再生障碍性贫血患者血清对脐血CD34~+细胞凋亡及其pAkt表达的影响
摘要
目的:比较不同方法分离脐血CD34+细胞的效果,并观察再生障碍性贫血患者血清对脐血CD34+细胞凋亡及其pAkt表达的影响。
方法:
1.采集脐血48份,室温(RT)下保存12、24、48小时,分别用密度离心法和羟乙基淀粉(HES)沉淀法分离脐血单个核细胞(MNC),再用免疫磁珠法纯化CD34+细胞,然后分别对分离前、后CD34+细胞进行计数和台朌蓝染色。
2.将收集的CD34+细胞随机分为三组,分别加入正常人、非重型再生障碍性贫血(NSAA)患者和重型再生障碍性贫血(SAA)患者血清培养24小时。
3.对各组培养后的CD34+细胞分别进行细胞计数、台盼蓝染色测细胞存活率。
4.流式细胞仪检测各组培养后的CD34+细胞的凋亡率。
5.细胞免疫化学法测各组培养后的CD34+细胞的pAkt蛋白表达。
结果:
1.脐血RT下保存48小时CD34+细胞回收率最高,其次为12小时,24小时回收率最低(P<0.01)。HES沉淀法的CD34+细胞回收率均明显高于密度离心法(P<0.01)。各组间细胞存活率差异无统计学意义(P>0.05)。
2.培养24小时,SAA组CD34+细胞浓度和细胞存活率最低,其次为NSAA组,正常对照组的CD34+细胞浓度和细胞存活率最高(P<0.01)。
3.培养24小时,SAA组的细胞凋亡率最高,其次为NSAA组,正常对照组的细胞凋亡率最低(P<0.01)。
4.培养24小时,细胞免疫化学结果显示,SAA组的pAkt蛋白水平最低,其次为NSAA组,正常对照组的pAkt蛋白水平最高(P<0.01)。
5.经同一份血清作用24小时的CD34+细胞凋亡率与细胞内pAkt水平相关性分析显示呈负相关性(r=-0.969,P<0.05)。
结论:
1. HES沉淀法优于密度离心法,且以RT下保存48小时分离为优。
2.再生障碍性贫血患者血清体外可诱导CD34+细胞凋亡,且SAA患者血清作用明显强于NSAA患者血清。
3.再生障碍性贫血患者血清可以下调CD34+细胞pAkt水平,且SAA患者血清作用明显强于NSAA患者血清。
4. pAkt可能参与再生障碍性贫血患者血清诱导CD34+细胞凋亡过程。
Objective: to compare the effect of separating CD34+ cells from cord blood with different methods, and to observe the effect of the serum from patients with aplasitc anemia on the apoptosis of CD34+ cells and the expression of pAkt.
Methods:
1. Collecting 48 cord blood units, separating mononuclear cells by density centrifugal method or hydroxyethyl starch precipitation method after they were kept at room temperature (RT) for 12h, 24h or 48h respectively, purifying CD34+ cells with immunomagnetic beads, counting the number of CD34+ cells before and after separation, and then assessing cell viability by typan blue staining.
2. The collected CD34+ cells were divided randomly into three groups, and were cultured with serum from healthy persons (controls, n=7), or from patients with severe AA (SAA, n=7) or non severe AA (NSAA, n=7) for 24h, respectively.
3. Counting the number of CD34+ cells of each group after incubation, and assessing their viabilities by typan blue staining.
4. Analysising the apoptosis of the CD34+ cells of each group with flow cytometry after incubation.
5. Detecting the expression of pAkt by immunocytochemical staining.
Results:
1. The recovery rates of CD34+ cells were the highest when kept for 48h at RT, lower for 12h, and the lowest for 24h (P<0.01). The recovery rates of CD34+ cells by HES precipitation method were higher than by density centrifugal method (P<0.01). There was no significant differences between the cell survival rates of each group (P>0.05).
2. After incubation for 24h, the CD34+ cell concentration and viability were the lowest in the SAA group, higher in the NSAA group, and the highest in the control group (P<0.01).
3. After incubation for 24h, the CD34+ cell apoptosis rates were the highest in the SAA group, lower in the NSAA group, and the lowest in the control group (P<0.01).
4. After incubation for 24h, the expression of pAkt was the lowest in the SAA group, higher in the NSAA group, and was the highest in the control group (P<0.01).
5. Correlation analysis indicated that there was negative correlation between the apoptosis rates of CD34+ cells and the expressions of pAkt in the cells cultured with the same serum(r=0.969, P<0.05).
Conclusions:
1. HES precipitation method is superior to density centrifugal method, and it is best to separate CD34+ cells when kept for 48h at RT.
2. The serum of patients with AA can induce the apoptosis of CD34+ cells, and the effect of SAA is stronger than that of NSAA.
3. The serum of patients with AA can downregulate the expression of pAkt, and the effect of SAA is stronger than that of NSAA.
4. pAkt protein may be involved in the apoptosis of CD34+ cells induced by the serum of patients with AA.
方法:
1.采集脐血48份,室温(RT)下保存12、24、48小时,分别用密度离心法和羟乙基淀粉(HES)沉淀法分离脐血单个核细胞(MNC),再用免疫磁珠法纯化CD34+细胞,然后分别对分离前、后CD34+细胞进行计数和台朌蓝染色。
2.将收集的CD34+细胞随机分为三组,分别加入正常人、非重型再生障碍性贫血(NSAA)患者和重型再生障碍性贫血(SAA)患者血清培养24小时。
3.对各组培养后的CD34+细胞分别进行细胞计数、台盼蓝染色测细胞存活率。
4.流式细胞仪检测各组培养后的CD34+细胞的凋亡率。
5.细胞免疫化学法测各组培养后的CD34+细胞的pAkt蛋白表达。
结果:
1.脐血RT下保存48小时CD34+细胞回收率最高,其次为12小时,24小时回收率最低(P<0.01)。HES沉淀法的CD34+细胞回收率均明显高于密度离心法(P<0.01)。各组间细胞存活率差异无统计学意义(P>0.05)。
2.培养24小时,SAA组CD34+细胞浓度和细胞存活率最低,其次为NSAA组,正常对照组的CD34+细胞浓度和细胞存活率最高(P<0.01)。
3.培养24小时,SAA组的细胞凋亡率最高,其次为NSAA组,正常对照组的细胞凋亡率最低(P<0.01)。
4.培养24小时,细胞免疫化学结果显示,SAA组的pAkt蛋白水平最低,其次为NSAA组,正常对照组的pAkt蛋白水平最高(P<0.01)。
5.经同一份血清作用24小时的CD34+细胞凋亡率与细胞内pAkt水平相关性分析显示呈负相关性(r=-0.969,P<0.05)。
结论:
1. HES沉淀法优于密度离心法,且以RT下保存48小时分离为优。
2.再生障碍性贫血患者血清体外可诱导CD34+细胞凋亡,且SAA患者血清作用明显强于NSAA患者血清。
3.再生障碍性贫血患者血清可以下调CD34+细胞pAkt水平,且SAA患者血清作用明显强于NSAA患者血清。
4. pAkt可能参与再生障碍性贫血患者血清诱导CD34+细胞凋亡过程。
Objective: to compare the effect of separating CD34+ cells from cord blood with different methods, and to observe the effect of the serum from patients with aplasitc anemia on the apoptosis of CD34+ cells and the expression of pAkt.
Methods:
1. Collecting 48 cord blood units, separating mononuclear cells by density centrifugal method or hydroxyethyl starch precipitation method after they were kept at room temperature (RT) for 12h, 24h or 48h respectively, purifying CD34+ cells with immunomagnetic beads, counting the number of CD34+ cells before and after separation, and then assessing cell viability by typan blue staining.
2. The collected CD34+ cells were divided randomly into three groups, and were cultured with serum from healthy persons (controls, n=7), or from patients with severe AA (SAA, n=7) or non severe AA (NSAA, n=7) for 24h, respectively.
3. Counting the number of CD34+ cells of each group after incubation, and assessing their viabilities by typan blue staining.
4. Analysising the apoptosis of the CD34+ cells of each group with flow cytometry after incubation.
5. Detecting the expression of pAkt by immunocytochemical staining.
Results:
1. The recovery rates of CD34+ cells were the highest when kept for 48h at RT, lower for 12h, and the lowest for 24h (P<0.01). The recovery rates of CD34+ cells by HES precipitation method were higher than by density centrifugal method (P<0.01). There was no significant differences between the cell survival rates of each group (P>0.05).
2. After incubation for 24h, the CD34+ cell concentration and viability were the lowest in the SAA group, higher in the NSAA group, and the highest in the control group (P<0.01).
3. After incubation for 24h, the CD34+ cell apoptosis rates were the highest in the SAA group, lower in the NSAA group, and the lowest in the control group (P<0.01).
4. After incubation for 24h, the expression of pAkt was the lowest in the SAA group, higher in the NSAA group, and was the highest in the control group (P<0.01).
5. Correlation analysis indicated that there was negative correlation between the apoptosis rates of CD34+ cells and the expressions of pAkt in the cells cultured with the same serum(r=0.969, P<0.05).
Conclusions:
1. HES precipitation method is superior to density centrifugal method, and it is best to separate CD34+ cells when kept for 48h at RT.
2. The serum of patients with AA can induce the apoptosis of CD34+ cells, and the effect of SAA is stronger than that of NSAA.
3. The serum of patients with AA can downregulate the expression of pAkt, and the effect of SAA is stronger than that of NSAA.
4. pAkt protein may be involved in the apoptosis of CD34+ cells induced by the serum of patients with AA.
引文
[1]Gluckman E, Broxmeyer HE, Aurebach AD,et al. Hematopoietic Reconstitution in a Patient with Fanconis Anemia by Means of Umbilical-cord Blood from an HLA-identical Sibling[J]. N Engl J Med, 1989, 321:1174.
[2]奚永志,唐佩弦.深入开展脐血干细胞的基础与临床研究.中华血液学杂志, 1999, 8(2):397.
[3]崔正言.干细胞研究的进展与应用前景.中国肿瘤生物治疗杂志, 2001, 8(4):239-242.
[4]沈伯均,隋星卫.脐血移植的评价和前景.中华儿科杂志, 1994, 32(6):381.
[5]Koller MR, Plasson MA, Manchel L, et al. Long-term culture initiating cell expansion is dependent on expansionmedium exchange combined with stromal and other accessory cell effects[J]. Blood, 1995, 86:1784.
[6]Regidor C, Posada M, Monteagudo D, et al. Umbilical cord blood banking for unrelated transplantation: evaluation of cell separation and storage methods[J ] . Exp Hematol, 1999, 27(3) :380-385.
[7]罗伟琼,陈惠珍,黄春柳等.脐血采集方法、影响因素分析及注意事项[J].中国热带医学, 2007, 7(4):647-648。
[8]Wong A, Yuen PM, Li K, et al. Cord blood collection before and after placental delivery: levels of nucleated cells, haematopoietic progenitor cells, leukocyte subpopulations and macroscopic clot [J]. Bone Marrow Transplantation, 2001, 27(2):133-138.
[9]Kenichi S, Mikitomo Y, Ryo S, et al. Cell processing for placental/umbilical cord blood banking. Jpn J Transfusion Med, 1998; 44:12-19.
[10]Shlebak AA, Marley SB, Roberts IA,et al. Optimal timing for processing and cryopreservation of umbilical cord haematopoietic stem cells for clinical transplantalion. Bone Marrow Transplant, 1999, 23: 131- 136.
[11] Bertolini F, Lazzari L, Lauri E, et al. Comparative study of different procedures for the collection and banking of umbilical cord blood [J]. J Hematother, 1995, 4 (1):22-36.
[12] Moldenhauer A, Wolf J, Habermann G, et al. Optimum storage conditions for cord blood-derived hematopoietic progenitor cells prior to isolation [J]. Bone Marrow Transplantation 2007, 40: 837-842.
[13] Majka M, Janowska-Wiezorek A, Ratajczak J, et al. Numerous growth factors, cytokines, and chemokines are secreted by human CD34(+) cells, myeloblasts, erythroblasts, and megakaryoblasts and regulate normal hematopoiesis in an acutocrine/paracrine manner [J ]. Blood 2001, 97:3075-3085.
[14] Kadereit S, Deeds LS, Haynesworth SE, et al, Expansion of LTC-ICs and maintenance of p21 and BCL-2 expression in cord blood CD34(+)/CD38(-) early progenitors cultured over human MSCs as a feeder layer [J ]. Stem cells 2002, 20:573-582.
[15]朱美玲,伍新尧,陈汝光等.不同分离方法分离脐血造血细胞效率的比较.中国输血杂志, 2006, 15(3):179.
[16]奚永志,李秀森.人正常骨髓CD34+造血细胞的形态学与细胞化学特征.中华血液学杂志, 1997, 18(3):131.
[17]罗伟琼,朱美玲,邹翠贤等.脐血有核细胞分离、冻存方法的研究.临床输血与检验, 2007: 9(1):55-56.
[18]蹇在伏,吴新华,陈方平等.两种不同方法分离脐血造血干细胞实验研究[ J].中国现代医学杂志, 2004, 14(19): 68-69.
[19]Regidor C, Posada M, Monteagudo D, et al. Umbilical cord blood banking for unrelated transplantation: evaluation of cell separation and storage methods. Exp Hemat, 1999, 27: 380-385.
[1]Marsh JC. Eur J Haemetol Suppl, 1996, 60: 75-79.
[2]刘红,陆德炎,杨锦媛等.重型再生障碍性贫血患者血清诱导D34+细胞凋亡及PML蛋白表达.中华血液学杂志, 2003, 24(11):596-597.
[3]Stephens L, Anderson K, Stokoe D, et al. Protein kinase B kinase that mediate phosphatidylinositol 3,4,5-trisphosphate-dependent activation of prokinase B [J].Science, 1998, 279(5351) 710-714.
[4]中华血液学学会第四届全国再生障碍性贫血学术会议纪要:再生障碍性贫血诊断标准和疗效标准.中华血液学杂志, 1987, 8: 486和封四.
[5]Scopes J, Bagnara M, Gordon2Smith EC, et al. Haemopoietic progenitor cells are reduced in aplastic anemia. Br J Haematol, 1994, 86: 427.
[6]Philpott NJ, Scopes J, Marsh JC, et al. Increased apoptosis in aplastic anemia bone marrow progenitor cells: possible pathophsiologic significance. Exp J Hematol, 1995; 23(14): 1642-1648.
[7]Callera F, Falcao RP, et al. Increased apoptotic cells in bone marrow biopsies from patients with aplastic anemia. Br J Haematol, 1997,98(1):18-20.
[8]Liu H, Mihara K, Kimura A, et al. Induction of apoptosis in CD34+cells by sera from patients with aplastic anemia. Hiroshima J Med Sci, 1999, 48(2): 57-63.
[9]刘红,吴芳颐,姜胜华等.二例重症再生障碍性贫血患者血清体外加速正常人CD34+细胞凋亡的研究.中华血液学杂志, 1998, 19: 514-517。
[10]Maciejeuski JP, Seller C, Sato T, et al. (1995) Increased expression of Fas antigen on bone marrow CD34+ cells of patients with aplastic anemia. British Journal of Heamatology, 91,245.
[11]Stephens L, andersoh K, Slokoe D, et al. Proetin kinase B Kinase thatmediale phosphalidvl inosiol 3,4,5 trisphosphale activalion of protein kinase B. Science[J], 1998, 279:710.
[12]Staal SP, Hartley JW, Rowe WP, et al. Isolation of transformingmurine leukemia viruses from mice with a high incidence of spontaneous lymphoma[J]. Proc Natl Acad Sci USA, 1977; 74(7):3065-3067.
[13]Alessi DR, Andjelkovic M, Caudwell B, et al. Mechanism of activation of protein kinase B by insulin and IGF-1. EMBO J, 1996, 15(23): 6541-6551.
[14]Alessi DR, Andjelkovic M, Caudwell B, et al. Mechanism of activation of protein kinase B by insulinand IGF-1[J]. EMBO J, 1996; 15(23):6541-6551.
[15]Welch H, Eguinoa A, Stephens LR, Hawkins PT. Protein kinase B andrac are activated in parallel within a phosphatidylinositide 3OH-kinase-controlled signaling pathway[J]. J Biol Chem, 1998; 273(18):11248-11256.
[16]Alessi DR, Andjelkovic M, Caudwell B, et al. Mechanism of activation of protein kinase B by insulin and IGF-1. EMBO J, 1996, 15(23):6541-6551.
[17]Balendran A, Casamayor A, Deak M, et al. PDK1 acquires PDK2 activity in the presence of a synthetic peptide derived from the carboxyl terminus of PRK2[J]. Curr Biol, 1999, 9(8): 393-404.
[18]Mao M et al. The Phosphatidylinositol 3-Kinase/AKT Kinase Pa-thway in Multiple Myeloma Plasma Cells:Roles in Cytokine-dependent Survival and Proliferative Responses. Cancer Res, 2000;60(23):6763-6770.
[19]Hong Liu, YaLing Qiu, et al. Involvement of protein kinase Cεin the negative regulation of AKT activation stimulated by granulocyte colony-stimulating factor. The journal of immunology. 2006, 176:2407-2413
[20]Dedhar S, Williams B, Hannigan G. Integrin-linked kinase(ILK):aregulator of integrin and growth factor signalling [J]. Trends Cell Biol, 1999, 9(8):319-323.
[21]Delcommenne M, Tan C, Gray V,et al. Phosphoinositide-3-OH k-inase-dependent regulation of glycogen synthase kinase 3 and pro-tein kinase B/AKT by the integrin-linked kinase [J]. Proc Natl Acad Sci USA, 1998, 95(19):11211-11216.
[22]Balendran A, Casamayor A, Deak M, et al. PDK1 acquires PDK2 activity in the presence of a synthetic peptide derived from the carboxyl terminus of PRK2[J]. Curr Biol, 1999, 9(8):393-404.
[23]Yamada T, Katagiri H, Asano T, et al. 3-phosphoinositide-dependent protein kinase 1, an Akt kinase, is involved in dephosphorylation of Thr-308 of Akt1 in Chinese hamster ovary cells[J]. J Biol Chem, 2001, 276(7):5339-5345.
[24]Schubert KM, Scheid MP, Duronio V. Ceramide inhibits protein kinase B/Akt by promoting dephosphorylation of serine473[J]. J Biol Chem, 2000, 275(18):13330-13335.
[25]Sato S, Fujita N, Tsuruo T. Modulation of Akt kinase activity by binding to Hsp90. Proc Natl Acad Sci USA, 2000, 97(20): 10832-10837.
[26]Yano S et al. Calcium promotes cell survival through CaM-K kinase activation of the protein-kinase-B pathway. Nature, 1998; 396: 584.
[27]Nam SY, Jung GA, Hur GC, et al. Upregulation of FLIP(S) by Akt, a possible inhibition mechanism of TRAIL-induced apoptosi-s in human gastric cancers [J]. Cancer Sci, 2003, 94(12):1066-1073.
[28]Dijkers PF, Medema RH, Lammers JW, et al. Expression of the pro-apoptotic Bcl-2 family member Bim is regulated by the fork head transcription factor FKHROL1. Curr Biol, 2000,10(19):1201～1204.
[29]Digicaylioglu Murat. Erythropoietin-mediated neuroprotection involves cross-talk between JaK2 and NF-kB signaling cascades[J]. Nature, 2001, 412:641-647.
[30]Romashkova JA, Makarov SS. NF-kappaB is a target of AKT inanti-apoptotic PDGF signalling. Nature, 1999, 401(6748):86-90.
[31]Zamzami N, Brenner C, Marzo I,et al. Subcellular and submito- chondrial mode of action of Bcl-2-like oncoproteins[J]. Oncogene, 1998, 16(17):2265-2282
[32]del-Peso L, Gonzalez-Garcia M, Page C, et al. Interleukin-3- induced phosphorylation of BAD through the protein kinase Akt. Science, 1997, 278(5338):687-689.
[33]Chu ZL, Mckinsey TA, Liu L, et al. Supression of tumor necrosis factor-induced cell death by inhibitor of apoptosis c-IAP2is u-nder NF-kB control [J]. Proc Natl Acad Sci USA, 1997, 94: 10057-10062.
[34]Dan HC, Sun M, Kaneko S, et al. Akt Phosphorylation and Stabilization of X-linked Inhibitor of Apoptosis Protein(XIAP)[J]. J Biol Chem, 2004, 279(7):5405-5412.
[35]Kim AH, Khursigara G, Sun X, et al. Akt phosphorylates and negatively regulates apoptosis signal-regulating kinase 1. Mol Cell Biol 2001, 21(3):893-900.
[1]Gluckman E, Broxmeyer HE, Aurebach AD,et al. Hematopoietic Reconstitution in a Patient with Fanconis Anemia by Means of Umbilical-cord Blood from an HLA-identical Sibling[J]. N Engl J Med, 1989, 321:1174.
[2]Koller MR, Plasson MA, Manchel 1, et al. Long-term culture initiating cell expansion is dependent on expansionmedium exchange combinedwith stromal and other accessory cell effects[J].Blood, 1995, 86:1784.
[3]奚永志,唐佩弦.深入开展脐血干细胞的基础与临床研究.中华血液学杂志,1999, 8(2): 397.
[4]崔正言.干细胞研究的进展与应用前景.中国肿瘤生物治疗杂志, 2001, 8(4):239-242.
[5]沈伯均,隋星卫.脐血移植的评价和前景.中华儿科杂志, 1994, 32(6):381.
[6]Regidor C, Posada M, Monteagudo D, et al. Umbilical cord blood banking for unrelated transplantation: evaluation of cell separation and storage methods [J ]. Exp Hematol, 1999, 27(3): 380-385.
[7]Gluckman E, Rocha V, Boyer Chammard A, et al. Outcome of cord blood transplantation from related and unrelated donors. Euro cord Transplant Group and the European Blood Marrow Transplantation Group [J ]. N Engl J Med, 1997, 337 (5) :373-381.
[8]罗伟琼,陈惠珍,黄春柳等.脐血采集方法、影响因素分析及注意事项[J].中国热带医学, 2007, 7(4):647-648.
[9]刘开彦,高志勇,姜永军。脐带血造血干细胞的采集、浓缩与低温冷冻保存。北京大学学报(医学版),2003;35(2):25-28.
[10]马秀峰.脐血采集中的伦理法律问题及护理对策[J].中华护理杂志, 2004,39(3):234.
[11]梅花,谭周敏,曾薇等.脐血采集质量影响因素分析[J].护理研究, 2005, 19(12B):2682-2683.
[12]罗小珍,陈惠珍,罗伟琼等.脐血采集质量与不同分娩方式的关系.中国妇幼健康研究, 2007, 18(3):206-207.
[13]罗志云,刘维飞,方建培等.两种脐血采集方法对新生儿的影响[J].南方护理学报, 1999, 6(4):4.
[14]Wong A, Yuen PM, Li K, et al . Cord blood collection before and after placental delivery: levels of nucleated cells, haematopoietic progenitor cells, leukocyte subpopulations and macroscopic clot [J ]. Bone Marrow Transplantation, 2001, 27(2) :133-138.
[15]罗伟琼,陈惠珍,黄春柳等.脐血采集方法、影响因素分析及注意事项[J].临床和实验医学, 2007,6(1): 69-70.
[16]Kenichi S, Mikitomo Y, Ryo S, et al. Cell processing for placental/ umbilical cord blood banking. Jpn J Transfusion Med, 1998;44:12-19.
[17]Shlebak AA, Marley SB, Roberts IA,et al. Optimal timing for processing and cryopreservation of umbilical cord haematopoietic stem cells for clinical transplantalion. Bone Marrow Transplant, 1999, 23:131-136.
[18]Bertolini F, Lazzari L, Lauri E, et al. Comparative study of different procedures for the collection and banking of umbilical cord blood [ J ] . J Hematother, 1995, 4 (1) :22-36.
[19]Moldenhauer A, Wolf J, Habermann G, et al. Optimum storage conditions for cord blood-derived hematopoietic progenitor cells prior to isolation [J ]. Bone Marrow Transplantation 2007, 40: 837-842.
[20]Majka M, Janowska-Wiezorek A, Ratajczak J, et al. Numerous growth factors, cytokines, and chemokines are secreted by human CD34(+) cells, myeloblasts, erythroblasts, and megakaryoblasts and regulate normal hematopoiesis in an acutocrine/paracrine manner [J ]. Blood 2001, 97: 3075-3085.
[21]Kadereit S, Deeds LS, Haynesworth SE, et al, Expansion of LTC-ICs and maintenance of p21 and BCL-2 expression in cord bloodCD34(+)/CD38(-) early progenitors cultured over human MSCs as a feeder layer [J ]. Stem cells 2002,20:573-582.
[22]Upratay Y, Boonmoh S, Promsuwicha O, et al. Collection and processing of umbilical cord blood for cryopreservation[J].J Med Assoc Thai, 2003, 86(11):1055-1062.
[23]Broxmeyer ME, et al. Cord blood ransplantation;an update. EXP Hematol.1994,22:675.
[24]Payne TA, et al. Phenotypic analysis of early hematopoietic progenitors in cord blood and determination of their correlation with clonogenic progenitor. BMT, 1995, 15;187.
[25]奚永志,李秀森等.人正常骨髓CD34+造血细胞的形态学与细胞化学特征.中华血液学杂志, 1997, 18(3): 131.
[26]许文荣,胡嘉波,朱伟等.脐血造血干/祖细胞的密度分离实验研究.临床检验杂志. 2001, 19(2): 73-76.
[27]杨景山.医学细胞化学与细胞生物技术.北京北京医科大学中国协和医科大学联合出版社,1990. 14-16.
[28]Kogler G, Callejas J. Hakenberg P et al. Hematopoiet is transplant potential of unrelated cord blood:critical issues. J Hematother, 1996.5(2):105.
[29]Campos L. Roubin N. Guyotae D, et al. Definition of optimal conditions for collection and cryopreservation of umbilical cord blood hematopoietic cells. Cryobiology. 1999, 2: 511-512.
[30]Regidor C, Posada M, Monteagudo D, et al. Umbilical cord blood banking for unrelated transplantation: evaluation of cell separation and storage methods. Exp Hemat, 1999, 27: 380-385.
[31]朱美玲,伍新尧,陈汝光等.不同分离方法分离脐血造血细胞效率的比较.中国输血杂志, 2002, 15(3):179-180.
[32]奚永志,李秀森等.人正常骨髓CD34+造血细胞的形态学与细胞化学特征.中华血液学杂志, 1997, 18(3): 131.
[33]罗伟琼,朱美玲,邹翠贤等.脐血有核细胞分离、冻存方法的研究.临床输血与检验, 2007: 9(1):55-56.
[34]Okuno Y, Iwasaki H, Huettner CS, et al. Differential regulation of the human and murine CD34 genes in hematopoietic stem cells. Proc Natl Acad Sci USA, 2002, 99: 6246-6251.
[35]张洪泉,傅曾锯,沈柏均.脐血造血细胞有分离与保存.中华血液学杂志, 1992, 13(4):180-182.
[36]Holyoale TL, Alcorn MJ. CD34 positive haemopoietic cells: biology and clinical applications. Blood Rev, 1994, 8:113.
[2]奚永志,唐佩弦.深入开展脐血干细胞的基础与临床研究.中华血液学杂志, 1999, 8(2):397.
[3]崔正言.干细胞研究的进展与应用前景.中国肿瘤生物治疗杂志, 2001, 8(4):239-242.
[4]沈伯均,隋星卫.脐血移植的评价和前景.中华儿科杂志, 1994, 32(6):381.
[5]Koller MR, Plasson MA, Manchel L, et al. Long-term culture initiating cell expansion is dependent on expansionmedium exchange combined with stromal and other accessory cell effects[J]. Blood, 1995, 86:1784.
[6]Regidor C, Posada M, Monteagudo D, et al. Umbilical cord blood banking for unrelated transplantation: evaluation of cell separation and storage methods[J ] . Exp Hematol, 1999, 27(3) :380-385.
[7]罗伟琼,陈惠珍,黄春柳等.脐血采集方法、影响因素分析及注意事项[J].中国热带医学, 2007, 7(4):647-648。
[8]Wong A, Yuen PM, Li K, et al. Cord blood collection before and after placental delivery: levels of nucleated cells, haematopoietic progenitor cells, leukocyte subpopulations and macroscopic clot [J]. Bone Marrow Transplantation, 2001, 27(2):133-138.
[9]Kenichi S, Mikitomo Y, Ryo S, et al. Cell processing for placental/umbilical cord blood banking. Jpn J Transfusion Med, 1998; 44:12-19.
[10]Shlebak AA, Marley SB, Roberts IA,et al. Optimal timing for processing and cryopreservation of umbilical cord haematopoietic stem cells for clinical transplantalion. Bone Marrow Transplant, 1999, 23: 131- 136.
[11] Bertolini F, Lazzari L, Lauri E, et al. Comparative study of different procedures for the collection and banking of umbilical cord blood [J]. J Hematother, 1995, 4 (1):22-36.
[12] Moldenhauer A, Wolf J, Habermann G, et al. Optimum storage conditions for cord blood-derived hematopoietic progenitor cells prior to isolation [J]. Bone Marrow Transplantation 2007, 40: 837-842.
[13] Majka M, Janowska-Wiezorek A, Ratajczak J, et al. Numerous growth factors, cytokines, and chemokines are secreted by human CD34(+) cells, myeloblasts, erythroblasts, and megakaryoblasts and regulate normal hematopoiesis in an acutocrine/paracrine manner [J ]. Blood 2001, 97:3075-3085.
[14] Kadereit S, Deeds LS, Haynesworth SE, et al, Expansion of LTC-ICs and maintenance of p21 and BCL-2 expression in cord blood CD34(+)/CD38(-) early progenitors cultured over human MSCs as a feeder layer [J ]. Stem cells 2002, 20:573-582.
[15]朱美玲,伍新尧,陈汝光等.不同分离方法分离脐血造血细胞效率的比较.中国输血杂志, 2006, 15(3):179.
[16]奚永志,李秀森.人正常骨髓CD34+造血细胞的形态学与细胞化学特征.中华血液学杂志, 1997, 18(3):131.
[17]罗伟琼,朱美玲,邹翠贤等.脐血有核细胞分离、冻存方法的研究.临床输血与检验, 2007: 9(1):55-56.
[18]蹇在伏,吴新华,陈方平等.两种不同方法分离脐血造血干细胞实验研究[ J].中国现代医学杂志, 2004, 14(19): 68-69.
[19]Regidor C, Posada M, Monteagudo D, et al. Umbilical cord blood banking for unrelated transplantation: evaluation of cell separation and storage methods. Exp Hemat, 1999, 27: 380-385.
[1]Marsh JC. Eur J Haemetol Suppl, 1996, 60: 75-79.
[2]刘红,陆德炎,杨锦媛等.重型再生障碍性贫血患者血清诱导D34+细胞凋亡及PML蛋白表达.中华血液学杂志, 2003, 24(11):596-597.
[3]Stephens L, Anderson K, Stokoe D, et al. Protein kinase B kinase that mediate phosphatidylinositol 3,4,5-trisphosphate-dependent activation of prokinase B [J].Science, 1998, 279(5351) 710-714.
[4]中华血液学学会第四届全国再生障碍性贫血学术会议纪要:再生障碍性贫血诊断标准和疗效标准.中华血液学杂志, 1987, 8: 486和封四.
[5]Scopes J, Bagnara M, Gordon2Smith EC, et al. Haemopoietic progenitor cells are reduced in aplastic anemia. Br J Haematol, 1994, 86: 427.
[6]Philpott NJ, Scopes J, Marsh JC, et al. Increased apoptosis in aplastic anemia bone marrow progenitor cells: possible pathophsiologic significance. Exp J Hematol, 1995; 23(14): 1642-1648.
[7]Callera F, Falcao RP, et al. Increased apoptotic cells in bone marrow biopsies from patients with aplastic anemia. Br J Haematol, 1997,98(1):18-20.
[8]Liu H, Mihara K, Kimura A, et al. Induction of apoptosis in CD34+cells by sera from patients with aplastic anemia. Hiroshima J Med Sci, 1999, 48(2): 57-63.
[9]刘红,吴芳颐,姜胜华等.二例重症再生障碍性贫血患者血清体外加速正常人CD34+细胞凋亡的研究.中华血液学杂志, 1998, 19: 514-517。
[10]Maciejeuski JP, Seller C, Sato T, et al. (1995) Increased expression of Fas antigen on bone marrow CD34+ cells of patients with aplastic anemia. British Journal of Heamatology, 91,245.
[11]Stephens L, andersoh K, Slokoe D, et al. Proetin kinase B Kinase thatmediale phosphalidvl inosiol 3,4,5 trisphosphale activalion of protein kinase B. Science[J], 1998, 279:710.
[12]Staal SP, Hartley JW, Rowe WP, et al. Isolation of transformingmurine leukemia viruses from mice with a high incidence of spontaneous lymphoma[J]. Proc Natl Acad Sci USA, 1977; 74(7):3065-3067.
[13]Alessi DR, Andjelkovic M, Caudwell B, et al. Mechanism of activation of protein kinase B by insulin and IGF-1. EMBO J, 1996, 15(23): 6541-6551.
[14]Alessi DR, Andjelkovic M, Caudwell B, et al. Mechanism of activation of protein kinase B by insulinand IGF-1[J]. EMBO J, 1996; 15(23):6541-6551.
[15]Welch H, Eguinoa A, Stephens LR, Hawkins PT. Protein kinase B andrac are activated in parallel within a phosphatidylinositide 3OH-kinase-controlled signaling pathway[J]. J Biol Chem, 1998; 273(18):11248-11256.
[16]Alessi DR, Andjelkovic M, Caudwell B, et al. Mechanism of activation of protein kinase B by insulin and IGF-1. EMBO J, 1996, 15(23):6541-6551.
[17]Balendran A, Casamayor A, Deak M, et al. PDK1 acquires PDK2 activity in the presence of a synthetic peptide derived from the carboxyl terminus of PRK2[J]. Curr Biol, 1999, 9(8): 393-404.
[18]Mao M et al. The Phosphatidylinositol 3-Kinase/AKT Kinase Pa-thway in Multiple Myeloma Plasma Cells:Roles in Cytokine-dependent Survival and Proliferative Responses. Cancer Res, 2000;60(23):6763-6770.
[19]Hong Liu, YaLing Qiu, et al. Involvement of protein kinase Cεin the negative regulation of AKT activation stimulated by granulocyte colony-stimulating factor. The journal of immunology. 2006, 176:2407-2413
[20]Dedhar S, Williams B, Hannigan G. Integrin-linked kinase(ILK):aregulator of integrin and growth factor signalling [J]. Trends Cell Biol, 1999, 9(8):319-323.
[21]Delcommenne M, Tan C, Gray V,et al. Phosphoinositide-3-OH k-inase-dependent regulation of glycogen synthase kinase 3 and pro-tein kinase B/AKT by the integrin-linked kinase [J]. Proc Natl Acad Sci USA, 1998, 95(19):11211-11216.
[22]Balendran A, Casamayor A, Deak M, et al. PDK1 acquires PDK2 activity in the presence of a synthetic peptide derived from the carboxyl terminus of PRK2[J]. Curr Biol, 1999, 9(8):393-404.
[23]Yamada T, Katagiri H, Asano T, et al. 3-phosphoinositide-dependent protein kinase 1, an Akt kinase, is involved in dephosphorylation of Thr-308 of Akt1 in Chinese hamster ovary cells[J]. J Biol Chem, 2001, 276(7):5339-5345.
[24]Schubert KM, Scheid MP, Duronio V. Ceramide inhibits protein kinase B/Akt by promoting dephosphorylation of serine473[J]. J Biol Chem, 2000, 275(18):13330-13335.
[25]Sato S, Fujita N, Tsuruo T. Modulation of Akt kinase activity by binding to Hsp90. Proc Natl Acad Sci USA, 2000, 97(20): 10832-10837.
[26]Yano S et al. Calcium promotes cell survival through CaM-K kinase activation of the protein-kinase-B pathway. Nature, 1998; 396: 584.
[27]Nam SY, Jung GA, Hur GC, et al. Upregulation of FLIP(S) by Akt, a possible inhibition mechanism of TRAIL-induced apoptosi-s in human gastric cancers [J]. Cancer Sci, 2003, 94(12):1066-1073.
[28]Dijkers PF, Medema RH, Lammers JW, et al. Expression of the pro-apoptotic Bcl-2 family member Bim is regulated by the fork head transcription factor FKHROL1. Curr Biol, 2000,10(19):1201～1204.
[29]Digicaylioglu Murat. Erythropoietin-mediated neuroprotection involves cross-talk between JaK2 and NF-kB signaling cascades[J]. Nature, 2001, 412:641-647.
[30]Romashkova JA, Makarov SS. NF-kappaB is a target of AKT inanti-apoptotic PDGF signalling. Nature, 1999, 401(6748):86-90.
[31]Zamzami N, Brenner C, Marzo I,et al. Subcellular and submito- chondrial mode of action of Bcl-2-like oncoproteins[J]. Oncogene, 1998, 16(17):2265-2282
[32]del-Peso L, Gonzalez-Garcia M, Page C, et al. Interleukin-3- induced phosphorylation of BAD through the protein kinase Akt. Science, 1997, 278(5338):687-689.
[33]Chu ZL, Mckinsey TA, Liu L, et al. Supression of tumor necrosis factor-induced cell death by inhibitor of apoptosis c-IAP2is u-nder NF-kB control [J]. Proc Natl Acad Sci USA, 1997, 94: 10057-10062.
[34]Dan HC, Sun M, Kaneko S, et al. Akt Phosphorylation and Stabilization of X-linked Inhibitor of Apoptosis Protein(XIAP)[J]. J Biol Chem, 2004, 279(7):5405-5412.
[35]Kim AH, Khursigara G, Sun X, et al. Akt phosphorylates and negatively regulates apoptosis signal-regulating kinase 1. Mol Cell Biol 2001, 21(3):893-900.
[1]Gluckman E, Broxmeyer HE, Aurebach AD,et al. Hematopoietic Reconstitution in a Patient with Fanconis Anemia by Means of Umbilical-cord Blood from an HLA-identical Sibling[J]. N Engl J Med, 1989, 321:1174.
[2]Koller MR, Plasson MA, Manchel 1, et al. Long-term culture initiating cell expansion is dependent on expansionmedium exchange combinedwith stromal and other accessory cell effects[J].Blood, 1995, 86:1784.
[3]奚永志,唐佩弦.深入开展脐血干细胞的基础与临床研究.中华血液学杂志,1999, 8(2): 397.
[4]崔正言.干细胞研究的进展与应用前景.中国肿瘤生物治疗杂志, 2001, 8(4):239-242.
[5]沈伯均,隋星卫.脐血移植的评价和前景.中华儿科杂志, 1994, 32(6):381.
[6]Regidor C, Posada M, Monteagudo D, et al. Umbilical cord blood banking for unrelated transplantation: evaluation of cell separation and storage methods [J ]. Exp Hematol, 1999, 27(3): 380-385.
[7]Gluckman E, Rocha V, Boyer Chammard A, et al. Outcome of cord blood transplantation from related and unrelated donors. Euro cord Transplant Group and the European Blood Marrow Transplantation Group [J ]. N Engl J Med, 1997, 337 (5) :373-381.
[8]罗伟琼,陈惠珍,黄春柳等.脐血采集方法、影响因素分析及注意事项[J].中国热带医学, 2007, 7(4):647-648.
[9]刘开彦,高志勇,姜永军。脐带血造血干细胞的采集、浓缩与低温冷冻保存。北京大学学报(医学版),2003;35(2):25-28.
[10]马秀峰.脐血采集中的伦理法律问题及护理对策[J].中华护理杂志, 2004,39(3):234.
[11]梅花,谭周敏,曾薇等.脐血采集质量影响因素分析[J].护理研究, 2005, 19(12B):2682-2683.
[12]罗小珍,陈惠珍,罗伟琼等.脐血采集质量与不同分娩方式的关系.中国妇幼健康研究, 2007, 18(3):206-207.
[13]罗志云,刘维飞,方建培等.两种脐血采集方法对新生儿的影响[J].南方护理学报, 1999, 6(4):4.
[14]Wong A, Yuen PM, Li K, et al . Cord blood collection before and after placental delivery: levels of nucleated cells, haematopoietic progenitor cells, leukocyte subpopulations and macroscopic clot [J ]. Bone Marrow Transplantation, 2001, 27(2) :133-138.
[15]罗伟琼,陈惠珍,黄春柳等.脐血采集方法、影响因素分析及注意事项[J].临床和实验医学, 2007,6(1): 69-70.
[16]Kenichi S, Mikitomo Y, Ryo S, et al. Cell processing for placental/ umbilical cord blood banking. Jpn J Transfusion Med, 1998;44:12-19.
[17]Shlebak AA, Marley SB, Roberts IA,et al. Optimal timing for processing and cryopreservation of umbilical cord haematopoietic stem cells for clinical transplantalion. Bone Marrow Transplant, 1999, 23:131-136.
[18]Bertolini F, Lazzari L, Lauri E, et al. Comparative study of different procedures for the collection and banking of umbilical cord blood [ J ] . J Hematother, 1995, 4 (1) :22-36.
[19]Moldenhauer A, Wolf J, Habermann G, et al. Optimum storage conditions for cord blood-derived hematopoietic progenitor cells prior to isolation [J ]. Bone Marrow Transplantation 2007, 40: 837-842.
[20]Majka M, Janowska-Wiezorek A, Ratajczak J, et al. Numerous growth factors, cytokines, and chemokines are secreted by human CD34(+) cells, myeloblasts, erythroblasts, and megakaryoblasts and regulate normal hematopoiesis in an acutocrine/paracrine manner [J ]. Blood 2001, 97: 3075-3085.
[21]Kadereit S, Deeds LS, Haynesworth SE, et al, Expansion of LTC-ICs and maintenance of p21 and BCL-2 expression in cord bloodCD34(+)/CD38(-) early progenitors cultured over human MSCs as a feeder layer [J ]. Stem cells 2002,20:573-582.
[22]Upratay Y, Boonmoh S, Promsuwicha O, et al. Collection and processing of umbilical cord blood for cryopreservation[J].J Med Assoc Thai, 2003, 86(11):1055-1062.
[23]Broxmeyer ME, et al. Cord blood ransplantation;an update. EXP Hematol.1994,22:675.
[24]Payne TA, et al. Phenotypic analysis of early hematopoietic progenitors in cord blood and determination of their correlation with clonogenic progenitor. BMT, 1995, 15;187.
[25]奚永志,李秀森等.人正常骨髓CD34+造血细胞的形态学与细胞化学特征.中华血液学杂志, 1997, 18(3): 131.
[26]许文荣,胡嘉波,朱伟等.脐血造血干/祖细胞的密度分离实验研究.临床检验杂志. 2001, 19(2): 73-76.
[27]杨景山.医学细胞化学与细胞生物技术.北京北京医科大学中国协和医科大学联合出版社,1990. 14-16.
[28]Kogler G, Callejas J. Hakenberg P et al. Hematopoiet is transplant potential of unrelated cord blood:critical issues. J Hematother, 1996.5(2):105.
[29]Campos L. Roubin N. Guyotae D, et al. Definition of optimal conditions for collection and cryopreservation of umbilical cord blood hematopoietic cells. Cryobiology. 1999, 2: 511-512.
[30]Regidor C, Posada M, Monteagudo D, et al. Umbilical cord blood banking for unrelated transplantation: evaluation of cell separation and storage methods. Exp Hemat, 1999, 27: 380-385.
[31]朱美玲,伍新尧,陈汝光等.不同分离方法分离脐血造血细胞效率的比较.中国输血杂志, 2002, 15(3):179-180.
[32]奚永志,李秀森等.人正常骨髓CD34+造血细胞的形态学与细胞化学特征.中华血液学杂志, 1997, 18(3): 131.
[33]罗伟琼,朱美玲,邹翠贤等.脐血有核细胞分离、冻存方法的研究.临床输血与检验, 2007: 9(1):55-56.
[34]Okuno Y, Iwasaki H, Huettner CS, et al. Differential regulation of the human and murine CD34 genes in hematopoietic stem cells. Proc Natl Acad Sci USA, 2002, 99: 6246-6251.
[35]张洪泉,傅曾锯,沈柏均.脐血造血细胞有分离与保存.中华血液学杂志, 1992, 13(4):180-182.
[36]Holyoale TL, Alcorn MJ. CD34 positive haemopoietic cells: biology and clinical applications. Blood Rev, 1994, 8:113.