediated IL-3 and IL-6 gene therapy may be of clinical relevance for the recovery of hematopoietic depression for patients after chemotherapy. Received: 4 March 1998 / Accepted: 20 May 1998     

成纤维细胞介导的人白介素6基因疗法增强机体免疫功能的实验研究

通过研究成纤维细胞介导的人ＩＬ－６基因疗法对机体免疫功能的影响，结果发现，高分泌ＩＬ－６成纤维细胞克隆体内移植后，能显著提高淋巴细胞增殖反应、ＩＬ－２和ＩＦＮ－ｒ产生水平以及ＮＫ和ＬＡＫ细胞的杀伤活性，当与ＩＬ－２合用时，对ＮＫ和ＬＡＫ活性的增强作用更加明显，表明成纤维细胞能显著增强机体免疫功能，可望为肿瘤生物治疗、造血功能重建等开辟新的途径。     

转IL—3基因的骨髓基质细胞体外以造血功能的促进作用

目的 探讨用逆转录病毒载体转入mIL-3基因的基质细胞系QXMSC1对骨央造血细胞前体的影响。方法 用含鼠IL－3基因的逆转录病毒载体感染骨央基质细胞系QXMSC1，获得IL－3高表达细胞系QXMSC1 IL－3用于实验，观察QXMSC1 IL－3细胞上清对骨央前体细胞CFU－GM、CFU－E、CFU－GEMM的影响，QXMSC1 IL－3基质细胞对长期培养起始细包的影响。结果 QXMSC1 IL－3培养上清对骨髓前体细胞、CFU－GM、CFU－E、CFU－GEMM有明显促进作用。QXMSC1 IL－3能明显增加有核细胞数和长期培养起始细胞数，诱导分化形成CFU－GM增多，阻断基质细胞与造血细胞相互接触，QXMSC1 IL－3促造血细胞增殖作用有所减弱。结论 基质QXMSC1 IL－3可能作为有效的基因载体进一步促进骨髓移植后或辐射损伤后的造血和免疫功能重建。     

A new method of gene transfer into hematopoietic progenitors using liquid culture with interleukin-3 and interleukin-6

The technique of gene transfer into hematopoietic cells is expected to offer a new form of therapeutics. As a result of studies on a gene-delivery system using granulocyte-macrophage colony-forming units (CFU-GM), a type of hematopoietic progenitor, we have established a technique for efficient gene transfer into CFU-GM. DGL, a retrovirus vector containing the SV40 promoter and the neomycin resistance gene, was constructed and found to transfer genes effectively into murine CFU-GM, which subsequently expressed the neomycin resistance gene. After gene transfer of murine non-adherent bone marrow cells precultured in liquid culture with recombinant murine IL-3 (rmIL-3) and recombinant human IL-6 (rhIL-6) for 6 days, gene transferred CFU-GM in bone marrow cells were able to proliferate 5-10-fold and the ratio of gene transferred CFU-GM to total CFU-GM reached 70-100% from less than 1% in the liquid culture with rmIL-3, rhIL-6 and neomycin for 6 days. Using this protocol, we have been able to obtain large amounts of highly concentrated gene-transferred CFU-GM for fundamental research on CFU-GM gene-delivery systems.     

Acute in vivo effects of IL-3 alone and in combination with IL-6 on the blood cells of the circulation and bone marrow.

Recombinant human IL-3 administered intravenously to rats as a single injection induced peripheral neutrophilia and monocytosis beginning at 4 to 6 hours after injection, peaking at 8 hours, and subsiding to normal by 12 to 24 hours. IL-3 did not induce an initial neutropenia such as accompanies endotoxin-, G-CSF-, and TNF-induced neutrophilia, or lymphopenia such as accompanies endotoxin-, IL-1-, and TNF-induced neutrophilia. The IL-3-induced peripheral neutrophilia was accompanied by a decrease in mature marrow neutrophils, indicating that the mechanism of neutrophilia was through marrow release rather than by demargination, which occurs after the administration of epinephrine or IL-6. The release of mature marrow neutrophils further suggests that IL-3 either has intrinsic neutrophil releasing activity or indirectly causes neutrophil release through the gene expression of a second cytokine. IL-3 induced a striking left-shifted myeloid hyperplasia in the bone marrow at 8 hours that morphologically was very similar to that observed after administration of endotoxin, a finding consistent with the hypothesis of previous investigators that endotoxin may in part act indirectly on hematopoietic cells by eliciting local marrow production of IL-3. Finally, IL-3 induced an increase in marrow pronormoblasts at 8 hours, consistent with the in vitro proliferative effect of IL-3 on erythroid stem cells. The combination of IL-3 and IL-6 induced a synergistic peripheral neutrophilia and monocytosis and a striking synergistic increase in marrow mast cells. The combination of IL-3 and IL-6 also induced an erythroid and left-shifted myeloid hyperplasia such as would be expected given the individual effects of these hematopoietic growth factors.     

Cytokines transduced bone marrow stromal cell lines promote immunohematopoietic reconstitution in mice after allogeneic bone marrow transplantation

Impaired immune reconstitution following allogeneic T-cell depleted bone marrow transplantation (allo-TCD-BMT) is a major obstacle to its clinical application. Stromal cell line QXMSC1, established from bone marrow cells of BALB/c(H-2d), was transfected with murine IL-3 and/ or IL-2 gene, and injected into lethally irradiated C57BL/6(H2b) mice. We evaluated its effects on immunologic and hematopoietic reconstitution after allo-TCD-BMT. The results showed that QXMSC1-IL-3 + IL-2 could significantly increase the numbers of hematopoietic primitive progenitors (CFU-S), committed progenitors (CFU-GM, and BFU-E), and lymphocytes (CD8+ cells, CD4+ cells, and B cells). Similarly, immune functions of recipient mice were significantly enhanced in the QXMSC1-IL-3 + IL-2 group. In addition, QXMSC1-IL-3 or QXMSC1-IL-2 also exerted apparent effects on accelerating immune reconstitution, but these effects were far less than that of QXMSC1-IL-3 + IL-2. Our results demonstrated that stromal cell-mediated IL-3 and IL-2 gene therapy may be a potent approach in promoting immunologic and hematopoietic reconstitution after allo-TCD-BMT.     

Letter to the EditorActivin A: A Possible Marker for Liver Transplant Outcome

Abstract

The influence of recombinant human IL-17 on granulocyte-macrophage (CFU-GM) and erythroid (BFU-E and CFU-E) progenitors and the release of IL-1α/β, IL-6 and erythropoietin (EPO) was estimated in the bone marrow cells obtained from normal and sub-lethally irradiated mice. In normal mice IL-17 increased CFU-GM and BFU-E and reduced CFU-E derived colonies numbers and augmented release of IL-6 and EPO. In irradiated mice the effects of IL-17 on hematopoietic progenitors were lineage-dependent, as well as dependent on their stage of differentiation and the time after the irradiation. IL-17 had no major effects on CFU-GM on day 1 and 3, but decreased their number on day 2, while enhanced both BFU-E and CFU-E on day 1 and 2 after irradiation, whereas on day 3 its effect on erythroid progenitors was again as observed in normal mice. After irradiation, IL-17 increased the release of IL-1α, IL-6 and EPO. The observed effects suggested the involvement of IL-17 in the regulation of hematopoiesis and indicated that its effects on both hematopoietic progenitors and cytokine release are dependent on the physiological/ pathological status of the organism.     

Megakaryocytopoiesis in vitro: from the stem cells' perspective

Megakaryocytopoiesis is a complex network regulated by different megakaryocyte (MK)-stimulating factors (i.e., thrombopoietin [TPO], stem cell factor [SCF], interleukin 3 [IL-3], IL-6, IL-11 and GM-CSF). Although all of these factors can affect human and murine megakaryocytopoiesis at different levels of MK development, the effect on very primitive hematopoietic stem cells (HSC) is not well understood. We have further characterized the in vitro biological activity of recombinant murine TPO, SCF and IL-3 on the maturation and proliferation of MK progenitors from different murine primitive hematopoietic cells in a fibrin clot system under serum-free conditions. Neither TPO nor SCF alone induced MK colony formation (CFU-MK) from Lin- Sca+ cells. However, isolated large and mature MKs were observed in the presence of TPO. In contrast, IL-3 exerted a potent effect on CFU-MK formation from Lin- Sca+ cells. On this population of HSC, a significant increase of large MK colonies with mature MK were obtained under those conditions in which TPO was combined with IL-3 or SCF plus IL-3. Similar results were obtained with murine bone marrow cells enriched by primitive progenitors from day 3 post-5-fluorouracil treated mice (5-FUBMC). In contrast, TPO-sensitive precursors were detected in fetal liver cells (FLC). These cells differentiate and proliferate to MK progenitors in the presence of TPO. A significant increase in the number of CFU-MK was induced when TPO was combined with either IL-3 or SCF. On these populations of primitive hematopoietic progenitors, IL-3 induced both the proliferation and differentiation of MK progenitors. Because erythropoietin and TPO share similarities between their molecules and their receptors, we studied whether these growth factors may modulate megakaryocytopoiesis from FLC. Flow cytometry analysis of FLC expressing erythroid markers demonstrated that these cells expressed c-Mpl receptor. In our in vitro studies, although EPO by itself did not induce MK colonies from FLC, it enhanced the proliferative activity of TPO. High ploidy and proplatelet-shedding MK were observed in Lin- Sca+ cells, 5-FUBMC and FLC stimulated with TPO alone or in combination with other MK-stimulating factors. Based on these observations, we propose that TPO, IL-3 and SCF constitute early MK-acting factors with differential proliferative and differentiative activities on murine stem cells. TPO by itself does not appear to be involved in the proliferation of MK progenitors from bone marrow HSC. TPO appears to induce in these cells the commitment toward MK differentiation. However, this growth factor may enhance the proliferative activity of IL-3. IL-3 is an early MK-stimulating factor able to induce in vitro the proliferation and differentiation of MK progenitors from HSC.     

小鼠胎盘间充质干细胞移植对造血系统自然衰老的影响

背景：造血系统作为人体重要组成部分,随着年龄的增长会出现功能不断衰退。 目的：观察小鼠胎盘间充质干细胞对小鼠造血系统自然衰老的延缓作用。 方法：取孕13.5 d Balb/c小鼠胎盘,制备胎盘间充质干细胞。6月龄Balb/c小鼠48只随机分均为细胞移植组和对照组。细胞移植组尾静脉输注胎盘间充质干细胞,每月1次,共6次;对照组输注生理盐水。第3个月进行外周血象、骨髓有核细胞计数、成纤维细胞集落培养、造血祖细胞集落培养和外源性脾集落形成单位计数检测;第6个月时增加骨髓切片观察、骨髓细胞重建造血能力测定。 结果与结论：细胞移植组小鼠一般情况优于对照组;干预第3个月,细胞移植组的骨髓有核细胞计数、巨噬系祖细胞、巨核系祖细胞多于对照组,其他指标无明显差别;干预第6个月,除外周血象仍无差别外,其他的上述指标均明显高于对照组;观察期内两组的上述指标均随时间延长呈下降趋势,但细胞移植组下降速度明显慢于对照组,差异有显著性意义(P < 0.05)。组织学观察发现,细胞移植组骨髓造血组织较对照组丰富,而对照组骨髓脂肪化显著增加;实验还发现细胞移植组骨髓细胞的造血重建能力明显优于对照组。结果提示,小鼠胎盘间充质干细胞能够延缓小鼠造血系统的衰退。