腺病毒介导的hVEGF165基因治疗对骨髓移植后骨髓造血干细胞恢复的影响及其意义

目的：探讨重组腺病毒Ad—EGFP／hVEGF165介导的血管内皮细胞生长因子在骨髓移植后小鼠体内的表达效率及对骨髓造血干细胞恢复的影响。方法：利用细菌内同源重组法快速构建复制缺陷型腺病毒Ad—EGFP／hVEGF165，经尾静脉注射给同基因骨髓移植BALB／c小鼠；在移植后不同时间，通过荧光显微镜、RT—PCR、免疫组化染色和ELISA方法检测外源基因在小鼠体内的表达；取移植后30d小鼠骨髓单个核细胞并计数，随后进行脾集落形成单位试验(CFU—S)以评价VEGF基因转移组小鼠骨髓造血干细胞数量恢复情况。结果：重组腺病毒AdEGFP／hVEGF165介导的VEGF基因转移广泛分布在骨髓移植小鼠心、肺、肝、脾、肾、小肠和骨髓组织；小鼠血浆中VEGF浓度高峰出现在移植后2d；接受重组腺病毒AdEGFP／hVEGF165基因干预治疗组小鼠骨髓单个核细胞计数及其形成的脾集落数量明显高于其他对照组。结论：腺病毒成功介导了hVEGF165基因在骨髓移植小鼠体内的稳定表达，明显促进骨髓移植小鼠骨髓造血干细胞数量的恢复．是骨髓单个核细胞数量增加的根本原因。     

神经导向因子Netrin-1促进骨髓间充质干细胞的血管生成作用

将携带增强型绿色荧光蛋白(EGFP)基因标记的Netrin-1基因重组腺病毒(Ad5-EGFP-Netrin-1)或空载体的腺病毒(Ad5-EGFP)转染到大鼠骨髓间充质干细胞后与Matrigel胶混匀共同移植到大鼠皮下,分别在移植后14d和28d观察Netrin-1和血管内皮细胞生长因子(VEGF)的蛋白表达以及微血管形成数量的情况.结果显示,Netrin-1能促进VEGF的表达和血管形成,移植的细胞数量越多而作用越强(均P＜0.05)     

60Coγ射线左半身照射对小鼠非照射区域骨髓造血组织VEGF表达的影响

目的探讨局部电离辐射对小鼠非照射区域骨髓造血组织血管内皮生长因子（VEGF）表达的影响。方法将6—8周龄雄性昆明小鼠随机分为正常对照组、全身照射组、全身屏蔽照射组以及左半身照射组，用铅屏蔽建立半身照射模型，以8．0Gy^60Coγ射线照射，观察小鼠外周血白细胞和骨髓有核细胞计数，检测血清SOD、MDA变化，用Western blotting和免疫组织化学结合激光共聚焦显微镜观察骨髓造血组织VEGF表达的改变。结果在半身照射条件下，小鼠外周血白细胞降低，未照射侧骨髓有核细胞计数减少，血清MDA升高，SOD降低（与正常对照组比，P〈0．01）；非照射区域骨髓造血组织VEGF表达明显减少，VEGF阳性细胞显著下降（与正常对照组比，P〈0．01）。结论局部电离辐射作用后，可导致非照射区域骨髓造血组织增殖抑制，VEGF表达显著减少，氧自由基激活可能参与了该损伤过程。     

犬骨髓成年多能祖细胞诱导分化血管内皮细胞的实验研究

目的内皮细胞移植对损伤组织的修复治疗至关重要,本研究旨在探讨骨髓成年多能干细胞(MAPCs)体内外诱导分化血管内皮细胞的可行性.方法采用Percoll密度梯度法分离培养MAPCs.应用10 ng/ml血管内皮细胞生长因子(VEGF)对骨髓MAPCs进行体外诱导分化2～3周,使其定向分化成为血管内皮细胞.采用细胞形态学、细胞免疫组化以确定诱导分化的效果.将标记BrdU的MAPCs自体移植于犬心肌内,观察局部微环境对于骨髓MAPCs的分化能力.结果应用10 ng/ml VEGF孵育骨髓MAPCs 2～3周,可见MAPCs分化为血管内皮细胞:细胞形态呈鹅卵石样;形成血管样结构;细胞vWF免疫染色阳性.移植于心肌内的MAPCs在体内形成新生血管,血管内皮BrdU染色与vWF染色均阳性.结论骨髓MAPCs可在体外VEGF诱导下或在体内微环境作用下分化为成熟血管内皮细胞.骨髓MAPCs可为损伤组织的移植修复治疗提供内皮细胞资源.     

老年大鼠缺血再灌注脑组织血管内皮生长表达及地塞米松对其表达的影响

目的　探讨局灶性脑缺血再灌注后血管内皮生长因子 (VEGF)蛋白表达的意义及地塞米松对其影响。　方法　采用老年雄性大鼠短暂性大脑中动脉闭塞 (MCAO)与再灌注模型 ,应用免疫组织化学方法观察大脑中动脉闭塞 90min再灌注 1～ 7d脑组织VEGF蛋白的表达及腹腔内注射地塞米松 ( 2mg·kg-1 ·d-1 ,持续 7d)对VEGF蛋白表达及脑组织含水量的影响。　结果　实验大鼠再灌注1h ,缺血半暗带神经元及同侧大脑中动脉供血区软脑膜开始表达VEGF ,1d达峰值 (P <0 0 1) ,前者于 1d后快速下降 ,后者维持高水平至 7d。缺血半暗带脑血管内皮细胞在 1～ 7d也有较丰富VEGF表达 (P <0 0 1)。地塞米松处理组缺血区的脑微血管内皮细胞VEGF表达明显减少 (P <0 0 1)。地塞米松在缺血再灌注 1～ 7d明显减少缺血脑组织含水量 (P <0 0 5)。　结论　局灶性脑缺血再灌注可诱导VEGF表达 ,地塞米松可抑制缺血再灌注脑组织血管内皮细胞VEGF表达     

急性白血病患者骨髓血管新生的研究及临床意义

目的　研究急性白血病 (AL)患者骨髓血管新生、骨髓液血管内皮生长因子 (VEGF)水平并探讨其临床意义。方法　采用免疫组化方法 ,以兔抗人Ⅷ相关抗原多克隆抗体标记骨髓内皮细胞 ,光学显微镜下计数骨髓全切片微血管数 ;用ELISA方法检测骨髓液VEGF水平。结果　骨髓微血管数在初发未治组 (2 2 82 /× 4 0 0视野 )明显高于正常对照组 (7 17/× 4 0 0视野 )及骨髓缓解组(8 5 7/× 4 0 0视野 ) (P值均 <0 0 0 1) ,而骨髓缓解组仍高于正常对照组 (P <0 0 5 ) ,初发未治组与复发难治组 (2 1 83/× 4 0 0视野 )之间的差异无显著性 (P >0 0 5 )。初发未治组中 ,骨髓微血管数在 2 3例急性淋巴细胞白血病 (ALL)患者 (2 3 0 9/× 4 0 0视野 )与 4 3例急性非淋巴细胞白血病 (ANLL)患者(2 2 37/× 4 0 0视野 )、FAB亚型M1～ 3 (2 2 91/× 4 0 0视野 )与M4～ 5患者 (2 1 4 6 /× 4 0 0视野 )之间的差异均无显著性 (P值均 >0 0 5 ) ,而骨髓微血管数与骨髓原始细胞百分数呈正相关性 (r =0 311,P <0 0 1)。骨髓液VEGF水平 ,在初发未治组 (188 88ng/L)显著高于骨髓缓解组 (78 74ng/L)和正常对照组 (79 5 2ng/L) (P值均 <0 0 1) ,而后两者之间的差异无显著性 (P >0 0 5 ) ;在ANLL组(2 70 12ng/L)显著高于ALL组 (1     

再生障碍性贫血患者术后贫血改善分析

目的：探讨血管内皮细胞生长因子（VEGF）与骨髓造血功能之间的关系。方法：采用双抗体夹心ELISA法检测再生障碍性贫血（AA）患者手术前后血清VEGF含量;采用全自动五分类细胞计数仪检测血常规;留取AA患者手术前后的骨髓液,涂片,进行瑞特染色后检测骨髓原始和幼稚细胞百分率。结果：AA患者术后VEGF水平较术前明显增高（P〈0．05）;贫血状况得到明显改善。结论：血清VEGF水平提高与改善骨髓造血状况关系密切。     

~(60)Co γ射线左半身照射对小鼠非照射区域骨髓造血组织VEGF表达的影响

目的探讨局部电离辐射对小鼠非照射区域骨髓造血组织血管内皮生长因子(VEGF)表达的影响。方法将6~8周龄雄性昆明小鼠随机分为正常对照组、全身照射组、全身屏蔽照射组以及左半身照射组,用铅屏蔽建立半身照射模型,以8.0Gy60Coγ射线照射,观察小鼠外周血白细胞和骨髓有核细胞计数,检测血清SOD、MDA变化,用Western blotting和免疫组织化学结合激光共聚焦显微镜观察骨髓造血组织VEGF表达的改变。结果在半身照射条件下,小鼠外周血白细胞降低,未照射侧骨髓有核细胞计数减少,血清MDA升高,SOD降低(与正常对照组比,P<0.01);非照射区域骨髓造血组织VEGF表达明显减少,VEGF阳性细胞显著下降(与正常对照组比,P<0.01)。结论局部电离辐射作用后,可导致非照射区域骨髓造血组织增殖抑制,VEGF表达显著减少,氧自由基激活可能参与了该损伤过程。     

激光心肌血运重建术与血管内皮生长因子基因治疗慢性缺血性心脏病的对比研究

目的:探索激光生物效应和血管内皮生长因子(VEGF)基因治疗促进缺血区血管再生的作用。方法:心肌缺血模型猪分别进行激光心肌血运重建术(TMLR)和PcDNA3·1VEGF165基因治疗,6周后行冠状动脉造影、单光子发射型计算机断层扫描(SPECT)、逆转录-聚合酶链反应(RT-PCR)病理学检查。结果:TMLR、基因治疗组冠状动脉造影显示侧支循环明显改善,SPECT显示缺血侧壁灌注显著改善,RT-PCR显示TMLR组VEGF基因有中度表达,基因治疗组有高度或中度表达,心肌血管面积、血管周长、血管数目明显增加(P<0·05);TMLR组血管管径≤25μm数目比基因组明显增加(P<0·05)。结论:TMLR或VEGF基因治疗可以增加心肌血管密度并改善缺血心肌的灌注;TMLR疗效优于VEGF心肌直接注射。     

自体造血干细胞移植治疗急性白血病的临床观察

目的：评价自体造血干细胞移植治疗急性白血病的疗效;观察多疗程中大剂量阿糖胞苷（Ara-C）治疗后外周血干细胞采集效果。方法：17例急性白血病患者行自体造血干细胞移植,其中骨髓移植4例,外周血造血干细胞移植13例,在外周血造血干细胞移植的患者中有4例在诱导和巩固治疗中采用中大剂量Ara—C2疗程或2疗程以上,观察不同组患者造血干细胞采集情况及造血重建情况,并监测患者的疗效。结果：外周血干细胞移植的患者采集的中位MNC数和中位CD34^＋细胞数明显高于骨髓组,差异有统计学意义（均P〈0．05）;使用中大剂量Ar〉C2疗程或2疗程以上的患者采集的中位MNC和中位CD34^＋细胞数与未使用的患者比较差异无统计学意义（P〉0．05）;外周血干细胞移植患者中性粒细胞〉0．5&#215;10^9／L和血小板〉20&#215;10^9／L的时间较骨髓移植患者短,差异有统计学意义（P〈0．01）;4例患者在复发状态行自体造血干细胞移植,移植后中位生存期9．5个月,缓解期行自体造血干细胞移植的移植后中位生存期81个月,差异有统计学意义（P〈0．05）;17例患者中位生存期（OS）84个月,中位无白血病生存期（DFS）81个月,患者3年无白血病存活率为（68．25&#177;11．23）％。结论：自体外周血造血干细胞移植比自体骨髓移植造血重建快;2疗程或2疗程以上的中大剂量Ara-C治疗的患者仍能采集到足量的外周造血干细胞;缓解期行自体造血干细胞移植可获得较好的OS和DFS。     

Enhancement of thymopoiesis after bone marrow transplant by in vivo interleukin-7

Bone marrow transplantation (BMT) is followed by a period of profound immune deficiency, during which new T lymphocytes are generated from either stem cells or immature thymic progenitors. Interleukin-7 (IL-7) induces proliferation and differentiation of immature thymocytes. We examined whether the in vivo administration of IL-7 to mice receiving BMT would alter thymic reconstitution. Lethally irradiated C57BL/6 mice received syngeneic BMT, followed by either IL-7 or placebo from days 5 to 18 post-BMT. At day 28, BMT recipients that had not received IL-7 had profound thymic hypoplasia (< 5% of normal), with relative increases in the numbers of immature thymocytes, decreased numbers of mature peripheral (splenic) T lymphocytes, and severely impaired T- and B-cell function. In contrast, transplanted mice treated with IL-7 had normalization of thymic cellularity, with normal proportions of thymic subsets and T-cell receptor beta variable gene (TCRV beta) usage, normal numbers of peripheral CD4+ T lymphocytes, and improved antigen- specific T- and B-cell function. In the BMT-IL-7 mice, there was an eightfold increase in the number of immature CD3-CD4-CD8- thymocytes in G2-M of the cell cycle, indicating that restoration of thymic cellularity was due to enhanced proliferation of immature thymic progenitors. Similar effects following IL-7 administration were also observed when donor bone marrow was depleted of mature T lymphocytes, indicating that IL-7 administration affected immature hematopoietic progenitors. IL-7 promotes thymic reconstitution after BMT, and may be useful in preventing post-BMT immune deficiency.     

Optimization of multidrug resistance 1 gene transfer confers chemoprotection to human hematopoietic cells engrafted in immunodeficient mice

OBJECTIVE: To investigate whether an optimization of MDR1 gene transfer protocol would result in stable hematopoietic stem cell (HSC) engraftment and myeloprotection in non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice after paclitaxel chemotherapy. METHODS: We transplanted freshly isolated CD34+ cells or MDR1-transduced CD34+ cells derived from human umbilical cord blood (UCB) into sublethally irradiated NOD/SCID mice. Twenty-eight days after transplantation, mice received paclitaxel chemotherapy and peripheral blood (PB) was collected for analysis of WBC, RBC and PLT counts once every week. RESULTS: We found that MDR1-transduced human hematopoietic cells could facilitate hematopoietic recovery and completely reconstitute hematopoiesis in mice as well as freshly isolated CD34+ cells. Mice transplanted with MDR1-transduced human hematopoietic cells were protected from paclitaxel chemotherapy with higher survival rate and higher level of WBC counts and RBC counts compared with mice transplanted with untransduced HSCs. We also demonstrated that hematopoietic cells transduced with MDR1 gene were enriched in vivo after paclitaxel chemotherapy determined by the higher percentage of human Rh-123(dull) CD45+ cells in bone marrow of mice. CONCLUSION: Our results demonstrated successful chemoprotection against myelosuppression in mice by MDR1-transduced repopulating human hematopoietic cells with an optimized transduction protocol.     

Rapid immune reconstitution and dendritic cell engraftment post-bone marrow transplantation with heterogeneous progenitors and GM-CSF treatment

OBJECTIVE: Bone marrow/hematopoietic stem cell transplantation (BMT) has been the treatment of choice for severe hematological diseases and cancers. Rapid host immune recovery following BMT is critical for reducing complications and improving therapeutic outcome. Here we report manipulations that facilitate rapid immune and dendritic cell (DC) reconstitution post-BMT for improvement in therapeutic outcome of BMT-based disease treatment. METHODS: Using lentiviral vector-modified or unmodified murine hematopoietic stem cells, we examined the engraftment efficiency and kinetics in immune reconstitution of unfractionated bone marrow cells (BM), lineage marker-negative (Lin-) hematopoietic progenitor cells (HPC), or purified Lin-Sca-1+ hematopoietic stem cells (HSC) at an equal hematopoietic progenitor number. RESULTS: Our study revealed that BM reconstituted host primary and secondary lymphoid tissues more efficiently and rapidly. Moreover, in a competitive BMT setting using lentiviral vector-engineered BM and HSC expressing GFP or DsRed respectively, we showed that GM-CSF treatment further enhanced DC reconstitution to therapeutic relevant level as early as 2 weeks post-BMT. On the other hand, Flt3 ligand was less effective in enhancing DC reconstitution till 3 weeks post-BMT. This accelerated DC engraftment by GM-CSF treatment correlated well with improved overall immune reconstitution and enhanced activation of antigen-specific T cells post-BMT. CONCLUSION: This study suggests that use of heterogeneous BM for transplantation facilitates more rapid immune reconstitution, especially in the presence of DC-stimulating cytokines. This improved immune reconstitution would provide additional therapeutic benefits for BMT-based immunotherapy and gene therapy of genetic disorders and cancers.     

Promotion of murine marrow alloengraftment and hematopoietic recovery across the major histocompatibility barrier by administration of recombinant human interleukin-1 alpha.

Irradiated C57BL/6 (H-2b) recipients of T-cell-depleted (TCD) BALB/c (H-2d) bone marrow (BM) and recombinant interleukin-1 alpha (IL-1 alpha) (1 microgram/d) had a significantly (P less than or equal to .006) higher 100-day actuarial survival rate, accelerated hematopoietic recovery, and higher levels of alloengraftment than a group of transplanted control mice treated identically, but given phosphate-buffered saline (PBS). To elucidate the mechanisms involved with IL-1 alpha-induced promotion of alloengraftment and hematopoietic recovery, we performed sequential splenic FACS studies on transplanted mice and secondary transfer studies in syngeneic mice given IL-1 alpha or PBS. Splenic phenotyping showed that recipients of IL-1 alpha had a higher proportion of donor granulocytes (52% v 19%) as compared with PBS controls as early as 7 days after bone marrow transplantation (BMT). On day 11 post-BMT, recipients of IL-1 alpha had a more than fourfold increase in splenocyte number, which included a higher percentage (90% v 59%) of donor cells, especially donor granulocytes (52% vs 32%), and a sevenfold increase in donor T cells as compared with controls. Host T-cell numbers were not affected. Taken together, these data suggest that IL-1 alpha stimulated bipotential (myeloid and lymphoid) donor cell engraftment. In a syngeneic BMT system, administration of IL-1 alpha resulted in a higher incidence of survival when recipients were transplanted with BM cells, indicating that IL-1 alpha administration probably either expanded or potentiated engraftment of a committed progenitor cell pool. Secondary transfer experiments using marrow from IL-1 alpha-treated mice showed that the number of day 12 colony-forming unit-spleen (CFU-S) cells was unaltered compared with untreated control mice, suggesting that more primitive, albeit committed, hematopoietic progenitor cells were not affected. We also examined the potential additive effects of IL-1 alpha and granulocyte-macrophage colony-stimulating factor (GM-CSF) administered in combination (for 14 days). Mice receiving a suboptimal amount of IL-1 alpha along with GM-CSF had significantly higher levels of donor alloengraftment (92%) with superior hematopoietic recovery, as compared with mice receiving either IL-1 alpha (57%) or GM-CSF (18%) alone.     

A comparison of postengraftment infectious morbidity and mortality after allogeneic partially T cell-depleted peripheral blood progenitor cell transplantation versus T cell-depleted bone marrow transplantation

OBJECTIVE: Postengraftment infections are a major cause of transplant-related morbidity and mortality following allogeneic hematopoietic stem cell transplantation (allo-SCT). Allogeneic peripheral blood progenitor cell transplantation (PBPCT) is associated with faster hematopoietic recovery compared to bone marrow transplantation (BMT) and unmanipulated PBPCT may be associated with fewer postengraftment infections. We set out to evaluate and compare the incidence, cause, and outcome of postengraftment infections following HLA-identical sibling T cell-depleted PBPCT vs T cell-depleted BMT between days 30 and 365 posttransplant. PATIENTS: Forty recipients of peripheral blood progenitor cells (PBPC) and 47 recipients of bone marrow (BM) were included. The two groups of patients were comparable with respect to their baseline characteristics. RESULTS: PBPC grafts contained significantly more CD34+ cells and PBPCT was associated with significantly faster neutrophil and lymphocyte recovery as compared to BMT. PBPC recipients experienced more chronic graft-vs-host disease (GVHD; 55% vs 34%; p=0.02). The number of definite and clinical infections per 100 patient days was comparable between recipients of PBPC and BM with similar contribution of causative microorganisms. At one year post SCT, 68% of PBPC recipients had experienced at least one CTC grade 3-4 infection vs 65% of BM recipients. Treatment-related mortality at one year from transplantation was 34% after PBPCT vs 30% after BMT, and no difference in infection-related mortality was observed. CONCLUSION: Postengraftment infectious morbidity and mortality were comparable between recipients of PBPC and BM despite a higher CD34+ cell content of PBPC grafts and faster lymphocyte recovery after PBPCT, which may in part be explained by the higher incidence of chronic GVHD.     

Bone marrow transplantation with interleukin-2-activated bone marrow followed by interleukin-2 therapy for acute myeloid leukemia in mice

We have investigated approaches to induce graft-versus-leukemia (GVL) effect in autologous bone marrow transplantation (ABMT) without graft-versus-host disease to improve survival and cure in leukemia. The present study shows that bone marrow transplantation (BMT) using syngeneic bone marrow activated with interleukin-2 (ABM) for 24 hours in vitro, followed by interleukin-2 (IL-2) therapy, was superior to BMT with fresh, syngeneic bone marrow (FBM) in terms of survival and cure in mice with acute myeloid leukemia (P less than .001) and led to normal hematopoietic reconstitution. Addition of IL-2 therapy after BMT with FBM did not improve the results over BMT with FBM alone (P = .98). These results suggest that the GVL effect of ABMT can be enhanced by using ABM for BMT followed by IL-2 therapy without compromising engraftment.     

Use of recombinant human interleukin-2 in conjunction with syngeneic bone marrow transplantation in mice as a model for control of minimal residual disease in malignant hematologic disorders

Unlike allogeneic bone marrow transplantation (BMT), autologous BMT is not accompanied by immune-mediated graft-versus-leukemia (GVL) effects; hence, the relapse rate observed after autologous BMT in malignant hematologic disorders is higher than that observed after allogeneic BMT. Autologous BMT represents a much safer medical procedure available for many patients in need in situations where allogeneic BMT is not feasible or risky. The present experiments were designed to investigate whether it might be possible to combine the therapeutic benefits of autologous BMT with additional immunotherapy after BMT. The tumor model used for investigating GVL effects was the murine B-cell leukemia (BCL1), a spontaneous, nonimmunogenic, highly lethal leukemia of BALB/c origin. BALB/c mice inoculated with 10(3) BCL1 leukemia cells were treated on day-1 with cyclophosphamide 100 mg/kg and transplanted with normal syngeneic BM cells on day 0. High-dose recombinant interleukin-2 (rIL-2) (100,000 Cetus units x 3/day intraperitoneally x 5 consecutive days) was initiated on day +1, +7, or +21 after BMT. Kinetics of lymphocyte reconstitution after syngeneic BMT indicated a steep increase in the absolute number of peripheral blood lymphocytes on days 17 through 24. All experimental groups were observed for relapse. Mice receiving no rIL-2 therapy relapsed and died within 50 days after BMT, whereas mice receiving rIL-2 showed long-term disease-free survival. Optimal time for administration of rIL-2 was noted at 3 weeks post-BMT, with 90% of the mice surviving with no evidence of disease for more than 1 year. Similarly, when 10(4) BCL1 cells were given 1 day after syngeneic BMT to simulate minimal residual disease after syngeneic BMT, rIL-2 therapy administered at 14 days post-BMT seemed effective in prolonging disease-free survival in contrast to the same regimen given at 1 day after BMT. Our data suggest that immunotherapy with rIL-2 should be further investigated as a new immunotherapeutic tool for decreasing the relapse rate after BMT for hematologic malignancies.     

Megakaryocyte growth and development factor stimulates enhanced platelet recovery in mice after bone marrow transplantation

Megakaryocyte growth and development factor (MGDF) is a recently characterized ligand for the cell surface receptor mpl. We have evaluated the effects of polyethylene glycollated recombinant human MGDF (PEG-rHuMGDF) on recovery of hematopoietic cells in mice following bone marrow transplantation (BMT) to support lethal irradiation. Mice treated with PEG-rHuMGDF (50 micrograms/kg/d) had accelerated recovery of platelet numbers compared with BMT mice treated with carrier or recombinant human granulocyte colony-stimulating factor (rHuG-CSF, 72 or 200 micrograms/kg/d). In contrast, PEG-rHuMGDF had no effect on white blood cell (WBC) or red blood cell (RBC) recovery. As previously reported, animals treated with rHuG-CSF had an enhanced recovery of WBC but not platelet or RBC levels. Interestingly, BMT receipient mice treated with the combination of PEG-rHuMGDF and rHuG-CSF showed simultaneous enhanced recovery of both leukocytes and platelets. PEGylated rHuMGDF was found to be considerably more potent than non- PEGylated rHuMGDF in this setting. PEG-rHuMGDF is an effective growth factor for enhancing platelet recovery in mice following BMT either alone or in combination with rHuG-CSF. It will be of interest to evaluate in a clinical setting the ratios of PEG-rHuMGDF and rHuG-CSF for simultaneous administration of these factors and accelerated recovery of both leukocytes and platelets.     

Prevention of corticosteroid-induced osteonecrosis in rabbits by intra-bone marrow injection of autologous bone marrow cells

Objectives. Femoral head osteonecrosis (ON) is a serious complicationof steroid administration. We evaluated bone marrow transplantation(BMT) for preventing corticosteroid-induced ON. Methods. Rabbits, injected with methylprednisolone (MPSL; 20mg/kg), were divided into four groups: (i) MPSL alone; MPSLinjection only, (ii) MPSL+needling; 2 days after MPSL injection,a hole (1.2 mm diameter) was drilled from the outer cortex 2.5cm distal to the proximal end of the greater trochanter, (iii)MPSL+saline; 2 days after MPSL injection, 2 ml saline was injecteddirectly into the bone marrow cavity, and (iv) MPSL+BMT; 2 daysafter MPSL injection, 1 x 10⁷/2 ml bone marrow cells (BMCs)were injected directly into the bone marrow cavity. Platelets,fibrinogen, prothrombin time and total cholesterol in peripheralblood were measured before and after treatment. Tissues werestained with haematoxylin and eosion and terminal deoxynucleotidyl-mediateddeoxyuridine triphosphate nick-end labelling stain and immunostainedfor VEGF, while cell proliferation and viability of whole BMCsin the femur were analysed by cell cycle analysis and [³H]-thymidineuptake. Results. The ON incidence in rabbits treated with MPSL alone,MPSL+needling and MPSL+saline was 72.7, 70.0 and 66.7%, respectively,while in the MPSL+BMT group, the incidence was 0%. Serologicalfindings in the MPSL+BMT group were almost normalized. VEGFand TUNEL staining were reduced in the MPSL+BMT group comparedwith all other groups. There were significantly fewer BMCs inG1 phase from the MPSL+BMT group than the other groups, whileuptake of [³H]-thymidine was significantly increased. Conclusion. Direct injection of autologous BMCs into femursprevents corticosteroid-induced ON following treatment withhigh-dose, short-term steroids. KEY WORDS: Corticosteroid, Osteonecrosis, Animal model, Bone marrow transplantation, Bone marrow cells Submitted 10 June 2007; revised version accepted 15 January 2008.