重组人粒细胞集落刺激因子促进骨髓间充质干细胞增殖

目的探讨重组人粒细胞集落刺激因子(rhG-CSF)对小鼠骨髓间充质干细胞(MSCs)增殖的影响。方法将昆明小鼠随机分为2组(n=15),G-CSF组采髓前皮下注射重组人粒细胞集落刺激因子(rhG-CSF)80μg·kg-1·d-1,连用5d,对照组皮下注射等量生理盐水。每组分别于最后一次注射后6h、12h及7d(168h)取小鼠骨髓,分离、培养MSCs,计数成纤维样细胞集落形成单位(CFU-F)的个数;应用流式细胞仪检测单个核细胞(MNCs)细胞周期及CFU-F细胞表面抗原特征。结果应用rhG-CSF后,培养MNCs所获得的CFU-F数目增加(P<0.01),CFU-F数与取材时间(6h、12h、7d)呈负相关(P<0.05),但12h取材者与7d取材者比较,CFU-F数目的差异无统计学意义(P>0.05)。MNCs培养形成的CFU-F,其细胞表面抗原呈CD34-、CD133-、CD90+、CD105+,分别占2.5%、3.1%、67.0%和78.0%。G-CSF组各时间点获得的MNCs,其G0/G1期细胞百分率较对照组低(P<0.05),而S+G2/M期增高(P<0.05),且6h取材者S+G2/M期细胞百分率明显高于12h和7d取材者(P<0.05)。结论rhG-CSF可促进骨髓MNCs进入细胞增殖周期,增殖的高峰在应用G-CSF后6h左右。     

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     

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。     

促血小板生成素对小鼠骨髓干细胞增殖的影响

目的 探讨促血小板生成素(TPO)对小鼠骨髓有核细胞(BMNC)的细胞周期及骨髓干细胞增殖的影响.方法 将昆明小鼠随机分为实验组与对照组,每组15只.实验组腹腔注射TPO50 μg·kg-1·d-1 ,连用5 d;对照组腹腔注射生理盐水0.1 ml·g-1·d-1.每组分别于最后1次注射后6 h、12 h及168 h收集BMNC,检测BMNC的细胞周期及表达CD34+的抗原绝对数;计数BMNC,以1×106细胞/cm2接种培养并计数原代成纤维样细胞集落形成单位(CFU-F).结果 应用TPO后的细胞周期显示,实验组在6 h和12 h取材BMNC的G0/G1期细胞百分率较对照组低12%～8%(P＜0.05),而S+G2期增高12%～8%(P＜0.05);实验组BMNC表达CD34+的抗原绝对数较对照组明显增加1～2倍(P＜0.05);实验组所获得CFU-F集落数比对照组增加3～9倍(P＜0.01).结论 TPO促进CD34+细胞增加的同时,还可促进小鼠BMNC进入细胞周期,促进CFU-F集落数增多,即促进骨髓间充质干细胞(MSC)增殖,有利于改善造血干细胞移植(HSCT)的效果.     

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.     

自体外周血干细胞移植治疗糖尿病足的疗效及其与CD34~+水平关系的研究

[目的]探讨自体外周血干细胞移植最佳动员方案及采集时机,同时观察自体外周血干细胞治疗糖尿病足的临床疗效。[方法]对2006年7月～2012年12月本科治疗的73例糖尿病足患者分别采用粒细胞集落刺激因子5、10μg/(kg.d)进行造血干细胞动员,分析粒细胞集落刺激因子动员天数、剂量与外周血白细胞、单个核细胞、CD34+细胞数的关系,在干细胞达到高峰时应用血细胞分离机分离出干细胞混悬液约50 ml,沿病变闭塞血管干走行区域注射。[结果]随着动员天数的增加,白细胞和单个核细胞、CD34+细胞数也随之增加,干细胞获得的效率与粒细胞集落刺激因子的剂量、动员时间有关,外周血中CD34+总数与单个核细胞总数呈正相关。患者注射后早期局部皮温明显升高,创面渗血增多,创面肉芽生长明显增快。患者在疼痛、间歇性跛行距离、踝肱指数(ABI)等方面的指标均有明显改善,4～6周复查DSA(数字减影血管造影)发现下肢闭塞血管有明显的侧支循环建立。[结论]自体外周血干细胞移植后患者有明显的侧支循环建立,局部血运改善,利于溃疡愈合,从而避免了截肢或最大限度降低了截肢平面。糖尿病足患者干细胞采集的最佳时机不能单凭动员天数和外周血白细胞数决定,而是由外周血单个核细胞数和CD34+的数量来决定。     

人粒细胞集落刺激因子动员外周血细胞45例分析

目的观察人粒细胞集落刺激因子(G-CSF)动员前后外周血细胞数量和标记的变化,评估G-CSF动员能力。方法统计2004年3月至2018年12月期间中山市人民医院45例下肢缺血性疾病患者应用G-CSF动员前后外周血中的白细胞、中性粒细胞、单核细胞、红细胞和血小板数量和CD34+标记变化,形成配对资料。结果动员前外周血中的白细胞、中性粒细胞、单核细胞、CD34+细胞绝对计数分别为(8.38±2.21)×10~9/L、(5.26±2.33)×10~9/L、(0.75±0.39)×10~9/L、(9.55±19.35)个/μl,G-CSF动员第3天上升至(40.60±12.90)×10~9/L、(34.88±11.74)×10~9/L、(1.86±0.77)×10~9/L、(33.45±47.42)个/μl,差异均有统计学意义(t=9.230、9.662、4.424、2.732,P=0.000、0.000、0.001、0.021);单核细胞占比动员前为(8.15±1.67)%,动员第3天反降至(4.89±2.13)%,差异有统计学意义(χ2=-5.116,P0.001)。停止动员后第3天外周血中的白细胞、中性粒细胞、单核细胞、CD34+细胞数量恢复到动员前水平。红细胞、血小板数量动员前后无明显变化,差异无统计学意义(t=1.428、0.538,P=0.184、0.602)。结论 G-CSF动员可以增加外周血中白细胞、中性粒细胞、单核细胞、CD34+细胞的数量,但动员单核细胞能力有限,而红细胞、血小板数量未受明显影响。     

粒细胞集落刺激因子减轻小鼠混合骨髓移植后移植物抗宿主病的实验研究

目的探讨同基因骨髓混合一定比例粒细胞集落刺激因子(G-CSF)动员的异基因骨髓移植能否减轻急性移植物抗宿主病(aGVHD).方法将BALB/c与BCF1(BALB/c×C57BL/6)小鼠或与G-CSF动员BCF1小鼠脾细胞按一定比例混合,腹腔注入BALB/c幼鼠,制备新生小鼠GVHD模型,结果以脾指数表示.成年雌性BALB/c小鼠接受60Co全身照射8.5Gy后进行移植,移植物为BALB/c与雄性BCF1或与G-CSF动员BCF.小鼠骨髓细胞按一定比例的混合,移植细胞总数60×105个/只.观察移植小鼠aGVHD典型症状、病理表现及存活率.ELISA法测定细胞因子含量,流式细胞术分析T细胞亚群变化.结果(1)注射BALB/c与BCF1小鼠脾细胞混合比例为21、11及异基因BCF1小鼠脾细胞的新生小鼠均发生GVHD;但G-CSF动员与否,GVHD发生程度差异有统计学意义.(2)21及11混合骨髓移植(MBMT)组小鼠有中到重度GVHD表现;经G-CSF动员的MBMT组小鼠8周存活率较未动员组明显提高(P＜0.05).(3)G-CSF动员供鼠后L3T4+细胞下降显著,L3T4+/Lyt2+比值明显低于未动员组(P＜0.01).(4)G-CSF动员供鼠后混合淋巴细胞反应(MLR)细胞培养上清中,IL-2、IFN-γ水平降低,IL-4水平升高.结论同基因骨髓混合一定量H-2半相合异基因骨髓移植可减轻GVHD的发生;G-CSF动员供鼠可进一步减轻MBMT后GVHD的发生.其机理可能与IL-2、IFN-γ下降、IL-4升高有关.     

骨髓干细胞转分化为肾祖细胞并参与修复急性肾脏损伤的研究

目的 观察骨髓干细胞是否可以向肾祖细胞转分化,成为肾脏祖细胞库的肾外来源;验证粒细胞集落刺激因子(G-CSF)是否可以促进骨髓干细胞向肾脏祖细胞的转分化,提高肾脏修复的效能.方法 6周龄全身表达绿色荧光蛋白(GFP)的C57BL/6J转基因小鼠提供骨髓,6~8周龄同种无荧光标记的C57BL/6J小鼠40只作为骨髓受体.骨髓移植前,受体小鼠接受致死剂量的γ放射线137Cs照射,骨髓重建情况经流式细胞仪检测确认.骨髓重建完毕后所有小鼠均接受单侧肾脏缺血再灌注损伤.干细胞动员效果及向肾脏归巢情况经流式细胞仪检测鉴定.损伤4、8周后取肾脏标本行免疫荧光组织化学染色,观察骨髓来源的肾脏祖细胞数以及骨髓细胞在微血管形成中的作用.损伤4周后通过组织切片免疫荧光组织化学方法观察并计数微血管细胞数.结果 G-CSF动员1 d后,分别为CD29、CD34、Sca-1、c-Kit、Flk-1阳性的干细胞占外周血非红系细胞的比例均高于对照组(P<0.05).损伤4周后,G-CSF动员组的肾脏中,骨髓来源并且分别表达Sca-1/GFP、CD29/GFP的干细胞的比例均高于对照组(P<0.05);在损伤4周及8周后,肾脏切片免疫荧光组织化学显示G-CSF动员肾脏中骨髓来源的肾祖细胞即Sca-1/GFP双阳性的细胞数量高于对照组.损伤4周后,动员组肾脏中表达CD31的微血管密度高于对照组(P<0.05).损伤4周后肾脏组织中存在CD105/GFP及α-SMA/GFP双阳性的细胞.结论 ①骨髓干细胞可以转分化为器官特异性干细胞-肾脏祖细胞;②G-CSF可以加速这一转分化的过程,并使损伤肾脏得到更好的修复.     

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.     

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.     

Effect of Stem Cell Factor and Granulocyte-Macrophage Colony-Stimulating Factor-Induced Bone Marrow Stem Cell Mobilization on Recovery from Acute Tubular Necrosis in Rats

Background: Acute tubular necrosis (ATN) is the most common reason for acute kidney injury (AKI), and there is still an absence of effective therapies. Objective: To assess the value of bone marrow cell mobilization by stem cell factor (SCF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) therapy in rats with gentamicin-induced ATN. Methods: ATN was induced in male Sprague–Dawley (SD) rats with five daily high-dose intraperitoneal injections of gentamicin. Subcutaneous injections of SCF and GM-CSF were administered simultaneously and these cytokines were observed on days 2, 5, 10, 17, 24, and 31. Peripheral blood and renal tissue CD34+ cell count, mortality rate, blood urea nitrogen (BUN), serum creatinine (SCr), creatinine clearance rate (CCr), and histopathologic lesion scores were determined. Twelve hours after bone marrow ablation (BMA) by lethal X-ray radiation, specific pathogen-free (SPF) ATN rats were given five daily injections of SCF and GM-CSF. BUN, SCr, and histopathologic lesion scores were evaluated on days 2, 5, and 10. Results: Peripheral blood CD34+ cell count increased significantly in ATN rats between 2 and 10 days after SCF and GM-CSF injection. Mortality was reduced from 34.7% in the ATN group to 18.6% in the ATN+CSF. In addition, cytokines administration significantly decreased SCr and BUN. Moreover, cytokines rapidly ameliorated tubular injury. There was no significant effect on ATN rats after BMA. Conclusions: This study demonstrated that SCF and GM-CSF effectively mobilized bone marrow cells in ATN rats, and cytokines administration partially prevented gentamicin-induced ATN. These results suggest that bone marrow stem cell (BMSC) mobilization may be an effective therapy for ATN.     

Diabetes Limits Stem Cell Mobilization Following G-CSF but Not Plerixafor

Previous studies suggest that diabetes impairs hematopoietic stem cell (HSC) mobilization in response to granulocyte colony-stimulating factor (G-CSF). In this study, we tested whether the CXCR4 antagonist plerixafor, differently from G-CSF, is effective in mobilizing HSCs in patients with diabetes. In a prospective study, individuals with and without diabetes (n = 10/group) were administered plerixafor to compare CD34⁺ HSC mobilization; plerixafor was equally able to mobilize CD34⁺ HSCs in the two groups, whereas in historical data, G-CSF was less effective in patients with diabetes. In a retrospective autologous transplantation study conducted on 706 patients, diabetes was associated with poorer mobilization in patients who received G-CSF with/without chemotherapy, whereas it was not in patients who received G-CSF plus plerixafor. Similarly in an allogeneic transplantation study (n = 335), diabetes was associated with poorer mobilization in patients who received G-CSF. Patients with diabetes who received G-CSF without plerixafor had a lower probability of reaching >50/μL CD34⁺ HSCs, independent from confounding variables. In conclusion, diabetes negatively impacted HSC mobilization after G-CSF with or without chemotherapy but had no effect on mobilization induced by G-CSF with plerixafor. This finding has major implications for the care of patients with diabetes undergoing stem cell mobilization and transplantation and for the vascular regenerative potential of bone marrow stem cells.     

干细胞因子联合粒细胞集落刺激因子动员骨髓干细胞促进单侧输尿管梗阻大鼠肾脏损伤的修复

目的 研究干细胞因子（SCF）联合粒细胞集落刺激因子（G-CSF）动员单侧输尿管梗阻（UUO）大鼠骨髓干细胞对肾间质中微血管、纤维化程度和肾功能的影响,并探讨其对微血管影响的可能机制。 方法 128只大鼠按数字随机法分为假手术组（Sham组）、SCF联合G-CSF动员组（SCF-G组）、UUO组、UUO+SCF-G组。于实验第 5、14、21、28天每组各随机抽取8只处死,检测Scr、肾间质CD34阳性表达细胞数目和Ⅷ因子阳性表达细胞数目、肾间质纤维化和间质病理损害积分、肾皮质血管内皮生长因子（VEGF）mRNA和血小板反应蛋白1（TSP-1）mRNA的表达。 结果 (1)UUO组2周时可见到肾间质纤维化伴肾小管周微血管的丢失。（2）UUO+SCF-G组肾间质干细胞归巢数目明显高于UUO组和Sham组（P < 0.05）。（3）UUO+SCF-G组肾小管周微血管指数减少出现的时间晚于UUO组（P < 0.05）。（4）第14、21、28天UUO+SCF-G组间质化纤维程度和肾小管损伤程度均轻于UUO组（P < 0.05）。（5）UUO+SCF-G组术后VEGF mRNA表达下调出现的时间晚于UUO组,且表达均高于同期UUO组 （P < 0.05)。（6）UUO+SCF-G组术后TSP-1 mRNA表达增高出现的时间晚于UUO组,且表达均低于同期UUO组（P < 0.05）。（7）在UUO组和UUO+SCF-G组中,肾小管周微血管指数与Scr、间质纤维化积分和肾小管间质病理积分均呈负相关;肾皮质VEGF mRNA表达与肾小管周微血管指数呈正相关;肾皮质TSP-1 mRNA表达与肾小管周微血管指数呈负相关。 结论 （1）UUO大鼠存在肾小管周微血管丢失,并与肾间质纤维化及间质病理损伤相关。（2）联合应用SCF和G-CSF动员骨髓干细胞可以归巢至受损的肾脏,有助于减少肾小管周微血管丢失,并进而减轻肾间质纤维化和间质损害,保护肾功能。（3）联合应用SCF和G-CSF可以上调肾皮质VEGF mRNA水平和下调TSP-1 mRNA水平,这可能是其促进内皮细胞修复及保护肾间质微血管损伤的机制之一。     

不同动员方案对异基因造血干细胞移植临床疗效的影响

目的 探讨异基因造血干细胞移植中不同动员方案的临床效果.方法 回顾性分析71例异基因外周血造血干细胞移植的临床资料,根据供者采用动员剂的不同分为G-CSF动员组(G组,有24例受者)和G-CSF联合GM-CSF动员组(G+M组,有47例受者).比较两组供者的动员效果及移植物细胞成分,观察受者术后造血功能重建的情况和GVHD的发生情况,观察供者应用动员剂后的不良反应.结果 动员4 d后,G组供者的外周血白细胞计数为(49.6±19.5)×109/L,明显高于G+M组供者的(25.4±10.4)×109/L(P＜0.05).两组间CD34+细胞占单个核细胞比例的差异无统计学意义(P＞0.05),但G+M组CD34+CD38-细胞占CD34+细胞的比例为(37.7±5.7)%,明显高于G组的(31.4±4.5)%(P＜0.05).两组供者经过1～3次采集均能获取足够的CD34+细胞,两组采集的供者淋巴细胞计数及其亚群分布的差异均无统计学意义(P＞0.05).两组受者间CD34+细胞、CD34+CD38-细胞及T淋巴细胞亚群输入量的差异均无统计学意义(P＞0.05).术后所有受者的造血功能均顺利重建.术后对受者进行2～55个月的随访,无论是急性还是慢性GVHD,其发病率和严重程度在两组间的差异均无统计学意义(P＞0.05).术后共有17例受者死于原发病复发,10例死于GVHD和感染等移植相关并发症,G组和G+M组分别有14例(58.3%)和31例(66.0%)受者存活.在使用动员剂后,供者出现的主要不良反应为骨骼肌酸痛和发热,多发生在用药后36 h,给予解热镇痛药后缓解.结论 单用G-CSF与联合应用G-CSF和M-CSF进行动员的临床效果相当,但后者对CD34+CD38-细胞的选择性较强,而在异基因造血干细胞移植输入较多的CD34+细胞和CD34+CD38-细胞有利于受者造血功能的快速重建.     

脐血单个核细胞体外扩增的实验研究

目的 探讨含有细胞因子的无血清培养基对脐血单个核细胞(MNC)体外培养后的扩增情况和用于移植的安全性.方法 从新鲜脐血中分离出的MNC,在含细胞因子的无血清培养体系中培养.分别将培养前和培养第10天时的造血细胞进行细胞计数、细胞活力分析、集落分析、流式细胞仪检测表面标记、彗星试验分析DNA的损伤情况、无菌性分析及移植至NOD/SCID小鼠体内等项研究.结果 经过体外短期培养,脐血中MNC、CD34+、CD133+、CD34+CXCR4+及CD34+ VLA-4+细胞扩增倍数均比培养前增高(P<0.05);半固体培养基可支持多系集落的生长;培养前和培养第10天时脐血细胞DNA损伤率均低于5%;无菌性分析提示细胞未受污染.将体外扩增后的脐血造血细胞移植入NOD/SCID小鼠体内,与新鲜脐血移植相比,小鼠的存活时间及植入能力的差异均无统计学意义(P>0.05).结论 脐血造血细胞体外扩增是解决脐血造血细胞数量不足的有效方法.脐血造血细胞经短期培养能为造血干细胞移植提供安全而具植入能力的造血细胞.     

Impact of CXCR4/CXCL12 Blockade on Normal Plasma Cells In Vivo

Plasma cells (PCs) are a major source of alloantibody in transplant patients and are resistant to current therapy. Because receptor–ligand interactions in stromal microenvironments play important roles in the localization, development, and survival of normal PCs, we hypothesized that interfering with CXCR4/CXCL12 interactions with plerixafor might cause PC depletion and enhance the efficacy of the proteasome inhibitor bortezomib. PCs in mouse spleen, bone marrow, and peripheral blood demonstrated CXCR4 expression. We then treated with plerixafor in doses ranging from 240 μg/kg in a single dose to a 1‐mg/kg daily dose for 10 days. CXCR4/CXCL12 blockade with plerixafor resulted in increased mobilization of PCs into the peripheral blood. Splenectomy completely abrogated this effect, suggesting that all plerixafor‐mobilized cells were from the spleen. The total number of PCs in the spleen and marrow remained constant despite treatment with plerixafor. Bortezomib caused a reduction in PCs, but adding plerixafor did not increase killing. We conclude that CXCR4/CXCL12 interactions are important for the retention of a subpopulation of PCs in the spleen, but this interaction has minimal effect on PCs in the marrow. The lack of enhancement of bortezomib‐mediated depletion suggests that factors other than CXCR4/CXCL12 interactions are responsible for drug resistance.