Exogenous administration of glial cell line-derived neurotrophic factor improves recovery after spinal cord injury

The aim of present study was to examine whether systemically delivered glial cell-derived neurotrophic factor (GDNF) was beneficial in reversing the spinal cord injury (SCI) in a spinal cord compression model. Rats were divided into three major groups: (1) sham operation (laminectomy only); (2) laminectomy+SCI+normal saline (1ml/kg, i.v.); (3) laminectomy+SCI+GDNF (50ng/kg, i.v.). Spinal cord injury was induced by compressing the spinal cord for 1min with an aneurysm clip calibrated to a closing pressure of 55g. GDNF or saline was administered immediately after SCI via the tail vein. Behavioral tests of motor function measured by maximal angle an animal could hold to the inclined plane were conducted at days 1-7 after SCI. The triphenyltetrazolium chloride staining and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling assay were also conducted after SCI to evaluate spinal cord infarction and apoptosis, respectively. Both GDNF and vascular endothelial growth factor (VEGF) in the injured spinal cord were assayed by immunofluorescence. It was found that systemically delivered GDNF, but not vehicle solution, significantly attenuated the SCI-induced hind limb dysfunction and spinal cord infarction and apoptosis. Both GDNF and VEGF could be detected in the injury spinal cord after GDNF, but not vehicle solution, therapy. The results indicate that GDNF treatment may be beneficial in reversing hind limb dysfunction by reducing spinal cord infarction and apoptosis in a spinal cord compression model.     

纳米粒子基因复合体应用于脊髓损伤治疗的实验研究

目的:观察壳聚糖纳米粒子胶质细胞源性神经营养因子基因复合体(CS-nano/pcD-NA3.1/GDNF)对脊髓损伤(SCI)的治疗作用.方法:Wistar大鼠170只,随机分为5组:A组(椎板切除+SCI-CS-nano/pcDNA3.1/GDNF,n=40),B组(椎板切除+SCI+GDNF基因治疗,n=40),C...     

Combined cord blood stem cells and gene therapy enhances angiogenesis and improves cardiac performance in mouse after acute myocardial infarction

BACKGROUND: Gene and stem cell therapies hold promise for the treatment of ischaemic cardiovascular disease. However, combined stem cell and angiogenic growth factor gene therapy for acute ischaemic myocardium has not been previously reported. This study hypothesized that combined stem cell and gene therapy would not only augment new vessels formation but also improve myocardial function in acute ischaemic myocardium. METHODS: Human angiopoietin-1 (Ang1) cDNA and VEGF(165) cDNA were ligated into AAV vector. The purified CD34(+) cells were obtained from human umbilical cord blood samples. Cord blood CD34(+) cells were transduced with AAV vector encoding either the human Ang1 (AAV-Ang1) or VEGF(165) (AAV-VEGF) cDNA alone, or both (AAV-Ang1 plus VEGF). Immediately after ligation of the left anterior descending coronary artery in male SCID mice, culture-expanded CD34(+) cells transduced with AAV-Ang1, AAV-VEGF or AAV-Ang1 plus VEGF were injected intramyocardially at the left anterior free wall. RESULTS: Western blot showed that Ang1 and VEGF protein expressions were enhanced in the CD34(+)cells transduced with AAV-Ang1 and AAV-VEGF, respectively. Infarct size significantly decreased and capillary density significantly increased after treatment with CD34(+)/AAV-Ang1 plus VEGF when compared with treatment by CD34(+) only. Combined therapy with CD34(+) and AAV-Ang1, CD34(+) and AAV-VEGF, CD34(+) and AAV-Ang1 plus VEGF, all showed significantly higher cardiac performance in echocardiography than the therapy with CD34(+) alone 4 weeks after myocardial infarction. CONCLUSIONS: Combined therapy with human umbilical cord blood CD34(+) cells and both Ang1 and VEGF genes reduced infarct size, attenuated the progression of cardiac dysfunction and increased capillary density in acute myocardial infarction in mice.     

神经营养因子对成年大鼠脊髓损伤后皮质脊髓束再生的影响

背景胶质细胞源性神经营养因子(GDNF)对脊髓运动神经元的存活有明显的保护作用,但GDNF对成年大鼠脊髓损伤后皮质脊髓束再生是否有促进作用仍不清楚.目的研究胶质细胞源性神经营养因子对脊髓损伤后皮质脊髓束再生的保护作用.设计随机对照实验.地点和材料本实验在第二军医大学神经生物实验室完成.实验动物采用雄性成年SD大鼠64只,体质量250～300 g.干预采用Nystrom法制备大鼠胸髓后路压迫损伤模型,压迫质量为50 g,时间为5 min,造成大鼠胸段脊髓急性重度损伤.脊髓损伤后24 h,对照组注射6μL阳离子脂质体DC-Chol和2μg pCDNA3的混合物,实验组将6μL DC-Chol和2μg重组质粒pEGFP-GDNF cDNA混合后注入大鼠损伤脊髓.主要观察指标应用RT-PCR技术和荧光显微镜检测GDNF体内转基因后在局部的表达,通过辣根过氧化物酶(HRP)顺行追踪技术和神经微丝(NF)免疫组化活性的变化来评价GDNF基因体内转染对大鼠皮质脊髓束再生的影响.结果GDNF基因体内转染1周后发现在基因注射局部有转录和蛋白水平表达,4周后在脊髓损伤周围仍检测到GDNF蛋白表达.脊髓损伤后4周,实验组脊髓损伤区皮质脊髓束HRP标记明显多于对照组,仅在实验组可见部分神经纤维穿过损伤区至远端5～9 mm.损伤区NF阳性轴突数(524.33±80.55/低倍视野)较对照组(309.84±56.65/低倍视野)明显增多(P＜0.01),GFAP阳性细胞数(186.68±16.25)明显少于对照组(239.78±21.44)(P＜0.01).结论阳离子脂质体介导GDNF体内转基因能促进近端皮质脊髓束再生和神经骨架蛋白的修复,提示神经营养因子基因治疗可用来治疗创伤性脊髓损伤.     

Calcium channel alpha-2-delta-1 protein upregulation in dorsal spinal cord mediates spinal cord injury-induced neuropathic pain states

Spinal cord injury (SCI) commonly results in the development of neuropathic pain, which can dramatically impair the quality of life for SCI patients. SCI-induced neuropathic pain can be manifested as both tactile allodynia (a painful sensation to a non-noxious stimulus) and hyperalgesia (an enhanced sensation to a painful stimulus). The mechanisms underlying these pain states are poorly understood. Clinical studies have shown that gabapentin, a drug that binds to the voltage-gated calcium channel alpha-2-delta-1 subunit (Ca_vα2δ-1) proteins is effective in the management of SCI-induced neuropathic pain. Accordingly, we hypothesized that tactile allodynia post SCI is mediated by an upregulation of Ca_vα2δ-1 in dorsal spinal cord. To test this hypothesis, we examined whether SCI-induced dysregulation of spinal Ca_vα2δ-1 plays a contributory role in below-level allodynia development in a rat spinal T9 contusion injury model. We found that Ca_vα2δ-1 expression levels were significantly increased in L4-6 dorsal, but not ventral, spinal cord of SCI rats that correlated with tactile allodynia development in the hind paw plantar surface. Furthermore, both intrathecal gabapentin treatment and blocking SCI-induced Ca_vα2δ-1 protein upregulation by intrathecal Ca_vα2δ-1 antisense oligodeoxynucleotides could reverse tactile allodynia in SCI rats. These findings support that SCI-induced Ca_vα2δ-1 upregulation in spinal dorsal horn is a key component in mediating below-level neuropathic pain states, and selectively targeting this pathway may provide effective pain relief for SCI patients.     

人脐血间充质干细胞促进脐血CD34+细胞在NOD/SCID小鼠体内植入的实验研究

目的探讨人脐血间充质干细胞(MSC)联合人脐血CD34+细胞移植,能否促进CD34+细胞在NOD/SCID小鼠体内植入及加速其造血恢复.方法NOD/SCID小鼠于60Co 2.5 Gy照射后24h内由尾静脉输注胎儿脐血CD34+细胞1×105/只(低细胞量移植组)或1×106/只(高细胞量移植组),联合移植组同时输注脐血MSC 1×106/只.动态观察移植后小鼠外周血白细胞、血红蛋白和血小板恢复情况,于移植后第8周用流式细胞术检测存活小鼠骨髓中人CD45+、CD45+CD3+、CD45+CD19+和CD45+CD33+细胞的含量.结果①低细胞量移植时,联合移植组的植入率明显高于单纯移植组,分别为26.02%和16.52%(P＜0.05);高细胞量移植时,联合移植组和单纯移植组的植入率相近,分别为43.71%和39.23%(P＞0.05).②高细胞量联合移植组和单纯移植组的存活率分别为80%和70%;低细胞量联合移植组和单纯移植组的存活率分别为70%和50%.③无论高细胞量还是低细胞量组,联合移植小鼠白细胞、血红蛋白和血小板的恢复明显早于单纯移植组.④移植8周后,小鼠骨髓中人CD45+CD19+、CD45+CD33+细胞含量在低细胞量移植时,联合移植组高于单纯移植组;但在高细胞量移植时,两组之间差异无统计学意义.CD45+CD41a+细胞的含量无论在低细胞量和高细胞量移植时,联合移植组均高于单纯移植组.各组小鼠骨髓中CD45+CD3+细胞的含量均较少,且各组之间差异无统计学意义.结论①低细胞量移植时,人脐血MSC联合移植可提高人脐血CD34+细胞在小鼠体内的植入率.②人脐血MSC与人脐血CD34+细胞联合移植,加速NOD/SCID小鼠各系造血恢复,提高移植小鼠存活率.③MSC联合移植可促进人脐血CD34+细胞在NOD/SCID小鼠体内向粒系、B淋巴系和巨核系定向分化.     

Effect of 17beta-estradiol on signal transduction pathways and secondary damage in experimental spinal cord trauma

Because studies have shown that 17beta-estradiol (E2) produces anti-inflammatory effects after various adverse circulatory conditions, we examined whether administration of E2 before spinal cord injury (SCI) has any salutary effects in reducing SCI. Spinal cord injury was induced by the application of vascular clips (force of 24 g) to the dura via a four-level T5-T8 laminectomy. To gain a better insight into the mechanism of action of the anti-inflammatory effects of E2, the following end points of the inflammatory process were evaluated: (1) spinal cord inflammation and tissue injury (histological score); (2) neutrophil infiltration (myeloperoxidase activity); (3) expression of iNOS, nitrotyrosine, and COX-2; (4) apoptosis (terminal deoxynucleotidyltransferase-mediated UTP end labeling staining and Bax and Bcl-2 expression); and (5) tissue TNF-alpha, IL-6, IL-1beta, and monocyte chemoattractant protein 1 levels. In another set of experiments, the pretreatment or posttreatment with E2 significantly ameliorates the recovery of limb function (evaluated by motor recovery score). To elucidate whether the protective effects of E2 were mediated via the estrogen receptors, we investigated the effect of an estrogen receptor antagonist, ICI 182,780, on the protective effects of E2. ICI 182,780 (500 microg/kg, s.c., 1 h before treatment with E2) significantly antagonized the effect of the E2 and abolished the protective effect against SCI. Taken together, our results clearly demonstrate that administration of E2 before SCI reduces the development of inflammation and tissue injury associated with spinal cord trauma.     

胎儿骨髓基质细胞与细胞因子支持人脐血单个核细胞体外扩增的研究

为了探讨胎儿骨髓基质细胞(FBMSC)联合细胞因子对脐血单个核细胞(MNC)体外扩增作用的影响及比较扩增前后脐血(CB)CD34 细胞上细胞表面趋化因子受体CXCR4和黏附分子CD49d (VLA4)的表达情况,将从新鲜CB标本中分离出的MNC分别接种于已建立的无血清培养体系,该体系分4组:A组为培养过程中不加细胞因子和基质细胞;B组为单用胚胎骨髓基质细胞支持;C组为单用细胞因子支持;D组为细胞因子和胚胎骨髓基质细胞联合支持。在0、6、10及14天检测细胞总数、CD34 细胞数及集落形成单位(CFU)数,同时检测CD34 细胞上CD49d 及CXCR4的表达数。结果表明:在体外14天培养过程中,各时间点D组CD34 细胞、CFU数及CD34 CXCR4 细胞和CD34 CD49d 细胞扩增数均高于A、B、C组(P<0.05);B、C和D组在各时间点各测量指标与A组比较具显著性差异(P<0.05);6天后,B组各测量指标与C组比较具显著性差异(P<0.05)。结论:胚胎骨髓基质细胞联合外源性细胞因子不仅可以支持脐血MNC的有效扩增,而且扩增后与趋化作用和与粘附作用相关的造血细胞亦较扩增前明显增加。单用细胞因子扩增会造成造血细胞的耗竭,单用基质细胞支持可扩增或维持造血细胞的量,但难以实现造血细胞的大量扩增,FBMSC联合外源性细胞因子可能是扩增造血干祖细胞的较理想方案。     

Human umbilical cord blood stem cells infusion in spinal cord injury: engraftment and beneficial influence on behavior

The use of human umbilical cord blood (hUCB)--a rich source of nonembryonic or adult stem cells--has recently been reported to ameliorate behavioral consequences of stroke. In this study, we tested whether human cord blood leukocytes also ameliorate behavioral impairments of spinal cord injury. Rats were divided into five groups: (1) laminectomy (without spinal cord injury) only; (2) laminectomy + cord blood infusion; (3) spinal cord injury + cord blood infused 1 day post injury; (4) spinal cord injury + cord blood infused 5 days post injury; and (5) spinal cord injury only. Spinal cord injury was induced by compressing the spinal cord for 1 min with an aneurysm clip calibrated to a closing pressure of 55 g. Open-field behavior was assessed 1, 2, and 3 weeks after intravenous injection of prelabeled human cord blood cells. Open-field test scores of spinal cord injured rats treated with human cord blood at 5 days were significantly improved as compared to scores of rats similarly injured but treated at day 1 as well as the otherwise untreated injured group. The results suggest that cord blood stem cells are beneficial in reversing the behavioral effects of spinal cord injury, even when infused 5 days after injury. Human cord blood-derived cells were observed in injured areas, but not in noninjured areas, of rat spinal cords, and were never seen in corresponding areas of spinal cord of noninjured animals. The results are consistent with the hypothesis that cord blood-derived stem cells migrate to and participate in the healing of neurological defects caused by traumatic assault.     

胶质源性神经营养因子对大鼠脊髓不完全损伤后运动功能的保护作用

目的：探讨胶质源性神经营养因子（GDN）对大鼠脊髓损伤后运动功能恢复的影响。方法：动物分为对照组、假手术组、生理盐水（NS）组及GDNF组,改良Allen方法致脊髓不完全损伤,蛛网膜下腔分别给予NS及GDNF20μl,伤后1d ,3d,7d,10d,14d及21d评定下肢运动功能（Tarlov评分及Rivlin斜板）。结果：假手术组与对照组、假手术组自身无明显变化（P＞0．05）,NS组及GDNF组伤后运动功能明显障碍。NS组14d、GDNF组7d以后脊髓功能明显改善,10d以后GDNF组运动功能改善显著超过NS组（P＜0．01）。结论：GDNF能够救损伤脊髓运动神经元,促进不完全脊髓损伤运动功能的恢复。     

Human dental pulp-derived stem cells promote locomotor recovery after complete transection of the rat spinal cord by multiple neuro-regenerative mechanisms

Spinal cord injury (SCI) often leads to persistent functional deficits due to loss of neurons and glia and to limited axonal regeneration after injury. Here we report that transplantation of human dental pulp stem cells into the completely transected adult rat spinal cord resulted in marked recovery of hind limb locomotor functions. Transplantation of human bone marrow stromal cells or skin-derived fibroblasts led to substantially less recovery of locomotor function. The human dental pulp stem cells exhibited three major neuroregenerative activities. First, they inhibited the SCI-induced apoptosis of neurons, astrocytes, and oligodendrocytes, which improved the preservation of neuronal filaments and myelin sheaths. Second, they promoted the regeneration of transected axons by directly inhibiting multiple axon growth inhibitors, including chondroitin sulfate proteoglycan and myelin-associated glycoprotein, via paracrine mechanisms. Last, they replaced lost cells by differentiating into mature oligodendrocytes under the extreme conditions of SCI. Our data demonstrate that tooth-derived stem cells may provide therapeutic benefits for treating SCI through both cell-autonomous and paracrine neuroregenerative activities.     

Ex vivo expansion of human umbilical cord blood-derived T-lymphocytes with homologous cord blood plasma

This study was designed to establish a more effective and safe culture system for adoptive immunotherapy by investigating the use of homologous cord blood plasma (HCBP) instead of fetal bovine serum (FBS), which has various limitations including ethical problems for the ex vivo expansion of human umbilical T lymphocytes. Fresh human umbilical mononuclear cell fractions were isolated by Ficoll-Hypaque density centrifugation. Nonadherent mononuclear cell fractions were cultured with anti-CD3 antibody (5 microg/ml), IL-2 (175 U/ml), and either 10% FBS or 10% HCBP. On day 8, the cellular proliferation rate and cell surface markers were assessed. There was no significant difference in proliferation when human umbilical cord blood T lymphocytes were grown in medium supplemented with FBS or HCBP (p > 0.05). In medium containing FBS, the proportion of CD3(+)CD4(+) (markers for helper T cell), CD3(+)CD8(+) (cytotoxic T cell), CD3(+)CD25(+) (activated T cell), CD3(+)CD38(+) (immature T cell), and CD3(+)CD45RO(+) (memory T cell) cells was significantly increased (p < 0.05), whereas proportion of CD3(+)CD45RA(+) (naive T cell) and CD16(+)CD56(+) (NK cell) cells was significantly decreased (p < 0.05). In HCBP supplemented medium, the proportion of CD3(+)CD8(+), CD3(+)CD25(+), CD3(+)CD45RA(+), and CD3(+)CD45RO(+) cells was significantly increased (p < 0.05). The proportion of CD3(+)CD4(+), CD3(+)CD45RO(+) and CD3(+)CD38(+) cells was significantly higher, but proportion of CD3(+)CD45RA(+) and CD3(+)CD8(+) cells was significantly lower in FBS compared with HCBP supplemented medium (p < 0.05). Our results support the feasibility of ex vivo expansion of human umbilical cord blood T lymphocytes in medium supplemented with HCBP for future adoptive cellular immunotherapy.     

脐血CD34^＋细胞体外扩增脐血巨核祖细胞的研究

本研究探讨人脐血CD34^＋细胞体外扩增的巨核祖细胞的生物学特性及其免疫原性变化，为体外扩增脐血巨核祖细胞的临床应用提供实验依据。采用Ficoll-Hapaque分离法分离人脐血单个核细胞，应用免疫磁珠法（MACS）再分离富集CD34^＋细胞，在含血小板生成素（TPO，50ng／ml）、白介素-1]（IL—11，50ng／ml）和肝素（25U／ml）的无血清液体培养体系中培养14天。用流式细胞术检测扩增产物免疫表型（CD34^＋、CD41a^＋、CD61^＋、CD34^＋CD41a^＋及CD34^＋CD61^＋）、巨核细胞凋亡率及其表面HLA Ⅰ、Ⅱ类分子的表达，并进行巨核细胞集落形成单位（CFU—Mk）的检测。结果显示：脐血CD34^＋细胞能够有效地向巨核细胞分化，CD41a^＋和CD61^＋细胞比例在培养第14天达到峰值，CD34^＋CD41^＋和CD34^＋CD61^＋细胞比例在扩增第7天达到最高峰[分别为（3．41±2．80）％和（1．89±1．43）％]；CFU—Mk大集落在扩增第7天达到高峰（（20．66±32．79）倍]，小集落在扩增第10天达到高峰[（435．62±482．65）倍]；在培养7、10和14天时巨核细胞的凋亡率分别为（19．48±9．64）％、（26．87±9．03）％和（52．46±11．74）％，其中培养7天和10天的凋亡率无显著性差异（P〉0．05），培养14天的凋亡率显著高于7天和10天（P均〈0．05）；巨核细胞表面HLA Ⅰ、Ⅱ类分子的表达随着扩增天数的延长逐渐降低，其中培养0到10天阶段下降明显．结论：采用TPO＋IL 11＋肝素组合．可以有效地扩增脐血巨核祖细胞；培养7天，CFU—Mk大集落扩增倍数、CD34^＋CD41^＋和CD34^＋CD61^＋细胞比例均达高峰，这是巨核祖细胞体外扩增的较佳培养时间。     

联合应用免疫抑制剂治疗大鼠急性脊髓损伤效果观察

目的探讨联合应用免疫抑制剂他克莫司(FK506)和FTY720对大鼠急性脊髓损伤的神经保护作用。方法采用Allen′s打击法制备大鼠脊髓损伤模型,84只大鼠分成4组:FK506+FTY720联合治疗组(A组)、FK506治疗组(B组),脊髓损伤组(C组)和正常对照组(D组)。A组通过尾静脉注射0.3 mg/kg FK506,并予鼻饲0.3 mg/kg FTY720,B组通过尾静脉注射0.5 mg/kg FK506,C组注射生理盐水。伤后6 h、24 h和48 h取伤段脊髓行HE染色分析和Tunel凋亡分析;伤后第1、3和7天行体感诱发电位(SEP)检测;伤后第1、3、7和14天行BBB评分和斜板试验评分。结果联合治疗组和FK506治疗组伤后各个时间取材点的脊髓损伤出血坏死区均轻于损伤组,在第6、24和48小时时间点脊髓Tunel神经细胞凋亡检测明显轻于损伤组,有显著差异性(P<0.05),但2组之间比较无显著差异性(P>0.05)。联合治疗组和FK506治疗组伤后各时间点SEP、BBB评分和斜板试验评分均优于损伤组,有显著性差异(P<0.05),但是2组之间差异无统计学意义(P>0.05)。结论大鼠急性脊髓损伤早期联合应用FK506和FTY720具有良好的保护神经功能和促进神经功能恢复的作用。     

Glycyrrhizin reduces secondary inflammatory process after spinal cord compression injury in mice

Glycyrrhizin, a major active constituent of liquorice root (Glycyrrhiza glabra), has a free radical scavenging property, and its effects were evaluated on an animal model of spinal cord injury (SCI) induced by the application of vascular clips (force of 24 g) to the dura via a four-level T5-T8 laminectomy. Spinal cord injury in mice resulted in severe trauma characterized by edema, tissue damage, and apoptosis (measured by terminal deoxynucleotidyltransferase-mediated dUTP-biotin end labeling staining, Bax, and Bcl-2 expression). Immunohistochemical examination demonstrated a marked increase in immunoreactivity for nitrotyrosine, iNOS, and poly(adenosine diphosphate-ribose) in the spinal cord tissue. Additionally, we demonstrate that these inflammatory events were associated with the activation of nuclear factor-kappaB. In contrast, the degree of (1) spinal cord inflammation and tissue injury (histological score), (2) nitrotyrosine and poly(adenosine diphosphate [ADP] ribose) formation, (3) iNOS expression, (4) nuclear factor-kappaB activation, and (5) apoptosis (terminal deoxynucleotidyltransferase-mediated dUTP-biotin end labeling, Bax, and Bcl-2) was markedly reduced in spinal cord tissue obtained from mice treated with glycyrrhizin extract (10 mg/kg, i.p., 30 min before and 1 and 6 h after SCI). In a separate set of experiments, we have clearly demonstrated that glycyrrhizin extract treatment significantly ameliorated the recovery of limb function (evaluated by motor recovery score). Taken together, our results clearly demonstrate that treatment with glycyrrhizin extract reduces the development of inflammation and tissue injury events associated with spinal cord trauma.     

不同时间移植人脐血CD34~+细胞可影响心肌梗死大鼠心功能及血管内皮细胞生长因子的分泌

背景:干细胞移植可以改善心脏功能,改善预后。目的:观察不同时间经静脉移植人脐血CD34+细胞对心肌梗死大鼠心功能及细胞因子分泌的影响。方法:结扎冠状动脉左前降支制备Wistar大鼠心肌梗死模型,于梗死后1,5,10,30d经尾静脉注入0.5mL人脐血CD34+细胞(实验组)或磷酸盐缓冲溶液(对照组)。结果与结论:与对照组相比,梗死后5,10d实验组大鼠左室射血分数明显升高(P<0.05),左室收缩末内径明显减小(P<0.05),左室后壁增厚率更高(P<0.05),毛细血管密度明显增加(P<0.05),且以梗死后10d移植大鼠心功能改善效果最明显(P<0.05)。梗死后10d实验组心肌局部血管内皮细胞生长因子最高(P<0.05)。说明大鼠心肌梗死后5,10d经静脉移植脐血CD34+细胞可明显改善心功能,梗死后10d移植血管内皮细胞生长因子分泌更多,血管生成更多,对心功能的改善更明显;同时说明脐血单个核细胞移植可能是通过增加血管内皮细胞生长因子分泌,提高毛细血管密度来改善心功能的。     

Identification of lymphomyeloid primitive progenitor cells in fresh human cord blood and in the marrow of nonobese diabetic-severe combined immunodeficient (NOD-SCID) mice transplanted with human CD34(+) cord blood cells.

Transplantation of genetically marked donor cells in mice have unambiguously identified individual clones with full differentiative potential in all lymphoid and myeloid pathways. Such evidence has been lacking in humans because of limitations inherent to clonal stem cell assays. In this work, we used single cell cultures to show that human cord blood (CB) contains totipotent CD34(+) cells capable of T, B, natural killer, and granulocytic cell differentiation. Single CD34(+) CD19(-)Thy1(+) (or CD38(-)) cells from fresh CB were first induced to proliferate and their progeny separately studied in mouse fetal thymic organotypic cultures (FTOCs) and cocultures on murine stromal feeder layers. 10% of the clones individually analyzed produced CD19(+), CD56(+), and CD15(+) cells in stromal cocultures and CD4(+)CD8(+) T cells in FTOCs, identifying totipotent progenitor cells. Furthermore, we showed that totipotent clones with similar lymphomyeloid potential are detected in the bone marrow of nonobese diabetic severe combined immunodeficient (NOD-SCID) mice transplanted 4 mo earlier with human CB CD34(+) cells. These results provide the first direct demonstration that human CB contains totipotent lymphomyeloid progenitors and transplantable CD34(+) cells with the ability to reconstitute, in the marrow of recipient mice, the hierarchy of hematopoietic compartments, including a compartment of functional totipotent cells. These experimental approaches can now be exploited to analyze mechanisms controlling the decisions of such primitive human progenitors and to design conditions for their ampification that can be helpful for therapeutic purposes.     

Glycogen synthase kinase-3 beta inhibition reduces secondary damage in experimental spinal cord trauma

Glycogen synthase kinase-3 (GSK-3) has recently been identified as an ubiquitous serine-threonine protein kinase that participates in a multitude of cellular processes and plays an important role in the pathophysiology of a number of diseases. The aim of this study was to investigate the effects of GSK-3beta inhibition on the degree of experimental spinal cord trauma induced by the application of vascular clips (force of 24 g) to the dura via a four-level T5-T8 laminectomy. Spinal cord injury (SCI) in mice resulted in severe trauma characterized by edema, neutrophil infiltration, production of a range of inflammatory mediators, tissue damage, and apoptosis. Treatment of the mice with 4-benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione (TDZD-8), a potent and selective GSK-3beta inhibitor, significantly reduced the degree of 1) spinal cord inflammation and tissue injury (histological score); 2) neutrophil infiltration (myeloperoxidase activity); 3) inducible nitric-oxide synthase, nitrotyrosine, and cyclooxygenase-2 expression; and 4) and apoptosis (terminal deoxynucleotidyl transferase dUTP nick-end labeling staining and Bax and Bcl-2 expression). In a separate set of experiments, TDZD-8 significantly ameliorated the recovery of limb function (evaluated by motor recovery score). Taken together, our results clearly demonstrate that treatment with TDZD-8 reduces the development of inflammation and tissue injury associated with spinal cord trauma.     

地中海贫血患儿血清群体反应性抗体对脐血CD34~+细胞增殖和凋亡的影响

本研究探讨特异性群体反应性抗体(panel reactive antibody,PRA)对脐血CD34+细胞的影响。取含PRA的β地中海贫血患儿血清,与脐血CD34+细胞、补体联合孵育,观察其对CD34+细胞的影响,并以[3H]TdR掺入法测定细胞DNA合成及流式细胞仪检测细胞凋亡。结果表明,PRA血清组细胞培养上清中乳酸脱氢酶水平高于对照组;PRA血清组脐血CD34+细胞DNA合成能力较对照组下降。流式细胞仪检测显示,各实验组间脐血CD34+细胞凋亡率差异无统计学意义。结论:特异性PRA血清对脐血CD34+细胞的增殖有抑制作用,补体可增强上述作用;特异性PRA血清对脐血CD34+细胞的凋亡无明显影响。