THE EFFECT OF STEM CELL FACTOR, INTERLEUKIN-6 AND ERYTHROPOIETIN ON EXPANSION OF CD34^+ CELLS FROM HUMAN UMBILICAL CORD BLOOD

CD34+ cells from human umbilical cord blood were purified by Dynal beads M-450 CD34 immunoselection system and cultured in the presence of various cytokines alone or in combination, including stem cell factor (SCF), interleukin-6 (IL-6) and erythropoietin (EPO). The results revealed that: (D In methylcellulose culture, the plating efficiencies of purified cord blood CD34+ cells were much different when stimulated by various cytokines. IL-6 alone had the lowest colo-ny yield, while the combination of SCF, IL-6 and EPO had the highest yield. ② In the suspension culture, IL-6 alone or IL-6 + EPO had little expanding effect on cord blood CD34+ celis, the other cytokine combinations could expand cord blood CD34+ celis at different Ievels. Among them, the combination of SCF, IL-6 and EPO had the maximal expanding effect on cord blood CD34+ celis, the number of progenitor celis peaked at day 21, about 29-fold increase and nucleated celis increased approximately 3676-fold at day 28. The expanding effect of

Expression of Caspase－3 in Cord Blood CD34^＋ Cells during Culture in vitro

Objective: To investigate the expression and significance of caspase-3 protein in CD34^+ cells from cord blood (CB) during culture in vitro with different growth factors. Methods: RT-PCR, Western blot and flow cytometry techniques were used to detect the expression of caspase-3 in CD34^+ CB cells during culture in vitro. Results: Caspase-3 mRNA was constitutively expressed at a low level in freshly isolated CD34^+ cells. The expression of caspase-3 mRNA and protein was upregulated when these cellswere first expanded in suspension culture with growth factors for 3 days. However, only the 32 kDa inactive caspase-3 proenzyme was detected in the freshly isolated CD34^+ cells as well as during the first 3 days expansion with cytokines. With longer culture time in vitro, especially in the presence of the combination of IL-3, IL-6 and GM-CSF, caspase-3 was activated and a cleavage product of 20 kDa became detectable.Conclusion: Caspase-3 is involved in apoptosis of primitive CB CD34^+ cells during expansion in vitro.

Transplantation of microencapsulated umbilical-cord-blood-derived hepatic-like cells for treatment of hepatic failure

AIM: To investigate intraperitoneal transplantation of microencapsulated hepatic-like cells from human umbilical cord blood for treatment of hepatic failure in rats. METHODS: CD34+ cells in umbilical cord blood cells were isolated by magnetic cell sorting. In the in vitro experiment, sorted CD34+ cells were amplified and induced into hepatic-like cells by culturing with a combination of fibroblast growth factor 4 and hepatocyte growth factor. Cultures without growth factor addition served as controls. mRNA and protein levels for hepatic-like cells were analyzed by reverse transcription-polymerase chain reaction, immunohistochemistry and immunofluorescence. In the in vivo experiment, the hepatic-like cells were encapsulated and transplanted into the abdominal cavity of acute hepatic failure (AHF) rats at 48 h after D-galactosamine induction of acute hepatic failure. Transplantation with PBS and unencapsulated hepatic-like cells served as controls. The mortality rate, hepatic pathological changes and serum biochemical indexes were determined. The morphology and structure of microcapsules in the greater omentum were observed. RESULTS: Human albumin, alpha-fetoprotein and GATA-4 mRNA and albumin protein positive cells were found among cultured cells after 16 d. Albumin level in culture medium was significantly increased after culturing with growth factors in comparison with culturing without growth factor addition (P < 0.01). Compared with the unencapsulated group, the mortality rate of the encapsulated hepatic-like cell-transplanted group was significantly lower (P < 0.05). Serum biochemical parameters, alanine aminotransferase, aspartate aminotransferase and total bilirubin in the encapsulated group were significantly improvement compared with the PBS control group (P < 0.01). Pathological staining further supported these findings. At 1-2 wk post-transplantation, free microcapsules with a round clear structure and a smooth surface were observed in peritoneal lavage fluid, surviving cells inside microcapsules were found by trypan blue staining, but some fibrous tissue around microcapsules was also detected in the greater omentum of encapsulated group by hematoxylin and eosin staining. CONCLUSION: Transplantation of microencapsulated hepatic-like cells derived from umbilical cord blood cells could preliminarily alleviate the symptoms of AHF rats.

Functional expression of CD95/Fas Antigen and Bcl-2 on Cord blood Hematopoietic Progenitor Cells

The cell surface expression and functional status of the CD95/Fas antigen on primitive hematopoietic progenitors isolated from human cord blood (CB) were studied. The CD34 + cells freshly isolated from CB displayed low CD95 expression. The combinations of cytokines such as SCF+FL could up regulate the expression of CD95 in vitro culture and tumor necrosis factor α (TNF α) and interon γ (IFN γ) further increased the CD95 expression induced by positive cytokines. The functional status of CD95 mediated apoptosis were analyzed by incubation of CD34 +CB cells in the presence of anti CD95 monoclonal antibodies (McAbs). The effects of anti CD95 McAbs were measured by viable cell counting, flow cytometry, LTIC and CFU C assays. A decrease of viable cells, CFU C and LTIC numbers were observed in the presence of anti CD95 McAbs and TNF α or IFN γ. However, growth factor deprivation or the early acting cytokine such as SCF and FL cross linking to CD95 caused low apoptosis of CD34 + cells. The correlation of increased intracytoplasmic levels of bcl 2 and the presence of CD95 on fresh CB CD34 + cells suggested that bcl 2 might be involved in protecting against CD95 mediated apoptosis of CB CD34 + cells.

Repetitive administration of cultured human CD34+cells improve adenine-induced kidney injury in mice

BACKGROUND There is no established treatment to impede the progression or restore kidney function in human chronic kidney disease(CKD).AIM To examine the efficacy of cultured human CD34+cells with enhanced proliferating potential in kidney injury in mice.METHODS Human umbilical cord blood(UCB)-derived CD34+cells were incubated for one week in vasculogenic conditioning medium.Vasculogenic culture significantly increased the number of CD34+cells and their ability to form endothelial progenitor cell colony-forming units.Adenineinduced tubulointerstitial injury of the kidney was induced in immunodeficient non-obese diabetic/severe combined immunodeficiency mice,and cultured human UCB-CD34+cells were administered at a dose of 1×106/mouse on days 7,14,and 21 after the start of adenine diet.RESULTS Repetitive administration of cultured UCB-CD34+cells significantly improved the time-course of kidney dysfunction in the cell therapy group compared with that in the control group.Both interstitial fibrosis and tubular damage were significantly reduced in the cell therapy group compared with those in the control group(P<0.01).Microvasculature integrity was significantly preserved(P<0.01)and macrophage infiltration into kidney tissue was dramatically decreased in the cell therapy group compared with those in the control group(P<0.001).CONCLUSION Early intervention using human cultured CD34+cells significantly improved the progression of tubulointerstitial kidney injury.Repetitive administration of cultured human UCB-CD34+cells significantly improved tubulointerstitial damage in adenine-induced kidney injury in mice via vasculoprotective and anti-inflammatory effects.

Construction and expression of retroviruses encoding dual drug resistance genes in human umbilical cord blood CD34^+ cells

A series of retroviral vectors encoding human mdrl gene alone as well as in combination with either human mgmt gene or human mutant Ser31-dftfrgene are engineered. The resultant retroviruses are used to transduce human umbilical cord blood CD34+ cells. It has been shown that expression of dual drug resistance genes in transduced cells confers a broad range of resistance to both kinds of corresponding drugs. These data suggest a rationale for the use of such double chemoresistance gene constructs in an in vivo model in which transduced hematopoietic cells will acquire multiple protection against the cytotoxic side effects of combination chemotherapy and may have future application in chemoprotection of normal tissues, thus killing tumor cells more effectively.

Effect of intracranial transplantation of CD34^+ cells derived from human umbilical cord blood in rats with cerebral ischemia

As a source of transplantable stem cells, the CD34^＋ subpopulation in human umbilical cord blood （HUCB） has been used extensively to treat some hematopoietic system diseases. However, whether CD34^＋ cells hold the therapeutic potential to cerebral ischemia is unknown. The purpose of this study was to observe the recovery of neural function after transplantation of CD34^＋ cells derived from HUCB into ischemic cerebral tissue in rats.

细胞因子扩增脐带血CD_(34)^+细胞中LTC-IC的变化

目的 :探讨脐带血造血干细胞的体外扩增及移植最佳时机。方法 :免疫磁珠法分离纯化脐带血 CD+ 3 4细胞 ,在细胞因子 FL T3配体、血小板生成素作用下 ,进行体外扩增 ,流式细胞仪检测培养体系中 CD+ 3 4 细胞数量的变化 ,甲基纤维素法检测 CFU - GM、长期培养起始细胞 ( L TC- IC)。结果 :经过 2周的培养 ,有核细胞扩增 ( 2 1± 5 )倍 ,CD+ 3 4 细胞扩增 ( 9± 3 )倍 ,CFU- GM扩增 ( 164± 5 1)倍 ,L TC- IC扩增 ( 6.5± 3 .1)倍 ,随着体外培养时间的延长 ,有核细胞、CD+ 3 4 细胞和 CFU- GM得到进一步扩增 ,但培养体系中已缺少 L TC- IC。结论 :FL T3配体联合血小板生成素能扩增具有造血干细胞特征的 L TC- IC细胞 ,培养时间以

Caspase-3在脐血CD_(34)^+造血干/祖细胞中的表达及意义

目的 :探讨脐血CD3 4+ 干 /祖细胞在不同细胞因子支持下的体外扩增过程中Caspase 3表达及意义。方法 :采用RT PCR、Westerblot和流式细胞仪分析技术测定脐血CD3 4+ 细胞在体外扩增过程中的生物学特性及Caspase 3的表达。 结果 :Caspase 3mRNA在新鲜分离的脐血CD3 4+ 细胞中低水平表达 ,在细胞因子支持下体外培养 3d ,扩增的CD3 4+ 细胞中Caspase 3mRNA和蛋白质表达上调 ,但在该两种细胞中仅能检测到分子量为32 0 0 0的无活性酶原形式的Caspase 3,随着体外培养时间的延长 ,在IL 3、IL 6和GM CSF组合条件下 ,Caspase 3被激活 ,可检测到分子量为 2 0 0 0 0的裂解片段。结论 :虽然造血干细胞的凋亡是个复杂的过程 ,但在脐血CD3 4+干 /祖细胞体外扩增过程中 ,Caspase

低温冷冻对脐血CD_(34)^+细胞和造血祖细胞增殖的影响

采用－８０℃低温冷冻法，观察冷冻前及冷冻后１、２、３周时单个核细胞（ＭＮＣ）的回收率，脐血造血前体细胞在体外的增殖力，并用流式细胞仪检测ＣＤ３４＋细胞的百分率。旨在探讨低温冷冻对脐血造血前体细胞的影响。结果表明：脐血冷冻后１、２、３周时ＭＮＣ的回收率分别为９３．６％，９１．５％和９０．７％（Ｐ＞０．０５）；细胞活力分别为９８％，９７％，９５％和９１％（Ｐ＞０．０５）；ＣＤ３４＋细胞依次为１．２０％、１．１８％、１．１６％和１．１７％；ＣＦＵ－ＧＭ，ＣＦＵ－Ｅ冷冻后１、２周时回收率达９７％和９５％（Ｐ＞０．０５），第３周时达８９％（Ｐ＞０．０５）。结果提示：长期冷冻可能导致冷冻之脐血祖细胞产率下降，但对代表造血干细胞的ＣＤ３４＋细胞影响则不大。

脐血CD_(34)^+细胞体外扩增和分化调节的研究

目的探讨脐血体外扩增的最佳造血生长因子组合及时间。 方法脐血CD34+ 细胞培养于含不同造血生长因子组合的无血清培养液。对其有核细胞数 (NC)、表面抗原以及集落形成能力进行监测。 结果同对照组相比 ,扩增组的NC和CD34 + 细胞均有不同程度的扩增 ,扩增高峰分别为第 10d和第 6d ;其中含SCF、IL - 3、IL - 6、TPO、Flt3-L、G -CSF及Epo的E组扩增的细胞数最多。第 6d ,CD34+ 细胞、NC及CFC总数分别扩增了 9.90、10 1.80和 31.18倍 ,第 10d分别累积扩增了 3.5 6、35 7.4 2和 73.11倍。而D组 (含SCF、IL - 3、IL - 6、TPO及Flt3-L)扩增的细胞中CD34+ 及CD34+CD38- 含量最高。提示E组细胞分化较D组明显。 结论体外扩增可望解决单份脐血造血干 祖细胞数量不足的问题 ,但扩增中应注意其过度分化 ,扩增时间以 6 ～ 1 0d为宜。

脐血CD_(34)^-单个核细胞来源间充质干细胞研究

目的 :探讨分离培养脐血CD-3 4 细胞来源间充质干细胞 (MSC)及研究其生物学特征。方法 :取足月妊娠健康产妇胎儿脐血 ,分离其中单个核细胞 (MNC) ,去除CD+3 4 细胞 ,体外用低糖型DMEM培养基培养。观察细胞形态、测定生长曲线、利用流式细胞仪对培养细胞进行表型测定、细胞周期分析、体外诱导分化实验以及检测造血因子的表达情况。结果 :脐血CD-3 4 细胞中可培养出间充质干细胞 ,可诱导向成骨和脂肪细胞分化并表达IL 6、SCF和SDF 1等造血生长因子。结论 :从足月妊娠健康产妇脐血CD-3 4 细胞可分离培养出间充质干细胞 ,具有与其它来源MSC类似的表型及分化潜能 ,在体外传代可保持其低分化状态并表达造血因子 。

脐血CD34+细胞的分离与纯化

脐血造血干细胞的分离是进行干细胞移植、体外扩增培养的关键。研究采用明胶自然沉降法、Ficoll分层法分离脐血MNC后,用MACS分离纯化CD34+细胞,计数并用流式细胞仪进行纯度分析。结果显示:每份脐血的采集量平均为95+52ml,3g/dl明胶自然沉降法所获MNC密度平均为(5.76±0.67)×107/ml;CD34+细胞密度平均为(5.53±1.16)×105/ml,较Fi-coll分层法高(P<0.01)。因此,3g/dl明胶自然沉降法是一种较理想的分离MNC的方法,用明胶沉降法和MACS相结合是分离脐血CD34+细胞的理想方法。

脐血 CD_(34)^+ 细胞扩增和改善其移植效率的实验研究

目的：探讨脐血ＣＤ３４＋细胞对造血生长因子的扩增效应及其植入成人骨髓基质的能力。方法：利用免疫磁珠法（ＭＡＣＳ）分离纯化脐血ＣＤ３４＋细胞，在液体培养体系中对其进行体外扩增，并利用铺展贴壁实验研究其移植效率。结果：脐血ＣＤ３４＋ＣＤ３８－细胞亚群的比例明显高于骨髓和外周血；在干细胞因子、白细胞介素（ＩＬ）－３、ＩＬ－６、粒细胞集落刺激因子、红细胞生成素存在的情况下，１０～１４天造血祖细胞可扩增近１００倍，细胞总数超过５００倍；但经２小时铺展贴壁后，仅有３６％的脐血ＣＤ３４＋细胞植入经照射预处理的成人骨髓基质，若经粒－巨噬系集落刺激因子和ＩＬ－３预处理６～８小时后则有６８．０％～８９．６％的ＣＤ３４＋细胞植入基质。结论：细胞因子有可能在体外扩增脐血ＣＤ３４＋细胞数量，改善其移植效率，使脐血祖细胞在“质”和“量”上满足临床移植的需要。

明胶酶A、B在脐血CD_(34)^+细胞及部分白血病细胞系中的表达

目的 研究骨髓及脐血CD34+ 细胞及白血病细胞系的明胶酶 ,即基质金属蛋白酶 9(MMP 9,明胶酶B)和基质金属蛋白酶 2 (MMP 2 ,明胶酶A)的产生 ,探讨它们在CD34+ 细胞迁移、归巢中的意义及在白血病发病中的作用。方法 通过MiniMACS磁珠分离技术分离脐血及骨髓的CD34+细胞 ,通过酶谱分析法测定脐血、骨髓CD34 + 细胞及白血病细胞系的无血清条件培养液中明胶酶的表达。结果 在脐血CD34+ 细胞的条件培养液中可测出相对分子质量为 92× 10 3的亮带 ,骨髓CD34+ 细胞的条件培养液中未测到亮带。部分白血病细胞系U937、KG 1a、HL 6 0可产生相对分子质量为 92×10 3和 72× 10 3的亮带 ,J6 1、J6 2细胞系可产生相对分子质量为 92× 10 3的亮带 ,而HEL、Namalva、CEM、K5 6 2、LCL H不产生亮带。结论 脐血CD34+ 细胞可产生MMP 9,而骨髓CD34+ 细胞不产生 ,脐血CD34+ 细胞具有较强的穿透基底膜的迁移能力并与MMP 9相关。在白血病细胞系中 ,部分髓系白血病细胞系产生MMP 9和MMP 2 ,而非髓系白血病细胞系不产生。

脐血CD_(34)^+细胞与单个核细胞的体外扩增特性研究

目的 探讨CD34+ 富集细胞和单个核细胞 (MNC)的体外扩增特性。方法 利用Min iMACS系统富集CD34+ 细胞 ,在相同条件下与同批MNC进行对照培养 ;观察了再次富选和MNC培养上清 (MNC SN)对CD34+ 富集细胞扩增的影响 ;并尝试了MNCCD34- 细胞的培养。结果 虽然CD34+ 富集细胞具有很高的扩增潜力 ,但在培养过程中 ,其集落密度和CD34 + 细胞含量却始终呈下降趋势 ,而MNC在培养中却出现了一个上升的趋势 ,集落密度和CD34+ 细胞含量分别由第 0天的 (4 12± 16 7) 10 5细胞和 (1.12± 0 .4 2 ) %增至第 7天的 (116 2± 5 6 6 ) 10 5细胞和 (4 .17± 1.4 4 ) % ;再次富选可以使培养过的CD34+ 富集细胞的总细胞和CD34+ 细胞扩增能力大大提高 ;MNCCD34- 细胞具有集落形成和转化为CD34+ 细胞的能力 ;MNC SN对CD34+ 富集细胞的集落形成有促进作用 ,而同时又对CD34+ 细胞有促分化作用。结论 CD34+ 富集细胞在体外大量扩增的同时存在大量分化 ,其在培养过程中产生的CD34-细胞对CD34+ 细胞的扩增有抑制作用 ;脐血MNC中大量的CD34- 细胞含有造血干 祖细胞 ,其分泌的细胞因子有促进CD34+ 细胞向较为成熟的集落形成祖细胞分化的作用。

脐血CD_(34)^+细胞扩增前后端粒酶活性及微卫星遗传稳定性的研究

目的 :通过检测脐血 CD+ 34 细胞扩增前后端粒酶活性及微卫星 DNA序列 ,探讨其遗传稳定性。方法 :用 Mini MACS磁分离技术分离脐血 CD+ 34 细胞 ,体外扩增 2周 ,PCR- EL ISA方法检测扩增前后细胞的端粒酶活性 ;用 PCR银染方法对脐血 CD+ 34 细胞扩增前后 5个微卫星序列进行比较分析。结果 :脐血 CD+ 34 细胞体外扩增过程中 ,端粒酶活性呈现先升后降的趋势 ,0天时 ,端粒酶活性相对值的中位数为 1 4 % ,第 7天明显增高 ,达 5 7% ,第 1 4天回落至 2 9% ;扩增前后 ,BAT- 2 5、Mfd4 1、D5 S4 36、D5 S396、D2 S1 2 35个微卫星位点未见改变。结论 :脐血 CD+ 34 细胞扩增过程中 ,端粒酶活性会暂时性升高 。