造血干细胞动员中外周血CD34+细胞的变化趋势及其对采集的影响

目的 探讨外周血造血干细胞动员过程中外周血CD34+细胞的变化趋势及其对采集时机和采集结果的影响.方法 2010年4月至2011年12月外周血造血干细胞动员及采集27例,其中自体13例,采用化疗联合粒细胞集落刺激因子(G-CSF) 10μg· kg-1·d-1动员方案;健康供者14例,单独应用G-SCF 7.5μg· kg-1·d-1动员方案.监测外周血CD34+细胞数,并与采集产物中获得的单个核细胞(MNC)及CD34+细胞量进行相关性分析.结果 健康供者中获得MNC(5.84±1.48)×108/kg,CD34+细胞(3.93±2.16)×106/kg.自体动员共获得MNC(6.58±3.72)×108/kg,CD34+细胞(3.98±3.06)×106/kg,仅1例自体动员、采集失败.自体动员外周血CD34+细胞在第4天达峰值,健康供者第5天仍处于上升阶段.外周血CD34+细胞百分比和绝对值与采集产物CD34+细胞呈明显正相关.采集当天CD34+细胞绝对值≥20/μ1时,单次采集获得CD34+细胞≥2×106/kg者的比例达76.2％(16/21).结论 外周血CD34+细胞计数是造血干细胞采集过程中的重要监测指标,对于把握采集时机和预测采集效果具有重要意义,CD34+细胞绝对值≥20/μ1可作为采集阈值.     

经抗CD158单克隆抗体修饰的供者自然杀伤细胞对受者树突状细胞的杀伤作用

目的 研究应用抗CD158a和CD158b单克隆抗体封闭供者杀伤细胞免疫球蛋白样受体(KIR)的抑制性受体KIR2DL1及KIR2DL3后,供者自然杀伤细胞(NK细胞)对受者树突状细胞(DC)的杀伤作用,及其减少移植物抗宿主病(GVHD)的作用.方法 对15例异基因全相合造血干细胞移植的供、受者进行HLA及KIR高分辨基因分析,采集供者骨髓及受者外周血单个核细胞,培养受者DC作为靶细胞,使用磁珠负选高纯度供者NK细胞作为效应细胞,用抗CD158a和CD158b单抗封闭供者KIR抑制性受体,并采用四甲基偶氮唑盐比色法分别检测抑制性KIR封闭前后NK细胞对靶细胞的杀伤作用.结果 在效靶比为10∶1下,供者NK细胞抑制性受体经抗体封闭后,对受者DC的杀伤活性明显提高,随着封闭抗体浓度的增加而增高,各浓度间比较,差异均有统计学意义(P＜0.05).当供、受者KIR配体匹配模式为宿主抗移植物(HVG)模式时,供者NK细胞对受者成熟DC的杀伤效果明显高于供、受者KIR基因全相合或移植物抗宿主(GVH)模式,在用不同浓度单抗封闭KIR抗体后,随着封闭抗体浓度的增加,杀伤效果增高更明显(P＜0.05).结论 利用抗CD158单抗封闭KIR可以提高供者来源NK细胞对受者DC的杀伤活性,输注异源反应性NK细胞可能作为一种新的治疗GVHD的方法.     

输注供者自然杀伤细胞对小鼠单倍型相合造血干细胞移植的影响

目的 探讨输注供者自然杀伤(NK)细胞对小鼠单倍型相合造血干细胞移植的影响.方法 选取C57BL/6(H-2b)雄性小鼠为供者、CB6F1(H-2d/b)雌性小鼠为受者.移植前制备供者的骨髓细胞(BMC)、脾细胞(SC)及脾NK细胞,NK细胞经体外培养扩增和激活;所有受者均接受直线加速器X线全身照射(TBI)预处理.TBI后将受者分为4组(每组10只),分别进行单倍型相合造血干细胞移植.单纯TBI组:TBI后不输注细胞,仅作为对照;单纯BMC输注组:输注5×106个BMC;诱发GVHD组:输注5×106个BMC+1.5×107个SC;NK细胞输注组:输注5 x 106个BMC+1.5×107个SC+1×107个NK细胞,并腹腔注射100 ng重组人白细胞介素2(rhIL-2)和1μg rhIL-15,持续7 d.移植后观察各组受者GVHD的发生情况,并对各组受者进行组织病理学、供者细胞嵌合度和免疫功能重建等检测.另取TBI后受者20只,设白血病复发组和白血病治疗组,每组10只.白血病复发组:输注5×106个BMC+1×107个SC+2×106个白血病细胞株EL9611;白血病治疗组:在白血病复发组的基础上再输注1 x 107个NK细胞,并腹腔注射100 ng rhIL-2和1μg rhIL-15,持续7 d.观察两组受者白血病复发情况和移植后100 d的存活率.结果 单纯BMC输注组受者无GVHD发生,NK细胞输注组受者GVHD的评分和组织病理学改变均较诱发GVHD组轻(P<0.05)f诱发GVHD组的免疫功能重建较NK细胞输注组延迟.白血病复发组和白血病治疗组移植后100 d的存活率分别为20%和90%,两组比较,差异有统计学意义(P<0.01).结论 输注激活的供者NK细胞可以减轻小鼠单倍型相合造血干细胞移植后的GVHD,减少白血病复发,促进免疫功能重建.     

第三方脐血预防异基因造血干细胞移植后移植物抗宿主病

目的 探讨无关供者和单倍型亲缘供者异基因造血干细胞移植(allo-HSCT)前输注第三方脐血预防移植物抗宿主病(GVHD)的效果.方法 2007年至2011年接受无关供者及单倍型亲缘供者allo-HSCT的受者共41例.脐血预防组(25例),于移植前1d予以HLA配型为4/6～6/6的第三方脐血细胞输注,平均输入有核细胞数为(1.64±0.49)×107/kg,次日再输入供者造血干细胞.其余患者作为对照组(16例).两组均采用抗胸腺细胞球蛋白+环孢素A+甲氨蝶呤+吗替麦考酚酯预防急性GVHD.比较两组间急性GVHD的发生率、严重程度以及移植相关死亡率.结果 脐血预防组和对照组aGVHD累积发生率分别为44.0％(11/25)和68.8％(11/16),两组比较,差异无统计学意义(x2=2.403,P＞0.05);Ⅲ～Ⅳ度aGVHD累积发生率分别为16.0％(4/25)和37.5％(6/16),两组比较,差异无统计学意义(x2=2.445,P＞0.05),100 d治疗相关病死率分别为12.0％(3/25)和12.5％(2/16),两组比较,差异无统计学意义(x2=0.002,P＞0.05).结论 在无关供者及单倍型亲缘供者allo-HSCT前应用第三方脐血细胞,可能有效预防GVHD的发生或减轻GVHD的严重度,但尚需要进一步设计扩大规模的随机对照临床试验验证其有效性.     

CD4+CD25+调节性T细胞与移植物抗宿主病

造血干细胞移植是治疗造血系统疾病的有效方法,但是发生率及死亡率都很高的移植物抗宿主病(GVHD)限制了这一治疗方法的广泛应用.目前免疫抑制药物虽然是最有效的治疗GVHD的办法,但是患者长期服用容易导致严重感染和肿瘤.因此,人们在寻找能够诱导抗原特异性的移植耐受的方法,以减少或减轻GVHD发生.目前发现体内存在一类CD4+CD25+免疫调节性T细胞(regulatory T cells,Treg细胞)在减少或减轻GVHD发生方面显示了一定的应用前景,本文就CD4+CD25+Treg细胞在GVHD防治中的应用前景进行了综述.     

供者使用粒系集落刺激因子后供髓对异基因骨髓移植受者的影响

目的　观察供者使用粒系集落刺激因子 (G CSF)后供髓对异基因骨髓移植受者造血重建及移植物抗宿主病 (GVHD)发生的影响。方法　用HLA相合 ,混合淋巴细胞培养 (MLC)阴性的供者骨髓 ,给受者行异基因骨髓移植 ,在预处理方案、GVHD的预防及支持治疗方法相同的条件下 ,比较研究组 30例供者应用G CSF 3～ 4μg·kg-1·d-1,连用 7d后采髓和对照组 15例供者常规采髓对受者异基因骨髓移植造血重建和GVHD发生的作用。结果　在类同采髓量中研究组所获单核细胞(MNC)、粒细胞巨噬细胞集落生成单位 (CFU GM )及CD34数显著大于对照组 (P <0 .0 1)。研究组受者移植后中性粒细胞计数 >0 .5× 10 9/L与血小板 >2 0× 10 9/L时间分别为 (16 .7± 3.2 )d与 (18.4± 3.0 )d ,对照组为 (2 2 .5± 5 .1)d与 (2 6 .3± 5 .90 )d ,P <0 .0 1,研究组造血重建加速。研究组受者急性Ⅱ Ⅳ度GVHD发生 1例 (发生率 3 .3% ) ,对照组发生率 2 6 .7% (P <0 .0 5 )。慢性GVHD两组差异无显著性 (P >0 .0 5 )。输入的T淋巴细胞总数无明显改变 ,通过比较 ,研究组受者CD4减少 ,CD8增加 (P <0 .0 1)。结论　HLA相合的异基因骨髓移植 ,使用经G CSF动员的供者骨髓 ,可使受者造血重建加快 ,尤其减轻重度急性GVHD ,这一作用可能与骨髓内T淋巴细胞亚群     

门静脉输注供者脾细胞诱导的供者特异性免疫低反应性

目的 探讨经门静脉输注供者脾细胞能否诱导皮肤移植小鼠产生供者特异性的免疫低反应性及其可能机制.方法 取Balb/c小鼠,随机分为空白对照组(经小鼠门静脉输注RPMI 1640培养液)、受者脾细胞组(经小鼠门静脉输注Balb/c小鼠脾细胞)、供者脾细胞组(经小鼠门静脉输注C57BL/6小鼠脾细胞)、空白移植对照组(经小鼠门静脉输注RPMI 1640培养液,7 d后移植C57BL/6小鼠的皮肤)、实验对照组(经小鼠门静脉输注Balb/c小鼠脾细胞,7 d后移植C57BL/6小鼠的皮肤)、实验组(经小鼠门静脉输注C57BL/6小鼠脾细胞,7 d后移植C57BL/6小鼠的皮肤)以及第三方移植组(经小鼠门静脉输注C57BL/6小鼠脾细胞,7 d后移植C3H小鼠的皮肤).记录空白移植对照组、实验对照组、实验组和第三方移植组移植皮肤的存活时间,并观察移植皮肤的病理学变化;脾细胞输注后7 d,分别获取空白对照组、受者脾细胞组和供者脾细胞组小鼠的外周血、脾脏和肝脏,用流式细胞仪测定样本中CD4+CD25+Foxp3+调节性T淋巴细胞(CD4+CD25+Foxp3+Treg细胞)的比例.结果 实验组移植皮肤的存活时间为(19.8±4.6)d,明显长于空白移植对照组、实验对照组和第三方移植组,但仍未达到长期存活.皮肤移植后7 d,空白移植对照组和实验对照组的移植皮肤呈现重度急性排斥反应的病理学改变,而实验组移植皮肤呈现中度急性排斥反应的病理学改变.供者脾细胞组外周血、肝脏和脾脏中CD4+CD25+Foxp3+Treg细胞比例明显高于空白对照组和受者脾细胞组.结论 门静脉输注供者脾细胞可特异性地延长供者皮肤移植物的存活时间,减轻移植物的排斥反应,该效应可能与受者体内的CD4+CD25+Foxp3+Treg细胞增加有关.     

非血缘关系造血干细胞移植61例分析

目的 探讨非血缘关系造血干细胞移植(URD-HSCT)的临床疗效,移植相关并发症及影响预后的危险因素.方法 回顾性分析61例接受URD-HSCT患者的临床资料.所有患者根据原发病分别给予非清髓性及清髓性预处理;供、受者HLA配型6/6抗原位点全相合21例,5/6相合5例,1个基因亚型不合24例,2个基因亚型不合11例;供、受者间ABO血型相合18例,不合43例;受者接受供者的有核细胞中位数为4.5×108/kg,CD34+细胞中位数为4.3×106/kg.术后移植物抗宿主病(GVHD)的预防采用以短程甲氨蝶呤+环孢素A+霉酚酸酯为基础的方案,49例加用抗CD25单克隆抗体,9例加用抗淋巴细胞或抗胸腺细胞免疫球蛋白;并常规采用促进造血功能恢复、抗感染等治疗.术后观察受者的造血功能重建、并发症以及预后情况.结果 61例患者中,59例术后经血型、染色体及DNA多态性检测证实供者细胞植活.术后23例受者发生Ⅱ～Ⅳ度急性GVHD,25例发生慢性GVHD;术后100 d内,48例受者发生细菌和(或)真菌感染,36例发生巨细胞病毒感染,以下呼吸道感染较多.术后有18例受者死亡,受者总的2年无病存活率为(68.0±6.4)%,其中12例因移植相关并发症死亡,移植相关死亡率19.7%;6例原发病复发的受者均死亡,复发率9.8%.其余受者经治疗后好转.结论 URD-HSCT是治疗造血系统恶性疾病的有效方法.急性GVHD和感染是严重影响移植疗效和预后的危险因素,需早期预防.     

移植肾CD20~+淋巴细胞浸润的临床意义

目的:探讨CD20+细胞在移植肾排斥反应中浸润程度与移植肾预后的关系。方法:选取93例肾移植后穿刺患者,肾活检组织标本行CD20免疫组化染色。并对病理结果行半定量分析,根据CD20+细胞在肾组织内浸润程度,分为阴性组48例(N组,CD20+细胞浸润占肾小管间质面积<10%)、中度浸润组25例(M组,CD20+细胞浸润占肾小管间质面积≥10%<50%)和重度浸润组20例(H组,CD20+细胞浸润占肾小管间质面积≥50%)。分析CD20+细胞浸润的程度与移植肾预后的相关性。随访内容包括患者穿刺活检后的临床资料,随访内容包括患者的肌酐、蛋白尿变化及移植肾的生存状况等指标。结果:三组患者在年龄、性别、组织活检时间以及移植的次数均没有明显差异(P>0.05);穿刺活检后12个月的肌酐N组[(276.79±240.78)μmol/L]低于M组[(360.16±290.30)μmol/L];M组低于H组[(466.50±330.53)μmol/L],P<0.05;CD20+浸润组的患者的4年生存率明显低于阴性组(P<0.05);穿刺活检后12个月内,N组穿刺后出现蛋白尿的概率明显低于M组和H组(P<0.05)。结论:CD20+细胞在肾移植内浸润的程度与移植肾预后有明显相关性。     

咪达唑仑和异丙酚对急性心肌梗死大鼠血清VEGF浓度及G-CSF药物动员骨髓干细胞效果的影响

目的 评价咪达唑仑和异丙酚对急性心肌梗死大鼠血清血管内皮生长因子(VEGF)浓度及粒细胞集落刺激因子(G-CSF)药物动员骨髓干细胞效果的影响.方法 雄性Wistar大鼠36只,体重250 ～ 280 g,采用结扎左冠状动脉前降支的方法制备急性心肌梗死模型,采用腹腔连续注射G-CSF 5d进行药物动员,于药物动员后第7天,按照随机数字表法,将大鼠随机分为G-CSF组(G组)、咪达唑仑组(M组)及异丙酚组(P组),每组12只.G组以0.5 ml/h的速率股静脉输注生理盐水6h;M组股静脉输注咪达唑仑0.05 mg·kg-·h-1 6 h;P组股静脉输注异丙酚5mg·kg-1 ·h-16 h.于给药完毕后经股静脉取血,采用流式细胞仅测定CD34+单核细胞( CD34+ MNC)和内皮祖细胞(EPCs)数目,采用ELISA法测定血清VEGF浓度.于心肌梗死后4周每组随机取6只大鼠测定左心室舒张末压(LVEDP)、最大收缩速率(+dp/dtmax)和最大舒张速率(- dp/max).结果 与G组比较,M组CD34+MNC及EPCs细胞数目增加,血清VEGF浓度升高,LVEDP下降,-dp/dtmax的绝对值升高(P＜0.05),P组LVEDP下降,- dp/dtmax的绝对值升高(P＜0.05);与P组比较,M组CD34+MNC及EPCs细胞数目增加,血清VEGF浓度升高,LVEDP下降,- dp/dtmax的绝对值升高(P＜0.05).结论 咪达唑仑可促进VEGF的释放,加强G-CSF动员骨髓干细胞的作用,改善急性心肌梗死后大鼠的心脏功能;异丙酚不能促进VEGF的释放及无骨髓干细胞动员的作用.     

The effect of G-CSF-stimulated donor marrow on engraftment and incidence of graft-versus-host disease in allogeneic bone marrow transplantation

Graft-versus-host disease (GVHD) and infection are major obstacles to successful allogeneic bone marrow transplantation (allo-BMT). In an attempt to improve the results of HLA-identical sibling BMT, we investigated the effect of accelerating hemopoietic reconstitution and reducing acute GVHD (aGVHD) in allo-BMT receiving G-CSF-stimulated donor marrow and the preliminary biological mechanism. The donors of 30 patients (study group) with leukemia were given G-CSF 3-4 microg/kg/d for 7 doses prior to marrow harvest. The results of subsequent engraftment in the recipients were compared with those of 18 patients without G-CSF (control group). Five donors themselves were studied to assess the effects of G-CSF on the hematopoietic progenitor cells and lymphocyte subsets in the bone marrow (BM). We observed that the stimulated BM yielded higher numbers of nucleated cells as well as CFU-GM and CD34+ cells (p<0.01), and that hemopoietic reconstitution was accelerated. The median number of days of granulocyte count exceeding 0.5x10(9)/L and platelet count exceeding 20x10(9)/L was 16 (range 10-23 d) and 18.5 (range 13-31 d), respectively (control group: median 22 d, range 13-29 d and median 23 d, range 17-34 d; p=0.001). The incidence of grade II-IV severe aGVHD was very low, with only 1 case (3.3%) with acute grade II aGVHD limited to the skin in the study group. Five of 18 patients in the control group manifested grade II-IV severe aGVHD (27.8%, p=0.02). The number of T-lymphocyte subsets in the harvested BM using G-CSF stimulation was changed. In the G-CSF-stimulated marrow group, CD4+ decreased and CD8+ increased significantly (p=0.02). The changes of progenitor cells and T-lymphocyte subsets in donors' BM from pre- and post-G-CSF stimulation showed that the percentage of CD4+ reduced (p=0.04) and that of CD8+ increased (p=0.06), while that of CD34+ also increased (p=0.002). The incidence of chronic GVHD and relapse had no significant difference between both groups. These results indicate that allo-BMT in BM G-CSF priming can accelerate engraftment and minimize the incidence of severe aGVHD. There is a trend in favor of improved transplantation-related mortality.     

供者应用粒细胞集落刺激因子促进异基因骨髓移植受者造血重建的临床研究

目的评价供者应用粒细胞集落刺激因子（G-CSF）以增加骨髓中CD34+细胞的效果和对异基因骨髓移植造血重建的影响。方法供者采髓前应用G-CSF250μg/d,连用7d,随后对15例白血病患者进行异基因骨髓移植,观察植入物单个核细胞、CD3     

CD34+细胞在干细胞旁分泌下向内皮细胞分化的研究

目的 探讨人脐血CD34+细胞在脐带间充质干细胞(UC-MSCs)旁分泌作用下向内皮细胞诱导分化的可行性.方法 收集20份脐血,体积(103.80±19.77)ml.免疫磁珠(MACS)分选CD34+细胞;取脐带用消化贴壁法获得UC-MSC.流式鉴定干细胞表型.实验分单纯培养组、诱导组、共培养组.结果 流式鉴定CD34+细胞纯度(95.02±3.81)%.培养14 d流式检测共培养组表达CD31、CD144、VWF分别为(65.43±5.61)%、(54.40±4.13)%、(47.53±3.96)%(与单纯培养组比较P＜0.05),部分表达CD34,阴性表达CD45,这与诱导组及成熟脐静脉内皮细胞表达率一致.结论 UC-MSCs旁分泌作用与外源性细胞因子都具有促分化作用,均能使脐血CD34+细胞向内皮细胞分化.

Abstract：

Objective To study whether the paracrine action of umbilical cord-derived mesenchymal stem cells (UC-MSC) can induce differentiation of human umbilical cord blood-derived CD34+ cells into endothelial cells in vitro. Methods The 20 fresh umbilical cord blood samples were collected with volume of (103. 80 ± 19. 77) ml. CD34+ cells were isolated from the mononuclear cells by magnetic activated cell sorting system (MACS) , and mesenchymal stem cells (MSCs) were isolated from umbilical cord by collagenase and trypsin digestion. Three groups were set up: CD34+ cells pure culture group, cytokineinduced group and two stem cells co-culture group with noncontact. Results The average purity of enriched CD34+ cells as assessed by FACS was (95. 02 ± 3. 81) %. Freshly isolated CD34+ cells were small and round which suspended in culture medium. Attached cord-like structure cells of CD34+ cells appeared after 7 days coculture with noncontacted MSC, and when the CD34+ cells grew into large number, they formed colonies. These cells expressed endothelial specific markers, including CD144 (54. 40 ±4. 13)% ,vWF (47. 53 ± 3. 96) % , CD31 (65. 43 ± 5. 61) % ( P ＜ 0. 05, as compared with CD34+ cells pure culture group) , partially expressed CD34 and, the leukocyte common antigen CD45 was negatively expressed.Conclusion Human umbilical cord blood-derived CD34+ cells could be induced into endothelial cells under the paracrine action of umbilical cord-derive mesenchymal stem cells which has the same effect of cytokines.     

粒细胞集落刺激因子和干细胞因子对骨髓单个核细胞的动员效率及其机制

目的 联合应用粒细胞集落刺激因子(G-CSF)和干细胞因子(SCF)动员骨髓单个核细胞,评价其动员效果,探讨CXCL12/CXCR4信号通路在骨髓单个核细胞动员中的作用及机制.方法 将昆明小鼠随机分为两组,治疗组皮下注射重组鼠G-CSF 100μg/(kg·d)和重组鼠SCF25μg/(kg·d),连续使用5d;对照组皮下注射等剂量的生理盐水.每组于不同时间点取小鼠骨髓,分离培养骨髓单个核细胞,计数成纤维样细胞集落形成单位(CFU-F)的个数;应用流式细胞仪分选CD34+ CXCR4+单个核细胞(MNCs);应用酶联免疫吸附试验(ELISA)法测定骨髓细胞外液CXCL12a的水平;采用逆转录-聚合酶链反应(RT-PCR)检测骨髓CXCL12 mRNA表达变化.结果 应用G-CSF/SCF后,骨髓及外周血中单个核细胞计数较对照组明显增加(P<0.01),CFU-F形成能力显著增强(P<0.05);流式分选表明CD34+ CXCR4+细胞占骨髓CD34+单个核细胞总数的(44.6±8.7)%;RT-PCR和EUSA检测示骨髓CXCL12 mRNA表达下降,骨髓细胞外液CXCL12蛋白也显著下降,两者变化一致.结论 G-CSF/SCF可有效地诱导骨髓单个核细胞动员,其机制可能是通过破坏骨髓CXCL12/CXCR4信号通路平衡,下调CXCL12/CXCR4间相互作用,以促进骨髓单个核细胞动员.     

动员自体骨髓干细胞对大鼠急性肾小管坏死的治疗

目的：观察粒细胞集落刺激因子联合干细胞因子动员骨髓干细胞的作用、骨髓干细胞是否具有向损伤肾组织归巢的能力及其在肾脏组织中的分布,初步探讨粒细胞集落刺激因子联合干细胞因子是否具有促进急性肾小管坏死修复的作用。方法：160只8～10周龄雄性SD大鼠随机分为4组：对照组,模型组、G-CSF＋SCF治疗组、G-CSF＋SCF对照组,检测：（1）外周血白细胞总数及单个核细胞中CD34＋细胞百分比的变化;（2）尿NAG酶检测;（3）肾脏组织病理学改变;（4）肾组织CD34＋细胞表达变化。结果：（1）G-CSF＋SCF治疗组和G-CSF＋SCF对照组外周血中白细胞数、CD34＋细胞百分比于第5天达高峰,与对照组、模型组相比,差异有统计学意义（P〈0.05）,以后逐渐下降;相应地,G-CSF＋SCF治疗组肾组织内CD34＋细胞较对照组、模型组也明显增多（P〈0.05）。（2）手术后第5、10、17天,G-CSF＋SCF治疗组尿NAG酶、肾脏病理学改变均明显好于模型组（P〈0.05）。第24天G-CSF＋SCF治疗组尿NAG酶、肾脏病理学改变基本恢复正常,而模型组仍异常。第31天各组间尿NAG酶、肾脏病理学改变其差异无统计学意义。结论：（1）粒细胞集落刺激因子和干细胞因子联合应用对缺血再灌注损伤诱发急性肾小管坏死大鼠的骨髓干细胞有显著的动员作用。（2）骨髓干细胞能在损伤的肾小管归巢和定居,并可能参与损伤肾组织的修复。（3）粒细胞集落刺激因子和干细胞因子联合应用能在一定程度上加速急性肾小管坏死后肾功能的修复。     

CD44+-ESA+在结肠癌干细胞分选中的应用

目的 分选结肠癌细胞株SW480细胞中的CD133+-CD44+-ESA+亚群细胞,并观察其致瘤性.方法 用流式细胞仪分选SW480细胞中CD133+-CD44+-ESA+、CD133--CD44+-ESA+及CD133--CD44--ESA-亚群细胞.将这3组细胞分别接种于NOD/SCID小鼠,每组5只,观察肿瘤生长...     

Mobilization of haematopoietic and non-haematopoietic cells by granulocyte-colony stimulating factor and vascular endothelial growth factor gene therapy in patients with stable severe coronary artery disease

OBJECTIVES: To investigate the mobilization of non-haematopoietic and haematopoietic cells from the bone marrow induced by intramyocardial VEGF gene therapy and G-CSF treatment alone or in combination in patients with chronic ischemic heart disease (IHD). Secondly, we tested the hypothesis that the quantity of circulating stem cells correlated with improvement in symptoms. DESIGN: We treated I) 16 patients with intramyocardial placebo injections, II) 16 patients with intramyocardial VEGF-A165 gene injections, III) 13 patients with low dose G-CSF, and IV) 16 patients with intramyocardial VEGF-A165 gene followed by high dose G-CSF. RESULTS: Circulating CD34+ cells and CD45 CD34 -cells increased by G-CSF in a dose-dependent manner, but did not increase with VEGF gene therapy. The CD45/CD34- cells subgroups increased in both G-CSF treated groups. No association was found between the concentration of mobilized stem cells in the circulation and improvement in symptoms. CONCLUSIONS: G-CSF mobilized both haematopoietic and non-haematopoietic cells from the bone marrow in a dose-dependent manner in patients with chronic IHD. However, even high levels of circulating stem cells did not improve symptoms of IHD.     

Mobilization of haematopoietic and non-haematopoietic cells by granulocyte-colony stimulating factor and vascular endothelial growth factor gene therapy in patients with stable severe coronary artery disease

Objectives. To investigate the mobilization of non-haematopoietic and haematopoietic cells from the bone marrow induced by intramyocardial VEGF gene therapy and G-CSF treatment alone or in combination in patients with chronic ischemic heart disease (IHD). Secondly, we tested the hypothesis that the quantity of circulating stem cells correlated with improvement in symptoms. Design. We treated I) 16 patients with intramyocardial placebo injections, II) 16 patients with intramyocardial VEGF-A₁₆₅ gene injections, III) 13 patients with low dose G-CSF, and IV) 16 patients with intramyocardial VEGF-A₁₆₅ gene followed by high dose G-CSF. Results. Circulating CD34+ cells and CD45 ? CD34 ? cells increased by G-CSF in a dose-dependent manner, but did not increase with VEGF gene therapy. The CD45 ? /CD34? cells subgroups increased in both G-CSF treated groups. No association was found between the concentration of mobilized stem cells in the circulation and improvement in symptoms. Conclusions. G-CSF mobilized both haematopoietic and non-haematopoietic cells from the bone marrow in a dose-dependent manner in patients with chronic IHD. However, even high levels of circulating stem cells did not improve symptoms of IHD.     

纯化自体外周血CD34+细胞移植治疗下肢重度缺血

目的 探讨纯化自体外周血CD34+细胞移植治疗下肢重度缺血的安全性、可行性和有效性.方法 2009年5月至2010年3月采用纯化自体外周血CD34+细胞移植治疗下肢重度缺血7例,其中血栓闭塞性脉管炎6例,结节性红斑伴血栓形成1例,年龄23～54岁,平均(39±11)岁;均不具备血管重建条件.经G-CSF动员后第5天采集外周血单个核细胞,分选获得纯化CD34+细胞,下肢肌肉局部注射,观察不良反应和缺血缓解情况.结果 7例患者均获技术和保肢成功,移植细胞数(7.1±2.3)×105/kg[(4.6×105～1×106)/kg].7例患者均获随访,随访时间6～14个月,平均(8±3)个月.术后1个月静息痛均明显缓解,Wong-Baker FACES疼痛评分由术前平均7.1±2.0(4～10)降至1.1±1.1(0～2),P=0.0000.无痛步行时间术前平均(4±4)min(1～10 min),术后3个月延长至(12±7)min(5～21 min),P=0.04,术后6个月(20±12)min(6～40 min),P=0.02.术前踝肱指数0.54±0.18(0.41～0.87),术后3个月提高至0.66±0.13(0.52～0.86),P=0.17,术后6个月0.72±0.13(0.56～0.91),P=0.07.6例溃疡中,3例直径＜2 cm者完全愈合,另3例直径＞2 cm者明显缩小.经皮氧分压术前(29±14)mmHg(10～52 mm Hg),术后3个月(46±14)mm Hg(27～63 mm Hg),P=0.04,术后6个月(57±10)mm Hg(41～66 mm Hg),P=0.001.无严重不良反应.结论 初步结果显示纯化自体外周血CD34+细胞移植治疗下肢重度缺血安全,可行,有效.

Abstract：

Objective To evaluate the safety, feasibility and efficacy of transplantation of purified peripheral blood CD34+ cells in treatment of critical ischemia of the lower extremities. Methods From May 2009 to March 2010, seven cases of critical ischemia of the lower extremities received purified peripheral blood CD34+ cells transplantation, among those 6 were caused by thromboangiitis obliterans and 1 by thrombosis coexistent with nodular erythema. Mean age was ( 39 ± 11 ) years ( range 23 - 54 ), and all patients were not suitable for surgical or endovascular revascularization. G-CSF was subcutaneously injected for 5 days before apheresis for peripheral blood mononuclear cells. Then CliniMACS system was used to isolate the CD34+ cells. If the number of CD34+ cells was between 105/kg and 106/kg , they were all intramuscular injected into patients' calf and foot. Results Technical success and limb salvage were achieved in all cases. The mean number of transplanted cells was (7. 1 ±2.3) × 105/kg [ range(4.6 ×105 -1 × 106 )/kg]. All cases were followed-up, ranging from 6 - 14 months (mean 8 ± 3 months). One month after transplantation, the rest pain was obviously relieved in all cases, and the Wong-Baker FACES pain rating scale score significantly decreased from 7. 1 ±2. 0(4 - 10)to 1. 1 ± 1.1 (0 -2) ,P =0. 0000. The pain-free walking distance was significantly improved from (4 ± 4) min (range 1 -10 min)to (12 ± 7 ) min (range 5 - 21min , P =0.04) at 3 months and(20.4 ± 12.5) min(range 6 -40 min, P = 0.02) at 6 months, respectively. The ankle-brachial index increased from 0. 54 ± 0. 18 ( range 0. 41 - 0. 87 ) to 0.66 ±0. 13(range 0. 52-0. 86 , P=0. 17)at 3 months and 0.72 ±0. 13(range 0.56 -0.91, P=0. 07)at 6 months, respectively. Of 6 cases with the toe ulcer, the ulcer was healed in 3 and apparently shrank in 3. Transcutaneous partial oxygen pressure rose from (29 ± 14) mm Hg(range 10 -52 mm Hg)to 46 ±14 mm Hg ( range 27 - 63 mm Hg, P = 0. 04) at 3 months and (57 ± 10) mm Hg( range 41 - 66 mm Hg, P =0.001) at 6 months, respectively. No serious complications were found either perioperatively or postoperatively. Conclusions Transplantation of purified peripheral blood CD34+ cells is safe, feasible and effective in the treatment of critical ischemia of the lower extremities.