Transplantation of placenta-derived mesenchymal stem cell-induced neural stem cells to treat spinal cord injury

Because of their strong proliferative capacity and multi-potency, placenta-derived mesenchymal stem cells have gained interest as a cell source in the field of nerve damage repair. In the present study, human placenta-derived mesenchymal stem ceils were induced to differentiate into neural stem cells, which were then transplanted into the spinal cord after local spinal cord injury in rats. The motor functional recovery and pathological changes in the injured spinal cord were observed for 3 successive weeks. The results showed that human placenta-derived mesenchymal stem cells can differentiate into neuron-like cells and that induced neural stem cells contribute to the restoration of injured spinal cord without causing transplant rejection. Thus, these cells promote the recovery of motor and sensory functions in a rat model of spinal cord injury. Therefore, human placenta-derived mesenchymal stem cells may be useful as seed cells during the repair of spinal cord injury.

Similarity on neural stem cells and brain tumor stem cells in transgenic brain tumor mouse models

Although it is believed that glioma is derived from brain tumor stem cells, the source and molecular signal pathways of these cells are still unclear. In this study, we used stable doxycycline-inducible transgenic mouse brain tumor models （c-myc/SV40Tag＋/Tet-on＋） to explore the malignant trans- formation potential of neural stem cells by observing the differences of neural stem cells and brain tumor stem cells in the tumor models. Results showed that chromosome instability occurred in brain tumor stem cells. The numbers of cytolysosomes and autophagosomes in brain tumor stem cells and induced neural stem cells were lower and the proliferative activity was obviously stronger than that in normal neural stem cells. Normal neural stem cells could differentiate into glial fibrillary acidic protein-positive and microtubule associated protein-2-positive cells, which were also negative for nestin. However, glial fibrillary acidic protein/nestin, microtubule associated protein-2/nestin, and glial fibrillary acidic protein/microtubule associated protein-2 double-positive cells were found in induced neural stem cells and brain tumor stem cells. Results indicate that induced neural stem cells are similar to brain tumor stem cells, and are possibly the source of brain tumor stem cells.

Are human dental papilla-derived stem cell and human brain-derived neural stem cell transplantations suitable for treatment of Parkinson’s disease?

Transplantation of neural stem cells has been reported as a possible approach for replacing impaired dopaminergic neurons. In this study, we tested the efficacy of early-stage human dental papilla-derived stem cells and human brain-derived neural stem cells in rat models of 6-hydroxydopamine-induced Parkinson＇s disease. Rats received a unilateral injection of 6-hydroxydopamine into right medial forebrain bundle, followed 3 weeks later by injections of PBS, early-stage human dental papilla-derived stem cells, or human brain-derived neural stem cells into the ipsilateral striatum. All of the rats in the human dental papilla-derived stem cell group died from tumor formation at around 2 weeks following cell transplantation. Postmortem examinations revealed homogeneous malignant tumors in the striatum of the human dental papilla-derived stem cell group. Stepping tests revealed that human brain-derived neural stem cell transplantation did not improve motor dysfunction. In apomorphine-induced rotation tests, neither the human brain-derived neural stem cell group nor the control groups （PBS injection） demonstrated significant changes. Glucose metabolism in the lesioned side of striatum was reduced by human brain-derived neural stem cell transplantation. [18F]-FP-CIT PET scans in the striatum did not demonstrate a significant increase in the human brain-derived neural stem cell group. Tyrosine hydroxylase （dopaminergic neuronal marker） staining and G protein-activated inward rectifier potassium channel 2 （A9 dopaminergic neuronal marker） were positive in the lesioned side of striatum in the human brain-derived neural stem cell group. The use of early-stage human dental papilla-derived stern cells confirmed its tendency to form tumors. Human brain-derived neural stem cells could be partially differentiated into dopaminergic neurons, but they did not secrete dopamine.

Overexpression of microRNA-124 promotes the neuronal differentiation of bone marrow-derived mesenchymal stem cells

microRNAs （miRNAs） play an important regulatory role in the self-renewal and differentiation of stem cells. In this study, we examined the effects of miRNA-124 （miR-124） overexpression in bone marrow-derived mesenchymal stem cells. In particular, we focused on the effect of overexpression on the differentiation of bone marrow-derived mesenchymal stem cells into neurons. First, we used GeneChip technology to analyze the expression of miRNAs in bone marrow-derived mesen- chymal stem cells, neural stem cells and neurons, miR-124 expression was substantially reduced in bone marrow-derived mesenchymal stem cells compared with the other cell types. We con- structed a lentiviral vector overexpressing miR-124 and transfected it into bone marrow-derived mesenchymal stem cells. Intracellular expression levels of the neuronal early markers [3-III tu- bulin and microtubule-associated protein-2 were significantly increased, and apoptosis induced by oxygen and glucose deprivation was reduced in transfected cells. After miR-124-transfected bone marrow-derived mesenchymal stem cells were transplanted into the injured rat spinal cord, a large number of cells positive for the neuronal marker neurofilament-200 were observed in the transplanted region. The Basso-Beattie-Bresnahan locomotion scores showed that the motor function of the hind limb of rats with spinal cord injury was substantially improved. These re- sults suggest that miR-124 plays an important role in the differentiation of bone marrow-derived mesenchymal stem cells into neurons. Our findings should facilitate the development of novel strategies for enhancing the therapeutic efficacy of bone marrow-derived mesenchymal stem cell transplantation for spinal cord injury.

In vivo tracking of neuronal-like cells by magnetic resonance in rabbit models of spinal cord injury

In vitro experiments have demonstrated that neuronal-like cells derived from bone marrow mesen- chymal stem cells can survive, migrate, integrate and help to restore the function and behaviors of spinal cord injury models, and that they may serve as a suitable approach to treating spinal cord injury. However, it is very difficult to track transplanted cells in vivo. In this study, we injected su- perparamagnetic iron oxide-labeled neuronal-like cells into the subarachnoid space in a rabbit model of spinal cord injury. At 7 days after cell transplantation, a small number of dot-shaped low signal intensity shadows were observed in the spinal cord injury region, and at 14 days, the number of these shadows increased on T2-weighted imaging. Perl＇s Prussian blue staining detected dot-shaped low signal intensity shadows in the spinal cord injury region, indicative of superpara- magnetic iron oxide nanoparticle-labeled cells. These findings suggest that transplanted neu- ronal-like cells derived from bone marrow mesenchymal stem cells can migrate to the spinal cord injury region and can be tracked by magnetic resonance in vivo. Magnetic resonance imaging represents an efficient noninvasive technique for visually tracking transplanted cells in vivo.

神经干细胞移植脑出血恢复期大鼠神经功能和促血管生成素1及受体的表达

背景:近年研究发现骨髓间充质干细胞经过长时间体外培养后,可自然分化为神经干细胞,从而再定向分化为神经细胞及神经胶质细胞,为帕金森病、脑梗死后遗症、小脑萎缩和脑发育不良等疾病的治疗提供了一种新的思路。目的:探讨神经干细胞移植对脑出血恢复期大鼠神经功能的影响及其作用机制。方法:将60只雄性SD大鼠按照随机数字表法分为正常组(18只)、脑出血组(21只)和移植组(21只),后2组大鼠采用Ⅶ型胶原酶诱导法建立大鼠脑出血模型,造模21 d后移植组大鼠通过尾静脉注射神经干细胞,脑出血组给予等量生理盐水。移植后第7,14,21天进行改良黏附物移除试验(MST)评分,评分结束后处死各组大鼠,采用RT-PCR法测定大鼠出血周围脑组织促血管生成素1 mR NA表达,采用Western Blotting法检测大鼠出血周围脑组织酪氨酸激酶受体2蛋白表达。结果与结论:与正常组相比,脑出血组和移植组各时点MST评分均明显降低,差异均有显著性意义(P<0.05);从移植后7 d开始移植组各时点的MST评分均明显高于脑出血组,差异均有显著性意义(P<0.05)。移植后7,14,21 d移植组和脑出血组促血管生成素1 m RNA和酪氨酸激酶受体2蛋白含量均明显高于正常组,且移植组升高更为明显,差异均有显著性意义(P均<0.05)。结果表明神经干细胞移植能够有效促进脑出血恢复期大鼠神经功能的恢复,其机制可能与增加出血周围脑组织促血管生成素及酪氨酸激酶受体2的含量有关。

骨髓基质细胞对中脑神经干细胞分化为神经元的诱导作用

目的:观察成年大鼠骨髓基质细胞诱导新生大鼠中脑神经干细胞分化为神经元的机制。方法:采用骨髓基质细胞和神经干细胞共培养方法,通过显微镜观察神经干细胞的分化状态;使用免疫组织化学技术,分析神经元在神经干细胞后代中所占的比例。结果:(1)骨髓基质细胞可诱导神经干细胞分化为高比例神经元;(2)骨髓基质细胞可促进神经元的存活。结论:骨髓基质细胞可提供神经干细胞分化为神经元和促进神经元存活的信号物质。

腺苷预处理浓度对局灶性脑缺血再灌注损伤大鼠内源性神经干细胞增殖的影响

目的探讨不同浓度腺苷预处理对脑缺血再灌注损伤大鼠内源性神经干细胞增殖的影响。方法成年雄性SD大鼠随机分为假手术组,模型对照组,0.9、1.2、1.5、1.8、2.1 mg.kg-1腺苷预处理组。用线拴法制备大鼠大脑中动脉闭塞模型。各腺苷预处理组于术前连续3 d分别腹腔注射0.9、1.2、1.5、1.8、2.1 mg.kg-1腺苷,假手术组及模型对照组在相同时间点腹腔注射等体积生理盐水。对各组大鼠进行神经功能缺失行为学评分,并观察其巢蛋白阳性细胞的表达。结果假手术组大鼠无神经功能缺损症状,偶可见巢蛋白阳性细胞;1.5、1.8、2.1 mg.kg-1腺苷预处理组与模型对照组比较,神经功能缺失症状得到明显改善,巢蛋白表达显著增加(P<0.01);1.5 mg.kg-1腺苷预处理组与1.8、2.1 mg.kg-1腺苷预处理组的神经功能评分及巢蛋白阳性表达比较,差异无统计学意义(P>0.05)。结论腺苷预处理浓度为1.5 mg.kg-1时,既能有效改善再灌注损伤引起的神经功能缺损症状,又可以很好地促进内源性神经干细胞的增殖。

切割穹窿海马伞海马提取液和银杏叶提取物联合诱导海马NSCs向胆碱能神经元的分化

为探讨切割穹窿海马伞海马提取液和银杏叶提取物(extract of ginkgo bilobo,EGb)在海马NSCs向胆碱能神经元定向分化中的作用,分别制备大鼠穹窿海马伞切割侧和正常侧海马提取液;将从鼠胚海马中分离扩增的NSCs球分成4组,应用不同的培养液促其分化:(1)联合组:含切割侧海马提取液和银杏内酯的DMEM/F12培养基;(2)提取液组:含切割侧海马提取液的DMEM/F12培养基;(3)EGb组:含银杏内酯的DMEM/F12培养基;(4)对照组:含正常侧海马提取液的DMEM/F12培养基。培养14d后行ChAT免疫荧光检测,计算ChAT阳性神经元的分化率,图像处理细胞面积和周长。结果显示联合组各项指标均明显优于其它各组(P<0.01);提取液组、EGb组各项指标也均优于对照组(P<0.05);细胞面积提取液组优于EGb组(P<0.05),细胞周长EGb组优于提取液组(P<0.05),两组ChAT阳性神经元分化率无明显差异(P>0.05)。上述提示切割穹窿海马伞的海马提取液和EGb联合应用可诱导海马NSCs分化为更多、更为成熟的胆碱能神经元。

中医药促进神经干细胞增殖与分化用于抗脑缺血损伤的研究进展

长期以来,运用传统中医药方法治疗脑缺血及其后遗症有着独特优势,在临床也取得了较好疗效,但具体作用机制和作用靶点还需进一步研究证明。神经干细胞可永久自我更新,具有多向分化潜能。中医药可通过多种途径促进神经干细胞的增殖与分化,从而有效治疗神经组织的缺血性损伤,为神经再生的治疗开辟了新方向,已成为抗脑缺血损伤研究的焦点。

GDNF和NO对局灶性脑缺血大鼠皮质和尾壳核神经干细胞增殖和分化的影响

目的观察胶质细胞源性神经营养因子(GDNF)对局灶性脑缺血大鼠皮质和尾壳核神经干细胞(NSCs)增殖分化以及nNOS阳性神经元分布的影响,探讨GDNF和NO对内源性NSCs增殖分化影响的作用机制。方法制作右侧局灶性脑缺血模型,左侧脑室注射GDNF,5-溴脱氧尿嘧啶(BrdU)标记DNA合成期(S期)细胞,免疫组化观察正常组、假手术组、脑缺血组、生理盐水组和GDNF组大鼠局灶性脑缺血90min后再灌注时间分别为3d、7d、14d、21d、28d的BrdU/nNOS、BrdU/NeuN、BrdU/GFAP双标阳性细胞。结果GDNF组较脑缺血组和生理盐水组行为学恢复明显;脑缺血后皮质和尾壳核BrdU和nNOS阳性细胞增多,BrdU/nNOS(38．23%±6．87%)、Br- dU/NeuN(52．38%±13．29%)、BrdU/GFAP(13．51%±8．78%)双标细胞阳性率明显增加,与其它组比较均有显著性差异(P＜0．05)。结论局灶性脑缺血激活内源性NSCs,GDNF可调节NO释放,促进NSCs增殖、分化。

大鼠胚胎脊髓源性神经干细胞的培养和分化

目的 探讨大鼠脊髓源性胚胎神经干细胞体外分离培养增殖及分化，为进一步的实验研究提供基础。方法 取孕14～16d的SD大鼠胚胎脊髓在条件培养基中培养增殖传代分化，用神经干细胞特异性抗体-巢蛋白（Nestin）鉴定克隆细胞，用特异性的单克隆抗体鉴定克隆球诱导分化后的细胞。用Brdu免疫荧光对神经干细胞传代能力检测。结果 获得了大量能自我增殖并分化的神经干细胞，分化后主要产生神经元特异性微管相关蛋白染色阳性的神经元质纤维酸性蛋白阳性的星型胶质细胞和少突胶质细胞三种神经组织细胞。Brdu免疫荧光显示神经球中绝大多数细胞为Brdu阳性细胞，其细胞核中可见荧光标记物。结论 大鼠胚胎脊髓能分离、培养出神经干细胞，并能诱导分化为神经元、星型胶质细胞和少突胶细胞。

神经再生素促进神经干细胞的生长和分化

目的：研究神经再生素对体外培养神经干细胞分化的促进作用及对其生长相关蛋白（GAP43）、神经丝蛋白（NF-H）表达的影响。方法：取出生3～5d的新生SD大鼠大脑皮层进行神经干细胞的体外培养、鉴定，神经干细胞与0、1、2mg／L的神经再生素（NRF）共培养8d，相差显微镜观察分析，应用Real-timePCR对与不同浓度的NRF（0、1、2、4、8rag／L）共培养8d的神经干细胞进行GAP43、NF-H的表达量检测。结果：成功培养出具有多向分化潜能的神经干细胞；神经再生素可明显促进神经干细胞的分化，并能在一定范围内随浓度递增而有效促进GAP43、NF-H的表达，且最佳作用浓度为4mg／L。结论：神经再生素可以促进神经干细胞的生长和分化。

氟西汀对慢性温和应激抑郁模型大鼠海马神经干细胞的影响

目的观察氟西汀对慢性温和应激抑郁模型大鼠海马神经干细胞的作用。方法 24只雄性大鼠分为对照组、模型组和氟西汀组,每组8只。采用慢性中等应激制作大鼠抑郁模型,敞箱实验法测定大鼠行为学变化,免疫组织化学技术检测巢蛋白(Nestin)表达以评价大鼠神经干细胞增殖情况。结果模型组大鼠的行为学得分较对照组明显降低,海马齿状回Nestin表达减少,与模型组比较,氟西汀治疗后大鼠的行为学得分升高,Nestin表达增加,差异均有统计学意义(P<0.05)。结论氟西汀能翻转慢性应激对大鼠行为的损害,这可能与其促进神经干细胞增殖有关。

SOX2及Nestin在Ⅰ型糖尿病大鼠不同时期正中隆起细胞的表达

目的:检测转录因子SOX2及巢蛋白(nestin)在Ⅰ型糖尿病大鼠不同时期正中隆起(median eminence,ME)细胞内的表达及其意义。方法:通过腹腔注射链脲佐菌素的方法,建立Ⅰ型糖尿病大鼠模型。模型组分为4、8、12周三组,每组10只,共30只;4周正常大鼠作为对照组。用免疫组织化学法检测SOX2及nestin在不同时期的Ⅰ型糖尿病大鼠及对照组大鼠的正中隆起细胞内的表达情况。结果:SOX2主要表达于正中隆起神经元的胞体,nestin主要表达于正中隆起神经元的胞体和突起。SOX2和nestin的表达均随着Ⅰ型糖尿病的时程而逐渐增加;在8周和12周大鼠SOX2在正中隆起的阳性表达百分比分别是73.84±3.29%、69.56±4.44%,与对照组(40.12±0.80%)比较具有显著性差异(P<0.05);而nestin在Ⅰ型糖尿病4、8、12周大鼠正中隆起的阳性表达百分比分别是86.42%±3.38%、81.39%±4.54%、66.30%±3.20%,与对照组(30.21%±1.72%)比较具有显著性差异(P<0.05),且其nestin阳性表达细胞的胞突及分支比对照组多而长。结论:SOX2及nestin在Ⅰ型糖尿病大鼠正中隆起细胞内表达增强,Ⅰ型糖尿病大鼠的血糖升高可能刺激大鼠正中隆起神经干细胞/祖细胞的增殖。

神经干细胞与雪旺细胞共移植后对阿尔茨海默病大鼠行为学及脑组织形态学的影响

目的探讨神经干细胞与雪旺细胞共移植后对阿尔茨海默病(AD)大鼠行为学及脑组织形态学的影响。方法建立AD大鼠模型,分别将神经干细胞、神经干细胞与雪旺细胞以及等量的生理盐水注入AD模型大鼠脑内。1个月后,与正常组一起进行morris水迷宫实验观察实验大鼠学习记忆能力的改变。35d后,处死实验鼠,通过HE染色观察各组实验鼠脑组织形态学改变。结果细胞移植后AD大鼠的学习记忆能力明显改善,与生理盐水对照组相比差异显著(P<0.01),具有统计学意义。共移植组表现最为明显,与正常组相比无明显差异(P>0.05)。与生理盐水对照组相比神经干细胞移植后海马与额叶受损细胞的恢复明显减轻,以共移植组的改变尤为明显,其形态学表现接近于正常组。结论雪旺细胞与神经干细胞共移植后AD大鼠的学习记忆能力增强,脑组织病理改变减轻,学习记忆能力明显改善,对AD大鼠的治疗作用优于神经干细胞单独移植组。

体外诱导人骨髓间充质干细胞分化为多巴胺能神经元

骨髓间充质干细胞(BMSCs)是存在于骨髓中的非造血干细胞,在体外一定条件下可向神经细胞分化。本实验通过密度梯度离心获取人骨髓中的单个核细胞,贴壁培养纯化BMSCs,并用脑源性神经营养因子(BDNF)、forskolin(FSK)和多巴胺(DA)联合对BMSCs进行诱导,电子显微镜观察诱导后细胞是否具有成熟神经元的超微结构特点;免疫细胞化学和RT-PCR检测DA能神经元分化过程中的标志物酪氨酸羟化酶(TH)的表达以及转录因子Nurr1、Ptx3、和Lmx1b的表达。结果显示:诱导2周后,电镜下可见细胞浆中有大量密集的呈扁平囊状的粗面内质网及其间的一些游离核糖体,以及神经丝。RT-PCR结果显示NSE(neuron specific enolase)、Nurr1、Ptx3、Lmx1b和TH的mRNA均有表达;免疫细胞化学表明诱导2周后TH阳性细胞的表达较诱导3d后明显提高。上述结果表明BDNF、FSK和DA可以在体外诱导人BMSCs定向分化为DA能神经元。

针刺和神经干细胞联合治疗脑瘫幼鼠的效果评价

目的研究针刺和神经干细胞联合治疗对脑瘫幼鼠脑神经细胞形态数目及学习记忆能力和肢体功能的影响并探讨可能机制。方法取新生7日龄SD幼鼠制备缺氧缺血性脑损伤性大鼠(HIBD)脑瘫模型,32只SD幼鼠随机分成4组:正常对照组(正常组)、模型组和治疗组(包括单纯针刺组和针刺+NSC组),每组8只,模型成功后(术后)第3天开始治疗。针刺部位为百会,颞1针,曲池,内关,足三里,涌泉。神经干细胞(NSC)取自新生SD大鼠海马组织分离培养,采用尾静脉注射,治疗3周(术后24 d)后进行效果评价,包括悬调实验,斜坡实验,Y迷宫测试。最后取脑组织进行免疫组织化学检测。结果悬调实验,斜坡实验,Y迷宫测试,海马正常神经细胞数目及脑皮质的凋亡指数和海马组织切片检查中,联合移植组与模型组和单纯针刺组相比,差异均有统计学意义(P<0.05)。结论联合治疗组比单纯针刺组能更好的提高脑瘫幼鼠的学习记忆能力,更好的改善其肢体功能,在维持其脑神经细胞数目和抑制脑皮质细胞凋亡方面有更好的作用。

神经干细胞的分离培养及超微结构研究

目的研究神经干细胞分离培养及其超微结构特征。方法从胚胎大鼠的大脑皮质、室管膜下区等神经组织分离神经干细胞,用无血清培养技术在体外进行培养、扩增和传代。采用免疫荧光技术,鉴定神经干细胞和分化的神经细胞,同时进行电镜结构观察。结果从胚胎大鼠的大脑皮质、室管膜下区等组织获得了大量神经干细胞,可自我增殖并多向分化。神经球表面呈微绒毛结构,内层细胞结合较为松散,可见凋亡小体。部分细胞分化为具有突起和轴突样结构的细胞。结论分离培养的神经干细胞具有自我增殖和多向分化潜能,并具有与功能相对应的超微形态结构。

功能性电刺激促进急性脑梗死大鼠脑部内源性神经干细胞增殖的研究

目的研究功能性电刺激（FES）对急性脑梗死大鼠行为学和内源性神经干细胞（NSCs）增殖的影响。探讨FES治疗改善脑梗死后神经功能的机制。方法54只成年雄性SD大鼠按随机数字表法分为FES治疗组、安慰刺激组和假手术组（每组各18只）。行大脑中动脉阻断（MCAO）制作局灶性脑梗死模型后第3天，FES治疗组开始接受FES治疗（10min／d，每天1次），安慰刺激组阻断动脉但不予电刺激。在FES治疗后3、7、14d评价大鼠行为学功能（平衡木行走测评、转棒上行走测评、网屏试验1，免疫组织化学法观察大鼠海马齿状回和室管膜下区NSCs巢蛋白（nestin）的表达水平．Western blot法检测梗死侧脑组织nestin总蛋白表达量。结果FES治疗组网屏试验评分在治疗后14d时低于安慰刺激组，比较差异有统计学意义（P〈0．05）。FES治疗组大鼠海马齿状回和室管膜下区nestin阳性细胞数和梗死侧脑组织nestin总蛋白表达量在治疗后7d、14d时均高于安慰刺激组和假手术组，比较差异有统计学意义（P〈0．05）。结论FES能促进急性脑梗死大鼠脑部内源性NSCs的增殖并改善大鼠行为学功能，这可能是FES治疗改善脑梗死后神经功能的机制之一。