脑挫裂伤致miR-9升高促进神经干细胞分化

目的：探讨颅脑损伤后miR-9表达的变化和对神经干细胞分化和增值的影响,为颅脑损伤后神经功能修复治疗提出新的思路。方法：通过RT-PCR技术检测miR-9在挫裂伤脑组织中的表达情况;培养胚胎来源神经干细胞,并通过免疫荧光鉴定神经干细胞及其分化;转染miR-9后,通过MTT测定神经干细胞的增殖情况,和流式细胞仪检测分化神经元所占比例。结果：miR-9在挫裂伤脑组织中表达显著上升。对神经干细胞过表达miR-9可显著促进细胞增殖,并诱导分化成神经元。结论：脑挫裂伤时miR-9显著升高,并具有着促进神经干细胞增值和诱导分化的作用,可为伤后神经功能修复提供新的治疗方法。     

骨髓基质干细胞诱导分化为神经元过程中miR-124和miR-128的表达

目的探讨骨髓基质干细胞诱导分化为神经元过程中miR-124和miR-128的表达变化及作用。方法采用全骨髓培养法体外分离培养获得骨髓基质干细胞,取传代培养至第3代的骨髓基质干细胞,在神经干细胞培养液及细胞因子等条件下诱导其分化为神经元,倒置显微镜下观察其形态变化,应用ABI公司的TaqManMicroRNAAssaysreal-timePCR技术,检测miR-124和miR-128在诱导分化过程中的表达。结果 miR-124分化后神经元的表达是未分化BMSCs的0.051倍(P0.05);miR-128分化后神经元的表达是未分化BMSCs的0.070倍(P0.05)。结论 miR-124和miR-128在骨髓基质干细胞诱导分化为神经元过程中可能起重要作用。     

Reconstitution of Telomerase Activity Extends the Proliferative Life-span and Maintains the Neurogenetic Potential of Mesenchymal Stem Cells Derived from Human Fetal Muscle

目的 :通过重建端粒酶活性延长胎儿肌肉源间充质干细胞寿命 ,并对其成神经潜能进行研究 ,为组织工程神经修复提供种子细胞。方法 :将人端粒酶催化亚基 (hTERT)基因通过脂质体转染法导入胎儿肌肉源间充质干细胞 ,RT PCR检测hTERTmRNA的表达 ,TRAP PCR检测细胞端粒酶活性。用bFGF诱导已重建端粒酶活性的肌肉源间充质干细胞向神经细胞分化 ,免疫荧光及免疫印迹法检测分化情况。结果 :转染hTERT的胎儿肌肉源间充质干细胞能稳定表达端粒酶活性。转染后传 75代的细胞经bFGF诱导仍维持着自我更新及向神经细胞分化的潜能 ,且无恶性转化倾向。结论 :重建端粒酶活性可延长胎儿肌肉源间充质干细胞寿命并维持自我更新及成神经潜能 ,为建立组织工程标准细胞系提供了新的实验手段     

厄贝沙坦对脑损伤大鼠神经细胞凋亡的影响

目的:研究血管紧张素I受体(AT1)抑制剂厄贝沙坦对侧位液压脑损伤模型大鼠神经细胞凋亡的影响。方法:利用改良的侧位液压损伤装置建立大鼠颅脑损伤(TBI)模型,术前及术后给予厄贝沙坦治疗,用激光多普勒测定局部脑区血流(r CBF)的变化,术前及术后1、3、5和7d利用神经功能评分评估大鼠神经功能损伤,利用TUNEL染色检测大鼠脑细胞凋亡情况,利用Western Blot检测大鼠脑组织损伤周围区域活性caspase 3的表达。结果:与正常值相比,TBI手术后损伤局部脑区r CBF下降至30%(P0.05),神经功能评分显著降低(P0.05),损伤区周围脑组织TUNEL阳性细胞明显增多,活性caspase 3的表达显著增加(P0.05)。厄贝沙坦治疗组大鼠r CBF显著高于单纯TBI组,梗死区面积显著缩小,神经功能得到明显改善,损伤区周围脑组织TUNEL阳性细胞和活性caspase 3表达下降(P均0.05)。结论:厄贝沙坦预处理能够通过抑制凋亡发挥神经保护作用。     

MicroRNA-9-1促进大鼠表皮干细胞分化为神经细胞

目的:探讨MicroRNA-9-1在体外诱导大鼠表皮干细胞向神经细胞分化过程中的作用。方法:构建大鼠MicroRNA-9-1慢病毒载体,并感染SD大鼠的表皮干细胞;实验分为感染组、未感染组和阴性对照组;采用β-巯基乙醇诱导感染后的SD大鼠表皮干细胞分化为神经细胞。倒置荧光显微镜下观察表皮干细胞感染后GFP荧光表达的情况;免疫细胞化学染色检测神经微管结合蛋白2(MAP-2)的表达水平;RT-PCR检测MAP-2mRNA的表达水平。结果:阳性克隆PCR检测证明大鼠MicroRNA-9-1慢病毒载体构建成功;感染后48 h,在倒置显微镜下观察到,感染组GFP荧光表达达到峰值,感染效率达到了(85.6±1.9)%;β-巯基乙醇诱导7 h时,感染组大部分表皮干细胞向神经细胞分化,且诱导效果显著好于未感染组和阴性对照组;MAP-2在蛋白((87.3±0.6)%)和mRNA水平(约2倍)的表达率显著高于其他各组(P0.05)。结论:MicroRNA-9-1慢病毒载体可以高效感染大鼠表皮干细胞,且可以促进表皮干细胞在β-巯基乙醇的诱导下向神经细胞分化。     

MiR-615在平行培养的神经干细胞与运动神经元的表达比较

目的探讨MiR-615在脊髓源性神经干细胞与运动神经元间的表达特征。方法通过免疫磁珠法分离纯化胚胎大鼠脊髓运动神经元;通过神经克隆球形成技术分离纯化胚胎大鼠脊髓源性神经干细胞。采用TaqMan miR-615Assay定量检测培养的脊髓源性神经干细胞与运动神经元中miR-615的表达差异。结果通过平行培养技术分别获得了纯化的脊髓源性神经干细胞与运动神经元。定量检测结果显示,miR-615在分离的运动神经元中较在脊髓源性神经干细胞中显著高表达。结论本文提示miR-615可能在神经干细胞定向分化为运动神经元过程中发挥重要的调节作用。     

三维神经干细胞分化模型在神经药物检测中的应用

将神经干细胞接种在透明质酸支架进行三维(3D)培养,使用传统平面(2D)培养做对比,经诱导培养基进行1,7,14 d诱导分化。采用细胞免疫组织化学和Real-time PCR技术检测神经干细胞特异性标记物巢蛋白(nestin)、神经元微管蛋白(tubulin)及胶质细胞胶原纤维酸性蛋白(glial fi brillary acidic protein,GFAP)在蛋白水平和m RNA水平上的变化;CCK-8和活细胞染色技术检测神经细胞的增殖能力及神经细胞膜损伤修复效果。结果显示,神经干细胞在3D和2D培养条件下经诱导培养基诱导14 d后,tubulin表达量明显增加,而GFAP表达量降低,3D效果更加明显。CCK-8和活细胞染色结果显示,干细胞在3D培养条件下较2D培养条件下其分化和分化后的神经细胞膜损伤修复效果显著。三维培养模型能够对神经细胞分化后的药物损伤模型起到更好的保护作用。因此认为,3D透明质酸–神经细胞分化模型是更适合于构建体外神经药物筛选及安全性检测的优势模型。     

骨髓间充质干细胞体外趋化神经前体细胞的机制

骨髓间充质干细胞(BMSC)和神经前体细胞(NPC)移植于脑组织损伤动物的实验证明这两类细胞移植后均能在体内迁徙,与周围细胞整合,促进神经功能修复。BMSC促进神经功能修复的机制之一被认为与其分泌一些细胞因子和趋化因子有关,但具体机制不十分明确。为从基质细胞衍生因子-1α(SDF-1α)及其唯一的受体CXCR4这对分子相互作用的机制上探讨BMSC移植的可能治疗作用,实验采用ELISA法检测了体外培养的BMSC上清液中SDF-1α的含量,体外微孔隔离室迁移实验发现NPC能在BMSC分泌的培养上清液中SDF-1α的作用下发生定向迁移,特异性抗CXCR4单抗能有效阻断NPC的定向迁移效应,证实了BMSC分泌的SDF-1α促进表达CXCR4的NPC向病灶处迁移可能是促进神经功能修复的机制之一,从而为干细胞移植治疗神经功能缺损提供了一个新的思路。     

内源性神经干细胞的应用及其基础研究

目前对于神经系统损伤所致的神经元丢失尚无有效的治疗方法,然而研究表明成年哺乳动物的内源性神经干细胞能产生新的神经元,这些神经干细胞迁移至损伤区域,分化为成熟神经元,填补和部分修复脑组织。近年来研究者们对内源性神经干细胞治疗潜在价值的探讨日益增多,尤其是有关缺血性脑损伤、帕金森病、阿尔兹海默病、创伤性脑外伤的治疗进展。本文就内源性神经干细胞的治疗潜在价值基础研究进展做一综述。     

过表达miR-155抑制BMP9诱导间充质干细胞C3H10T1/2成骨分化

目的:研究过表达miR-155对BMP9诱导间充质干细胞C3H10T1/2成骨分化的影响。方法:(1)用重组腺病毒Ad-BMP9(BMP9)诱导C3H10T1/2细胞成骨分化,定量PCR(qPCR)检测miR-155的表达,RT-PCR检测Runx2和ALP的表达。(2)miR-155和BMP9共同处理C3H10T1/2细胞,qPCR检测miR-155的表达,ALP活性和染色检测早期成骨能力。(3)miR-155和BMP9共同处理C3H10T1/2细胞,诱导分化14d茜素红S染色检测晚期成骨能力。(4)miR-155和BMP9共同处理C3H10T1/2细胞,qPCR检测成骨分化相关基因Runx2、OSX、COL1A1、ALP、OCN和OPN的表达。(5)miR-155和BMP9共同处理C3H10T1/2细胞,Western blot检测p-Smad1/5/8、OCN和OPN蛋白水平的表达。(6)qPCR和Western blot分别检测HIF1α和VEGF的mRNA表达水平和蛋白质表达水平。(7)应用荧光素酶报告基因对miR-155的靶基因进行筛选和验证。结果:在BMP9诱导C3H10T1/2细胞成骨分化过程中,过表达miR-155降低ALP活性及染色;减少钙盐沉积;成骨分化相关基因Runx2、OSX、COL1A1、ALP、OCN和OPN表达降低;抑制p-Smad1/5/8、OCN和OPN蛋白水平的表达;HIF1α和VEGF的mRNA和蛋白表达水平减少。在对靶基因的检测中,过表达miR-155可以抑制HIF1α蛋白水平的表达,但对其mRNA水平无明显影响。结论:miR-155过表达减弱BMP9诱导间充质干细胞C3H10T1/2成骨分化,可能是通过抑制Smad/BMP信号通路发挥作用,也有可能是通过抑制靶基因HIF1α的表达来发挥作用。     

Altered expression levels of miRNAs in serum as sensitive biomarkers for early diagnosis of traumatic injury

MicroRNAs (miRNAs) are small non-coding RNAs of approximately 22 nucleotides in length which regulate gene expression negatively and play important roles in many pathological processes. It has been demonstrated that circulating miRNAs hold promise to serve as practicable molecular markers for diverse physiological and pathological conditions. In this investigation, we chose partial hepatectomy (PH) as traumatic injury model. There were significantly differential expression of miRNAs in rat serum post-traumatic injury (21 miRNAs were more than twofold up-regulated). Especially, the expression of miR-9 showed the highest up-regulated (>70-fold), and it possessed the characteristics of biomarker that was more sensitive than aspartate aminotransferase and alanine aminotransferase and C-reactive protein for traumatic liver injury. There was also a prominent increase in the expression levels of miR-9 in different brain areas after traumatic injury. Our data suggest that serum miR-9 may serve as promising biomarker for traumatic injury with high sensitivity. Furthermore, these findings may help to elucidate the complex network which mediates stress response to traumatic injury.     

NAAGS基因修饰神经干细胞在创伤性颅脑损伤治疗中的研究

目的：探讨移植NAAG合酶（NAAG synthetase,NAAGS）基因修饰的神经干细胞（Neural Stem Cells,NSCs）能否促进创伤性颅脑损伤大鼠神经功能的恢复。方法：利用电穿孔转染大鼠NSCs,通过脑立体定向仪分别将PBS（模型组）、NSCs（NSCs组）、转基因NSCs（NAAGS＋NSCs组）移植到创伤性颅脑损伤（Traumatic Brain Injury,TBI）大鼠局部损伤灶边缘,通过NSS评分评价移植后大鼠神经功能的变化以及用TUNEL法检测NSCs的凋亡情况,并采用放射免疫法分析脑组织中促炎因子水平。结果：Nss评分结果显示NAAGs＋NSCs组和NSCs组在第7、14、21天神经功能评分均低于模型组（P〈0．05）;NAAGS＋NSCs组在第14和21天神经功能评分低于NSCs组（P〈0．05）;在各时间点细胞移植组比模型组的神经细胞凋亡数明显减少;转基因NSCs移植能明显降低TBI脑组织中促炎因子水平。结论：转基因NSCs移植后可以合成NAAGS促进TBI大鼠神经功能的恢复。     

Simvastatin treatment promotes proliferation of human dental pulp stem cells via modulating PI3K/AKT/miR-9/KLF5 signalling pathway

Simvastatin serves as an effective therapeutic potential in the treatment of dental disease via alternating proliferation of dental pulp stem cells. First, western-blot and real-time quantitative PCR were used to detect the effect of simvastatin or LY294002 on the expression levels of AKT, miR-9 and KLF5, or determine the effect of miR-9. Simvastatin, KLF5 and AKT significantly enhanced the proliferation of pulp stem cells, whilst this effect induced by simvastatin was suppressed by LY294002, AKT siRNA, KLF5 siRNA and miR-9, and simvastatin dose-dependently upregulated the expression of PI3K. Furthermore, simvastatin upregulated PI3K and p-AKT expression in a concentration-dependent manner. LY294002 abrogated the upregulation of p-AKT expression levels induced by simvastatin, and LY294002 induced the miR-9 expression and simvastatin dose-dependently inhibited the expression of miR-9, by contrast, LY294002 reduced the KLF5 expression and simvastatin dose-dependently promoted the expression of KLF5. And using computational analysis, KLF5 was found to be a candidate target gene of miR-9, and which was further verified using luciferase assay. Finally, the level of KLF5 in cells was much lower following the transfection with miR-9 and KLF5 siRNA, and the level of AKT mRNA in cells was significantly inhibited after transfection with AKT siRNA than control. These findings suggested simvastatin could promote the proliferation of pulp stem cells, possibly by suppressing the expression of miR-9 via activating the PI3K/AKT signalling pathway, and the downregulation of miR-9 upregulated the expression of its target gene, KLF5, which is directly responsible for the enhanced proliferation of pulp stem cells.     

Novel Anti-Apoptotic MicroRNAs 582-5p and 363 Promote Human Glioblastoma Stem Cell Survival via Direct Inhibition of Caspase 3, Caspase 9, and Bim

Glioblastoma is the most common and lethal primary brain tumor. Tumor initiation and recurrence are likely caused by a sub-population of glioblastoma stem cells, which may derive from mutated neural stem and precursor cells. Since CD133 is a stem cell marker for both normal brain and glioblastoma, and to better understand glioblastoma formation and recurrence, we looked for dys-regulated microRNAs in human CD133+ glioblastoma stem cells as opposed to CD133+ neural stem cells isolated from normal human brain. Using FACS sorting of low-passage cell samples followed by microRNA microarray analysis, we found 43 microRNAs that were dys-regulated in common in three separate CD133+ human glioblastomas compared to CD133+ normal neural stem cells. Among these were several microRNAs not previously associated with cancer. We then verified the microRNAs dys-regulated in glioblastoma using quantitative real time PCR and Taqman analysis of the original samples, as well as human GBM stem cell and established cell lines and many human specimens. We show that two candidate oncogenic microRNAs, miR-363 and miR-582-5p, can positively influence glioblastoma survival, as shown by forced expression of the microRNAs and their inhibitors followed by cell number assay, Caspase 3/7 assay, Annexin V apoptosis/fluorescence activated cell sorting, siRNA rescue of microRNA inhibitor treatment, as well as 3′UTR mutagenesis to show luciferase reporter rescue of the most successful targets. miR-582-5p and miR-363 are shown to directly target Caspase 3, Caspase 9, and Bim.     

MicroRNA-146a inhibits glioma development by targeting Notch1

Dysregulated epidermal growth factor receptor (EGFR) signaling through either genomic amplification or dominant-active mutation (EGFR(vIII)), in combination with the dual inactivation of INK4A/ARF and PTEN, is a leading cause of gliomagenesis. Our global expression analysis for microRNAs revealed that EGFR activation induces miR-146a expression, which is further potentiated by inactivation of PTEN. Unexpectedly, overexpression of miR-146a attenuates the proliferation, migration, and tumorigenic potential of Ink4a/Arf(-/-) Pten(-/-) Egfr(vIII) murine astrocytes. Its ectopic expression also inhibits the glioma development of a human glioblastoma cell line in an orthotopic xenograft model. Such an inhibitory function of miR-146a on gliomas is largely through downregulation of Notch1, which plays a key role in neural stem cell maintenance and is a direct target of miR-146a. Accordingly, miR-146a modulates neural stem cell proliferation and differentiation and reduces the formation and migration of glioma stem-like cells. Conversely, knockdown of miR-146a by microRNA sponge upregulates Notch1 and promotes tumorigenesis of malignant astrocytes. These findings indicate that, in response to oncogenic cues, miR-146a is induced as a negative-feedback mechanism to restrict tumor growth by repressing Notch1. Our results provide novel insights into the signaling pathways that link neural stem cells to gliomagenesis and may lead to new strategies for treating brain tumors.     

miR-124 promotes neural differentiation in mouse bulge stem cells by repressing Ptbp1 and Sox9

Hair follicle stem cells (HFSCs) are able to differentiate into neurons and glial cells. Distinct microRNAs (miRNAs) regulate the proliferation and differentiation of HFSCs. However, the exact role of miR-124 in the neural differentiation of HFSCs has not been elucidated. HFSCs were isolated from mouse whisker follicles. miR-9, let-7b, and miR-124, Ptbp1 , and Sox9 expression levels were detected by real-time polymerase chain reaction (RT-PCR). The influence of miR-124 transfection was evaluated using immunostaining. We demonstrated that miR-124 and let-7b expression levels were significantly increased after the neural differentiation. Sox9 and Ptbp1 were identified as the target of miR-124 in the HFSCs. During neural differentiation and miR-124 mimicking, Ptbp1 and Sox9 levels were decreased. Moreover, the miR-124 overexpression increased MAP2 (58.43 ± 11.26) and NeuN (48.34 ± 11.15) proteins expression. The results demonstrated that miR-124 may promote the differentiation of HFSCs into neuronal cells by targeting Sox9 and Ptbp1.     

Hydrogen exerts neuroprotection by activation of the miR-21/PI3K/AKT/GSK-3β pathway in an in vitro model of traumatic brain injury

Few studies have explored the effect of hydrogen on neuronal apoptosis or impaired nerve regeneration after traumatic brain injury, and the mechanisms involved in these processes are unclear. In this study, we explored neuroprotection of hydrogen-rich medium through activation of the miR-21/PI3K/AKT/GSK-3β pathway in an in vitro model of traumatic brain injury. Such model adopted PC12 cells with manual scratching. Then, injured cells were cultured in hydrogen-rich medium for 48 hours. Expression of miR-21, p-PI3K, p-Akt, p-GSK-3β, Bax and Bcl-2 was measured using RT-qPCR, Western blot analysis and immunofluorescence staining. Rate of apoptosis was determined using TUNEL staining. Neuronal regeneration was assessed using immunofluorescence staining. The results showed that hydrogen-rich medium improved neurite regeneration and inhibited apoptosis in the injured cells. Scratch injury was accompanied by up-regulation of miR-21, p-PI3K, p-Akt and p-GSK-3β. A miR-21 antagomir inhibited the expression of these four molecules, while a PI3K blocker only affected the three proteins and not miR-21. Both the miR-21 antagomir and PI3K blocker reversed the protective effect of hydrogen. In conclusion, hydrogen exerted a neuroprotective effect against neuronal apoptosis and impaired nerve regeneration through activation of miR-21/PI3K/AKT/GSK-3β signalling in this in vitro model of traumatic brain injury.     

Lentivirus-mediated microRNA-26a-modified neural stem cells improve brain injury in rats with cerebral palsy

This study is launched to investigate the effect of lentivirus-mediated microRNA-26a (miR-26a)-modified neural stem cells (NSCs) in brain injury in rats with cerebral palsy (CP). The successfully constructed miR-26a lentivirus expression vector and empty vector virus were used to modify NSCs. The model of CP with ischemia and anoxia was established in rats. NSCs and miR-26a-NSCs were stereoscopically injected into the cerebral cortex of the modeled rats, respectively. The survival and migration of NSCs infected with recombinant lentivirus expressing green fluorescence in vivo was observed under a light microscope. The neurobehavioral functions, morphology, and ultrastructure of cerebral cortex and hippocampus, apoptosis of brain cells, expression of apoptosis-related protein caspase-3 and Bax, together with the expression of the glial fibrillary acidic protein (GFAP) in cerebral cortex and hippocampus were determined. Expression of miR-26a in NSCs infected with plVTHM-miR-26a increased significantly. After NSCs transplantation, the neurobehavioral status of CP rats was improved, the degree of brain pathological injury was alleviated, the apoptotic index of cells in cerebral cortex and hippocampus and the expression of the apoptotic protein (caspase-3 and Bax) were decreased, the expression of GFAP were significantly decreased. After miR-26a-NSCs transplantation, these aforementioned results further improved or decreased. Our study suggests that miR-26a-modified NSCs mediated by lentivirus can improve brain injury, inhibit apoptosis of brain cells and activation of astrocytes in CP rats.