外周造血干细胞纯化、体外扩增及多药耐药基因转染的实验研究
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摘要
目的：本研究的目的是寻找最佳细胞因子组合，以及利用RetroNectin提高逆转录病毒介导的多药耐药基因的转染效率，从而为临床治疗做准备。
实验材料和方法：外周血标本均来自2001年15月至2002年5月在天津医科大学附属肿瘤医院住院接受自体外周血干细胞移植的恶性肿瘤患者。标本共22份，男女患者各11人，平均年龄38.8岁。采用CE(卡铂+足叶乙甙)+G-CSF+GM-CSF方案动员后采集。利用磁性细胞分离器对造血干细胞进行分选后，在不同细胞因子组合下进行体外培养：①对照组，不加任何细胞因子；②SCF+IL-3+GM-CSF+TPO；③FL+GM-CSF+IL-4；④SCF+IL-3+FL+TPO；⑤SCF+IL-3+IL-6+FL+GM-CSF；⑥SCF+IL-3+FL+TPO+EPO。观察培养不同时间的细胞总数、CD34+细胞比例及集落形成能力。利用逆转录病毒作载体将多药耐药基因导入造血干细胞，在转染中予细胞因子支持，并利用RetroNectin以提高转染效率。通过Rhodamine 123排出法、RT－PCR、竞争性PCR、集落形成和抗性实验、细胞增殖实验(MTT)检测基因的转染效率及效果。
结果：22份外周血标本中CD34+细胞的比例为1.3±0.28％，分选后CD34+细胞的纯度为90.3±4.35％，回收率为62.39±5.21％。第6组细胞因子组合(SCF+IL-3+FL+TPO
天津医科大学博士学位论文
十EPO)支持下，CD34+细胞的增殖能力最强，所占比例最
高。在该组细胞因子组合支持下，利用RetroNectin可使逆转
录病毒介导的基因转染效率超过20%，转染组的耐药性较未
转染组增强。
结论:在SCF+IL一3+FL+TPO+EPO的支持下，利
用RetroNectin可以使逆转录病毒介导的多药耐药基因的转
染效率达到20%，基本满足了临床治疗的需要。
Objective: Our research was designed to investigate the appropriate combination of hematopoietic growth factors (HGF) and increase MDRl transfer efficiency of retrovirus-mediated transduction into peripheral blood stem cells (PBSC).
Materials and methods: From May 2001 to May 2002, we enrolled 22 patients with malignant tumors eligible for autologous peripheral blood stem cell transplantation in Tianjin cancer hospital. 11 patients were male, and the others were female. The median age was 38.8 years. PBSC were harvested after mobilization with CBP + Vp16 + G-CSF + GM-CSF. CD34+ cells were isolated by using a high-gradient magnetic cell sorting system (MACS), and expanded with different HGF combination in a liquid culture system. There were six groups: (1)control, without any HGF; (2)SCF + IL-3 + GM-CSF + TPO; (3)FL + GM-CSF + IL-4; (4)SCF + IL-3 + FL + TPO; (5)SCF + IL-3 + IL-6 + FL + GM-CSF; (6)SCF + IL-3 + FL + TPO + EPO. We characterized the expanded cells by total cells counting, flow cytometry and colony forming units. MDRl was transferred into PBSC by retrovirus in the presence of HGF and RetroNectin. We examined the transfection and expression of MDRl by Rhodamine 123 efflux assay, RT-PCR, competitive
PCR, colony forming units culture after exposure to Taxol and MTT colorimetric assay.
Results: The proportion of CD34+ cells in mobilized PB was l.3± 0.28%. Overall yield was 62.39±5.21% and overall purity was 90.3±4.35%. The proliferation and clonogenic potential of CD34+ cells were significantly higher in the presence of SCF + 1L-3 + FL + TPO + EPO, and the percentage of CD34+ cells in total cells were highest. MDR1 was tranducted into PBSC by retrovirus in the presence of HGF and RetroNectin. Gene tansfer efficiency was over 20%, and the resistance to Taxel and Vincristime was higher.
Conclusion: The gene transfer efficiency was over 20% in the presence of SCF + IL-3 + FL + TPO + EPO and RetroNectin. The efficiency of this gene transfer system provides a foundation for ameliorating combination chemotherapy toxicity in clinical trial.
实验材料和方法：外周血标本均来自2001年15月至2002年5月在天津医科大学附属肿瘤医院住院接受自体外周血干细胞移植的恶性肿瘤患者。标本共22份，男女患者各11人，平均年龄38.8岁。采用CE(卡铂+足叶乙甙)+G-CSF+GM-CSF方案动员后采集。利用磁性细胞分离器对造血干细胞进行分选后，在不同细胞因子组合下进行体外培养：①对照组，不加任何细胞因子；②SCF+IL-3+GM-CSF+TPO；③FL+GM-CSF+IL-4；④SCF+IL-3+FL+TPO；⑤SCF+IL-3+IL-6+FL+GM-CSF；⑥SCF+IL-3+FL+TPO+EPO。观察培养不同时间的细胞总数、CD34+细胞比例及集落形成能力。利用逆转录病毒作载体将多药耐药基因导入造血干细胞，在转染中予细胞因子支持，并利用RetroNectin以提高转染效率。通过Rhodamine 123排出法、RT－PCR、竞争性PCR、集落形成和抗性实验、细胞增殖实验(MTT)检测基因的转染效率及效果。
结果：22份外周血标本中CD34+细胞的比例为1.3±0.28％，分选后CD34+细胞的纯度为90.3±4.35％，回收率为62.39±5.21％。第6组细胞因子组合(SCF+IL-3+FL+TPO
天津医科大学博士学位论文
十EPO)支持下，CD34+细胞的增殖能力最强，所占比例最
高。在该组细胞因子组合支持下，利用RetroNectin可使逆转
录病毒介导的基因转染效率超过20%，转染组的耐药性较未
转染组增强。
结论:在SCF+IL一3+FL+TPO+EPO的支持下，利
用RetroNectin可以使逆转录病毒介导的多药耐药基因的转
染效率达到20%，基本满足了临床治疗的需要。
Objective: Our research was designed to investigate the appropriate combination of hematopoietic growth factors (HGF) and increase MDRl transfer efficiency of retrovirus-mediated transduction into peripheral blood stem cells (PBSC).
Materials and methods: From May 2001 to May 2002, we enrolled 22 patients with malignant tumors eligible for autologous peripheral blood stem cell transplantation in Tianjin cancer hospital. 11 patients were male, and the others were female. The median age was 38.8 years. PBSC were harvested after mobilization with CBP + Vp16 + G-CSF + GM-CSF. CD34+ cells were isolated by using a high-gradient magnetic cell sorting system (MACS), and expanded with different HGF combination in a liquid culture system. There were six groups: (1)control, without any HGF; (2)SCF + IL-3 + GM-CSF + TPO; (3)FL + GM-CSF + IL-4; (4)SCF + IL-3 + FL + TPO; (5)SCF + IL-3 + IL-6 + FL + GM-CSF; (6)SCF + IL-3 + FL + TPO + EPO. We characterized the expanded cells by total cells counting, flow cytometry and colony forming units. MDRl was transferred into PBSC by retrovirus in the presence of HGF and RetroNectin. We examined the transfection and expression of MDRl by Rhodamine 123 efflux assay, RT-PCR, competitive
PCR, colony forming units culture after exposure to Taxol and MTT colorimetric assay.
Results: The proportion of CD34+ cells in mobilized PB was l.3± 0.28%. Overall yield was 62.39±5.21% and overall purity was 90.3±4.35%. The proliferation and clonogenic potential of CD34+ cells were significantly higher in the presence of SCF + 1L-3 + FL + TPO + EPO, and the percentage of CD34+ cells in total cells were highest. MDR1 was tranducted into PBSC by retrovirus in the presence of HGF and RetroNectin. Gene tansfer efficiency was over 20%, and the resistance to Taxel and Vincristime was higher.
Conclusion: The gene transfer efficiency was over 20% in the presence of SCF + IL-3 + FL + TPO + EPO and RetroNectin. The efficiency of this gene transfer system provides a foundation for ameliorating combination chemotherapy toxicity in clinical trial.
引文
[1] 王晓，裴雪涛．造血干／祖细胞研究进展．生物学通报，2000；35(10)：1-3．
[2] Lopez M, Beaujean F. Positive selection of autologous peripheral blood stem cells. Baillieres Best Pract Res Clin Haematol. 1999; 12(1-2):71-86.
[3] Maurer AM, Liu Y, Caen JP, et al. Ex vivo expansion of megakaryocytic cells. Int J Hematol. 2000; 71(3):203-10.
[4] Keil F, Elahi F, Greinix HT, et al. Ex vivo expansion of long-term culture initiating marrow cells by IL-10, SCF, and IL-3.Transfusion. 2002; 42(5):581-7.
[5] 崔秀珍，邵莹，任宝柱，等．化疗加G-CSF和GM-CSF联合动员自体外周血干细胞．中华血液学杂志．
[6] Peschle C, Botta R, Muller R, et al. Purification and functional assay of pluripotent hematopoietic stem cells. Rev Clin Exp Hematol. 2001; 5(1):3-14.
[7] Rizzo JD, Elias AD, Stiff PJ, et al. Autologous stem cell transplantation for small cell lung cancer. Biol Blood Marrow Transplant. 2002; 8(5):273-80.
[8] Tomas JF, Perez-Carrion R, Escudero A, et al. Results of a pilot study of 40 patients using high-dose therapy with hematopoietic rescue after standard-dose adjuvant therapy for
high-risk breast cancer. Bone Marrow Transplant. 1997;19(4) :331-6．
[9] Tokuda Y, Ohta M, Okumura A, et al. High-dose chemotherapy with autologous hematopoietic stem-cell transplantation in breast cancer. Cancer Chemother Pharmacol. 1997;40 Suppl:S94-9．
[10] Montemurro F, Ueno NT, Rondon G, et al. High-dose chemotherapy with hematopoietic stem-cell transplantation for breast cancer: current status, future trends. Clin Breast Cancer. 2000 Oct;1(3) :197-209．
[11] Baynes RD, Dansey RD, Klein JL, et al. High-dose chemotherapy and hematopoietic stem cell transplantation for breast cancer: past or future? Semin Oncol. 2001;28(4) :377-88．
[12] Koscielniak E, Morgan M, Treuner J. Soft tissue sarcoma in children: prognosis and management. Paediatr Drugs. 2002;4(1) :21-8．
[13] Matthay KK. Intensification of therapy using hematopoietic stem-cell support for high-risk neuroblastoma. Pediatr Transplant. 1999;3 Suppl 1:72-7．
[14] Hayashi C, Omuro Y, Okamoto R, et al. Three times high-dose chemotherapy and peripheral blood stem cell transplantation rescue for patient with refractory ovarian germ cell tumor. Nippon Naika Gakkai Zasshi. 2002;91(12) :3500-2．
[15] Ferrandina G, Pierelli L, Perillo A, et al. Lymphocyte recovery in advanced ovarian cancer patients after high-dose chemotherapy and peripheral blood stem cell plus growth factor
support: clinical implications. Clin Cancer Res. 2003;9(1) :195-200．
[16] Vose JM, Bierman PJ, Lynch JC, et al. Transplantation of highly purified CD34+Thy-1+ hematopoietic stem cells in patients with recurrent indolent non-Hodgkin's lymphoma. Biol Blood Marrow Transplant. 2001;7(12) :680-7．
[17] Moore MA. Cytokine and chemokine networks influencing stem cell proliferation, differentiation, and marrow homing. J Cell Biochem Suppl. 2002;38:29-38．
[18] Bensinger WI. Recent developments in hematopoietic stem cell transplantation for multiple myeloma. Int J Hematol. 2003;77(3) :232-8．
[19] Wu AG, Michejda M, Mazumder A, et al. Analysis and characterization of hematopoietic progenitor cells from fetal bone marrow, adult bone marrow, peripheral blood, and cord blood. Pediatr Res. 1999;46(2) : 163-9．
[20] 达万明，裴雪涛．外周血干细胞移植．北京：人民卫生出 版社，2000：8
[21] Gunsilius E, Gastl G, Petzer AL. Hematopoietic stem cells. Biomed Pharmacother. 2001;55(4) : 186-94．
[22] Stadtmauer EA, Schneider CJ, Silberstein LE. Peripheral blood progenitor cell generation and harvesting. Semin Oncol. 1995;22(3) :291-300．
[23] Demirer T, Rowley S, Buckner CD, et al. Peripheral-blood stem-cell collections after paclitaxel, cyclophosphamide, and recombinant human granulocyte colony-stimulating factor in
patients with breast and ovarian cancer. J Clin Oncol. 1995;13(7) :1714-9．
[24] 石松凯，周生余，韩晓红，等．环磷酰胺联合重组人粒 细胞集落刺激因子动员自体外周血干细胞．中华血液学杂志． 1998；19：425-7．
[25] Balduzzi A, Perseghin P, Dassi M, et al. Peripheral blood stem cell collection in children with acute leukemia: effectiveness of the 'DIAVE' mobilizing regimen. Bone Marrow Transplant. 2002;30(7) :413-6．
[26] Preti RA, Lazarus HM, Winter J, et al. Tumor cell depletion of peripheral blood progenitor cells using positive and positive/negative selection in metastatic breast cancer.Cytotherapy. 2001;3(2) :85-95．
[27] Pafumi C, Bosco P, Cavallaro A, et al. Two CD34+ stem cells from umbilical cord blood enrichment methods. Pediatr Hematol Oncol. 2002 ;19(4) :239-45．
[28] Xiao J, Wu YJ, Zhang ZN, et al. Application of Double Immunomagnetic Positive Sorting to Ex Vivo Expansion of Marrow CD34(+)CD59(+) Cells from Patients with Paroxysmal Nocturnal Hemoglobinuria. Zhongguo Shi Yan Xue Ye Xue Za Zhi. 2003;11(2) : 179-83．
[29] Kruger W, Gruber M, Hennings S, et al. Purging and haemopoietic progenitor cell selection by CD34+ cell separation. Bone Marrow Transplant. 1998;21(7) :665-71．
[30] Nieto Y, Shpall EJ. CD34+ blood stem cell transplantation. In: Blood stem cell transplantation. London: Martin Dunitz,
1998;73-170．
[31] Croop JM, Cooper R, Seshadri R, et al. Large-scale mobilization and isolation of CD34+ cells from normal donors. Bone Marrow Transplant. 2000;26(12) :1271-9．
[32] Gryn J, Shadduck RK, Lister J, et al. Factors affecting purification of CD34(+) peripheral blood stem cells using the Baxter Isolex 300i. J Hematother Stem Cell Res. 2002;11(4) :719-30．
[33] Pei X. Stem cell engineering: the new generation of cellular therapeutics. Int J Hematol. 2002 Aug;76 Suppl 1:155-6．
[34] Engelhardt M, Douville J, Behringer D, et al. Hematopoietic recovery of ex vivo perfusion culture expanded bone marrow and unexpanded peripheral blood progenitors after myeloablative chemotherapy. Bone Marrow Transplant. 2001;27(3) :249-59．
[35] Yao M, Fouillard L, Lemoine FM, et al. Ex vivo expansion of CD34-positive peripheral blood progenitor cells from patients with non-Hodgkin's lymphoma: no evidence of concomitant expansion of contaminating bc12/JH-positive lymphoma cells. Bone Marrow Transplant. 2000;26(5) :497-503．
[36] Stiff P, Chen B, Franklin W, et al. Autologous transplantation of ex vivo expanded bone marrow cells grown from small aliquots after high-dose chemotherapy for breast cancer. Blood. 2000;95(6) :2169-74．
[37] Wang X, Pei X, Li L. Studies on the properties of dendritic cells from cord blood CD34+ cells and peripheral blood
monocytes. Zhonghua Xue Ye Xue Za Zhi. 1999;20(11) :583-5．
[38] Salmon P, Arrighi JF, Piguet V, et al. Transduction of CD34+ cells with lentiviral vectors enables the production of large quantities of transgene-expressing immature and mature dendritic cells. J Gene Med. 2001;3(4) :311-20．
[39] Bontkes HJ, De Gruijl TD, Schuurhuis GJ, et al. Expansion of dendritic cell precursors from human CD34(+) progenitor cells isolated from healthy donor blood; growth factor combination determines proliferation rate and functional outcome. J Leukoc Biol. 2002;72(2) :321-9．
[40] Hirohata S, Yanagida T, Tomita T, et al. Bone marrow CD34+ progenitor cells stimulated with stem cell factor and GM-CSF have the capacity to activate IgD-B cells through direct cellular interaction. J Leukoc Biol. 2002;71(6) :987-95．
[41] Suzuki M, Harashima A, Okochi A, et al. Transforming growth factor-beta( 1) augments granulocyte-macrophage colony-stimulating factor-induced proliferation of umbilical cord blood CD34(+) cells with an associated tyrosine phosphorylation of STAT5． Exp Hematol. 2002;30(10) : 1132-8．
[42] Xiao W, Koizumi K, Nishio M, et al. Tumor necrosis factor-alpha inhibits generation of glycophorin A+ cells by CD34+ cells. Exp Hematol. 2002;30(11) : 1238-47．
[43] 苏丽萍．Flt3／FL结构、功能及对脐血造血干／祖细胞体外 扩增作用的研究．国外医学输血及血液学分册． 2001；24(4) ：315-8．
[44] Petzer AL, Hogge DE, Landsdorp PM, et al. Self-renewal
of primitive human hematopoietic cells (long-term-culture-initiating cells) in vitro and their expansion in defined medium. Proc Natl Acad Sci U S A. 1996;93(4) :1470-4．
[45] Cartron G, Binet C, Herault O, et al. Proliferation of human progenitor cells in a long-term culture system is more efficiently sustained by the addition of Flt-3 ligand or megakaryocyte growth and development factor than by Kit ligand. Int J Hematol. 2003;77(2) : 133-41．
[46] Piacibello W, Sanavio F, Severino A, et al. Engraftment in nonobese diabetic severe combined immunodeficient mice of human CD34(+) cord blood cells after ex vivo expansion: evidence for the amplification and self-renewal of repopulating stem cells. Blood. 1999;93(11) :3736-49．
[2] Lopez M, Beaujean F. Positive selection of autologous peripheral blood stem cells. Baillieres Best Pract Res Clin Haematol. 1999; 12(1-2):71-86.
[3] Maurer AM, Liu Y, Caen JP, et al. Ex vivo expansion of megakaryocytic cells. Int J Hematol. 2000; 71(3):203-10.
[4] Keil F, Elahi F, Greinix HT, et al. Ex vivo expansion of long-term culture initiating marrow cells by IL-10, SCF, and IL-3.Transfusion. 2002; 42(5):581-7.
[5] 崔秀珍，邵莹，任宝柱，等．化疗加G-CSF和GM-CSF联合动员自体外周血干细胞．中华血液学杂志．
[6] Peschle C, Botta R, Muller R, et al. Purification and functional assay of pluripotent hematopoietic stem cells. Rev Clin Exp Hematol. 2001; 5(1):3-14.
[7] Rizzo JD, Elias AD, Stiff PJ, et al. Autologous stem cell transplantation for small cell lung cancer. Biol Blood Marrow Transplant. 2002; 8(5):273-80.
[8] Tomas JF, Perez-Carrion R, Escudero A, et al. Results of a pilot study of 40 patients using high-dose therapy with hematopoietic rescue after standard-dose adjuvant therapy for
high-risk breast cancer. Bone Marrow Transplant. 1997;19(4) :331-6．
[9] Tokuda Y, Ohta M, Okumura A, et al. High-dose chemotherapy with autologous hematopoietic stem-cell transplantation in breast cancer. Cancer Chemother Pharmacol. 1997;40 Suppl:S94-9．
[10] Montemurro F, Ueno NT, Rondon G, et al. High-dose chemotherapy with hematopoietic stem-cell transplantation for breast cancer: current status, future trends. Clin Breast Cancer. 2000 Oct;1(3) :197-209．
[11] Baynes RD, Dansey RD, Klein JL, et al. High-dose chemotherapy and hematopoietic stem cell transplantation for breast cancer: past or future? Semin Oncol. 2001;28(4) :377-88．
[12] Koscielniak E, Morgan M, Treuner J. Soft tissue sarcoma in children: prognosis and management. Paediatr Drugs. 2002;4(1) :21-8．
[13] Matthay KK. Intensification of therapy using hematopoietic stem-cell support for high-risk neuroblastoma. Pediatr Transplant. 1999;3 Suppl 1:72-7．
[14] Hayashi C, Omuro Y, Okamoto R, et al. Three times high-dose chemotherapy and peripheral blood stem cell transplantation rescue for patient with refractory ovarian germ cell tumor. Nippon Naika Gakkai Zasshi. 2002;91(12) :3500-2．
[15] Ferrandina G, Pierelli L, Perillo A, et al. Lymphocyte recovery in advanced ovarian cancer patients after high-dose chemotherapy and peripheral blood stem cell plus growth factor
support: clinical implications. Clin Cancer Res. 2003;9(1) :195-200．
[16] Vose JM, Bierman PJ, Lynch JC, et al. Transplantation of highly purified CD34+Thy-1+ hematopoietic stem cells in patients with recurrent indolent non-Hodgkin's lymphoma. Biol Blood Marrow Transplant. 2001;7(12) :680-7．
[17] Moore MA. Cytokine and chemokine networks influencing stem cell proliferation, differentiation, and marrow homing. J Cell Biochem Suppl. 2002;38:29-38．
[18] Bensinger WI. Recent developments in hematopoietic stem cell transplantation for multiple myeloma. Int J Hematol. 2003;77(3) :232-8．
[19] Wu AG, Michejda M, Mazumder A, et al. Analysis and characterization of hematopoietic progenitor cells from fetal bone marrow, adult bone marrow, peripheral blood, and cord blood. Pediatr Res. 1999;46(2) : 163-9．
[20] 达万明，裴雪涛．外周血干细胞移植．北京：人民卫生出 版社，2000：8
[21] Gunsilius E, Gastl G, Petzer AL. Hematopoietic stem cells. Biomed Pharmacother. 2001;55(4) : 186-94．
[22] Stadtmauer EA, Schneider CJ, Silberstein LE. Peripheral blood progenitor cell generation and harvesting. Semin Oncol. 1995;22(3) :291-300．
[23] Demirer T, Rowley S, Buckner CD, et al. Peripheral-blood stem-cell collections after paclitaxel, cyclophosphamide, and recombinant human granulocyte colony-stimulating factor in
patients with breast and ovarian cancer. J Clin Oncol. 1995;13(7) :1714-9．
[24] 石松凯，周生余，韩晓红，等．环磷酰胺联合重组人粒 细胞集落刺激因子动员自体外周血干细胞．中华血液学杂志． 1998；19：425-7．
[25] Balduzzi A, Perseghin P, Dassi M, et al. Peripheral blood stem cell collection in children with acute leukemia: effectiveness of the 'DIAVE' mobilizing regimen. Bone Marrow Transplant. 2002;30(7) :413-6．
[26] Preti RA, Lazarus HM, Winter J, et al. Tumor cell depletion of peripheral blood progenitor cells using positive and positive/negative selection in metastatic breast cancer.Cytotherapy. 2001;3(2) :85-95．
[27] Pafumi C, Bosco P, Cavallaro A, et al. Two CD34+ stem cells from umbilical cord blood enrichment methods. Pediatr Hematol Oncol. 2002 ;19(4) :239-45．
[28] Xiao J, Wu YJ, Zhang ZN, et al. Application of Double Immunomagnetic Positive Sorting to Ex Vivo Expansion of Marrow CD34(+)CD59(+) Cells from Patients with Paroxysmal Nocturnal Hemoglobinuria. Zhongguo Shi Yan Xue Ye Xue Za Zhi. 2003;11(2) : 179-83．
[29] Kruger W, Gruber M, Hennings S, et al. Purging and haemopoietic progenitor cell selection by CD34+ cell separation. Bone Marrow Transplant. 1998;21(7) :665-71．
[30] Nieto Y, Shpall EJ. CD34+ blood stem cell transplantation. In: Blood stem cell transplantation. London: Martin Dunitz,
1998;73-170．
[31] Croop JM, Cooper R, Seshadri R, et al. Large-scale mobilization and isolation of CD34+ cells from normal donors. Bone Marrow Transplant. 2000;26(12) :1271-9．
[32] Gryn J, Shadduck RK, Lister J, et al. Factors affecting purification of CD34(+) peripheral blood stem cells using the Baxter Isolex 300i. J Hematother Stem Cell Res. 2002;11(4) :719-30．
[33] Pei X. Stem cell engineering: the new generation of cellular therapeutics. Int J Hematol. 2002 Aug;76 Suppl 1:155-6．
[34] Engelhardt M, Douville J, Behringer D, et al. Hematopoietic recovery of ex vivo perfusion culture expanded bone marrow and unexpanded peripheral blood progenitors after myeloablative chemotherapy. Bone Marrow Transplant. 2001;27(3) :249-59．
[35] Yao M, Fouillard L, Lemoine FM, et al. Ex vivo expansion of CD34-positive peripheral blood progenitor cells from patients with non-Hodgkin's lymphoma: no evidence of concomitant expansion of contaminating bc12/JH-positive lymphoma cells. Bone Marrow Transplant. 2000;26(5) :497-503．
[36] Stiff P, Chen B, Franklin W, et al. Autologous transplantation of ex vivo expanded bone marrow cells grown from small aliquots after high-dose chemotherapy for breast cancer. Blood. 2000;95(6) :2169-74．
[37] Wang X, Pei X, Li L. Studies on the properties of dendritic cells from cord blood CD34+ cells and peripheral blood
monocytes. Zhonghua Xue Ye Xue Za Zhi. 1999;20(11) :583-5．
[38] Salmon P, Arrighi JF, Piguet V, et al. Transduction of CD34+ cells with lentiviral vectors enables the production of large quantities of transgene-expressing immature and mature dendritic cells. J Gene Med. 2001;3(4) :311-20．
[39] Bontkes HJ, De Gruijl TD, Schuurhuis GJ, et al. Expansion of dendritic cell precursors from human CD34(+) progenitor cells isolated from healthy donor blood; growth factor combination determines proliferation rate and functional outcome. J Leukoc Biol. 2002;72(2) :321-9．
[40] Hirohata S, Yanagida T, Tomita T, et al. Bone marrow CD34+ progenitor cells stimulated with stem cell factor and GM-CSF have the capacity to activate IgD-B cells through direct cellular interaction. J Leukoc Biol. 2002;71(6) :987-95．
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