Bone marrow stromal cells induce BMP2/4 production in oxygen-glucose-deprived astrocytes, which promotes an astrocytic phenotype in adult subventricular progenitor cells

Bone morphogenetic proteins (BMPs) affect cell proliferation and differentiation. Astrocytes in ischemic brain are highly responsive to bone marrow stromal cell (BMSC) treatment. We investigated the effects of BMSCs on astrocytes cultured under oxygen- and glucose-deprived conditions, which in part simulate in vivo stroke conditions, to test the hypothesis that BMSCs alter astrocytic expression of BMPs which may contribute to neurological functional recovery of stroke. Quantitative real-time RT-PCR showed that the expression of BMP2/4 mRNAs decreased within ischemic astrocytes, In contrast, BMP2/4 mRNA was significantly increased after cocultured with BMSCs. Western blotting also confirmed this increase at the protein level in the medium of ischemic astrocytes after coculture with BMSCs. As a source of neural stem and progenitor cells, cultured subventricular zone (SVZ) neurospheres exposed to medium obtained from ischemic astrocytes cocultured with BMSCs were significantly enriched in cells expressing the astrocytic marker glial fibrillary acidic protein (GFAP), but not at the expense of beta-III-tubulin-positive SVZ neuroblasts. The expression of BMP2/4 subsequently increased the phosphorylation of downstream effector Smad1 and the expression of notch signal pathway-induced protein Hes1 in cultured SVZ neurospheres. BMP antagonist Noggin blocked the elevation of phosphorylated Smad1 and the expression of Hes1 as well as reducing the percentage of astrocytic SVZ progenitor cells. Our results indicate that BMSCs increase BMP2/4 expression in ischemic astrocytes. These changes enhance subventricular progenitor cell gliogenesis by activating relevant signaling pathways. BMSC-stimulated signaling of endogenous astrocytes may alter the ischemic environment, promoting remodeling of brain and hence, improve functional recovery after stroke.     

Transfection of Noggin in bone marrow stromal cells (BMSCs) enhances BMSC-induced functional outcome after stroke in rats

Early intervention with intravenous administration of bone marrow stromal cells (BMSCs) reduces infarction size and ameliorates functional deficits in rat ischemia models. Noggin, an inhibitor of bone morphogenetic protein (BMP), has been demonstrated to provide protection from ischemic disease. In the present work, we hypothesize that administering Noggin-transfected BMSCs enhances BMSC-induced brain repair after cerebral ischemia. We compared the effects of BMSCs alone and Noggin-transfected BMSCs (Noggin-BMSCs) systematically delivered into the middle cerebral artery occlusion (MCAo) rat model. Noggin expression in BMSCs was achieved using adenoviral infection together with a green fluorescent protein (GFP) vector to monitor transduction efficiency and facilitate posttransplantation tracking. BMSCs and Noggin-BMSCs were intravenously injected into the rats 6 hr after MCAo. At 7 days after MCAo, the GFP-expressing BMSCs and Noggin-BMSCs were found primarily in the ischemic penumbra, which indicated that the intravenously delivered cells survived and reached in the lesion site. Both BMSC and Noggin-BMSC treatment significantly promoted neurogenesis in the ipsilateral subventrical zone (SVZ), reduced infarct volume, and led to functional improvement compared with the control group. Moreover, these beneficial effects were significantly greater in the Noggin-BMSC-treated group compared with BMSCs alone treatment (P < 0.05). Noggin expression in the ischemic hemisphere was significantly increased in the Noggin-BMSC-treated group as revealed by enzyme-linked immunosorbent assay (ELISA) at 7 days after MCAo compared with BMSC-treated and control groups (P < 0.05). These results indicate that transfection of Noggin in BMSCs enhances BMSC-induced neuroprotective effects when administered intravenously during the acute phase after stroke.     

神经生长因子、Noggin基因转染对骨髓间充质干细胞分化的影响

目的 研究外源性神经生长因子(NGF]、Noggin转染骨髓间充质干细胞(BMSCs)的可行性,并观察基因修饰的细胞向神经元方向的分化情况.方法 采用贴壁法分离纯化BMSCs,通过细胞表面标志及成脂诱导鉴定细胞.Ad-GFP-NGF、Ad-GFP-Noggin单独及联合转染BMSCs.通过Westernblot、免疫细胞化学观察目的 蛋白表达.通过免疫组化观察转染后BMSCs向神经元方向分化情况.结果贴壁法获得的细胞具有BMSCs表型,能分化为脂肪细胞.未转染组和Ad-GFP转染组少量表达NGF,不表达Noggin.NGF、Noggin单独及联合转染BMSCs均能高效表达目的 蛋白.NGF、Noggin转染的BMSCs可分化为具有神经元形态,并表达神经丝蛋白(NF-H)的细胞,联合转染组NF-H阳性细胞比例最高.结论 贴壁法能有效纯化BMSCs,Ad-GFP-NGF、Ad-GFP-Noggin单独及联合转染BMSCs安全并能高效表达目的 蛋白,NGF、Noggin转染的BMSCs 体外培养能向神经元样细胞方向分化,两种蛋白联合修饰能增强这种分化作用.     

神经生长因子、Noggin基因转染对骨髓间充质干细胞分化的影响

目的 研究外源性神经生长因子(NGF]、Noggin转染骨髓间充质干细胞(BMSCs)的可行性,并观察基因修饰的细胞向神经元方向的分化情况.方法 采用贴壁法分离纯化BMSCs,通过细胞表面标志及成脂诱导鉴定细胞.Ad-GFP-NGF、Ad-GFP-Noggin单独及联合转染BMSCs.通过Westernblot、免疫细胞化学观察目的 蛋白表达.通过免疫组化观察转染后BMSCs向神经元方向分化情况.结果贴壁法获得的细胞具有BMSCs表型,能分化为脂肪细胞.未转染组和Ad-GFP转染组少量表达NGF,不表达Noggin.NGF、Noggin单独及联合转染BMSCs均能高效表达目的 蛋白.NGF、Noggin转染的BMSCs可分化为具有神经元形态,并表达神经丝蛋白(NF-H)的细胞,联合转染组NF-H阳性细胞比例最高.结论 贴壁法能有效纯化BMSCs,Ad-GFP-NGF、Ad-GFP-Noggin单独及联合转染BMSCs安全并能高效表达目的 蛋白,NGF、Noggin转染的BMSCs 体外培养能向神经元样细胞方向分化,两种蛋白联合修饰能增强这种分化作用.     

神经生长因子、Noggin基因转染对骨髓间充质干细胞分化的影响

目的 研究外源性神经生长因子(NGF]、Noggin转染骨髓间充质干细胞(BMSCs)的可行性,并观察基因修饰的细胞向神经元方向的分化情况.方法 采用贴壁法分离纯化BMSCs,通过细胞表面标志及成脂诱导鉴定细胞.Ad-GFP-NGF、Ad-GFP-Noggin单独及联合转染BMSCs.通过Westernblot、免疫细胞化学观察目的 蛋白表达.通过免疫组化观察转染后BMSCs向神经元方向分化情况.结果贴壁法获得的细胞具有BMSCs表型,能分化为脂肪细胞.未转染组和Ad-GFP转染组少量表达NGF,不表达Noggin.NGF、Noggin单独及联合转染BMSCs均能高效表达目的 蛋白.NGF、Noggin转染的BMSCs可分化为具有神经元形态,并表达神经丝蛋白(NF-H)的细胞,联合转染组NF-H阳性细胞比例最高.结论 贴壁法能有效纯化BMSCs,Ad-GFP-NGF、Ad-GFP-Noggin单独及联合转染BMSCs安全并能高效表达目的 蛋白,NGF、Noggin转染的BMSCs 体外培养能向神经元样细胞方向分化,两种蛋白联合修饰能增强这种分化作用.     

Chemokine, vascular and therapeutic effects of combination Simvastatin and BMSC treatment of stroke

We investigated the additive therapeutic effect of the combination treatment of stroke with sub-therapeutic doses of Simvastatin, a HMG-CoA reductase inhibitor, and bone marrow stromal cells (BMSCs). Rats were administered Simvastatin (0.5 mg/kg), BMSCs (1 × 10⁶) or combination of Simvastatin and BMSCs starting at 24 h after stroke. Combination treatment significantly improved neurological outcome, enhanced angiogenesis and arteriogenesis, and increased the number of engrafted-BMSCs in the ischemic brain. The number of engrafted-BMSCs and arteriogenesis was significantly correlated with functional outcome. Simvastatin significantly increased stromal cell-derived factor-1 (SDF1) expression in the ischemic brain and chemokine (CXC motif) receptor-4 (CXCR4) in BMSCs, and increased BMSC migration to RBMECs and astrocytes. Combination treatment of stroke upregulates the SDF1/CXCR4 axis and enhances BMSC migration into the ischemic brain, amplifies arteriogenesis and angiogenesis, and improves functional outcome after stroke.     

Update on Therapeutic Mechanism for Bone Marrow Stromal Cells in Ischemic Stroke

Cerebral ischemia is a major cause of morbidity and mortality in the aged population, as well as a tremendous burden on the healthcare system. Despite timely treatment with thrombolysis and percutaneous intravascular interventions, many patients are often left with irreversible neurological deficits. Bone marrow stromal cells (BMSCs), also referred to as mesenchymal stem cells (MSCs), are a type of nonhematopoietic stem cells which exists in bone marrow mesh, with the potential to self-renew. Unlike cells in the central nervous system, BMSCs differentiate not only into mesodermal cells, but also endodermal and ectodermal cells. Moreover, it has been reported that BMSCs develop into cells with neural and vascular markers and play a role in recovery from ischemic stroke. These findings have fuelled excitement in regenerative medicine for neurological diseases, especially for ischemic stroke. There is now preclinical evidence to suggest that BMSCs grafted into the brain of ischemic models abrogate neurological deficits. Based on the overwhelming evidence from animal studies as well as in clinical trials, BMSC transplantation is considered a promising strategy for treatment of ischemic stroke. The goal of this review is to present an integrated consideration of molecular mechanisms in a chronological fashion and discuss an optimal BMSC delivery route for ischemic stroke.     

人β-NGF及BDNF基因共转染对大鼠骨髓基质细胞分化的影响

目的 探讨人β-神经生长因子(β-NGF)和脑源性神经营养因子(BDNF)基因共转染对大鼠骨髓基质细胞(BMSCs)分化的影响. 方法 梯度离心法分离培养大鼠胫股骨骨髓中BMSCs,用Src癌基因同源区3抗体(SH3)的免疫细胞化学染色鉴定BMSCs.按照3μL脂质体/1μg(3 μL)pSVCEP NGF/BDNF-CAT质粒的比例共转染第一代BMSCs,并转染pEGFP-C1质粒作为转染的标记.BMSCs培养4周后,荧光显微镜观察增强型绿色荧光蛋白(EGFP)的表达并计算转染率,细胞免疫荧光染色后用激光扫描共聚焦显微镜观察表达产物以及BMSCs分化情况. 结果 体外培养能够获得纯化的原代和传代BMSCs,传代后仍然保持增殖和分化功能;免疫组化SH3染色阳性证实为纯化的BMSCs.人β-NGF/BDNF基因共转染BMSCs后,BMSCs能够稳定和持续表达NGF和BDNF;BMSCs也能表达Nestin、NSE、NF-M和GFAP. 结论 经脂质体介导的外源性目的 基因NGF/BDNF cDNA均能分别和共同在BMSCs中成功表达,基因转染后的BMSCs能诱导分化为神经前体细胞或神经样细胞.     

Intraventricular nerve growth factor infusion improves cerebral blood flow and stimulates doublecortin expression in two infants with hypoxic-ischemic brain injury

OBJECTIVE AND IMPORTANCE: Hypoxic-ischemic brain injuries in childhood are associated with poor neurological outcome. Recently, experimental and clinical works show that nerve growth factor (NGF) reduces neurological deficits and promotes endothelial cells proliferation and angiogenesis following hypoxic-ischemic brain injuries. After brain stroke, new neurons express a protein, called doublecortin (DCX), which indicates a new marker for neurogenesis and migrating neuroblasts. This study investigates the effects of intraventricular NGF administration in two infants with severe hypoxic-ischemic brain damage and its role in both the cerebral perfusion and neurogenesis. CLINICAL PRESENTATION: Two infants, aged 8 and 13 months, with hypoxic-ischemic brain damage, secondary to prolonged cardiorespiratory arrest, were treated with intraventricular NGF administration. Before the therapy, both infants were comatose, aphasic and showed flaccid tetraparesis. After 1 month NGF treatment, their neurological conditions improved, electro-encephalography (EEG) examinations showed increased alpha/theta ratio, and single photon emission computed tomography (SPECT) works demonstrated a better cerebral perfusion. The DCX expression in the cerebrospinal fluid (CSF) increased concomitantly with increasing levels of NGF. INTERVENTION: The drug utilized was 2.5S NGF purified and lyophilized from male mouse submaxillary glands. The NGF administration was started 4 months after ischemic brain injury. NGF (0.1 mg) was administered via the external drainage catheter into the right cerebral ventricle once a day for 10 consecutive days. CONCLUSION: Our study shows that the intraventricular NGF administration improves the cerebral perfusion and stimulates the pathway of neurogenesis differentiation with the activation of DCX biosynthesis.     

Down-regulation of neurocan expression in reactive astrocytes promotes axonal regeneration and facilitates the neurorestorative effects of bone marrow stromal cells in the ischemic rat brain

The glial scar, a primarily astrocytic structure bordering the infarct tissue inhibits axonal regeneration after stroke. Neurocan, an axonal extension inhibitory molecule, is up-regulated in the scar region after stroke. Bone marrow stromal cells (BMSCs) reduce the thickness of glial scar wall and facilitate axonal remodeling in the ischemic boundary zone. To further clarify the role of BMSCs in axonal regeneration and its underlying mechanism, the current study focused on the effect of BMSCs on neurocan expression in the ischemic brain. Thirty-one adult male Wistar rats were subjected to 2 h of middle cerebral artery occlusion followed by an injection of 3 x 10(6) rat BMSCs (n = 16) or phosphate-buffered saline (n = 15) into the tail vein 24 h later. Animals were sacrificed at 8 days after stroke. Immunostaining analysis showed that reactive astrocytes were the primary source of neurocan, and BMSC-treated animals had significantly lower neurocan and higher growth associated protein 43 expression in the penumbral region compared with control rats, which was confirmed by Western blot analysis of the brain tissue. To further investigate the effects of BMSCs on astrocyte neurocan expression, single reactive astrocytes were collected from the ischemic boundary zone using laser capture microdissection. Neurocan gene expression was significantly down-regulated in rats receiving BMSC transplantation (n = 4/group). Primary cultured astrocytes showed similar alterations; BMSC coculture during reoxygenation abolished the up-regulation of neurocan gene in astrocytes undergoing oxygen-glucose deprivation (n = 3/group). Our data suggest that BMSCs promote axonal regeneration by reducing neurocan expression in peri-infarct astrocytes.     

Growth-associated protein 43 and neural cell adhesion molecule expression following bone marrow-derived mesenchymal stem cell transplantation in a rat model of ischemic brain injury

BACKGROUND:Transplantation of bone marrow-derived mesenchymal stem cells(BMSCs)improves motor functional recovery,but the mechanisms remain unclear.OBJECTIVE: To investigate expression of growth-associated protein 43(GAP-43)and neural cell adhesion molecule following BMSC transplantation to the lateral ventricle in rats with acute focal cerebral ischemic brain damage.DESIGN,TIME AND SETTING: A randomized,controlled,animal experiment using immunohistochemistry was performed at the laboratories of Department of Neurology,Renmin Hospital of Wuhan University and Doctoral Scientific Research Work Station of C-BONS PHARMA,Hubei Province,China,from January 2007 to December 2008.MATERIALS: Monoclonal mouse anti-rat 5-bromo-2-deoxyuridine and neural cell adhesion molecule antibodies were purchased from Sigma,USA;monoclonal mouse anti-rat GAP-43 antibody was purchased from Wuhan Boster,China.METHODS: Rat models of right middle cerebral artery occlusion were established using the thread method.At 1 day after middle cerebral artery occlusion,20 μL culture solution,containing 5 × 105 BMSCs,was transplanted to the left lateral ventricle using micro-injection.MAIN OUTCOME MEASURES: Scores of neurological impairment were measured to assess neural function.Expression of GAP-43 and neural cell adhesion molecule at the lesion areas was examined by immunohistechemistry.RESULTS: GAP-43 and neural cell adhesion molecule expression was low in brain tissues of the sham-operated group,but expression increased at the ischemic boundary(P < 0.05).Transplantation of BMSCs further enhanced expression of GAP-43 and neural cell adhesion molecule(P < 0.05)and remarkably improved neurological impairment of ischemic rats(P< 0.05).CONCLUSION: BMSC transplantation promoted neurological recovery in rats by upregulating expression of GAP-43 and neural cell adhesion molecule.     

小分子干扰RNA介导RhoA沉默优化骨髓间充质干细胞的培养

以往骨髓间充质干细胞的培养方法存在衰老和分化率低等问题。 目的：检测是否可以通过沉默RhoA基因的方法优化骨髓间充质干细胞培养。 方法：体外培养SD大鼠骨髓间充质干细胞,经小分子干扰RNA转染以沉默RhoA基因表达,分为3组培养：干细胞组(未转染小分子干扰RNA)、经随机打乱顺序的小分子干扰RNA 转染干细胞组、经小分子干扰RNA 转染的干细胞组。用RT-PCR,Western blot检测骨髓间充质干细胞在转染前后RhoA基因和蛋白的表达。应用细胞生长曲线、MTT比色法观察细胞生长的优化作用,采用流式细胞术测定细胞周期分布的变化。 结果与结论：与干细胞组、经随机打乱顺序的小分子干扰RNA 转染干细胞组比较,经小分子干扰RNA 转染的干细胞组RhoA基因和蛋白表达量明显降低(P < 0.05),细胞的生长速度明显增快,细胞周期G0/G1期减少,S期细胞数增多(P < 0.05)。说明通过沉默RhoA基因的方法可以促进骨髓间充质干细胞增殖,优化培养方法。     

骨髓基质细胞移植缺血性脑梗死大鼠后神经营养因子的表达

背景：骨髓基质细胞在适宜条件下可分化为神经元和星形胶质细胞,分泌可溶性分子促进神经元存活。 目的：观察骨髓基质细胞移植后缺血性脑梗死大鼠脑组织神经营养因子表达情况及其对神经功能的影响。 方法：改良的Longa栓线法制作大鼠大脑中动脉缺血模型,1 h后再灌注,24 h后治疗组尾静脉注射3×106骨髓基质细胞,盐水对照组尾静脉注射1 mL生理盐水,空白对照组不进行注射。 结果与结论：在梗死后第7,14天治疗组的神经损伤评分明显低于对照组(P < 0.05);治疗组神经生长因子和脑源性神经营养因子表达在各时间点均高于对照组(P < 0.05)。结果显示静脉移植骨髓基质细胞可改善缺血性脑梗死大鼠神经功能,移植骨髓基质细胞后缺血脑组织中神经生长因子及脑源性神经营养因子表达促进了神经功能的恢复。     

骨髓基质干细胞移植对缺血性卒中大鼠PSD-95表达的影响

目的 应用骨髓基质干细胞（BMSCs）治疗缺血性卒中大鼠,观察BMSCs的治疗效果,检测突触后密度蛋白-95（PSD-95）的表达水平,进而研究BMSCs治疗缺血性卒中的机制.方法 将40只成年雌性SD大鼠制备成大脑中动脉缺血2h再灌注24h动物模型,随机分为梗死对照组和BMSCs组,每组20只.每组再按梗死后3,7d分为2个亚组,每组10只.梗死对照组于缺血再灌注24 h后经尾静脉注射PBS液1ml,BMSCs组同时经尾静脉注射BMSCs 3×106.所有大鼠于梗死后1,3,7d分别进行神经功能评分,应用免疫组化法测定PSD-95表达水平,用TUNEL测定凋亡细胞水平.结果 （1）神经功能评分：梗死后3,7 d BMSCs组神经功能评分明显低于梗死对照组,差异有统计学意义（t分别为2.138,3.417;P＜0.05）.（2） PSD-95表达：BMSCs组在梗死后3d时PSD-95表达较梗死对照组的表达有增多,但差异无统计学意义;BMSCs组在梗死后7d时PSD-95表达明显多于梗死对照组,且差异有统计学意义（t=6.013,P＜0.05）.（3）TUNEL细胞凋亡染色：梗死后3d时梗死对照组大鼠缺血侧可见许多凋亡细胞,显多于BMSCs组,且差异有统计学意义（t=4.978,P＜ 0.05）.结论 BMSCs移植能促进缺血性卒中大鼠的神经功能的恢复.BMSCs移植后能明显增加缺血性卒中大鼠PSD-95的表达,减少细胞的凋亡,对缺血性卒中有保护作用.     

Noggin improves ischemic brain tissue repair and promotes alternative activation of microglia in mice

We previously reported that bone morphogenetic proteins (BMPs) and their endogenous antagonist noggin are expressed in the brain weeks after an ischemic insult. Here, to define their roles in ischemic brain tissue repair and remodeling, we infused recombinant BMP7 or noggin into the ipsilateral ventricle of mice for 2 weeks starting 2 weeks after transient middle cerebral artery occlusion (MCAO). Four weeks after MCAO, we measured ischemic brain volume, functional recovery, and molecules related to neurogenesis and angiogenesis such as synaptophysin, GAP-43, and VEGF. Noggin-treated mice but not BMP7-treated mice showed preserved ipsilateral brain volume and reduced neurological deficits compared with artificial cerebrospinal fluids (aCSF)-treated mice. Noggin treatment also decreased glial scar thickness, increased levels of GAP-43 and VEGF protein, and increased the number of Iba1-positive activated microglia in the ipsilateral brain. Furthermore, noggin treatment decreased M1 markers (IL-1β, TNF-α, IL-12, CCL2 and CD86) and increased M2 markers (IL-1ra, IL-10, arginase 1, CD206 and Ym1) of activated microglia, suggesting a shift from M1 to M2 phenotypes. These results suggest that noggin improves functional recovery from ischemic stroke and enhances alternatively activated microglia, thereby promoting tissue repair and remodeling.     

Transplantation of bone marrow stromal cells for peripheral nerve repair

Cell transplantation using bone marrow stromal cells (BMSCs) to alleviate neurological deficits has recently become the focus of research in regenerative medicine. Evidence suggests that secretion of various growth-promoting substances likely plays an important role in functional recovery against neurological diseases. In an attempt to identify a possible mechanism underlying the regenerative potential of BMSCs, this study investigated the production and possible contribution of neurotrophic factors by transected sciatic nerve defect in a rat model with a 15 mm gap. Cultured BMSCs became morphologically homogeneous with fibroblast-like shape after ex vivo expansion. We provided several pieces of evidence for the beneficial effects of implanted fibroblast-like BMSCs on sciatic nerve regeneration. When compared to silicone tube control animals, this treatment led to (i) improved walking behavior as measured by footprint analysis, (ii) reduced loss of gastrocnemius muscle weight and EMG magnitude, and (iii) greater number of regenerating axons within the tube. Cultured fibroblast-like BMSCs constitutively expressed trophic factors and supporting substances, including nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), ciliary neurotrophic factor (CNTF), collagen, fibronectin, and laminin. The progression of the regenerative process after BMSC implantation was accompanied by elevated expression of neurotrophic factors at both early and later phases. These results taken together, in addition to documented Schwann cell-like differentiation, provide evidence indicating the strong association of neurotrophic factor production and the regenerative potential of implanted BMSCs.     

绿色荧光蛋白标记兔骨髓间充质干细胞的成骨及成脂潜能

背景：为便于进一步追踪骨髓间充质干细胞,常需对其进行标记。 目的：采用绿色荧光蛋白标记兔骨髓间充质干细胞,观察标记后其诱导成骨及成脂能力。 设计、时间及地点：细胞观察实验,于2007-12/2008-10在华中科技大学同济医学院附属同济医院骨科实验室进行。 材料：4月龄健康新西兰大耳白兔6只,购自华中科技大学同济医学院动物实验中心。 方法：无菌条件下取兔双侧股骨,应用密度梯度离心法及贴壁法分离纯化骨髓间充质干细胞。取传至第3代细胞,用脂质体介导法将绿色荧光蛋白质粒转入,经G418筛选得到稳定转染的细胞,分别进行成骨、成脂诱导。 主要观察指标：骨髓间充质干细胞的形态、表面标志表达、绿色荧光蛋白标记情况,骨髓间充质干细胞成骨、成脂分化潜能。 结果：经原代及传代培养的骨髓间充质干细胞多呈纺锤形或梭形,成纤维细胞样,流式细胞仪分析骨髓间充质干细胞CD44呈阳性表达,CD34呈阴性。经G418筛选后,镜下可见大量发出绿色荧光的骨髓间充质干细胞。骨髓间充质干细胞成骨诱导21 d后有较多的钙盐沉积,茜素红染色呈红色;成脂诱导3 d后,细胞内有小脂滴出现,2 周后油红O染色示有大量脂质沉积。 结论：绿色荧光蛋白标记的兔骨髓间充质干细胞经诱导培养后具有多向分化潜能。     

Bone marrow stromal cells as a therapeutic treatment for ischemic stroke

Cerebral ischemia remains the most frequent cause of death and quality-of-life impairments due to neurological deficits, and accounts for the majority of total healthcare costs. However, treatments for cerebral ischemia are limited. Over the last decade, bone marrow stromal cell (BMSC) therapy has emerged as a particularly appealing option, as it is possible to help patients even when initiated days or even weeks after the ischemic insult. BMSCs are a class of multipotent, self-renewing cells that give rise to differentiated progeny when implanted into appropriate tissues. Therapeutic effects of BMSC treatment for ischemic stroke, including sensory and motor recovery, have been reported in pre-clinical studies and clinical trials. In this article, we review the recent progress in BMSC-based therapy for ischemic stroke, focusing on the route of delivery and pre-processing of BMSCs. Selecting an optimal delivery route is of particular importance. The ideal approach, as well as the least risky, for translational applications still requires further identification. Appropriate preprocessing of BMSCs or combination therapy has the benefit of achieving the maximum possible restoration. Further pre-clinical studies are required to determine the time-window for transplantation and the appropriate dosage of cells.     

Hes1 Knockdown Exacerbates Ischemic Stroke Following tMCAO by Increasing ER Stress-Dependent Apoptosis via the PERK/ eIF2a/ATF4/CHOP Signaling Pathway

Apoptosis induced by endoplasmic reticulum(ER)stress plays a crucial role in mediating brain damage after ischemic stroke.Recently,Hes1(hairy and enhancer of split 1)has been implicated in the regulation of ER stress,but whether it plays a functional role after ischemic stroke and the underlying mechanism remain unclear.In this study,using a mouse model of ischemic stroke via transient middle cerebral artery occlusion(tMCAO),we found that Hes1 was induced following brain injury,and that siRNA-mediated knockdown of Hes1 increased the cerebral infarction and worsened the neurological outcome,suggesting that Hes1 knockdown exacerbates ischemic stroke.In addition,mechanistically,Hes1 knockdown promoted apoptosis and activated the PERK/eIF2a/ATF4/CHOP signaling pathway after tMCAO.These results suggest that Hes1 knockdown promotes ER stress-induced apoptosis.Furthermore,inhibition of PERK with the specific inhibitor GSK2606414 markedly attenuated the Hes1 knockdown-induced apoptosis and the increased cerebral infarction as well as the worsened neurological outcome following tMCAO,implying that the protection of Hes1 against ischemic stroke is associated with the amelioration of ER stress via modulating the PERK/eIF2a/ATF4/CHOP signaling pathway.Taken together,these results unveil the detrimental role of Hes1 knockdown after ischemic stroke and further relate it to the regulation of ER stress-induced apoptosis,thus highlighting the importance of targeting ER stress in the treatment of ischemic stroke.     

Neurotrophic and growth factor gene expression profiling of mouse bone marrow stromal cells induced by ischemic brain extracts

Treatment of rodents after stroke with bone marrow stromal cells (BMSCs) improves functional outcome. However, the mechanisms underlying this benefit have not been ascertained. This study focused on the contribution of neurotrophic and growth factors produced by BMSCs to therapeutic benefit. Rats were subjected to middle cerebral artery occlusion and the ischemic brain extract supernatant was collected to prepare the conditioned medium. The counterpart normal brain extract from non‐ischemic rats was employed as the experimental control. Using microarray assay, we measured the changes of the neurotrophin associated gene expression profile in BMSCs cultured in different media. Furthermore, real‐time RT‐PCR and fluorescent immunocytochemistry were utilized to validate the gene changes. The morphology of BMSCs, cultured in the ischemic brain‐conditioned medium for 12 h, was dramatically altered from a polygonal and flat appearance to a fibroblast‐like long and thin cell appearance, compared to those in the normal brain‐conditioned medium and the serum replacement medium. Forty‐four neurotrophin‐associated genes in BMSCs were identified by microarray assay under all three culture media. Twelve out of the 44 genes (7 neurotrophic and growth factor genes, 5 receptor genes) increased in BMSCs cultured in the ischemic brain‐conditioned medium compared to the normal brain‐conditioned medium. Real time RT‐PCR and immunocytochemistry validated that the ischemic brain‐conditioned medium significantly increased 6/7 neurotrophic and growth factor genes, compared with the normal brain‐conditioned medium. These six genes consisted of fibroblast growth factor 2, insulin‐like growth factor 1, vascular endothelial growth factor A, nerve growth factor beta, brain‐derived neurotrophic factor and epidermal growth factor. Our results indicate that transplanted BMSCs may work as ‘small molecular factories’ by secreting neurotrophins, growth factors and other supportive substances after stroke, which may produce therapeutic benefits in the ischemic brain.