粒细胞集落刺激因子对脑缺血大鼠骨髓干细胞分布及脑保护作用的影响

目的 研究人重组粒细胞集落刺激因子(rG-CSF)对骨髓干细胞(BMSCs)在脑缺血大鼠血液和脑组织中的分布变化及抗脑缺血损伤的影响.方法 将106只SD大鼠按随机数字表法分为假手术组(10只)、模型组(48只)、rG-CSF组(48只),后两组再分为术后2、3、7、14 d亚组,每个亚组12只.用改良线栓法制备大鼠局灶性脑缺血模型.rG-CSF组于术前3 d和术后2 d皮下注射rG-CSF 10μg/kg,假手术组和模型组给予等量生理盐水,均每日1次.术后各时间点进行神经功能评分;取腹主动脉血,测定外周血白细胞计数(WBC)及CD34+细胞计数;观察脑组织病理改变;并用免疫组化法测定脑组织CD34+细胞表达.结果 ①制模后2 d大鼠神经功能评分即显著降低,随后逐渐升高;rG-CSF组术后7 d和14 d神经功能评分(分)较模型组显著增高(7 d:11.86±0.69比10.53±0.76,14 d:13.38±0.52比12.38±0.52,均P<0.01).②制模后2 dSb周血WBC和CD34+细胞计数即显著增加,3 d达峰值,7 d和14 d降低;除14 d CD34+细胞计数外,rG-CSF组其余时间点WBC和CD34+细胞计数均较模型组明显增加[WBC(×109/L)2 d:11.75±1.76比8.07±1.27,3 d:13.07±1.70比10.88±1.78,7 d:8.63±1.36比5.58±1.57,14 d:6.98±0.98比4.87±0.92;CD34+细胞计数(个/μl)2 d:8.83±2.14比3.17±0.75,3 d:13.50±1.87比5.00±1.55,7 d:5.33±1.21比2.33±1.21,P<0.05或P<0.013.③制模后2 d大鼠脑组织CD34+细胞表达即明显增强,7 d达峰值,14 d降低;rG-CSF组各时间点CD34+表达[吸光度(A)值]均较模型组显著增加(2 d:43.21±4.41比22.04±2.95,3 d:45.79±1.76比25.69±2.44,7 d:52.09±2.86比33.04±2.62,14 d:29.73±1.99比16.91±2.95,均P<0.01).④rG-CSF组脑组织病理损伤较模型组减轻,以14 d改善明显.结论 脑缺血可引起BMSCs进入外周血及向脑组织归巢,其在外周血和脑组织的变化呈先增多再减少的特点,分别于缺血后3 d和7 d达峰值;rG-CSF可使进入外周血和脑组织的BMSCs明显增加.BMSCs动员对脑缺血损伤的保护作用明显,且随动员后时间的延长呈增强趋势.

Abstract：

Objective To explore the influence of recombination granulocyte colony stimulating factor (rG-CSF)on mobilization and distribution of bone marrow stem cells (BMSCs) in blood and brain tissue,and its role in protecting brain in rats with cerebral ischemia.Methods One hundred and six SpragueDawley(SD)rats were divided into sham-operated group (n=10),model group(n=48),rG-CSF group (n=48) according to the method of random digital table,and rats in model and rG-CSF groups were divided into four subgroups:i.e.2,3,7 and 14 days subgroups,with 12 rats in each subgroup.Middle cerebral artery occlusion(MCAO)model was reproduced with nylon thread.In rats of rG-CSF group rG-CSF (10 btg/kg)was administered by subcutaneous injection 3 days before and 2 days after operation respectively,once a day.Rats in sham-operated and model groups were administered with normal saline in the same volume,once a day.At the corresponding time after operation,general neural function score(GNFS)of rats was measured.Blood was collected through abdominal aorta,then white blood cell (WBC) and CD34+ cells in peripheral blood were counted.Brain pathologic changes were observed,and expression of CD34+ cells in rats brain tissue was determined by using immunohistochemical method.Results ①GNFS was lower obviously in 2-day model group compared with that in sham-operated group,and then increased gradually.At 7 days and 14 days after operation,GNFS in rG-CSF group was higher significantly than that in model group (7 days:11.86±0.69 vs.10.53±0.76,14 days:13.38±0.52 vs.12.38±0.52,both P<0.01).②WBC and CD34+ cells in peripheral blood in model group increased obviously,with the highest level appeared at 3 days and lowered at 7 days and 14 days.Increase of WBC and CD34+ cells in rats of rG-CSF group was more obvious than that of model group at each time point except CD34+ in 14 days group [WBC (×109/L)2 days:11.75±1.76 vs.8.07±1.27,3 days:13.07±1.70 vs.10.88±1.78,7 days:8.63±1.36 vs.5.58士1.57,14 days:6.98士0.98 vs.4.87士0.92;CD34'(cells/t~1)2 days:8.83±2.14 vs.3.17±0.75,3 days:13.50±1.87 vs.5.00±1.55,7 days:5.33±1.21 vs.2.33±1.21,P<0.05 or P<0.01].③Expression of CD34+ cells in the brain of rats in 2-day model group increased significantly,and the highest level appeared at 7 days and decreased at 14 days.Absorbance (A) value of CD34+ cells expression in rat brains of each rG-CSF group was more significant than that in model group(2 days:43.21±4.41 vs.22.04±2.95,3 days:45.79±1.76 vs.25.69±2.44,7 days:52.09±2.86 vs.33.04±2.62,14 days:29.73±1.99 vs.16.91±2.95,all P<0.01).④ The signs of injury to brain in pathological examination were less obvious in 14 days rG-CSF group.Conclusion BMSCs could be induced to enter peripheral blood and "home" to brain tissue after cerebral ischemia.It was showed that BMSCs increased in number at first and then decreased in peripheral blood and brain,the peak number was found on 3rd day in peripheral blood and 7th day in brain.Mobilization with rG-CSF could increase the number of BMSCs in peripheral blood and brain tissue.The effect of mobilization of BMSCs on protecting brain was significant after cerebral ischemia,and effect appeared to be more pronounced with prolongation of mobilization.     

中青年原发性高血压患者循环内皮祖细胞CD34+水平与颈动脉粥样硬化的相关性研究

Objective To explore the relationship between the level of circulating endothelial progenitor cells (EPCs) CD34+with the Framingham cardiovascular risk factors, or with the carotid artery intima-madia thickness (IMT), and to evaluate the value of circulating EPCs CD34+level as a cytologicalmarker of early vascular lesion in youth and middle aged essential hypertension (EH) patients.Methods A total of 62 patients with EH aged between 25 to 45 were enrolled as study group and 20 healthy people were enrolled as control group.EH patients were stratified with cardiovascular risk factors according to Framingham risk factors score into low-risk group with 18 cases, mid-risk group with 14 cases, high-risk group with 17 cases, and extremely high-risk group with 13 cases.The level of circulating EPCs CD34+,carotid artery IMT were respectively measured.The relationship between the level of circulating EPCsCD34+ and Framingham cardiovascular risk factors score, carotid artery IMT was analyzed.Results The level of circulating EPCs CD34+ was gradually decreased with an increase of the Framingham risk factors score in each hypertensive subgroup [low-risk group:(0.12±0.02)%, mid-risk group:(0.07±0.03)%,high-risk group:(0.04±0.03)%, extremely high-risk group:(0.01±0.01)%], and they were significantly lower than that in control group [(0.15±0.03)%], and there was a significant difference among hypertensive subgroups (P＜0.05 or P＜0.01).Carotid artery IMT was significantly thicker among hypertensive subgroups [low-risk group:(0.80±0.07)mm, mid-risk group:(1.11±0.08)mm, high-risk group: (1.26±0.10)mm, extremely high-risk group:(1.45±0.09)mm], and there was a significant difference between each hypertensive group and that of control group [(0.73±0.08)mm, all P＜0.01].There was also statistical significance among hypertensive subgroups(P＜0.05 or P＜0.01).There was a negative correlation between the level of circulating EPCs CD34+and Framingham risk factors score (r=-0.875, P＜0.01), and also a negative correlation with carotid artery IMT (r=-0.852, P＜0.01).Conclusion There was a significant correlation between the level of circulating EPCs CD34+with Framingham risk factors score and also carotid artery IMT in EH patients.Circulating EPCs CD34+could be a cytological marker of early vascular lesion in hypertension patients.     

中青年原发性高血压患者循环内皮祖细胞CD34+水平与颈动脉粥样硬化的相关性研究

Objective To explore the relationship between the level of circulating endothelial progenitor cells (EPCs) CD34+with the Framingham cardiovascular risk factors, or with the carotid artery intima-madia thickness (IMT), and to evaluate the value of circulating EPCs CD34+level as a cytologicalmarker of early vascular lesion in youth and middle aged essential hypertension (EH) patients.Methods A total of 62 patients with EH aged between 25 to 45 were enrolled as study group and 20 healthy people were enrolled as control group.EH patients were stratified with cardiovascular risk factors according to Framingham risk factors score into low-risk group with 18 cases, mid-risk group with 14 cases, high-risk group with 17 cases, and extremely high-risk group with 13 cases.The level of circulating EPCs CD34+,carotid artery IMT were respectively measured.The relationship between the level of circulating EPCsCD34+ and Framingham cardiovascular risk factors score, carotid artery IMT was analyzed.Results The level of circulating EPCs CD34+ was gradually decreased with an increase of the Framingham risk factors score in each hypertensive subgroup [low-risk group:(0.12±0.02)%, mid-risk group:(0.07±0.03)%,high-risk group:(0.04±0.03)%, extremely high-risk group:(0.01±0.01)%], and they were significantly lower than that in control group [(0.15±0.03)%], and there was a significant difference among hypertensive subgroups (P＜0.05 or P＜0.01).Carotid artery IMT was significantly thicker among hypertensive subgroups [low-risk group:(0.80±0.07)mm, mid-risk group:(1.11±0.08)mm, high-risk group: (1.26±0.10)mm, extremely high-risk group:(1.45±0.09)mm], and there was a significant difference between each hypertensive group and that of control group [(0.73±0.08)mm, all P＜0.01].There was also statistical significance among hypertensive subgroups(P＜0.05 or P＜0.01).There was a negative correlation between the level of circulating EPCs CD34+and Framingham risk factors score (r=-0.875, P＜0.01), and also a negative correlation with carotid artery IMT (r=-0.852, P＜0.01).Conclusion There was a significant correlation between the level of circulating EPCs CD34+with Framingham risk factors score and also carotid artery IMT in EH patients.Circulating EPCs CD34+could be a cytological marker of early vascular lesion in hypertension patients.     

抗CD44单抗IM7对慢性粒细胞白血病CD34+细胞黏附和迁移能力影响的体外研究

Objective To explore the effects of anti-CD44 monoclonal antibody-IM7 on the in vitro adhesion and migration of chronic myeloid leukemia stem cell(CML-LSC) and its mechanism. Methods CD34+ CD38- CD123+ leukemic stem cells (LSC) from 20 newly-diagnosed chronic myeloid leukemia ( CML) patients BM cells and CD34+ CD38- hematopoietic stem cells (HSC) from 20 full-term newborn cord blood cells were isolated with EasySepTM magnet beads. The CD44 expression of the LSC and HSC was detected by flow cytometry (FCM), and the adhesion and migration ability of the LSC and HSC pre- and post-incubated with IM7 in vitro by MTT assay and transendothelial migration assay, respectively. Results ①After incubated with IM7, the LSC and HSC CD44 expression rates were (86.60±2.10)% vs. (25.40±1.70)% (P<0.05), respectively. ②The adhesive ability of the LSC to endothelial cells was decreased markedly after incubated with IM7, the OD value (A570) changing from pre- incubation of (0.62±0.11) to post-incubation of (0.34±0.07), while there was little change of A570 in the HSC group. ③The migration abilityof the LSC group was inhibited evidently after incubated with IM7, the inhibition rate being 46%～63%, while little change of that in HSC group was detected. ④The adhesive ability of the LSC group to marrow stromal cells was decreased markedly after incubated with IM7, while little change was found in that of HSC group. Conclusion The anti-CD44 monoclonal antibody-IM7 can effectively inhibit the adhesion and migration abilities of the LSC in vitro, which might provide a theoretical evidence for targeting therapy.     

抗CD44单抗IM7对慢性粒细胞白血病CD34+细胞黏附和迁移能力影响的体外研究

Objective To explore the effects of anti-CD44 monoclonal antibody-IM7 on the in vitro adhesion and migration of chronic myeloid leukemia stem cell(CML-LSC) and its mechanism. Methods CD34+ CD38- CD123+ leukemic stem cells (LSC) from 20 newly-diagnosed chronic myeloid leukemia ( CML) patients BM cells and CD34+ CD38- hematopoietic stem cells (HSC) from 20 full-term newborn cord blood cells were isolated with EasySepTM magnet beads. The CD44 expression of the LSC and HSC was detected by flow cytometry (FCM), and the adhesion and migration ability of the LSC and HSC pre- and post-incubated with IM7 in vitro by MTT assay and transendothelial migration assay, respectively. Results ①After incubated with IM7, the LSC and HSC CD44 expression rates were (86.60±2.10)% vs. (25.40±1.70)% (P<0.05), respectively. ②The adhesive ability of the LSC to endothelial cells was decreased markedly after incubated with IM7, the OD value (A570) changing from pre- incubation of (0.62±0.11) to post-incubation of (0.34±0.07), while there was little change of A570 in the HSC group. ③The migration abilityof the LSC group was inhibited evidently after incubated with IM7, the inhibition rate being 46%～63%, while little change of that in HSC group was detected. ④The adhesive ability of the LSC group to marrow stromal cells was decreased markedly after incubated with IM7, while little change was found in that of HSC group. Conclusion The anti-CD44 monoclonal antibody-IM7 can effectively inhibit the adhesion and migration abilities of the LSC in vitro, which might provide a theoretical evidence for targeting therapy.     

血浆外泌体样小体免疫调节作用的初步研究

Objective To identify the exosomes-like vesicles from the plasma and study their biologic characteristics and regulatory effect.Methods The exosomes-like vesicles were purified from healthy donors plasma with a series of high-speed centrifugations and ultrafiltration.Morphology was identified by transmission electron microscopy and biologic characteristics by Western blot and flow cytometry.CIM+T ceils and CD4+ CD25+CD127 low Treg cells were purified from peripheral blood mononuclear cells(PBMCs)by Magnetic cell sorting.After exosomes-like vesicles cultured with CD4+ T cells or CD4+ CD25+CD127low Treg cells,cell proliferation and apoptosis were assayed.Phosphorylated β-catenin level in Wnt signaling by phosflow.Results Exosomes-like vesicles from plasma were similar to previously described exosomes in shapes and size and expressed exosome marker proteins CD63 and CD81 as well as the MHC-Ⅱ molecule.costimulatory molecules CD86 etc.Mter co-cultured with CD4+ T cells,exosomes- like vesicles inhibited the proliferation of the T cells in a dose-dependent manner.After Treg cells cultured with exosomes-like vesicles for 14days,the survival rate of the Treg cells was 57.07%,while that of the control Treg was 30.91%.Frizzled receptors 2,3,4and LRP6 gene mRNA expressed(the relative gray value was 48.50、34.84、23.85、49.73)in the Treg cells by RT-PCR,and Wnt molecular expressed in exosomes-like vesicles.After Treg ceils cocultured with exosomes-like vesicles,the MFI of phosphorylated β-catenin decreased(from 20.06±2.99 to 12.41±2.08),and the expression of Bcl-2 mRNA was upregulated significantly(the relative gray value from 0.45 to 84.97).Conclusions Exosomes-like vesicles existed in human plasma and express immune regulatory molecules.They can suppress the proliferation of activated CD4+ T cels induce their apoptosis and prolong the survival of natural Treg cells via Wnt signaling pathway.     

血浆外泌体样小体免疫调节作用的初步研究

Objective To identify the exosomes-like vesicles from the plasma and study their biologic characteristics and regulatory effect.Methods The exosomes-like vesicles were purified from healthy donors plasma with a series of high-speed centrifugations and ultrafiltration.Morphology was identified by transmission electron microscopy and biologic characteristics by Western blot and flow cytometry.CIM+T ceils and CD4+ CD25+CD127 low Treg cells were purified from peripheral blood mononuclear cells(PBMCs)by Magnetic cell sorting.After exosomes-like vesicles cultured with CD4+ T cells or CD4+ CD25+CD127low Treg cells,cell proliferation and apoptosis were assayed.Phosphorylated β-catenin level in Wnt signaling by phosflow.Results Exosomes-like vesicles from plasma were similar to previously described exosomes in shapes and size and expressed exosome marker proteins CD63 and CD81 as well as the MHC-Ⅱ molecule.costimulatory molecules CD86 etc.Mter co-cultured with CD4+ T cells,exosomes- like vesicles inhibited the proliferation of the T cells in a dose-dependent manner.After Treg cells cultured with exosomes-like vesicles for 14days,the survival rate of the Treg cells was 57.07%,while that of the control Treg was 30.91%.Frizzled receptors 2,3,4and LRP6 gene mRNA expressed(the relative gray value was 48.50、34.84、23.85、49.73)in the Treg cells by RT-PCR,and Wnt molecular expressed in exosomes-like vesicles.After Treg ceils cocultured with exosomes-like vesicles,the MFI of phosphorylated β-catenin decreased(from 20.06±2.99 to 12.41±2.08),and the expression of Bcl-2 mRNA was upregulated significantly(the relative gray value from 0.45 to 84.97).Conclusions Exosomes-like vesicles existed in human plasma and express immune regulatory molecules.They can suppress the proliferation of activated CD4+ T cels induce their apoptosis and prolong the survival of natural Treg cells via Wnt signaling pathway.     

Effects of Electroacupuncture Combined with Repetitive Transcranial Magnetic Stimulation on the Expression of Nestin in Neural Stem Cell after Focal Cerebral Ischemia in Adult Rats*

Objective: To investigate the influence of electroacupuncture (EA) combined with repetitive transcranial magnetic stimulation(rTMS) on the temporal profile of nestin expression after induction of focal cerebral ischemia in adult rats and to explore the mechanism of EA combined with rTMS in treating ischemic brain injury. Method: The model of transient focal ischemia was produced by occlusion of middle cerebral artery. Seventy-five Wistar rats were randomly divided into normal group, model group, EA group, rTMS group and EA+rTMS group. The neurologic impairment rating and ability of learning and memory were observed at the 7th、14th and 28th d after infarction respectively. Meanwhile, Western blotting was used to observe the number of nestin expression positive cells. Result: Nestin-positive cells were found in cortex, subgranular zone(SGZ), subventricular zone (SVZ) of the ipsilateral side at different time points after cerebral ischemia. The number of nestin-positive cells peaked at the 7th d, began to decrease at the 14th d and was significantly higher in EA+rTMS group than that in model group(P<0.05), then almost reached normal at the 28th d. The improvement of neural motor function deficits as well as the indexes of learning and memory were more obvious in EA+rTMS group compared with model group(P<0.01, P<0.05). These effects were most obvious in EA+rTMS group compared with the EA and rTMS group(P<0.05). Conclusion: EA and rTMS possess the potency of building up and can increase the number of nestin-positive cells in some brain regions after focal cerebral ischemia, which might be one of the important mechanisms of EA combined with rTMS in treating ischemia brain injury.     

多发性硬化患者外周血CD8+CD28-调节性T细胞的变化

Objective To investigate the levels of peripheral blood CD8+ CD28- regulatory lymphocytes and their clinical values in the patients with multiple sclerosis (MS). Method From October 2005 to August 2008, 51 patients with active rehpsing-remitting MS were enrrolled from Department of Neurology of the First Affil-iated Hospital of Wenzliou Medical College. The diagnostic criteria for MS were the 2005 revisions to the "McDon-ald criteria". All the admitted patients received 1 g of methylprednisoione per day intravenously for 5 days, fol-lowed by 60 mg prednisone per day orally for 12 days,and tapered in 6 weeks. Fourteen patients were reevaluated after corticosteroid therapy. Twenty healthy individuals ,as normal controls,matched for age and sex with the MS patients were also enrolled in this study. The percentages of peripheral blood T cells (CD8+ CD28-, CD8+CD28+, CD8+, CD4+ CD8-) were measured by flow cytometric analysis. Parametric statistical analysis were per-formed using standard methods, and linear regression analysis was conducted using Pearson correlation test. Re-sults (1)Compared with controls,the patients with active MS had significantly lower percentage of CD8+ CD28-T cells [(18.48±9.89)% vs. (24.48±4.86)%, P <0.01], and higher percentage of CD8+ CD28+ T cells [(12.23±4.31) % vs. (8.55±3.49) %, P <0.01]. (2)The percentage of CD8+ CD28- T cells was negative-ly correlated with that of CD4+ CDS- T cells (r = -0.488, P < 0.01). (3) After corticosteroid therapy, the per-eentage of peripheral blood CD8+ CD28- / CD8+ CD28+ T cells didn' t significantly decrease or increase in 14 ac-tive MS patients (P > 0.05). Conclusions The decrease of peripheral blood CD8+ CD28- regulatory T cells might be associated with the pathogenesis of MS, and CD8+ CD28- regulatory T cells perhaps played their roles through CD4+ T cells. Corticosteroid therapy could not reverse the levels of CD8+ CD28- T cells.     

动员后外周血中CD34+细胞去除前后对改善肢体缺血疗效的研究

目的对动员的外周血单个核细胞（PBMNC）和动员的去除CD34^＋细胞的PBMNC移植治疗裸鼠下肢缺血的疗效进行比较，以探讨PBMNC的治疗机制。方法经过单采获得G-CSF动员的PBMNC后，一部分通过CD34磁珠抗体分选得到去除CD34^＋细胞的PBMNC。动员的PBMNC和动员的去除CD34^＋细胞的PBMNC荧光标记后按1×10^6细胞或相应体积的PBS分别局部肌肉注射移植到裸鼠缺血下肢。观察下肢血流灌注以及毛细血管密度。用ELISA法检测下肢肌肉的血管内皮生长因子（VEGF）表达，并进一步观察表达的VEGF是否由移植细胞分泌。结果PBMNC移植后缺血下肢血流明显恢复，毛细血管密度明显增加，但去除CD34^＋细胞的PBMNC移植组疗效有所下降。细胞移植后4周，PBMNC组的血流灌注由（20．3±4．2）％恢复为（96．4±5．6）％，对照组仅恢复为（71．3±4．4）％（P〈0．01），去除CD34^＋细胞组恢复为（83．8±5．2）％（P〈0．05）。PBMNC组血管密度为（521±47）／mm^2，去除CD34^＋细胞组为（3964-21）／mm^2（P〈0．05），但仍高于对照组[（276±43）／mm^2]（P〈0．05）。在PBMNC组可以观察到移植的细胞整合到缺血的毛细血管壁。缺血肌肉VEGF的表达明显升高，其共表达VEGF和移植的单个核细胞。结论移植G-CSF动员的PBMNC不但可以通过干细胞整合到血管壁的机制促进血管生长，还可以通过提供细胞因子的机制促进血管生长。去除CD34^＋细胞削弱了动员的PBMNC移植治疗肢体缺血的血管新生效应。     

血缘关系供者外周血干细胞移植中移植物细胞组分对造血重建移植物抗宿主病的影响

目的 探讨HLA全相合血缘关系供者外周血干细胞移植(PBSCT)中移植物细胞组分对恶性血液病患者移植后造血重建、移植物抗宿主病(GVHD)的影响.方法 回顾性分析我科107例接受HLA全相合血缘关系供者PBSCT的恶性血液病患者,其移植物细胞组分与移植后患者造血重建、GVHD的关系.结果 移植物各细胞组分与粒细胞重建时间无关;单个核细胞(MNC)、CD34+细胞数与血小板重建时间呈负相关(r值分别为-0.32和-0.21,P值均＜0.05).CD34+、CD34+CD38-细胞数与急性GVHD发生呈负相关(r分别为-0.24和-0.29,P值均＜0.05).淋巴细胞各亚群数量与急性GVHD发生均无明显关系.CD25+ CD4+、CD3+、CD4+ CD3+细胞数及CD4+/CD8+细胞比值与慢性GVHD发生均呈正相关(P值均＜0.05),且相关系数均大于0.4,其中CD25+ CD4+细胞数与慢性GVHD相关系数高达0.78.CD34+、CD34+ CD38-细胞数与慢性GVHD发生无明显关系.结论PBSCT中输入MNC、CD34+、CD34+ CD38-细胞数增加到一定阈值后,增加细胞数并不能进一步有效促进患者造血重建,反而有可能因输入淋巴细胞数增加而增加患者慢性GVHD、广泛慢性GVHD的发生率.     

体外扩增小鼠造血干/祖细胞及重建造血功能的研究

目的 探讨体内中性粒细胞的快速植入及长期重建造血的能力。方法 分离经氟尿嘧啶处理的雄性BDF1小鼠骨髓单个核细胞，以MoFlo细胞分选系统分离纯化CD34^ /c-kit^ 造血干，祖细胞，体外应用骨髓间充质干细胞共培养和阶段扩增结合的方法分别对分离的单个核细胞和CD34^ /c-kit^ 细胞进行体外扩增，并将扩增的有核细胞移植给经亚致死剂量照射的雌性小鼠。结果 以骨髓间充质干细胞为培养基质细胞，结合分阶段扩增方法，有效提高了各种细胞的扩增倍数，其中单个核细胞扩增的总有核细胞、CD34^ 细胞、GM-CFC和HPP-CFC扩增倍数分别为10．8，4．8，65．9和38．8，CD34^ /c-kit^ 细胞扩增的以上4种细胞扩增倍数分别为76．1，2．9，71．7和51．8；扩增细胞移植后可快速产生体内中性粒细胞的植入，并在2个月后的移植小鼠中仍可检测到移植的造血细胞。结论 与骨髓间充质干细胞的共培养，为造血干，祖细胞的有效扩增提供了良好的环境，分阶段扩增法加快了体外造血干，祖细胞的扩增和成熟，为移植后体内中性粒细胞的快速植入创造了条件。     

自体外周血干细胞动员中测定CD34^＋Thy—1＋细胞的意义

目的：确切评估动员后外周血干细胞（ＰＢＳＣ）水平的变化，及时指导临床选择最佳采血时机。方法：用流式细胞术测定化疗和粒细胞集落刺激因子（Ｇ－ＣＳＦ）联合动员时外周血ＣＤ３４＋Ｔｈｙ－１＋细胞含量的变化，同时用体外集落培养方法评价外周血祖细胞（ＰＢＰＣｓ）的克隆形成能力。结果：动员后循环血中ＣＤ３４＋Ｔｈｙ－１＋细胞、ＣＤ３４＋细胞和克隆形成细胞（ＣＦＣ）含量分别增高４８．６倍、５０．０倍和５３．１倍，高峰时间在化疗后第１２～１４天（注射Ｇ－ＣＳＦ的第６～８天）；外周血单个核细胞中ＣＤ３４＋Ｔｈｙ－１＋细胞、ＣＤ３４＋细胞的比例分别增高１３．８倍和１０．５倍；动员的早期阶段，ＣＤ３４＋细胞中Ｔｈｙ－１＋细胞比例最高。结论：联合应用化疗和Ｇ－ＣＳＦ对ＰＢＰＣｓ，尤其对早期干／祖细胞具有显著动员作用；用流式细胞术检测ＣＤ３４＋Ｔｈｙ－１＋细胞可及时指导临床准时采集ＰＢＳＣ。     

脐带间充质干细胞对CD34+细胞在小鼠体内归巢的影响及其机制研究

目的 探讨脐带来源间充质干细胞(MSC)对脐血来源CD34+细胞在NOD/SCID小鼠体内归巢的影响及其可能的机制.方法 将CD34+细胞与MSC细胞共移植入经放射线照射后的NOD/SCID小鼠,采用流式细胞术和RT-PCR检测移植后20 h NOD/SCID小鼠骨髓及脾脏中人CD34+细胞,计算其相应的骨髓和脾脏的归巢效率.将脐血CD34+细胞与脐带MSC体外共培养,检测MSC细胞对CD34+细胞趋化功能的影响;并于培养4、7 d检测培养后CD34+细胞表面CD49e、CD31、CD62L、CD11a等归巢相关黏附分子表达情况.结果 ①移植后20 h采用流式细胞术成功在小鼠骨髓和脾脏中检测到人CD45+细胞.共移植组CD34+细胞骨髓归巢率[(7.2±1.1)%]高于单移植组[(5.4±0.9)%](P<0.05).②RT-PCR结果 显示共移植组小鼠骨髓细胞和脾脏细胞,单移植组小鼠脾脏细胞扩增得到人GAPDH基因片段,而单移植组小鼠骨髓细胞未见明显扩增条带.③MSC存在时,CD34+细胞的体外迁移能力为(35.7±5.8)%,显著高于CD34+细胞自发迁移率[(3.5±0.6)%,P<0.05].④CD34+细胞与MSC体外共培养后细胞表面CD49e、CD31和CD62L黏附分子的表达水平高于CD34+细胞单独培养组.结论 MSC细胞与CD34+细胞共移植有利于CD34+细胞向骨髓、脾脏等造血器官归巢,这可能与MSC促进CD34+细胞迁移以及维持CD34+细胞表面归巢相关黏附分子的表达相关.     

粒系集落刺激因子对外周血T淋巴细胞亚群的影响及与CD34+细胞动员效果的相关性

本研究观察粒系集落刺激因子(G—CSF)作为造血干细胞动员剂对外周血T淋巴细胞亚群的影响及与CD34^ 细胞动员效果的关系。对26例行自体造血干细胞移植患在G—CSF动员前后收集外周血标本，用流式细胞术检测动员前后CD3^ 、CD3^ CD4^ 、CD3^ CD8^ 、CD3^ CD4^ CD8^ 及CD3^ CD4^-CD8细胞绝对数量的变化并与外周血CD34^ 细胞的动员效果进行相关性分析。结果表明：GCSF动员后外周血CD3^ 、CD3^ CD4^ 、CD3^ CD4^ CD8^ 及CD3^ CD4^-CD8细胞的绝对数量分别增加2.23，2．62，2.99及10．96倍，而CD3^ CD4^ CD8^ 细胞的变化无统计学意义(P=0．243)。各亚群细胞的变化与CD34^ 细胞动员效果比较，仅CD3^ CD4 CD8细胞的变化与CD34^ 细胞动员效果间具有良好的相关性，r=0．796，P=0.000。结论：G—CSF将造血干细胞由骨髓动员到外周血的同时，使外周血中T细胞亚群的绝对数量发生不同程度的变化。在各T淋巴细胞亚群中CD3^ CD8^-细胞的增加与CD34^ 细胞的动员效果间具有统计学意义的相关性。     

Lenograstim versus filgrastim in mobilization before autologous hematopoietic stem cell transplantation in patients with multiple myeloma and lymphoma - Single center experience

ObjectivePeripheral blood stem cell transplantation is frequently used in the treatment of various hematological malignancies after intensive chemotherapy. The primary aim of our study is to compare the amount of collected CD34+ cells and engraftment times in patients mobilized with filgrastim or lenograstim.Material and MethodsDemographic and clinical data of multiple myeloma (MM) and lymphoma patients who underwent autologous transplantation and mobilized with G-CSF (filgrastim or lenograstim) without chemotherapy were collected retrospectively.ResultsOne hundred eleven MM and 58 lymphoma patients were included in the study. When mobilization with filgrastim and lenograstim was compared in MM patients, there was no significant difference in neutrophil and thrombocyte engraftment times of lenograstim and filgrastim groups (p = 0.931 p = 0.135, respectively). Similarly, the median number of CD34+ cells collected in patients receiving filgrastim and lenograstim was very similar (4.2 × 10⁶/kg vs 4.3 × 10⁶/kg, p = 0.977). When compared with patients who received lenalidomide before transplantation and patients who did not receive lenalidomide, the CD34+ counts of the two groups were similar. However, neutrophil and platelet engraftment times in the group not receiving lenalidomide tended to be shorter (p = 0.095 and p = 0.12, respectively). When lymphoma patients mobilized with filgrastim and lenograstim were compared, neutrophil engraftment time (p = 0.498), thrombocyte engraftment time (p = 0.184), collected CD34+ cell counts (p = 0.179) and mobilization success (p = 0.161) of the groups mobilized with filgrastim and lenograstim were similar.ConclusionThe superiority of the two agents to each other could not be demonstrated. Multi-center prospective studies with larger numbers of patients are needed.     

幼儿外周血造血干／祖细胞采集方法的建立

本研究探索采集体重低于20kg幼儿外周血造血干／祖细胞的方法及安全性。在采集外周血造血干／祖细胞时，在患儿股静脉处放置7F双腔导管作为采血及回输通道，采用COBE Spectra血细胞分离机的自动干／祖细胞采集程序及管路，在预冲管路完成后，向管路中灌注体外辐照的异体悬浮红细胞，采集后仅回输管路中的红细胞成分。对采集物计数单个核细胞、CD34^＋细胞和细胞存活率。结果表明：对7例低体重幼儿采集外周血造血干／祖细胞13次均获得成功。获得单个核细胞平均数为4．44×10^8／kg[（3．46—6．45）×10^8／kg]，CD34^＋细胞平均计数为2．20×10^6／kg[（1．34—3．79）×10^6／kg]，细胞存活率98．45％（97％-100％）。在采集过程中患儿生命体征平稳。结论：采用COBE Spectra血细胞分离机，可以在保持低体重幼儿生命体征平稳的前提下获得含有足够数量CD34^＋的单个核细胞。     

Predicting hematopoietic stem cell mobilization failure in patients with multiple myeloma: a simple method using day 1 CD34+ cell yield

Early and reliable prediction of the likelihood of achieving adequate stem cell collection for autologous stem cell transplantation (ASCT) in patients with multiple myeloma (MM) would improve collection efficiency, prevent unnecessary aphereses, and permit appropriate treatment alterations. No previous study has reported a threshold CD34+ cell collection quantity on Day 1 or 2 of leukapheresis that could predict successful stem cell collection. We performed a retrospective analysis of all MM patients undergoing first attempt of stem cell collection at our institution from 2001 through 2008. Recursive partitioning analysis was used to identify Day 1 or Day 1+2 CD34+ collection quantity that predicted failure to reach target ≥ 2 × 10(6) CD34+ cells/kg within five days of collection. Totally, 172 patients were included in the analysis. Patients underwent mobilization with G-CSF or G-CSF+ chemotherapy. 23 of 172 patients (13.4%) failed to collect sufficient (≥ 2 × 10(6) CD34+ cells/kg) CD34+ cells after five days of apheresis: 22 of 29 who collected ≤ 0.70 × 10(6) CD34+ cells/kg and 1 of 143 who collected > 0.70 × 10(6) CD34+ cells/kg (75.9% vs. 0.7%, P < 0.001) on Day 1. Collection failure occurred in 23 of 30 patients who collected ≤ 1.54 × 10(6) CD34+ cells/kg and 0 of 142 who collected >1.54 × 10(6) CD34+ cells/kg (76.7% vs. 0%, P < 0.001) on Days 1 + 2. Day 1 CD34+ cell collection quantity identifies patients unlikely to achieve adequate collection for ASCT. Patients who collect ≤ 0.70 × 10(6) CD34+ cells/kg on day 1 could be considered for treatment modifications to improve CD34+ collection, such as early administration of plerixafor or large volume apheresis.     

The number of CD34+ cells mobilized into the peripheral blood can predict the quality of subsequent collections

PBPC were mobilized using a variety of chemotherapy regimens plus G-CSF in a group of 126 consecutive patients. Data are presented that show a close correlation between the number of CD34+ cells mobilized into the peripheral blood (PB) and the number of CD34+ cells subsequently collected by leukapheresis (R = 0.904). On the basis of this correlation, a regression formula was calculated that could give an estimate of the total number of CD34+ cells likely to be collected by leukapheresis from a given number of CD34+ cells per microliter PB. An easy-to-read table has been compiled to show how this type of analysis can be applied to predict the likely dose of CD34+ cells that will be obtained by leukapheresis over a wide range of patient weights.