骨髓间充质干细胞向胶质瘤迁移机制的实验研究

目的探讨血管内皮生长因子(VEGF)在大鼠骨髓间充质干细胞(BMSCs)向C6胶质瘤迁移中的作用。方法直接贴壁法分离培养骨髓间充质干细胞。建立体外迁移模型检测大鼠重组VEGF164诱导BMSCs迁移的效果。利用磷酸肌醇3-激酶(PI3K)特异性抑制剂LY294002,分析PI3K在VEGF164诱导BMSCs迁移过程中的作用。检测C6细胞诱导BMSCs迁移的效果,分析VEGF在这一过程中的作用。结果通过直接贴壁法获得了纯化的BMSCs。20 ng/ml VEGF164能诱导BMSCs发生定向迁移,这一迁移过程能被LY294002所抑制。在加入VEGF中和抗体后,向C6细胞发生迁移的BMSCs数量减少。结论 VEGF164可以诱导BMSCs发生定向迁移,PI3K参与了这一过程的信号转导。VEGF是诱导BMSCs向C6胶质瘤定向迁移的细胞因子之一。     

不同来源人羊膜间充质干细胞的混合培养

背景：人羊膜间充质干细胞可诱导分化为神经样细胞,但在培养过程中发现干细胞增殖的数量不足。 目的：为了获得足够数量的人羊膜间充质干细胞,观察人羊膜间充质干细胞混合培养的生长情况及经碱性成纤维细胞生长因子诱导人羊膜间充质干细胞向神经样细胞的分化情况。 方法：将两个不同胎盘来源的羊膜分离培养、混合,采用细胞分离和培养技术获取人羊膜间充质干细胞,分离人羊膜间充质干细胞后传代培养,加入碱性成纤维细胞生长因子诱导分化。当细胞传至第3代时,随机取培养的6份人羊膜间充质干细胞(半量)为A组,6份人羊膜间充质干细胞(半量)为B组,将A、B组各剩余的半量人羊膜间充质干细胞混合为C组。3组细胞浓度均为1.0×107 L-1。以活细胞计数和MTT比色法比较3组细胞扩增数量,以免疫组织化学法检测羊膜间充质干细胞神经胶质细胞标志物、神经元特异性烯醇化酶和巢蛋白的表达。 结果与结论：混合人羊膜间充质干细胞之间有互相促增殖的作用。人羊膜间充质干细胞具有较强的可朔性,经碱性成纤维细胞生长因子诱导的人羊膜间充质干细胞可表达神经胶质细胞标志物、神经元特异性烯醇化酶和巢蛋白。     

辛伐他汀对高糖高脂诱导人骨髓间充质干细胞凋亡的影响

背景：研究表明,他汀类药物能够促进骨髓间充质干细胞的增殖与黏附能力,抑制高糖高脂培养下骨髓间充质干细胞的凋亡。 目的：观察辛伐他汀对高糖高脂诱导条件下人骨髓间充质干细胞凋亡的影响。 方法：将0.001,0.01,0.1,1.0 μmol/L辛伐他汀分别与高糖高脂诱导条件下人骨髓间充质干细胞培养48 h,以正常培养骨髓间充质干细胞及高糖高脂诱导条件下培养的骨髓间充质干细胞为对照。倒置显微镜下观察细胞形态,MTT法比较不同浓度辛伐他汀对高糖高脂环境下骨髓间充质干细胞存活率的影响,应用流式细胞术检测细胞凋亡,加入PI3K/Akt信号转导通路抑制剂LY294002后辛伐他汀对骨髓间充质干细胞凋亡的影响。 结果与结论：与高糖高脂诱导组比较,辛伐他汀0.01,0.1,1.0 µmol/L组骨髓间充质干细胞存活率均升高(P < 0.01),其中辛伐他汀浓度在0.1 μmol/L时骨髓间充质干细胞存活率升高最显著(P < 0.01);同时流式细胞仪检测结果显示,辛伐他汀0.01,0.1,1.0 µmol/L组细胞凋亡率下降(P < 0.01),其中0.1 µmol/L组凋亡率下降最显著(P < 0.01)。0.1 µmol/L辛伐他汀对骨髓间充质干细胞的影响可被LY294002阻断。说明辛伐他汀能抑制高糖高脂诱导条件下骨髓间充质干细胞的凋亡,其机制可能与PI3K/Akt信号途径有关。     

体外诱导羊膜来源间充质干细胞分化为血管内皮细胞

背景：羊膜来源间充质干细胞植入机体不同类型组织后是否可以分化为相应组织靶细胞呢？ 目的：检测血管内皮细胞生长因子在体外诱导人羊膜间充质干细胞分化为血管内皮细胞的可行性。 方法：分离培养羊膜间充质干细胞,鉴定其表面抗原表达,用含体积分数2%胎牛血清以及50 μg/L血管内皮生长因子的条件培养基诱导,诱导后细胞通过内皮细胞标志物血管内皮生长因子受体2以及v-WF染色鉴定。 结果与结论：羊膜间充质干细胞表面抗原CD29、CD44、CD105阳性,CD34、CD45、CD106以及HLA-DR阴性。诱导后细胞形态明显改变,内皮细胞标志物血管内皮细胞生长因子受体2以及v-WF染色结果阳性。提示羊膜间充质干细胞在体外具有分化为血管内皮细胞的能力。     

体外诱导人骨髓间充质干细胞向肝样细胞分化：肝细胞生长因子和表皮细胞生长因子的作用*

背景：研究证实人骨髓间充质干细胞能够诱导分化为肝样细胞,但其具体生物学特性及分化机制尚不清楚,且诱导分化培养体系尚不成熟。 目的：探讨肝细胞生长因子和表皮细胞生长因子体外诱导人骨髓间充质干细胞向肝样细胞分化的可行性。 方法：取食管癌手术患者肋骨,采用密度梯度离心联合贴壁筛选法获取人骨髓间充质干细胞,流式细胞仪检测细胞表面分子的表达。取第3代人骨髓间充质干细胞,分为4组,肝细胞生长因子组加入20 μg/L肝细胞生长因子,表皮细胞生长因子组加入20 μg/L表皮细胞生长因子,联合组同时加入上述两种生长因子,空白对照组不加任何生长因子。倒置显微镜下观察细胞形态的变化,诱导7,14 d RT-PCR检测甲胎蛋白与白蛋白mRNA的表达。 结果与结论：人骨髓间质干细胞不表达造血细胞标志CD34,CD45,强表达β1-整合素CD29和基质受体CD44。肝细胞生长因子组、表皮细胞生长因子组、联合组诱导后,细胞形态由长梭形变为类圆形或多角形,诱导第7,14天甲胎蛋白、白蛋白 mRNA均呈阳性表达;空白对照组未见多角形细胞,甲胎蛋白、白蛋白 mRNA均呈阴性表达。证明肝细胞生长因子、表皮细胞生长因子以及二者联合均具有诱导人骨髓间充质干细胞向肝样细胞分化的能力,至于二者联合是否能增强其分化能力尚待进一步免疫细胞化学染色定量分析予以验证。     

表皮生长因子对非黏附骨髓间充质干细胞克隆形成效率的影响

背景：目前普遍认为骨髓间充质干细胞是一类高度黏附的成纤维样细胞,但也有研究显示处于非黏附状态的骨髓间质细胞也具有自我复制及多向分化潜能。 目的：探讨表皮生长因子对骨髓中存在的非黏附骨髓间充质干细胞产生成纤维细胞集落形成单位的影响。 设计、时间及地点：细胞学体外观察,于2007-07/12在南京医科大学骨与干细胞研究中心完成。 材料：4周龄雄性C57/BL6小鼠6只,由南京医科大学实验动物中心提供。表皮生长因子为美国Sigma公司产品。 方法：全骨髓法体外分离培养小鼠骨髓间充质干细胞,调整细胞浓度为2×109 L-1。收集第3代细胞,分别接种于向脂肪细胞、成骨细胞、软骨细胞分化的培养液中进行诱导。取30 mL骨髓间充质干细胞悬液,平均分到两管中,表皮生长因子处理组加入终浓度为10 μg/L表皮生长因子,空白对照组加入普通完全培养基,采用反复转移非黏附骨髓细胞培养法,每天将非黏附骨髓细胞转移到新的培养皿,原来的皿中为总骨髓来源的贴壁细胞,转移第1次定义PO1,第2次为PO2,依次类推,培养12 d终止。 主要观察指标：骨髓间充质干细胞的鉴定结果,亚甲基蓝染色显示每皿细胞克隆的形成情况,计算克隆形成区域占皿底面积的百分比及克隆数目。 结果：非黏附骨髓间充质细胞在第4天转移到新的培养皿后,继续培养仍能够贴壁生长,并逐渐扩增形成克隆;诱导后能够分化为脂肪细胞、成骨细胞和软骨细胞。给予表皮生长因子处理后,无论是总骨髓来源的贴壁细胞,还是反复转移的非黏附骨髓间充质干细胞,均能够不断产生成纤维细胞集落形成单位,且数量明显多于空白对照组(P < 0.05)。表皮生长因子处理组总骨髓来源的贴壁细胞、PO1~PO5非黏附骨髓间充质干细胞所形成的贴壁细胞数分别是空白对照组的1.54倍,4.25倍,2.51倍,1.56倍,1.25倍,2.01倍;所产生的克隆数分别是空白对照组的1.2倍,1.8倍,1.4倍,1.4倍,1.1倍。 结论：表皮生长因子能有效促进骨髓中的非黏附骨髓间充质干细胞在悬浮状态下增殖,明显提高了骨髓间充质干细胞的富集效率。     

表皮生长因子干预小鼠非黏附骨髓间充质干细胞成纤维细胞集落形成及向神经元样细胞的分化**

背景：非黏附骨髓间充质干细胞在体外可以不断形成成纤维细胞集落形成单位，并且可诱导分化为脂肪细胞、成骨细胞和软骨细胞，表现出一定的多分化潜能。 目的：探讨表皮生长因子对非黏附骨髓间充质干细胞形成成纤维细胞集落的影响，及其在体外向神经细胞分化的能力。 方法：分离小鼠双侧股骨、胫骨，全骨髓法分离总骨髓细胞，采用反复转移非黏附的骨髓细胞培养法纯化非黏附骨髓间充质干细胞。取第5代总骨髓细胞和非黏附骨髓间充质干细胞，加入含表皮生长因子、碱性成纤维细胞生长因子的神经细胞诱导液培养2周。观察非黏附骨髓细胞产生成纤维细胞集落形成单位的能力，表皮生长因子对成纤维细胞集落形成单位的影响，甲苯胺蓝和免疫细胞化学染色鉴定相关蛋白的表达。 结果与结论：总骨髓细胞、反复转移的非黏附骨髓间充质干细胞均能够不断产生成纤维细胞集落形成单位。给予表皮生长因子处理后，非黏附骨髓间充质干细胞成纤维细胞集落形成单位的效率明显增高。诱导2周后，免疫细胞化学染色显示，总骨髓细胞和非黏附骨髓间充质干细胞均表达神经细胞特异性蛋白NeuN和NF-200；经甲苯胺蓝染色在部分细胞中可见神经元特异性标记尼氏体。证实表皮生长因子可有效促进小鼠非黏附骨髓间充质干细胞形成成纤维细胞集落形成单位的效率，经反复转移的非黏附骨髓间充质干细胞能够诱导分化为神经细胞。     

体外羊膜间充质干细胞分离培养及向神经元样细胞的分化

背景：胎盘组织作为寻找人类间充质干细胞的新来源,已成为备受人们关注的研究热点。从胎盘组织中羊膜层分离、培养细胞,因其来源广泛、不受伦理限制等优越性而具有广阔的应用前景。 目的：观察人羊膜间充质干细胞体外分离培养的方法,及向神经元样细胞分化的能力。 方法：用酶消化法从羊膜中分离和培养间充质干细胞,并通过形态的均一性及流式细胞术检测其表面标志以鉴定纯度。应用DMEM-LG+20 g/L DMSO+100 µmol/L BHA+10 μg/L碱性成纤维细胞生长因子诱导分化,通过免疫荧光染色鉴定诱导后的细胞。 结果与结论：可从羊膜组织成功分离培养出间充质干细胞,细胞贴壁生长,在体外短期内可以稳定增殖和传代。具有与骨髓间充质干细胞相似的表面标志,体外可以诱导分化为神经元样细胞,胶质纤维酸性蛋白与神经元特异性烯醇化酶免疫荧光染色均为阳性。提示羊膜间充质干细胞可以在体外分离培养并诱导分化为神经元样细胞,羊膜组织可以作为干细胞研究以及神经组织疾病细胞治疗的一个全新的细胞来源。     

干细胞生长因子和粒细胞集落刺激因子与心肌梗死大鼠体内骨髓间充质干细胞的迁移*

背景：外周静脉移植间充质干细胞只有1%~5%的移植细胞能归巢到心肌梗死区域。 目的：观察干细胞生长因子、粒细胞集落刺激因子对骨髓间充质干细胞归巢的影响。 方法：采用贴壁培养法分离培养SD大鼠骨髓间充质干细胞,取传至3~5代细胞。建立SD大鼠急性心肌梗死模型,干细胞生长因子组、粒细胞集落刺激因子组、干细胞生长因子+粒细胞集落刺激因子组在骨髓间充质干细胞移植前3 d和移植后3 d单独或混合皮下注射干细胞生长因子、粒细胞集落刺激因子,骨髓间充质干细胞组不注射细胞因子。 结果与结论：荧光显微镜下观察,骨髓间充质干细胞迁移至心肌梗死组织,骨髓间充质干细胞组、干细胞生长因子组、粒细胞集落刺激因子组迁移至心肌梗死区的骨髓间充质干细胞数量没有明显的区别(P > 0.05),干细胞生长因子+粒细胞集落刺激因子组的骨髓间充质干细胞数量明显高于其他3组(P < 0.05)。免疫荧光组织化学显示,植入的部分骨髓间充质干细胞表达心肌特异蛋白cTnI。结果说明干细胞生长因子和粒细胞集落刺激因子两种细胞因子联合应用可以促进骨髓间充质干细胞归巢至心肌梗死区域,在体内微环境的诱导下,骨髓间充质干细胞能够转化为心肌样细胞。     

体外培养人骨髓间充质干细胞向表皮细胞分化及表皮细胞角蛋白的表达

背景：动物实验已经证实骨髓间充质干细胞体外诱导可分化为表皮细胞。 目的：观察体外培养条件下人骨髓间充质干细胞向表皮细胞的分化及表皮细胞角蛋白表达。 方法：采用Ficoll-Paque密度梯度离心法提取人胚胎骨髓间充质干细胞,以免疫细胞化学及流式细胞仪测定细胞表面CD33、CD34标记物进行鉴定。取第3代骨髓间充质干细胞以30%条件培养基诱导其向表皮细胞分化,免疫细胞化学染色观察诱导后细胞形态与细胞角蛋白水平变化。 结果与结论：采用密度梯度离心法从人胚胎骨髓中分离培养得到细胞成分均一的骨髓间充质干细胞。骨髓间充质干细胞经体外诱导后,出现细胞角蛋白19表达阳性细胞,说明骨髓间充质干细胞在体外诱导后可能发生向表皮细胞分化。     

Responses and signaling pathways in human optic nerve head astrocytes exposed to hydrostatic pressure in vitro

In this study, we examined the effects of mechanical stress induced by elevated hydrostatic pressure (HP) on the migration of human optic nerve head (ONH) astrocytes, using an in vitro model that follows repopulation of a cell-free area (CFA) created on a monolayer of cultured astrocytes. alpha-Tubulin staining detected phenotypic changes in astrocytes exposed to HP. The influence of proliferation in closure of the CFA was determined by incorporation of BrdU under 1.5-cm H2O, control pressure (CP), and 10-cm H2O HP with or without 5-fluorouracil. Under control and experimental conditions, closure of the CFA occurred mostly by migration and less by proliferation. Exposure to 10-cm H2O HP induced faster closure of the CFA at 1, 3, and 5 days. The signaling pathways involved in responses to HP were determined using genistein, tyrphostin A25, AG1478, and AG1295, inhibitors of receptor tyrosine kinases; wortmannin and LY294002, inhibitors of phosphatidyl inositol 3-kinase (PI-3K); and SC58236, an inhibitor of inducible cyclooxygenase-2 (COX2). Genistein and tyrphostin A25 blocked HP-induced migration at 1, 3, and 5 days, but did not affect closure of the CFA under CP. AG1478 and AG1295 blocked HP-induced migration and partially inhibit closure of the CFA under CP. LY294002 blocked HP-induced migration. SC58236 markedly inhibited closure of the CFA under CP by inhibiting COX2 activity. Exposure to HP, a physical stress, induced faster closure of the CFA via activation of members of the epidermal growth factor receptor (EGFR) family and PI-3K pathways. Under CP, closure of the CFA in response to denudation, a form of injury, is due to activation of COX2 in ONH astrocytes.     

重组人白细胞介素-2玻璃体注射慢性高眼压大鼠神经节细胞层神经元的凋亡

BACKGROUND: The majority of studies addressing neuroprotection have focused on drugs, proteins and cytokines. A recent study reports that recombinant human interleukin-2 (rhIL-2) achieves good therapeutic effects, but the underlying mechanisms remain poorly understood. OBJECTIVE: To observe the neuroprotection of recombinant human interleukin-2 (rhIL-2) by intravitreous injection on the retinal ganglion cells (RGCs) following chronic elevated IOP model in Wistar rats. DESIGN, TIME AND SETTING: A randomized, controlled, animal experiment was performed at the Center Laboratory, Second Xiangya Hospital, Central South University, China, from July 2008 to March 2009. MATERIALS: LY294006 and AG490 were provided by Invitrogen, USA. Rabbit anti-AKT1 polyclonal antibody and rabbit anti-STAT3 polyclonal antibody were purchased from American Basic Gene Associate Bioscience, USA. METHODS: Chronic elevated IOP model of Wistar rats were established by cauterizing the limbal episcleral veins to block the reflux of aqueous humor by 532nm viridis-lite diode laser in right eyes. Intravitreous injection of 2µl PBS, rhIL-2 or rhIL-2 LY294002, AG490 mixed liquor for eyes were administered 1 week after chronic elevated IOP model established. Ocular tissue was immunostained to investigate ED-1 expressions. Rats were sacrificed after intravitreous injection 3 days, 14 days, 21 days to identify RGC, and the 3% fluorogold was used to the epithalamus 7 days before sacrifice. Western blot analysis Akt1 and STAT3 protein in the retina after intravitreous injection 3h,12h, 96h. MAIN OUTCOME MEASURES: ED-1 expression of activated microglia/macrophages was determined by immunofluorescence. The RGCs levels were detected using fluorogold staining. The efficacy of the intraocular rhIL-2 and rhIL-2, LY294002, AG490 mixed liquor injections in inhibiting PI3K/Akt、Jak/Stat3 signal transductions were confirmed by Western blot analysis. RESULTS: Numerous ED-1 positive macrophages were seen in the vitreous and along the inner retinal surface in rhIL-2 treatment group. The number of RGCs was much higher than that in control groups and PBS group (P<0.01). The numbers of RGCs in rhIL-2 and LY294002, AG490 mixed liquor group was as much lower than that in rhIL-2 group (P<0.01). Western blotting analysis showed very little Akt1 and STAT3 protein in the retina after 3h intravitreous injections of rhIL-2 in rats, started to increase rapidly at 12 reperfusion,96h decreased slightly. LY294002 and AG490 prevented the Akt1 and STAT3 protein expression in the retina. CONCLUTION: rhIL-2 has neuroprotective effection after intravitreous injection. It can decrease or avoid RGCs layer damage in chronic glaucoma model of rats. Intravitreal injection of rhIL-2 activates macrophages, which derived factors might enhance the RGCs survival. After intravitreal injection of rhIL-2 PI3K/Akt、Jak/Stat3 signal transductions may play an important role following a chronic elevated IOP model, and LY294002, AG490 can block this effect.     

Phosphoinositide 3-kinase promotes adult subventricular neuroblast migration after stroke

Rodent adult subventricular zone (SVZ)-derived progenitor cells abandon the rostral migratory stream (RMS)/olfactory complex postmiddle cerebral artery occlusion (MCAo) and migrate into compromised tissue, possibly playing a role in brain recovery. Using SVZ tissue explants from the adult rat, we investigated the role of the phosphoinositide 3-kinase (PI3K) signal transduction pathway in the migration of SVZ cells. Stroke significantly (P <.01) increased migratory speed (198 +/- 39 microm/day) of neuroblasts out of the SVZ explants compared with the speed (99 +/- 20 microm/day) in the normal SVZ (nSVZ) explants within the first 3 days of incubation. Three-dimensional laser scanning confocal microscopy revealed formation of neuroblast encompassing chain-like astrocyte structures extruding from both normal and stroke explants. Western blots showed that stroke SVZ (sSVZ) explants increased Akt phosphorylation. Treatment of sSVZ explants with the selective PI3K inhibitor LY294002 significantly (P <.01) attenuated neuroblast migration and Akt phosphorylation, whereas treatment with LY294002 did not affect the number of bromodeoxyuridine (BrdU)- and caspase-3-immunoreactive cells, indicating that stroke-enhanced neuroblast migration is independent of cell proliferation and survival. PI3K catalyzes phosphatidylinositol-3,4,5-triphosphate (PIP(3)) which facilitates Akt phosphorylation. Thus, our data demonstrate that the PI3K/Akt signal transduction pathway mediates neuroblast migration after stroke.     

Leptin and its intracellular signaling pathway maintains the neurosphere

Leptin is associated with the maintenance of epidermal growth factor (EGF)-reactive neural lineage cells, including the neural progenitors. One-day treatment with leptin (10, 100, or 1000 ng/ml) followed by EGF treatment increased the number of small-sized and mid-sized colonies compared with the nonleptin treatment. Leptin prevented the inactivation of the phosphatidylinositol 3-kinase (PI3 K) and extracellular signal regulated kinase (ERK) pathways in neurosphere cells cultured in the non-EGF medium. Bromodeoxyuridine (BrdU) incorporation into the neurosphere cells induced by leptin was suppressed by LY294002, a PI3 K inhibitor, but not by U0126, a MEK1/2 inhibitor, which activates ERK1/2, although U0126 decreased phosphorylated extracellular signal regulated kinase levels. These results suggest that leptin maintains the self-renewal ability and EGF reactivity of immature neural lineage cells and the signal is mediated, at least in part, by the PI3 K pathway.     

Nerve growth factor regulates human choroidal, but not retinal, endothelial cell migration and proliferation

We have previously shown that sympathetic denervation results in significant blood vessel growth of the choroid and retina. The mechanism of this growth remains unclear. Since sympathetic denervation can result in increased nerve growth factor (NGF) levels, it was the goal of this study to determine if choroidal and retinal endothelial cells in culture would respond to nerve growth factor and if nerve growth factor promote endothelial cell migration and proliferation, two components of angiogenesis. Western blotting with phospho-specific antibodies, cell migration, and cell proliferation assays were employed to determine NGF effects on both choroidal and retinal cell growth. NGF treatment produced phosphorylation of TrkA in choroidal and retinal endothelial cells. NGF stimulation resulted in activation of ERK1/2, Akt, and Src in choroidal endothelial cells, while little phosphorylation was noted following NGF treatment in retinal endothelial cells. NGF increased choroidal endothelial cell migration by 50% over control and this was inhibited by pretreatment with LY294002 (PI3K inhibitor), Akt inhibitor, and MMP2/9 inhibitor. KT5823, PD98059, and PP2 did not affect choroidal cell migration. NGF also produced a 47% increase in choroidal endothelial cell proliferation, which was blocked by PP2, LY294002, Akt inhibitor, KT5823, and PD98059. NGF stimulation did not alter retinal endothelial cell migration or proliferation. Thus, it appears that increased NGF levels that may be noted after sympathectomy are capable of producing some aspects of vascular remodeling via different signaling cascades in choroidal endothelial cells in culture.     

EGF和AG1478对GL15细胞增殖、侵袭和GFAP表达的影响

目的探讨表皮生长因子受体(EGFR)信号通路对人胶质母细胞瘤GL15细胞系细胞生物学特性影响的分子机制。方法Gimsa染色分析GL15细胞核型,甲基四唑兰(MTT)和Transwell分别检测表皮生长因子(EGF),AG1478对GL15细胞增殖和侵袭力的影响,逆转录酶-多聚酶链反应(RT-PCR)和Western blot分别检测EGF和AG1478作用后GL15细胞胶质纤维酸性蛋白(GFAP)mRNA和蛋白表达。结果Gimsa染色表明EGFR所在的7号染色体过度扩增。外源性的EGF双向调节GL15细胞增生和生长,小剂量(50ng/ml,100ng/ml)EGF能促进细胞增生和提高细胞侵袭能力以及GFAPmRNA和蛋白表达,而大剂量(200ng/ml)则能降低其增殖和侵袭能力,AG1478则能明显拮抗EGF活性。结论GL15胶质母细胞瘤细胞系的生物学活性与EGFR所在的7号染色体过度扩增密切相关。EGF可双向调节GL15细胞系的生长,AG1478能不完全抑制EGFR的活性。EGFR是基因治疗胶质母细胞瘤的理想靶点。     

Factors affecting directional migration of bone marrow mesenchymal stem cells to the injured spinal cord

Microtubule-associated protein 1B plays an important role in axon guidance and neuronal migration. In the present study, we sought to discover the mechanisms underlying microtu- bule-associated protein 1B mediation of axon guidance and neuronal migration. We exposed bone marrow mesenchymal stem cells to okadaic acid or N-acetyl-D-erythro-sphingosine （an inhibitor and stimulator, respectively, of protein phosphatase 2A） for 24 hours. The expression of the phosphorylated form of type I microtubule-associated protein 1B in the cells was greater after exposure to okadaic acid and lower after N-acetyl-D-erythro-sphingosine. We then injected the bone marrow mesenchymal stem cells through the ear vein into rabbit models of spinal cord contusion. The migration of bone marrow mesenchymal stem cells towards the injured spinal cord was poorer in cells exposed to okadaic acid- and N-acetyl-D-erythro-sphingosine than in non-treated bone marrow mesenchymal stem cells. Finally, we blocked phosphatidylinosi- tol 3-kinase （PI3K） and extracellular signal-regulated kinase 1/2 （ERK1/2） pathways in rabbit bone marrow mesenchymal stem cells using the inhibitors LY294002 and U0126, respectively. LY294002 resulted in an elevated expression of phosphorylated type I microtubule-associated protein 1B, whereas U0126 caused a reduction in expression. The present data indicate that PI3K and ERKI/2 in bone marrow mesenchymal stem cells modulate the phosphorylation of micro- tubule-associated protein 1B via a cross-signaling network, and affect the migratory efficiency of bone marrow mesenchymal stem cells towards injured spinal cord.     

sEHi对颈动脉狭窄患者内皮祖细胞增殖及PI3K/Akt信号通路的影响

目的 探讨可溶性环氧化物水解酶抑制剂(sEHi)AUDA 调控颈动脉狭窄(CS)患者外周血内皮祖细胞(EPCs)增殖的分子机制。方法 从CS患者外周血分离、培养内皮祖细胞,收集培养至第7 d的细胞,分为未处理组、AUDA组、PI3K抑制剂(LY294002)组和AUDA+ LY294002组,取无颈动脉狭窄患者的EPCs作为对照组,MTT法检测EPCs的增殖能力,Western blot法检测EPCs Akt磷酸化的水平。结果 对照组EPCs增殖能力较未处理组增强,AUDA组较未处理组、AUDA+ LY294002组、LY294002组EPCs增殖能力增强,未处理组、AUDA+ LY294002组较LY294002组内皮祖细胞增殖能力增强; Western blot结果显示AUDA可以促进EPCs P-Akt蛋白的表达,而LY294002可以抑制上述作用。结论 AUDA可能通过活化PI3K /Akt信号通路来促进内皮祖细胞的增殖。     

Heparin-binding epidermal growth factor-like growth factor stimulates cell proliferation in cerebral cortical cultures through phosphatidylinositol 3'-kinase and mitogen-activated protein kinase

Heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF) stimulates cell proliferation in the adult mammalian brain, but the mechanism involved is unknown. To address this issue we treated mouse brain cerebral cortical cultures enriched in neuronal precursors with full-length HB-EGF, its HB or EGF-like domain alone, or both domains in combination. Labeling of cultures with bromodeoxyuridine (BrdU), a marker of cell proliferation, was increased approximately 10% by the HB domain and approximately 20% by the EGF-like domain, and the effects of the two domains were additive. Full-length HB-EGF was most effective (approximately 50% increase) in stimulating BrdU incorporation. Preincubation with heparinase III or with Na-chlorate abolished cell proliferation induced by HB-EGF, consistent with dependence on cell-surface heparan sulfate proteoglycans. The effect of HB-EGF was also blocked by the EGF receptor (EGFR/ErbB1) inhibitors PD153035 and PD158780, implicating EGFR in HB-EGF-induced cell proliferation. The phosphatidylinositol 3'-kinase (PI3K) inhibitors LY294002 and wortmannin, and the MAPK/extracellular signal-regulated kinase (ERK) kinase (MEK) inhibitors U0126 and PD98059, reduced HB-EGF-induced BrdU incorporation into cultures, and HB-EGF enhanced phosphorylation of Akt and ERK, implying a role for PI3K/Akt and MEK/ERK signaling in HB-EGF-stimulated cell proliferation. These findings help to clarify the molecular mechanisms through which HB-EGF operates.