人脐带间充质干细胞对百草枯中毒肺损伤小鼠模型的治疗作用

目的:探讨脐带间充质干细胞(UCMSC)治疗百草枯中毒引起的小鼠肺损伤的可行性。方法:小鼠腹腔一次性注射百草枯制备百草枯中毒小鼠模型,24 h后尾静脉注射UCMSC,分别于治疗后7和21 d取材,观察UCMSC对急性肺损伤和慢性肺纤维化的治疗作用。结果:UCMSC移植对40和50 mg/kg百草枯染毒组急性肺损伤有效,动物死亡率显著降低,但对60 mg/kg百草枯染毒组动物无效。UCMSC治疗对慢性肺纤维化有显著治疗作用,治疗组动物体重恢复早,死亡率降低,肺纤维化评分降低。RT-PCR结果显示,UCMSC移植3 h有人特异性线粒体基因的表达,但24 h后未检测到。结论:UCMSC对百草枯中毒性急慢性肺损伤有一定的治疗作用,这种作用可能是通过旁分泌机制实现的。     

Optimal Route for Human Umbilical Cord Blood-Derived Mesenchymal Stem Cell Transplantation to Protect Against Neonatal Hyperoxic Lung Injury: Gene Expression Profiles and Histopathology

The aim of this study was to determine the optimal route of mesenchymal stem cell (MSC) transplantation. To this end, gene expression profiling was performed to compare the effects of intratracheal (IT) versus intravenous (IV) MSC administration. Furthermore, the therapeutic efficacy of each route to protect against neonatal hyperoxic lung injury was also determined. Newborn Sprague-Dawley rats were exposed to hyperoxia (90% oxygen) from birth for 14 days. Human umbilical cord blood-derived MSCs labeling with PKH26 were transplanted through either the IT (5×10⁵) or IV (2×10⁶) route at postnatal day (P) 5. At P14, lungs were harvested for histological, biochemical and microarray analyses. Hyperoxic conditions induced an increase in the mean linear intercept and mean alveolar volume (MAV), indicative of impaired alveolarization. The number of ED-1 positive cells was significantly decreased by both IT and IV transplantations. However, IT administration of MSCs resulted in a greater decrease in MAV and ED-1 positive cells compared to IV administration. Moreover, the number of TUNEL-positive cells was significantly decreased in the IT group, but not in the IV group. Although the IT group received only one fourth of the number of MSCs that the IV group did, a significantly higher number of donor cell-derived red PKH 26 positivity were recovered in the IT group. Hyperoxic conditions induced the up regulation of genes associated with the inflammatory response, such as macrophage inflammatory protein-1 α, tumor necrosis factor-α and inter leukin-6; genes associated with cell death, such as p53 and caspases; and genes associated with fibrosis, such as connective tissue growth factor. In contrast, hyperoxic conditions induced the dwon-regulation of vascular endothelial growth factor and hepatocyte growth factor. These hyperoxia-induced changes in gene expression were decreased in the IT group, but not in the IV group. Thus, local IT MSC transplantation was more effective than systemic IV MSC administration in protecting against neonatal hyperoxic lung injury.     

脐带血来源间充质干细胞体外分离培养条件的优化

脐带血间充质干细胞（umbilical cord blood mesenchymal stem cells,UCB-MSCs）不仅可以作为滋养层细胞支持造血干细胞在体外的大规模扩增,在造血移植过程中还能够降低并发症的发生率以及加速造血重建功能的恢复．但是,目前UCB-MSCs的原代分离培养成功率一般只有30％左右,为进一步提高该成功率,利用正交实验方法对UCB-MSCs体外培养的主要影响因素：细胞的接种密度、细胞因子的组合及用量、是否添加血清和滋养层细胞,进行逐层筛选,并对培养出的间充质干细胞进行了流式细胞分析和向成骨、软骨及脂肪方向的诱导分化检测,以期获得UCB-MSCs培养的最佳方法．实验结果表明,细胞的接种密度是UCB-MSCs培养最显著的影响因素（P〈0．1）,接种密度越大,MSCs越容易生长,能够培养出MSCs的几率就越大,其次为细胞因子,添加细胞因子能有效地刺激MSCs的生长．在高接种密度的基础上,添加细胞因子IL-3（15μg／L）和GM-CSF（5μg/L）,可大大提高UCB-MSCs体外原代培养的成功率,从30％左右提高到90％以上．流式细胞检测结果显示,所分离培养的细胞表达间充质干细胞的抗原（CD13^＋、CD29^＋、CD44^＋、CD105^＋、CD166^＋）,不表达造血细胞的抗原（CD34^-、CD45^-、HLA-DR^-）,并能够向成骨、软骨及脂肪方向分化,这与源于骨髓的间充质干细胞相一致．所建立的培养方法能够为UCB-MSCs的临床应用提供大量优质的种子细胞．     

脐带间充质干细胞移植治疗脊髓损伤的临床研究

目的:探讨脐带间充质干细胞移植治疗脊髓损伤的疗效及安全性。方法:40例脊髓损伤患者给予脐带间充质干细胞移植治疗,移植方法采用静脉输注联合腰穿鞘内注射的方法。术后随访1年余定期观察患者临床症状及各项指标的变化并进行综合分析。移植过程中为促进干细胞的生长和分化,根据患者病情及身体状况给予相应的康复功能锻炼。结果:与入院时比较,脐带间充质干细胞移植治疗3、6、12个月后,不完全性脊髓损伤患者针刺觉评分、轻触觉评分、运动评分均有明显改善(P<0.05或0.01),完全性脊髓损伤患者针刺觉评分、轻触觉评分、运动评分均无明显变化(P>0.05),两组残损分级均无明显改善(P>0.05)。移植后各项生化指标正常,未出现严重的并发症和明显的不良反应。结论:脐带间充质干细胞移植治疗脊髓损伤近期疗效明显,可以改善患者的临床症状,提高患者的生存质量,是一种值得借鉴的治疗方法。     

Human Umbilical Cord Mesenchymal Stem Cells Derived Exosomes Promote Neurological Function Recovery in a Rat Spinal Cord Injury Model

Neurochemical Research - Spinal cord injury (SCI) often leads to personal and social-economic consequences with limited therapeutic options. Exosomes derived from human umbilical cord mesenchymal...     

Proangiogenic Compositions of Microvesicles Derived from Human Umbilical Cord Mesenchymal Stem Cells

Introduction & Objective

Microvesicles (MVs) derived from mesenchymal stem cells (MSCs) have been shown to promote angiogenesis. This study was aimed to shed a light on the mechanisms by analyzing the angiogenesis-promoting compositions of MSC-MVs. Also we try to figure out the impact of hypoxia on angiogenesis.

Methods

MVs were isolated from the culture supernatants of MSCs under hypoxia/normoxia and serum-deprivation condition. The morphological features of MVs were revealed by an electron microscope and the origin of the MVs was identified by a bead-bound assay. An antibody array was used to analyze the expression of angiogenic cytokines from MVs and the parent MSCs as well. The major candidate factors were screened and the results were validated by immune blotting.

Results

MSC-MVs were around 80 nm in diameter. They expressed CD29, CD44, and CD73, but not CD31 and CD45. Antibody array showed that both MSCs and MVs expressed many angiogenesis-promoting biomolecules, including interleukin-6 (IL-6), basic fibroblast growth factors (bFGF), and recptor of urokinase-type plasminogen activator (UPAR). MSC-MVs contained angiogenin, vascular endothelial growth factor (VEGF), monocyte chemotactic protein-1 (MCP-1) and the receptor-2 for vascular endothelial growth factor at higher levels than the parent MSCs. Under hypoxic condition most cytokines were expressed in greater quantity than normoxic in MSCs while in MVs there was no significant difference between hypoxic and normoxic conditions except UPAR, Angiogenin, VEGF, IGF, Tie-2/TEK, and IL-6 which were higher in MVs under hypoxic conditions than those in normoxic condition.

Conclusion

Upon serum-deprivation condition, MSCs could secrete MVs that contain a variety of factors contributing to their angiogenesis-promoting function. And among them, Angiogenin, VEGF, MCP-1, VEGF R2 might be of greater importance than the other cytokines. Also UPAR, Angiogenin, VEGF, IGF, Tie-2/TEK, IL-6 might be responsible for hypoxia-augmented proangiogenic effects of MVs.     

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 ,可作为组织工程的种子细胞和具有促进造血作用     

脐带间充质干细胞与再生障碍性贫血

再生障碍性贫血(aplastic anemia,AA)是由于物理、化学、生物或不明因素作用使骨髓造血干细胞和骨髓微环境严重受损,造成骨髓造血功能降低或衰竭,以全血细胞减少为主要表现的一组综合征。间充质干细胞(MSC)是属于中胚层的一类多能干细胞,主要存在于结缔组织和器官间质中。不仅具有多向分化潜能,还有多种免疫调节作用。许多研究表明MSCs抑制同种异体效应T淋巴细胞增殖,还能下调T淋巴细胞表面的活化分子CD25、CD38、CD69的表达。MSCs对DCs的分化、成熟和活化具有抑制作用,改变DCs的细胞因子分泌,使成熟的DCl分泌TNF-a减少,DC2分泌IL-10增加,从而抑制T淋巴细胞增殖。MSCs能够抑制IL-2和IL-15介导的NK细胞增殖,使IL-2刺激NK细胞分泌的IFN-y减少。通过这些机制来干预再生障碍性贫血,同时研究发现MSCs免疫原性较低。尤其脐带MSC,因为其独特优势,目前已经成为干细胞干预治疗AA的研究热点。     

Mechanism of Human Umbilical Cord Mesenchymal Stem Cells Derived-Extracellular Vesicle in Cerebral Ischemia-Reperfusion Injury

Neurochemical Research - Mesenchymal stem cells (MSCs)-derived extracellular vesicles (EVs) are implicated in cerebral ischemia reperfusion (I/R) injury process. In this study, after extraction and...     

人脐带间充质干细胞的安全性评价

干细胞治疗作为某些难治性疾病的新型治疗手段,在组织修复、自身免疫疾病和退行性疾病治疗中具有重要的临床价值.本研究以裸鼠为动物模型,通过裸鼠皮下成瘤、克隆形成和端粒酶活性等试验,评估人脐带间充质干细胞的安全性,为干细胞用于临床治疗的安全性提供参考.裸鼠成瘤性试验结果显示,阴性对照组和试验组成瘤数均为0,阳性对照组成瘤数为...     

人外周血单个核细胞与脐带间充质干细胞共培养的AFM研究

采用原子力显微镜与倒置显微镜在细胞层次上观察了人外周单个核细胞(PBMCs)与同种异源脐带间充质干细胞(hUC-MSCs)共培养的过程,并在单细胞水平上分析了共培养前后人外周单个核细胞的形貌和生物物理性质。结果发现:共培养后贴壁人外周单个核细胞的形态发生了很大的改变,并且表面分布着大小不一的颗粒状聚合物。利用AFM高空间分辨的力位移曲线测量系统,发现共培养72h后培养上清中人外周单个核细胞、贴壁的人外周单个核细胞的粘滞力分别是单纯培养72h的人外周单个核细胞的2倍、5倍,而细胞的硬度分别是单纯培养人外周单个核细胞的1.5倍、2倍。CCK-8检测提示,共培养过程中,干细胞的生长与外周血单个核细胞的生长出现了竞争作用。通过AFM探测人外周单个核细胞与脐带间充质干细胞共培养的可视化数据,有助于更好地了解间充质干细胞与外周血单个核细胞的相互作用。     

Human Umbilical Cord Mesenchymal Stem Cells and Derived Hepatocyte-Like Cells Exhibit Similar Therapeutic Effects on an Acute Liver Failure Mouse Model

Mesenchymal stem cells (MSCs) have exhibited therapeutic effects in multiple animal models so that are promising liver substitute for transplantation treatment of end-stage liver diseases. However, it has been shown that over-manipulation of these cells increased their tumorigenic potential, and that reducing the in vitro culture time could minimize the risk. In this study, we used a D-galactosamine plus lipopolysaccharide (Gal/LPS)-induced acute liver failure mouse model, which caused death of about 50% of the mice with necrosis of more than 50% hepatocytes, to compare the therapeutic effects of human umbilical cord MSCs (hUCMSCs) before and after induction of differentiation into hepatocyte (i-Heps). Induction of hUCMSCs to become i-Heps was achieved by treatment of the cells with a group of growth factors within 4 weeks. The resulted i-Heps exhibited a panel of human hepatocyte biomarkers including cytokeratin (hCK-18), α-fetoprotein (hAFP), albumin (hALB), and hepatocyte-specific functions glycogen storage and urea metabolism. We demonstrated that transplantation of both cell types through tail vein injection rescued almost all of the Gal/LPS-intoxicated mice. Although both cell types exhibited similar ability in homing at the mouse livers, the populations of the hUCMSCs-derived cells, as judged by expressing hAFP, hCK-18 and human hepatocyte growth factor (hHGF), were small. These observations let us to conclude that the hUCMSCs was as effective as the i-Heps in treatment of the mouse acute liver failure, and that the therapeutic effects of hUCMSCs were mediated largely via stimulation of host hepatocyte regeneration, and that delivery of the cells through intravenous injection was effective.     

人脐带间充质干细胞的分离培养与鉴定简

目的：建立并优化人脐带间充质干细胞分离纯化方法,并对其表面标志与多向分化潜能进行鉴定。方法：收集健康足月产胎儿脐带组织,采用组织块贴壁法进行原代培养,流式细胞仪对其表面标志进行检测,通过向成骨成脂分化对其多向分化潜能进行鉴定,RT-PCR对其干细胞特性基因Oct4、Nanog、Sox2、Nestin进行检测。结果：采用组织块贴壁法可在2周左右获得大量间充质干细胞,培养的细胞经流式细胞仪检测,高表达CD29、CD44、CD105、CD106,低表达CD34、CD45;经成骨成脂诱导2周后可分化为成骨细胞和成脂细胞,RT-PCR检测发现原代细胞表达Oct4、Nanog、Sox2、Nestin基因。结论：人脐带间充质干细胞可在体外扩增培养,具有多向分化潜能,可作为组织工程种子细胞来源。     

人滑膜间充质干细胞与人脐带间充质干细胞的生物学性状比较

该文旨在比较人滑膜间充质干细胞(human synovial mesenchymal stem cells,hSMSCs)与人脐带间充质干细胞(human umbilical cord mesenchymal stem cells,hUC-MSCs)的生物学性状.流式细胞仪鉴定hSMSCs和hUC-MSCs.比较两种间...     

Effects of Bone Marrow Mesenchymal Stem Cells on Hematopoietic Recovery and Acute Graft-Versus-Host Disease in Murine Allogeneic Umbilical Cord Blood Transplantation Model

To investigate the effect of bone marrow mesenchymal stem cells (MSC) on hematopoietic recovery and acute graft-versus-host disease (GVHD) in a murine allogeneic umbilical cord blood transplantation (allo-UCBT) model. MSCs were obtained from C57/BL mouse bone marrow. The MSC phenotypes were identified by flow cytometry (FCM), and their ability to differentiate into osteoblasts and adipocytes was tested. Once murine allo-UCBT and aGVHD models were established, mice were divided into five groups: (1) total body irradiation (TBI) group, each mouse receiving 0.3 ml sterile saline infusion after TBI and used as control; (2) UCB group, receiving 2 × 10⁶ umbilical cord blood mononuclear cells (UCB-MNC) after TBI; (3) UCB+MSC group, receiving 2 × 10⁶ UCB-MNC and 2 × 10⁷ MSC after TBI; (4) UCB+SC group, receiving 2 × 10⁶ UCB-MNC and 2 × 10⁶ spleen cells after TBI; and (5) UCB+SC+MSC group, receiving 2 × 10⁶ UCB-MNC, 2 × 10⁷ MSC and 2 × 10⁶ spleen cells after TBI. To evaluate the engraftment of HSC, the white blood cells, red blood cells, and platelets counts were tested at different time points after transplantation, and the ratio of chimerism was identified by FCM. The acute GVHD clinical scores, recipient mice survival, and the histopathological analyses were used to evaluate the effect of MSC on acute GVHD. MSCs were successfully obtained in vitro and FCM analysis showed that these cells are highly positive for CD90.2, CD44, and negative for CD34, CD45, and they are capable to differentiate into osteoblasts and adipocytes after being induced. Compared to UCB group, the UCB+MSC mice had shorter duration of myelosuppression and higher percentage of donor-derived cells which was up to 22.87 ± 4.3 % and the white blood cell (WBC), red blood cell (RBC), and platelet counts started to increase by day 6 after transplantation. Moreover, the average survival time for UCB+MSC mice was 25.0 ± 10.55 days, while for the UCB group it was 15.5 ± 12.50 days. The UCB+SC mice showed fatigue, loss of appetite, weight loss, arched back, and hair ruffling on day 13 post transplantation. Approximately 50 % of mice showed skin ulcers, had diarrhea and other manifestations of acute GVHD, and all mice were died within 20 days. Histopathological analysis confirmed grade II–IV GVHD manifestation. In addition to transient weight loss, some UCB+SC+MSC mice developed arched back, hair ruffling, diarrhea and other manifestations of acute GVHD. The clinical scores in UCB+SC+MSC mice with acute GVHD (grade I–II or without GVHD) were lower than UCB+SC group (P < 0.05). Bone marrow MSCs can promote hematopoietic recovery and decrease the incidence of acute GVHD in murine allo-UCBT model.     

Intravitreal Transplantation of Human Umbilical Cord Blood Stem Cells Protects Rats from Traumatic Optic Neuropathy

Objectives

To treat traumatic optic neuropathy (TON) with transplantation of human umbilical cord blood stem cells (hUCBSC) and explore how transplanted stem cells participate in the neuron repairing process.

Methods

A total of 195 Sprague-Dawley rats were randomly assigned to three groups: sham-surgery, optic nerve injury, and stem cell transplant group. Optic nerve injury was established in rats by directly clamping the optic nerve for 30 seconds. hUCBSC was microinjected into the vitreous cavity of injured rats. Optic nerve function was evaluated by flash visual evoked potentials (F-VEP). Apoptosis in retina tissues was detected by TUNEL staining. GRP78 and CHOP gene expression was measured by RT-PCR.

Results

After injury, transplantation of hUCBSC significantly blunted a reduction in optic nerve function indicated by smaller decreases in amplitude and smaller increases in peak latency of F-VEP waveform compared to the injury alone group. Also, significant more in retinal ganglion cell (RGC) count and less in RGC apoptosis were detected after transplantation compared to injured rats. The protective effect correlated with upregulated GRP78 and downregulated CHOP mRNA expression.

Conclusion

Intravitreal transplantation of hUCBSCs significantly blunted a reduction in optic nerve function through increasing RGC survival and decreasing retinal cell apoptosis. The protective role of transplantation was associated with upregulation of GRP78 expression and downregulation of CHOP expression in retinal cells.     

不同数目骨髓间充质干细胞移植对大鼠肺损伤的抑制作用

目的研究不同数目的骨髓间充质干细胞（mesenchymal stem cells,MSCs）静脉移植对野百合碱（monocrotaline,MCT）诱导大鼠肺损伤的抑制作用。方法全骨髓贴壁法培养Wistar大鼠MSCs,取第3～5代细胞进行移植。健康雄性Wistar大鼠20只由颈外静脉移植MSCs,按移植细胞个数分为5×105组、1×106组、5×106组和对照（生理盐水）组（n=5）,测定移植前、移植后5 min,30 min及24 h的RVSP。另40只随机分组（n=10）：①MCT/MSCs 5×105组;②MCT/MSCs 1×106组;③MCT组;④对照组,腹腔注射60 mg/kgM CT（对照组注射等量生理盐水）,同时分别移植MSCs 5×105个、1×106个或等量PBS液体。4周后检测大鼠右心室收缩压（right ventricularsystolic pressure,RVSP）、RV/（LV＋S）重量比值;肺组织苏木素-伊红染色、地衣红染色和平滑肌Actin免疫组织化学染色。统计数据采用SPSS 11.0软件,对各组数据进行单因素方差分析检验。结果少于1×106个MSCs颈外静脉移植是安全的。MSCs颈外静脉移植4周后,MCT/MSCs 1×106组RVSP（35.6±8.4）mmHg与MCT组（47.2±10.5）mmHg相比明显下降（P〈0.05）,心室比0.3572±0.0923明显低于MCT组0.4454±0.0935（P〈0.05）,而MCT/MSCs 5×105组RVSP为（42.5±11.3）mmHg,心室比0.4003±0.0725,与MCT组相比无明显下降（两者P〉0.05）;病理染色可见肺组织肺小动脉中,MCT/MSCs 1×106组中膜厚度分布为（19.2±3.8）%,较MCT组（26.4±4.9）%明显变薄（P〈0.05）;而MCT/MSCs 5×105组（23.3±3.6）%较MCT组相比无显著差异（P〉0.05）。结论 MSCs颈外静脉移植对MCT诱导的肺损伤具有抑制作用,1×106个细胞较5×105个细胞移植抑制作用显著。     

骨髓间充质干细胞在氨气致大鼠吸入性肺损伤的疗效观察

目的:观察骨髓间充质干细胞在氨气致大鼠吸入性肺损伤的疗效。方法:随机选取69只普通SD大鼠,随机将这些大鼠分为正常组(n=23)、地塞米松治疗组(n=23)、干细胞治疗组三组(n=23),正常组给予尾静脉注射生理盐水,地塞米松治疗组给予注射地塞米松,干细胞治疗组给予干细胞注射。结果:干细胞治疗组大鼠治疗后1 d、3 d的肺组织病理学评分及肺W/D、BALF白细胞数量及蛋白浓度、TNF-α、IL-8水平均显著低于地塞米松治疗组(P0.05),均显著高于正常组(P0.05)。结论:骨髓间充质干细胞在氨气致大鼠吸入性肺损伤的疗效较地塞米松显著。