神经干细胞移植后在视神经损伤大鼠视网膜内表达BDNF的研究

目的探讨神经干细胞(NSC)移植入视神经损伤(ONI)的SD大鼠玻璃体下腔后在视网膜内表达脑源性神经营养因子(BDNF)的情况,为进一步探索NSC移植治疗视神经损伤提供实验依据。方法体外培养NSC,其上清液采用酶联免疫吸附实验(ELISA)定量分析BDNF含量。取34只SD大鼠,其中4只作为正常对照,另外30只制作成右眼ONI模型并随机等分为N组和P组。ONI术后随即向N组大鼠右眼玻璃体下腔注入定量NSC,P组注入等量PBS,并采用半定量RT-PCR方法检测正常大鼠视网膜及N组、P组大鼠术后第3天、1周、2周、3周、4周时视网膜BDNFmRNA的表达水平,行统计学分析。结果正常视网膜内可见BDNF表达;N组与P组大鼠视网膜内表达BDNF的量除第1周差异无统计学意义外,余时间段N组均高于P组。结论NSC能促使视神经损伤大鼠视网膜高表达BDNF,NSC移植治疗视神经损伤值得进一步深入研究。     

Penile erectile dysfunction after brachial plexus root avulsion injury in rats

Our previous studies have demonstrated that some male patients suffering from brachial plexus injury, particularly brachial plexus root avulsion, show erectile dysfunction to varying degrees. However, the underlying mechanism remains poorly understood. In this study, we evaluated the erectile function after establishing brachial plexus root avulsion models with or without spinal cord injury in rats. After these models were established, we administered apomorphine （via a sub- cutaneous injection in the neck） to observe changes in erectile function. Rats subjected to simple brachial plexus root avulsion or those subjected to brachial plexus root avulsion combined with spinal cord injury had significantly fewer erections than those subjected to the sham operation. Expression of neuronal nitric oxide synthase did not change in brachial plexus root avulsion rats. However, neuronal nitric oxide synthase expression was significantly decreased in brachial plexus root avulsion ＋ spinal cord injury rats. These findings suggest that a decrease in neuronal nitric oxide synthase expression in the penis may play a role in erectile dysfunction caused by the combi- nation of brachial plexus root avulsion and spinal cord injury.     

Adipose-derived mesenchymal stem cells accelerate nerve regeneration and functional recovery in a rat model of recurrent laryngeal nerve injury

Medialization thyroplasty or injection laryngoplasty for unilateral vocal fold paralysis cannot restore mobility of the vocal fold. Recent studies have shown that transplantation of mesenchymal stem cells is effective in the repair of nerve injuries. This study investigated whether adipose-derived stem cell transplantation could repair recurrent laryngeal nerve injury. Rat models of recurrent laryngeal nerve injury were established by crushing with micro forceps. Adipose-derived mesenchymal stem cells(ADSCs; 8 × 105) or differentiated Schwann-like adipose-derived mesenchymal stem cells(d ADSCs; 8 × 105) or extracellular matrix were injected at the site of injury. At 2, 4 and 6 weeks post-surgery, a higher density of myelinated nerve fiber, thicker myelin sheath, improved vocal fold movement, better recovery of nerve conduction capacity and reduced thyroarytenoid muscle atrophy were found in ADSCs and d ADSCs groups compared with the extracellular matrix group. The effects were more pronounced in the ADSCs group than in the d ADSCs group. These experimental results indicated that ADSCs transplantation could be an early interventional strategy to promote regeneration after recurrent laryngeal nerve injury.     

Human umbilical cord blood stem cells and brain-derived neurotrophic factor for optic nerve injury:a biomechanical evaluation

Treatment for optic nerve injury by brain-derived neurotrophic factor or the transplantation of human umbilical cord blood stem cells has gained progress, but analysis by biomechanical indicators is rare. Rabbit models of optic nerve injury were established by a clamp. At 7 days after injury, the vitreous body received a one-time injection of 50 μg brain-derived neurotrophic factor or 1 × 106 human umbilical cord blood stem cells. After 30 days, the maximum load, maximum stress, maximum strain, elastic limit load, elastic limit stress, and elastic limit strain had clearly improved in rabbit models of optical nerve injury after treatment with brain-derived neurotrophic factor or human umbilical cord blood stem cells. The damage to the ultrastructure of the optic nerve had also been reduced. These findings suggest that human umbilical cord blood stem cells and brain-derived neurotrophic factor effectively repair the injured optical nerve, improve biomechanical properties, and contribute to the recovery after injury.     

SPIO、GFP双标BDNF基因修饰中脑神经干细胞的研究

目的建立超顺磁氧化铁（SPIO）、绿色荧光蛋白（GFP）双标脑源性神经营养因子（BDNF）基因修饰中脑神经干细胞。方法以质粒pcDNA3-BDNF、pEGFPN1共转染第3代大鼠胚胎中脑神经干细胞,并用SPIO标记。荧光显微镜检测GFP的表达;免疫细胞化学、Westernblot鉴定BDNF的表达;普鲁士蓝染色、透射电镜鉴定SPIO标记。结果 GFP在基因转染12h后开始表达,24h明显增加,48h达顶峰。免疫细胞化学、Westernblot表明：细胞成功表达BDNF。普鲁士蓝染色显示：SPIO标记的中脑神经干细胞内有大量蓝染铁颗粒,细胞标记率达100%。透射电镜显示：SPIO颗粒位于吞饮小泡和细胞质内。结论成功建立SPIO、GFP双标BDNF基因修饰中脑神经干细胞,为进一步开展帕金森病的细胞移植治疗研究奠定基础。     

Human periodontal ligament stem cells repair mental nerve injury

Human periodontal ligament stem cells are easily accessible and can differentiate into Schwann cells. We hypothesized that human periodontal ligament stem cells can be used as an alternative source for...     

Differentiation of endogenous neural stem cells in adult versus neonatal rats after brachial plexus root avulsion injury

An experimental model of brachial plexus root avulsion injury of cervical dorsal C5-6 was established in adult and neonatal rats.Real-time PCR showed that the levels of brain-derived neurotrophic factor,nerve growth factor and neurotrophin-3 in adult rats increased rapidly 1 day after brachial plexus root avulsion injury,and then gradually decreased to normal levels by 21 days.In neonatal rats,levels of the three neurotrophic factors were decreased on the first day after injury,and then gradually increased from the seventh day and remained at high levels for an extended period of time.We observed that greater neural plasticity contributed to better functional recovery in neonatal rats after brachial plexus root avulsion injury compared with adult rats.Moreover, immunohistochemical staining showed that the number of bromodeoxyuridine/nestin-positive cells increased significantly in the spinal cords of the adult rats compared with neonatal rats after brachial plexus root avulsion injury.In addition,the number of bromodeoxyuridine/glial fibrillary acidic protein-positive cells in adult rats was significantly higher than in neonatal rats 14 and 35 days after brachial plexus injury.Bromodeoxyuridine/β-tubulin-positive cells were not found in either adult or neonatal rats.These results indicate that neural stem cells differentiate mainly into astrocytes after brachial plexus root avulsion injury.Furthermore,the degree of neural stem cell differentiation in neonatal rats was lower than in adult rats.     

Combining acellular nerve allografis with brain- derived neurotrophic factor transfected bone marrow mesenchymal stem cells restores sciatic nerve injury better than either intervention alone

In this study, we chemically extracted acellular nerve allografts from bilateral sciatic nerves, and repaired 10-mm sciatic nerve defects in rats using these grafts and brain-derived neurotrophic factor transfected bone marrow mesenchymal stem cells. Experiments were performed in three groups： the acellular nerve allograft bridging group, acellular nerve allograft ＋ bone marrow mesenchymal stem cells group, and the acellular nerve allograft ＋ brain-derived neurotrophic factor transfected bone marrow mesenchyrnal stem cells group. Results showed that at 8 weeks after bridging, sciatic functional index, triceps wet weight recovery rate, myelin thickness, and number of myelinated nerve fibers were significantly changed in the three groups. Variations were the largest in the acellular nerve allograft ＋ brain-derived neurotrophic factor transfected bone marrow mesenchymal stem cells group compared with the other two groups. Experimental findings suggest that chemically extracted acellular nerve allograft combined nerve factor and mesenchymal stem cells can promote the restoration of sciatic nerve defects. The repair effect seen is better than the single application of acellular nerve allograft or acellular nerve allograft combined mesenchymal stem cell transplantation.     

Protective effect of brain-derived neurotrophic factor and neurotrophin-3 overexpression by adipose-derived stem cells combined with silk fibroin/chitosan scaffold in spinal cord injury

ABSTRACT

Objectives: Spinal cord injury (SCI) is a most debilitating traumatic injury, and cytotherapy is a promising alternative treatment strategy. Here we investigated the effect and mechanism of adipose-derived stem/stromal cells (ASCs) with overexpressing brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT3) (BDNF-NT3) in combination with silk fibroin/chitosan scaffold (SFCS) in SCI.

Methods: Female Sprague-Dawley rats were used as an SCI model. SFCS,SFCS and ASCs, or ASCs overexpressing NT3, BDNF, and BDNF-NT3 were implanted into SCI rats. Basso, Beattie, and Bresnahan score, pathological changes, and spinal cord tissue and nerve fiber morphology were observed and assayed. GAP-43, GFAP, and caspase-3 expression was determined using immunohistochemistry and western blotting.

Results: Smoother spinal cords, less scar tissue, and lower inflammatory activity were found in the SFCS, SFCS and ASCs, ASCs with NT3, BDNF, and BDNF-NT3 overexpression treatment than in the untreated SCI rat groups. Increasing formation of nerve fibers was observed in the above groups in order. GAP-43 expression significantly increased, while GFAP and caspase-3 expression significantly decreased. These results indicated obvious alleviation in pathological changes and BDNF-NT3 overexpression in ASCs combined with SFCS treatment in SCI rats.

Conclusion: Thus, BDNF-NT3 overexpression from ASCs with SFCS had synergistic neuroprotective effects on SCI and may be a treatment option for SCI.     

腺病毒介导BDNF基因转移对大鼠创伤性脑损伤后iNOS表达及细胞凋亡的影响

研究重组腺病毒介导的脑源性神经营养因子（brain derived neurotrophic factor,BDNF)基因转移对创伤性脑损伤（TBI）后诱导型一氧化氮合酶（inducible nitric oxide synthase,iNOS)表达及细胞凋亡的影响。将重组腺病毒载体4μl注入承受单侧大脑皮质重锤打击的大鼠海马,对照组注射病毒缓冲液。伤后3h及1,3,7,14d利用免疫组化单标／双标染色。原位杂交／组化染色及DNA末端原位标记等方法,检测伤侧大脑皮质和海马各多区iNOS、BDNF及凋亡相关信号表达的改变。与对照组相比,同伤组大脑皮质及海马各区iNOS阳性细胞于伤后3h开始显著增多,7d达高峰。多数iNOS阳性细胞同时呈现凋亡相关蛋白阳性反应或TUNEL阳性反应,但很少同时表达BDNF mRNA。注射病毒载体组后3,7d,海马CA1区和DH区表达iNOS、凋亡相关蛋白的细胞及凋亡细胞显著减少（P均＜0．01）,而表达BDNF mRNA的神经元显著增多。提示,TBI诱导海马细胞表达iNOS及诱导海马细胞凋亡;腺病毒介导的BDNF基因转移通过抑制iNOS表达、增加BDNF表达及减少细胞凋亡的机制保护海马神经元。     

骨髓间充质干细胞尾静脉移植脊髓损伤大鼠脑源性神经营养因子及神经生长因子的表达

背景：骨髓间充质干细胞移植对脊髓损伤有治疗作用,但其机制尚不完全清楚。 目的：应用免疫组织化学方法观察骨髓间充质干细胞静脉移植损伤脊髓局部脑源性神经营养因子及神经生长因子的表达,分析骨髓间充质干细胞移植治疗大鼠脊髓损伤的作用途径。 方法：运用改良Allen法制备T10脊髓外伤性截瘫大鼠模型,假手术组6只,脊髓损伤组24只随机分为对照组和骨髓间充质干细胞移植组。骨髓间充质干细胞移植组、假手术组接受骨髓间充质干细胞单细胞悬液1 mL(1×106 cells)自大鼠尾静脉缓慢注射移植,对照组静脉注射PBS 1 mL。 结果与结论：脊髓损伤后损伤局部的脑源性神经营养因子、神经生长因子表达增加,骨髓间充质干细胞静脉注射移植后能促进脊髓损伤局部脑源性神经营养因子、神经生长因子更进一步的表达,这可能是促进神经结构及神经功能恢复的因素之一。     

Combination of bone marrow mesenchymal stem cells and brain-derived neurotrophic factor for treating spinal cord injury

BACKGROUND:Because bone marrow mesenchymal stem cells (BMSCs) do not secrete sufficient brain-derived neurotrophic factor (BDNF), the use of exogenous BDNF could improve microenvironments in injured regions for BMSCs differentiation. OBJECTIVE:To analyze recovery of the injured spinal cord following BMSCs venous transplantation in combination with consecutive injections of BDNF. DESIGN, TIME AND SETTING:A randomized, controlled animal experiment was performed at the Central Laboratory of First Hospital and Anatomical Laboratory, Fujian Medical University from October 2004 to May 2006.MATERIALS:Human BDNF was purchased from Sigma, USA. METHODS:A total of 44 New Zealand rabbits were randomly assigned to model (n = 8), BDNF (n = 12), BMSC (n = 12), and BMSC+BDNF (n = 12) groups. Spinal cord (L2) injury was established with the dropping method. The model group rabbits were injected with 1 mL normal saline via the ear margin vein; the BDNF group was subdurally injected with 100 μg/d human BDNF for 1 week; the BMSC group was injected with 1 mL BMSCs suspension (2 × 106/mL) via the ear margin vein; and the BMSC+BDNF group rabbits were subdurally injected with 100 μg/d BDNF for 1 week, in addition to BMSCs suspension via the ear margin vein. MAIN OUTCOME MEASURES:BMSCs surface markers were detected by flow cytometry. BMSCs differentiation in the injured spinal cord was detected by immunofluorescence histochemistry. Functional and structural recovery, as well as morphological changes, in the injured spinal cord were respectively detected by Tarlov score, horseradish peroxidase retrograde tracing, and hematoxylin & eosin staining methods at 1, 3, and 5 weeks following transplantation. RESULTS:Transplanted BMSCs differentiated into neuronal-like cells in the injured spinal cord at 3 and 5 weeks following transplantation. Neurological function and pathological damage improved following BMSC + BDNF treatment compared with BDNF or BMSC alone (P < 0.01 or P < 0.05). CONCLUSION:BMSCs venous transplantation in combination with BDNF subdural injection benefits neuronal-like cell differentiation and significantly improves structural and function of injured spinal cord compared with BMSCs or BDNF alone.     

Functional recovery and microenvironmental alterations in a rat model of spinal cord injury following human umbilical cord blood-derived mesenchymal stem cells transplantation

BACKGROUND: Transplantation of human umbilical cord blood-derived mesenchymal stem cells (MSCs) has been shown to benefit spinal cord injury (SCI) repair. However, mechanisms of microenvironmental regulation during differentiation of transplanted MSCs remain poorly understood. OBJECTIVE: To observe changes in nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and interleukin-8 (IL-8) expression following transplantation of human umbilical cord-derived MSCs, and to explore the association between microenvironment and neural functional recovery following MSCs transplantation.DESIGN, TIME AND SETTING: A randomized, controlled, animal experiment was performed at the Department of Orthopedics, First Affiliated Hospital of Soochow University from April 2005 to March 2007. MATERIALS: Human cord blood samples were provided by the Department of Gynecology and Obstetrics, First Affiliated Hospital of Soochow University. Written informed consent was obtained. METHODS: A total of 62 Wister rats were randomly assigned to control (n = 18), model (n = 22, SCI + PBS), and transplantation (n = 22, SCI + MSCs) groups. The rat SCI model was established using the weight compression method. MSCs were isolated from human umbilical cord blood and cultured in vitro for several passages. 5-bromodeoxyuridine (BrdU)-labeled MSCs (24 hours before injection) were intravascularly transplanted. MAIN OUTCOME MEASURES: The rats were evaluated using the Basso, Beattie and Bresnahan (BBB) locomotor score and inclined plane tests. Transplanted cells were analyzed following immunohistochemistry. Enzyme-linked immunosorbant assay was performed to determine NGF, BDNF, and IL-8 levels prior to and after cell transplantation.RESULTS: A large number of BrdU-positive MSCs were observed in the SCI region of the transplantation group, and MSCs were evenly distributed in injured spinal cord tissue 1 week after transplantation. BBB score and inclined plane test results revealed significant functional improvement in the transplantation group compared to the model group (P< 0.05), which was maintained for 2-3 weeks. Compared to the model group, NGF and BDNF levels were significantly increased in the injured region following MSCs transplantation at 3 weeks (P < 0.05), but IL-8 levels remained unchanged (P > 0.05).CONCLUSION: MSCs transplantation increased NGF and BDNF expression in injured spinal cord tissue. MSCs could promote neurological function recovery in SCI rats by upregulating NGF expression and improving regional microenvironments.     

BDNF-GFP转染神经干细胞修复大鼠脊髓损伤：发挥干细胞与神经因子的双重效应？

摘要 背景：神经干细胞移植入大鼠脊髓损伤模型可以促进功能恢复,基因治疗已被广泛用于治疗脊髓损伤。 目的：确定BDNF-GFP转染后神经干细胞移植对大鼠脊髓损伤的修复效果。 设计,时间和背景：本实验是在中国医科大学基础医学院发育生物学实验室与2009年5月至2010年1月完成。 材料：10只新生Wistar大鼠和88只2-3个月大,雌雄不限的Wistar大鼠。 方法：以携带BDNF-GFP基因的腺病毒转染神经干细胞。88只Wistar大鼠中假手术组8只, 80只大鼠制成T9左侧横断模型,并随机分成四组：BDNF和GFP修饰的神经干细胞移植组,GFP修饰的神经干细胞移植组;单纯神经干细胞移植组和模型组。在各神经干细胞移植组,脊髓损伤后向横断处显微注射等体积细胞,模型组在相同的部位注射等体积的PBS。 主要观察指标： BBB评分检测脊髓损伤模型运动功能恢复情况;制备脊髓损伤模型2周后取材,免疫组化评估BDNF-GFP转染的神经干细胞移植后的细胞学特点;制备脊髓损伤模型2、4、6、8周Real-time PCR检测脊髓横断处BDNF表达情况。 结果： BDNF-GFP转染后神经干细胞在脊髓半切模型中存活并表达BDNF和GFP,移植该细胞后的大鼠体内高表达具有生物活性的BDNF,且脊髓损伤动物运动功能较对照组明显恢复。 结论：移植BDNF-GFP转染后神经干细胞可能是一种修复脊髓损伤的有效的方法。 关键词：神经干细胞,脑源性神经营养因子;绿色荧光蛋白;脊髓损伤;移植。     

神经干细胞移植对大鼠视神经部分损伤后视觉诱发电位的影响

目的观察研究神经干细胞(NSCs)移植入视神经部分损伤SD大鼠后闪光视觉诱发电位(F-VEP)的变化。方法24只健康成年SD大鼠随机分为NSCs移植组(N组)和对照组(C组),每组12只大鼠,两组均使用精确校准方法在大鼠右眼造成部分视神经损伤,左眼为正常对照。从胚胎SD大鼠海马分离NSCs,利用细胞培养和体内移植技术,将培养后的NSCs注入视神经损伤后N组大鼠玻璃体内,C组大鼠视神经损伤眼玻璃体内注入同等体积的PBS。以上两组分6个时间段,即损伤前、损伤时、损伤后1周、2周、3周、4周分别检测损伤视神经眼的F-VEP,记录P1波幅及峰潜时,并进行统计分析。结果N组及C组P1波幅随时间延长均不同程度降低,但前者趋势较后者缓和。自第2周开始N组波幅均高于C组,且差异有显著性；N组及C组P1峰潜时均随时间变化,在第3周时达到最长,第4周时P1峰潜时有缩短；自第1周开始N组峰潜时均较C组缩短,且差异均有显著性意义。结论NSCs移植入视神经部分损伤大鼠可部分改善视神经传导功能。     

Transplantation of human umbilical cord blood mesenchymal stem cells to treat a rat model of traumatic brain injury

In the present study, human umbilical cord blood mesenchymal stem cells were injected into a rat model of traumatic brain injury via the tail vein. Results showed that 5-bromodeoxyuridine-labeled cells aggregated around the injury site, surviving up to 4 weeks post-transplantation. In addition, transplantation-related death did not occur, and neurological functions significantly improved. Histological detection revealed attenuated pathological injury in rat brain tissues following human umbilical cord blood mesenchymal stem cell transplantation. In addition, the number of apoptotic cells decreased. Immunohistochemistry and in situ hybridization showed increased expression of brain-derived neurotrophic factor, nerve growth factor, basic fibroblast growth factor, and vascular endothelial growth factor, along with increased microvessel density in surrounding areas of brain injury. Results demonstrated migration of transplanted human umbilical cord blood mesenchymal stem cells into the lesioned boundary zone of rats, as well as increased angiogenesis and expression of related neurotrophic factors in the lesioned boundary zone.     

Neural stem cells over-expressing brain-derived neurotrophic factor promote neuronal survival and cytoskeletal protein expression in traumatic brain injury sites

Cytoskeletal proteins are involved in neuronal survival.Brain-derived neurotrophic factor can increase expression of cytoskeletal proteins during regeneration after axonal injury.However,the effect of neural stem cells genetically modified by brain-derived neurotrophic factor transplantation on neuronal survival in the injury site still remains unclear.To examine this,we established a rat model of traumatic brain injury by controlled cortical impact.At 72 hours after injury,2 × 10~7 cells/m L neural stem cells overexpressing brain-derived neurotrophic factor or naive neural stem cells(3 m L) were injected into the injured cortex.At 1–3 weeks after transplantation,expression of neurofilament 200,microtubule-associated protein 2,actin,calmodulin,and beta-catenin were remarkably increased in the injury sites.These findings confirm that brain-derived neurotrophic factor-transfected neural stem cells contribute to neuronal survival,growth,and differentiation in the injury sites.The underlying mechanisms may be associated with increased expression of cytoskeletal proteins and the Wnt/β-catenin signaling pathway.     

Downregulation of microRNA‐124‐3p promotes subventricular zone neural stem cell activation by enhancing the function of BDNF downstream pathways after traumatic brain injury in adult rats

AimsIn this study, the effect of intracerebral ventricle injection with a miR‐124‐3p agomir or antagomir on prognosis and on subventricular zone (SVZ) neural stem cells (NSCs) in adult rats with moderate traumatic brain injury (TBI) was investigated.MethodsModel rats with moderate controlled cortical impact (CCI) were established and verified as described previously. The dynamic changes in miR‐124‐3p and the status of NSCs in the SVZ were analyzed. To evaluate the effect of lateral ventricle injection with miR‐124‐3p analogs and inhibitors after TBI, modified neurological severity scores (mNSSs) and rotarod tests were used to assess motor function prognosis. The variation in SVZ NSC marker expression was also explored. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of predicted miR‐124‐3p targets was performed to infer miR‐124‐3p functions, and miR‐124‐3p effects on pivotal predicted targets were further explored.ResultsAdministration of miR‐124 inhibitors enhanced SVZ NSC proliferation and improved the motor function of TBI rats. Functional analysis of miR‐124 targets revealed high correlations between miR‐124 and neurotrophin signaling pathways, especially the TrkB downstream pathway. PI3K, Akt3, and Ras were found to be crucial miR‐124 targets and to be involved in most predicted functional pathways. Interference with miR‐124 expression in the lateral ventricle affected the PI3K/Akt3 and Ras pathways in the SVZ, and miR‐124 inhibitors intensified the potency of brain‐derived neurotrophic factor (BDNF) in SVZ NSC proliferation after TBI.ConclusionDisrupting miR‐124 expression through lateral ventricle injection has beneficial effects on neuroregeneration and TBI prognosis. Moreover, the combined use of BDNF and miR‐124 inhibitors might lead to better outcomes in TBI than BDNF treatment alone.