课题背景：课题组目前取得的研究成果包括：①建立优化体外扩增骨髓间充质干细胞的培养体系，达到临床级应用规模，即(1~2)×106/kg受者体质量。②初步证实骨髓间充质干细胞移植适宜的数量范围为（1~5）×106/kg受者体质量，造血干细胞回输前先期回输体外扩增的骨髓间充质干细胞更有利于造血的重建。③就单独输注间充质干细胞联合有效化疗显示的临床效果，正在进行进一步深入的动物实验。

应用要点：①选题上，目前对间充质干细胞支持造血的临床应用研究主要集中于在造血干细胞移植后或与造血干细胞共移植治疗，以缩短造血功能的恢复时间。而化疗后单独输注体外扩增的骨髓间充质干细胞治疗白血病在国内尚无报道，进一步拓宽了骨髓间充质干细胞在治疗血液病中的应用。②方法上，采用密度梯度离心法分离细胞后，体外以无血清培养扩增骨髓间充质干细胞，在保证细胞纯度的同时，避免了含动物血清培养过程中可能的未知病毒等病原体感染人类，应用于临床更为安全。

偏倚或不足：作为在国内骨髓间充质干细胞单独用于白血病治疗的首次报道，主要不足之处是病例数少，仅1例。由于缺乏相类似的病例作比较，以及尚无足够的理论依据，此法适用性及安全性均有待进一步验证与观察。

摘要
目的：回顾性分析化疗后骨髓抑制期输注体外扩增的异基因骨髓间充质干细胞治疗低增生性复发难治性急性髓细胞白血病的可行性与安全性。
方法：①53岁女性患者1例，2002-01在南京医科大学第一附属医院确诊为急性髓细胞性白血病M0型，予HA、EA等诱导方案化疗后达完全缓解，在随后的4年中给予包括MA、中剂量Ara-C、DA、TA在内的多次巩固治疗以维持完全缓解。2006-01中性粒细胞缺乏，白血病复发。同年3月再次给予TAE方案诱导治疗未缓解，骨髓持续低增生，并持续粒细胞缺乏达半年。②患者有一位HLA配型完全相合的同胞姐姐，为行异体造血干细胞移植，于2006-07再次入院，患者及其家属对治疗知情同意，实验经医院伦理委员会批准。③患者于2006-08-23予CAG方案化疗，即阿柔比星10 mg/d，×8 d；阿糖胞苷15 mg/次，2次/d，×14 d；2006-08-23~2006-09-25给予洁欣300 μg/d。化疗过程中骨髓抑制进一步加重，并出现霉菌性败血症等重症感染。抗感染治疗的同时，于CAG化疗结束后第2天输注经体外扩增的异基因骨髓间充质干细胞，细胞数为1.4×106/kg受者体质量，以促进骨髓造血恢复支持治疗。
结果：异基因骨髓间充质干细胞输注后第15天，中性粒细胞数达到半年多来第1次大于0.5×109 L-1。输注1个月后，外周血白细胞数5.5×109 L-1，中性粒细胞数4.64×109 L-1，血红蛋白量94 g/L，血小板数25×109 L-1，外周血象渐恢复正常水平。复查骨髓涂片显示骨髓三系增生活跃，原始细胞占0.4%，骨髓流式细胞仪检查MRD阴性，骨髓达完全缓解，感染症状逐渐控制。随访半年，患者持续完全缓解，未有移植物抗宿主病等并发症出现。
结论：CAG方案联合输注体外扩增的异体骨髓间充质干细胞有助于衰竭骨髓的造血重建，对长期反复化疗后出现低增生性复发难治急性白血病患者的治疗是一个新的有前途的方法，但其安全性与有效性需要通过临床试验进一步证实。
关键词：骨髓间充质干细胞；急性白血病；粒细胞集落刺激因子；预激

陆世丰，陆化，刘澎，沈文怡，洪鸣，张建富，杨慧，李建勇.CAG预激方案与异基因骨髓间充质干细胞输注联合治疗低增生复发难治急性髓细胞白血病1例[J].中国组织工程研究与临床康复，2008，12(16):3006-3010
[www.zglckf.com/zglckf/ejournal/upfiles/08-16/16k-3006(ps).pdf]

CAG regimen combined with infusion of allogenetic bone marrow mesenchymal stem cells for treatment of hypoplastic relapsed acute myelogenous leukemia: One case report

Abstract

AIM：To analyze the practicability and safety of the infusion of expanded in vitro human bone marrow-derived mesenchymal stem cells (MSCs) for a refractory and relapsed acute myeloid leukemia (AML) with hypo-proliferative bone marrow after chemotherapy.
METHODS: A 53-year-old woman was admitted to the First Affiliated Hospital of Nanjing Medical University in January 2002 and diagnosed with acute myeloid leukemia type M0, according to the French-American-British (FAB) classification. Complete remission (CR) was achieved after two courses of standard AML induction chemotherapy, consisting of homoharringtonine, etoposide, and Ara-C (cytarabine). An additional course of intensive chemotherapy (mitoxantrone, intermediate-dose cytarabine, daunomycin, and pirarubicin) was carried out for four years. In January 2006, a bone marrow examination revealed a hypocellular marrow with 47.6% blast cells. In March 2006, although she received a TAE regimen (pirarubicin, cytarabine, and etoposide), she did not achieve CR. A hypocellular marrow and severe agranulocytosis was maintained for approximately six months. Since she had a HLA-identical sibling sister, the patient was again admitted to hospital in July 2006 for human leukocyte antigen-identical allogeneic stem cell transplantation. Informed consents were obtained from the patient and her family members and the protocol was approved by Hospital Ethics Committee. On August 23rd 2006, she received a CAG regimen which consisted of aclarubicin (10 mg/d for 8 days), Ara-C (15 mg/d, twice daily for 14 days), and granulocyte colony-stimulating factor (G-CSF, 300 μg/d from August 23rd to September 25th). During the course of chemotherapy of CAG, the patient developed severe invasive fungal infection. The patient received anti-infection treatment and infusion of expanded in vitro allogeneic MSCs from her human leukocyte antigen-identical sibling sister on the second day of the CAG regimen. The patient received a total of 1.4×106 mesenchymal cells/kg body mass to promote marrow hematogenesis recovery.
RESULTS: Fifteen days after MSC infusion, neutrophil granulocytes (NG) rose above 0.5×109 L-1. One month after MSC infusion, white blood cells count was 5.5×109 L-1, NG was 4.64×109 L-1, haemoglobin levels were 94 g/L, and platelets were 25×109 L-1. Peripheral blood gradually recovered to normal levels. Bone marrow examination revealed 0.4% blast cells with tri-lineage engraftment, while flow cytometry examination detected no micro-residual disease (MRD). She achieved CR again and recovered from severe invasive fungal infection. In the follow-up for 6 months, the patient maintained status of CR without any evidence of leukemia relapse or graft-versus-host disease.
CONCLUSION: It is suggested that the combination of CAG regimen based on G-CSF priming, followed by infusion of MSCs is a promising new approach for the therapy of refractory and relapsed acute myeloid leukemia with long-term agranulocytosis. However, safety and efficacy need further validation by clinical trials.

Lu SF, Lu H, Liu P, Shen WY, Hong M, Zhang JF, Yang H, Li JY.CAG regimen combined with infusion of allogenetic bone marrow mesenchymal stem cells for treatment of hypoplastic relapsed acute myelogenous leukemia: One case report.Zhongguo Zuzhi Gongcheng Yanjiu yu Linchuang Kangfu 2008;12(16):3006-3010(China)
[www.zglckf.com/zglckf/ejournal/upfiles/08-16/16k-3006(ps).pdf]