Neural progenitor cells derived from fibroblasts induced by small molecule compounds under hypoxia for treatment of Parkinson’s disease in rats

Neural progenitor cells(NPCs) capable of self-renewal and differentiation into neural cell lineages offer broad prospects for cell therapy for neurodegenerative diseases. However, cell therapy based on NPC transplantation is limited by the inability to acquire sufficient quantities of NPCs. Previous studies have found that a chemical cocktail of valproic acid, CHIR99021, and Repsox(VCR) promotes mouse fibroblasts to differentiate into NPCs under hypoxic conditions. Therefore, we used VCR(0.5 mM valproic acid, 3 μM CHIR99021, and 1 μM Repsox) to induce the reprogramming of rat embryonic fibroblasts into NPCs under a hypoxic condition(5%). These NPCs exhibited typical neurosphere-like structures that can express NPC markers, such as Nestin, SRY-box transcription factor 2, and paired box 6(Pax6), and could also differentiate into multiple types of functional neurons and astrocytes in vitro. They had similar gene expression profiles to those of rat brain-derived neural stem cells. Subsequently, the chemically-induced NPCs(ciNPCs) were stereotactically transplanted into the substantia nigra of 6-hydroxydopamine-lesioned parkinsonian rats. We found that the ciNPCs exhibited long-term survival, migrated long distances, and differentiated into multiple types of functional neurons and glial cells in vivo. Moreover, the parkinsonian behavioral defects of the parkinsonian model rats grafted with ciNPCs showed remarkable functional recovery. These findings suggest that rat fibroblasts can be directly transformed into NPCs using a chemical cocktail of VCR without introducing exogenous factors, which may be an attractive donor material for transplantation therapy for Parkinson’s disease.

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, 20μL culture solution, containing 5×10^5 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 immunohistochemistry. 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.

Alterations in the polysialylated neural cell adhesion molecule and retinal ganglion cell density in mice with diabetic retinopathy

AIM：To investigate the impact of polysialylated neural cell adhesion molecule（PSA-NCAM）on the survival of retinal ganglion cells（RGCs）in the experimentally induced diabetes in mice.METHODS：Diabetes was induced in 2.5 months old Swiss Webster mice by intraperitoneal injection of streptozotocin（STZ,90 mg/kg）once daily for two consecutive days.Examination of the proteins of interest in the retinas from diabetic mice at 2mo after diabetes induction was performed using immunohistochemistry and Western blot analysis.RGCs were counted in the wholemounted retinas,and Brn3a marker was used.RESULTS：Examination of retinas from diabetic mice at 2mo after diabetes induction revealed a considerable reduction in RGC density.Our experiments also demonstrated a redistribution of PSA-NCAM in the retina of diabetic animals.PSA-NCAM immunoreactivity was diminished in the inner part of the retina where RGCs were located.In contrast,an enhanced PSA-NCAM immunoreactivity was detected in the outer layers of the retina.PSA-NCAM signal was co-localized with glial fibrillary acidic protein immunoreactivity in the Müller cell branches.Previous studies have shown that matrix metalloproteinase-9（MMP-9）is responsible for the reduction in PSA-NCAM levels in neuronal cells.The reduced levels of PSA-NCAM in inner layers（nerve fiber layer,ganglion cell layer）were accompanied by the increased expression of MMP-9.In contrast,in the outer retinal layers,the expression of MMP-9 was much less pronounced.CONCLUSION：MMP-9 induces PSA-NCAM shedding in the inner part of the retina and the decreased level of PSA-NCAM in the inner part of the retina might be,at least in part,responsible for the loss of RGCs in diabetic mice.

Nerve bundle formation during the promotion of peripheral nerve regeneration:collagenⅥ-neural cell adhesion molecule 1 interaction

The formation of nerve bundles,which is partially regulated by neural cell adhesion molecule 1(NCAM1),is important for neural network organization during peripheral nerve regeneration.However,little is known about how the extracellular matrix(ECM)microenvironment affects this process.Here,we seeded dorsal root ganglion tissue blocks on different ECM substrates of peripheral nerve ECM-derived matrixgel,Matrigel,laminin 521,collagen I,and collagen IV,and observed well-aligned axon bundles growing in the peripheral nerve ECM-derived environment.We confirmed that NCAM1 is necessary but not sufficient to trigger this phenomenon.A protein interaction assay identified collagen VI as an extracellular partner of NCAM1 in the regulation of axonal fasciculation.Collagen VI interacted with NCAM1 by directly binding to the FNIII domain,thereby increasing the stability of NCAM1 at the axolemma.Our in vivo experiments on a rat sciatic nerve defect model also demonstrated orderly nerve bundle regeneration with improved projection accuracy and functional recovery after treatment with 10 mg/m L Matrigel and 20μg/m L collagen VI.These findings suggest that the collagen VI-NCAM1 pathway plays a regulatory role in nerve bundle formation.This study was approved by the Animal Ethics Committee of Guangzhou Medical University(approval No.GY2019048)on April 30,2019.

Cognitive disorder and changes in cholinergic receptors, N-methyl-D aspartate receptors, neural cell adhesion molecule, and brain-derived neurotrophic factor following brain injury

BACKGROUND： Learning and memory damage is one of the most permanent and the severest symptoms of traumatic brain injury; it can seriously influence the normal life and work of patients. Some research has demonstrated that cognitive disorder is closely related to nicotine cholinergic receptors, N-methyl-D aspartate receptors, neural cell adhesion molecule, and brain-derived neurotrophic factor. OBJECTIVE： To summarize the cognitive disorder and changes in nicotine cholinergic receptors, N-methyl-D aspartate receptors, neural cell adhesion molecule, and brain-derived neurotrophic factor following brain injury. RETRIEVAL STRATEGY： A computer-based online search was conducted in PUBMED for English language publications containing the key words ＂brain injured, cognitive handicap, acetylcholine, N-methyl-D aspartate receptors, neural cell adhesion molecule, brain-derived neurotrophic factor＂ from January 2000 to December 2007. There were 44 papers in total. Inclusion criteria： ① articles about changes in nicotine cholinergic receptors, N-methyl-D aspartate receptors, neural cell adhesion molecule, and brain-derived neurotrophic factor following brain injury; ② articles in the same researching circle published in authoritative journals or recently published. Exclusion criteria： duplicated articles. LITERATURE EVALUATION： References were mainly derived from research on changes in these four factors following brain injury. The 20 included papers were clinical or basic experimental studies. DATA SYNTHESIS： After craniocerebral injury, changes in these four factors in brain were similar to those during recovery from cognitive disorder, to a certain degree. Some data have indicated that activation of nicotine cholinergic receptors, N-methyl-D aspartate receptors, neural cell adhesion molecule, and brain-derived neurotrophic factor could greatly improve cognitive disorder following brain injury. However, there are still a lot of questions remaining; for example, how do these factors change at different time points after brain injury, and what is the relationship between associated factors and cognitive disorder. CONCLUSION： It is necessary to comprehensively study some associated factors, to analyze their changes and their relationship with cognitive disorder following brain injury, and to investigate their effects at different time points after brain injury.

Molecular and cellular changes in the post-traumatic spinal cord remodeling after autoinfusion of a genetically-enriched leucoconcentrate in a mini-pig model

Post-traumatic spinal cord remodeling includes both degenerating and regenerating processes,which affect the potency of the functional recovery after spinal cord injury(SCI).Gene therapy for spinal cord injury is proposed as a promising therapeutic strategy to induce positive changes in remodeling of the affected neural tissue.In our previous studies for delivering the therapeutic genes at the site of spinal cord injury,we developed a new approach using an autologous leucoconcentrate transduced ex vivo with chimeric adenoviruses(Ad5/35)carrying recombinant cDNA.In the present study,the efficacy of the intravenous infusion of an autologous genetically-enriched leucoconcentrate simultaneously producing recombinant vascular endothelial growth factor(VEGF),glial cell line-derived neurotrophic factor(GDNF),and neural cell adhesion molecule(NCAM)was evaluated with regard to the molecular and cellular changes in remodeling of the spinal cord tissue at the site of damage in a model of mini-pigs with moderate spinal cord injury.Experimental animals were randomly divided into two groups of 4 pigs each:the therapeutic(infused with the leucoconcentrate simultaneously transduced with a combination of the three chimeric adenoviral vectors Ad5/35‐VEGF165,Ad5/35‐GDNF,and Ad5/35‐NCAM1)and control groups(infused with intact leucoconcentrate).The morphometric and immunofluorescence analysis of the spinal cord regeneration in the rostral and caudal segments according to the epicenter of the injury in the treated animals compared to the control mini-pigs showed:(1)higher sparing of the grey matter and increased survivability of the spinal cord cells(lower number of Caspase-3-positive cells and decreased expression of Hsp27);(2)recovery of synaptophysin expression;(3)prevention of astrogliosis(lower area of glial fibrillary acidic protein-positive astrocytes and ionized calcium binding adaptor molecule 1-positive microglial cells);(4)higher growth rates of regeneratingβIII-tubulin-positive axons accompanied by a higher number of oligodendrocyte transcription factor 2-positive oligodendroglial cells in the lateral corticospinal tract region.These results revealed the efficacy of intravenous infusion of the autologous genetically-enriched leucoconcentrate producing recombinant VEGF,GDNF,and NCAM in the acute phase of spinal cord injury on the positive changes in the post-traumatic remodeling nervous tissue at the site of direct injury.Our data provide a solid platform for a new ex vivo gene therapy for spinal cord injury and will facilitate further translation of regenerative therapies in clinical neurology.

Expression changes of nerve cell adhesion molecules L1 and semaphorin 3A after peripheral nerve injury

The expression of nerve cell adhesion molecule L1 in the neuronal growth cone of the central nervous system is strongly associated with the direction of growth of the axon, but its role in the regeneration of the peripheral nerve is still unknown. This study explored the problem in a femoral nerve section model in rats. L1 and semaphorin 3A m RNA and protein expressions were measured over the 4-week recovery period. Quantitative polymerase chain reaction showed that nerve cell adhesion molecule L1 expression was higher in the sensory nerves than in motor nerves at 2 weeks after injury, but vice versa for the expression of semaphorin 3A. Western blot assay results demonstrated that nerve cell adhesion molecule L1 expression was higher in motor nerves than in the sensory nerves at the proximal end after injury, but its expression was greater in the sensory nerves at 2 weeks. Semaphorin 3A expression was higher in the motor nerves than in the sensory nerves at 3 days and 1 week after injury. Nerve cell adhesion molecule L1 and semaphorin 3A expressions at the distal end were higher in the motor nerves than in the sensory nerves at 3 days, 1 and 2 weeks. Immunohistochemical staining results showed that nerve cell adhesion molecule L1 expression at the proximal end was greater in the sensory nerves than in the motor nerves; semaphorin 3A expression was higher in the motor nerves than in the sensory nerves at 2 weeks after injury. Taken together, these results indicated that nerve cell adhesion molecules L1 and semaphorin 3A exhibited different expression patterns at the proximal and distal ends of sensory and motor nerves, and play a coordinating role in neural chemotaxis regeneration.

ADAM10 facilitates rapid neural stem cell cycling and proper positioning within the subventricular zone niche via JAMC/RAP1Gap signaling

The mechanisms that regulate neural stem cell(NSC)lineage progression and maintain NSCs within diffe rent domains of the adult neural stem cell niche,the subventricular zone are not well defined.Quiescent NSCs are arranged at the apical ventricular wall,while mitotically activated NSCs are found in the basal,vascular region of the subventricular zone.Here,we found that ADAM 10(a disintegrin and metalloproteinase 10)is essential in NSC association with the ventricular wall,and via this adhesion to the apical domain,ADAM10 regulates the switch from quiescent and undiffe rentiated NSC to an actively prolife rative and differentiating cell state.Processing of JAMC(junctional adhesion molecule C)by ADAM 10 increases Rap1 GAP activity.This molecular machinery promotes NSC transit from the apical to the basal compartment and subsequent lineage progression.Understanding the molecular mechanisms responsible for regulating the proper positioning of NSCs within the subventricular zone niche and lineage progression of NSCs could provide new targets for drug development to enhance the regenerative prope rties of neural tissue.

Neuronal conversion from glia to replenish the lost neurons

Neuronal injury,aging,and cerebrovascular and neurodegenerative diseases such as cerebral infarction,Alzheimer’s disease,Parkinson’s disease,frontotemporal dementia,amyotrophic lateral sclerosis,and Huntington’s disease are characte rized by significant neuronal loss.Unfo rtunately,the neurons of most mammals including humans do not possess the ability to self-regenerate.Replenishment of lost neurons becomes an appealing therapeutic strategy to reve rse the disease phenotype.Transplantation of pluripotent neural stem cells can supplement the missing neurons in the brain,but it carries the risk of causing gene mutation,tumorigenesis,severe inflammation,and obstructive hydrocephalus induced by brain edema.Conversion of neural or non-neural lineage cells into functional neurons is a promising strategy for the diseases involving neuron loss,which may overcome the above-mentioned disadvantages of neural stem cell therapy.Thus far,many strategies to transfo rm astrocytes,fibroblasts,microglia,Muller glia,NG2 cells,and other glial cells to mature and functional neurons,or for the conversion between neuronal subtypes have been developed thro ugh the regulation of transcription factors,polypyrimidine tra ct binding protein 1(PTBP1),and small chemical molecules or are based on a combination of several factors and the location in the central nervous system.However,some recent papers did not obtain expected results,and discrepancies exist.Therefore,in this review,we discuss the history of neuronal transdifferentiation,summarize the strategies for neuronal replenishment and conversion from glia,especially astrocytes,and point out that biosafety,new strategies,and the accurate origin of the truly co nverted neurons in vivo should be focused upon in future studies.It also arises the attention of replenishing the lost neurons from glia by gene therapies such as up-regulation of some transc ription factors or downregulation of PTBP1 or drug interfe rence therapies.

TPO、CD56、Galectin-3辅助诊断甲状腺乳头状癌的价值分析

目的:分析甲状腺过氧化物酶(TPO)、神经细胞黏附分子56(CD56)和β-半乳糖凝集素-3(Galectin-3)辅助诊断甲状腺乳头状癌的价值。方法:选取2018年3月—2020年12月于克拉玛依市中心医院接受手术切除的PTC患者104例作为研究对象,分析患者PTC癌变组织及距离癌灶>2 cm的癌旁组织TPO、CD56、Galectin-3表达情况,评估TPO、CD56、Galectin-3单项及联合检测辅助诊断PTC的效能。结果:TPO、CD56、Galectin-3单项及联合诊断PTC的特异度比较,差异无统计学意义(P>0.05);三项联合诊断PTC的准确度高于TPO、CD56,Galectin-3及TPO均高于CD56,差异有统计学意义(P<0.05);Galectin-3、三项联合诊断PTC的灵敏度高于TPO、CD56,TPO高于CD56,差异有统计学意义(P<0.05)。结论:TPO、CD56、Galectin-3三项联合检测可用于辅助诊断PTC。

3D培养诱导胚胎成纤维细胞为神经前体样细胞

目的探讨3D培养微环境和小分子组合(ATPV)对胚胎成纤维细胞诱导为神经前体样细胞的影响。方法在0.5%琼脂糖低粘附3D培养微环境和ATPV处理下,将小鼠胚胎成纤维细胞形成3D球体,分别通过RT-PCR、免疫荧光染色和碱性磷酸酶染色的方法检测神经前体细胞标记基因的表达水平。结果在3D环境下培养,随着成球培养时间的增长,小鼠胚胎成纤维细胞NPC特异性标记基因的表达明显上升,表现出神经前体样细胞的特征,并且3D ATPV培养微环境明显提高了小鼠胚胎成纤维细胞向神经前体样细胞的转化效率。结论小鼠胚胎成纤维细胞在3D和3D ATPV微环境下,聚集成球培养后逐渐获得了干细胞特征并诱导为神经前体样细胞,暗示着成球培养和小分子微环境可以给小鼠胚胎成纤维细胞创造一种良性的细胞聚集微环境,诱导神经前体样细胞的产生,这将为神经退行性病变的细胞替代疗法带来新的细胞来源。

小分子化合物组合改善多能干细胞向脑类器官定向分化的效率

目的优化多能干细胞(PSCs)定向分化为脑类器官的条件,提高人脑类器官的分化效率。方法利用人胚胎干细胞(hESCs)系H9向人脑类器官诱导分化体系,在早期脑类器官分化到神经祖细胞的发育阶段添加小分子化合物组合,通过形态学观察不同分化阶段脑类器官中的神经祖细胞形成的效率、细胞凋亡的情况以及分化成神经元的效率,通过RT-qPCR检测标志性基因的表达量,综合评估添加的小分子化合物组合对脑类器官形成的影响。结果在脑类器官神经祖细胞发育的关键阶段(1 d~14 d),培养基中依次添加多索啡(dorsomorphine)、A83-01、GSK-3β抑制剂CHIR99021和SMAD抑制剂SB-431542,可使神经祖细胞阶段的相关标志性基因的表达量显著提高,促进特异性神经管样结构形成,脑类器官中心区域的细胞凋亡减少。结论通过使用上述4种小分子化合物组合,可明显提高早期脑类器官的形成效率,减少脑类器官中的细胞凋亡、促进神经元形成,减少培养过程中的组织结构异质性。

Down syndrome cell adhesion molecule and its functions in neural development

The nervous system is a complex network with many types of neurons and numerous synaptic connections.The present knowledge on how neurons recognize specific targets and form such an intricate network is still limited.The Down syndrome cell adhesion molecule （DSCAM） belongs to the immunoglobulin superfamily and contributes to defects in the central nervous system in Down syndrome patients.DSCAM plays important roles in neural development,including dendritic patterning and self-avoidance,axon guidance and branching,axon target recognition and synaptic formation.However,the functional mechanisms and the underlying signaling pathways are still largely unknown.Here the functions of DSCAM in neural development were reviewed.Future research for better understanding DSCAM function and the relevance of DSCAM to human diseases was also discussed.

Intraductal papillary neoplasm of the bile duct in liver cirrhosis with hepatocellular carcinoma

A case of intraductal papillary neoplasm of the bile duct (IPNB) arising in a patient with hepatitis B-related liver cirrhosis with hepatocellular carcinoma (HCC) is reported. A 76-year-old man was admitted to our hospital with recurrent HCC. Laboratory data showed that levels of carcinoembryonic antigen and carbohydrate antigen 19-9 were elevated. He died of progressive hepatic failure. At autopsy,in addition to HCCs,an intraductal papillary proliferation of malignant cholangiocytes with fibrovascular cores was found in the dilated large bile ducts in the left lobe,and this papillary carcinoma was associated with an invasive mucinous carcinoma (invasive IPNB). Interestingly,extensive intraductal spread of the cholangiocarcinoma was found from the reactive bile ductular level to the interlobular bile ducts and septal bile ducts and to the large bile ducts in the left lobe. Neural cell adhesion molecule,a hepatic progenitor cell marker,was detected in IPNB cells. It seems possible in this case that hepatic progenitor cells located in reactive bile ductules in liver cirrhosis may have been responsible for the development of the cholangiocarcinoma and HCC,and that the former could have spread in the intrahepatic bile ducts and eventually formed grossly visible IPNB.

Pre-degenerated peripheral nerves co-cultured with bone marrow-derived cells: a new technique for harvesting high-purity Schwann cells

Schwann cells play an important role in the peripheral nervous system, especially in nerve repair following injury, so artificial nerve regen- eration requires an effective technique for obtaining purified Schwann cells. In vivo and in vitro pre-degeneration of peripheral nerves have been shown to obtain high-purity Schwann cells. We believed that in vitro pre-degeneration was simple and controllable, and available for the clinic. Thus, we co-cultured the crushed sciatic nerves with bone marrow-derived cells in vitro. Results demonstrated that, 3 hours after injury, a large number of mononuclear cells moved to the crushed nerves and a large number of bone marrow-derived cells infiltrated the nerve segments. These changes promoted the degradation of the nerve segments, and the dedifferentiation and proliferation of Schwann cells. Neural cell adhesion molecule and glial fibrillary acidic protein expression were detected in the crushed nerves. Schwann cell yield was 9.08 ± 2.01 ×104/mg. The purity of primary cultured Schwann cells was 88.4 ± 5.79%. These indicate a successful new method for ob- taining Schwann cells of high purity and yield from adult crushed sciatic nerve using bone marrow-derived cells.

Review: Neuronal Differentiation Protocols of Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) are self-renewing cells found in almost all postnatal organs and tissues in the perivascular region. These cells present multiple characteristics that make them candidates to be applied in cell therapy for neurodegenerative diseases, such as their secretory action, migration to the lesion area, and immunomodulatory potential. These cells have a high capacity for mesodermal differentiation;however, numerous studies have shown that MSCs can also differentiate into neurons. However, despite positive results in multiple trials in which undifferentiated MSCs transplanted into animal models of neurodegenerative diseases, some studies suggest that the therapeutic effects obtained are enhanced by the use of MSCs differentiated towards the neuronal lineage before transplant. In this sense, there are several methods to induce in vitro reprogramming of MSCs towards the neuronal lineage, including chemical substances, growth factors, cocultures with neural lineage cells, transfection of genes, miRNAs, etc., and small molecules stand out. Therefore, this article compares multiple experimental tests in which these inducers promote neuronal differentiation of MSCs and identify those methods that originate an optimal neuronal differentiation. The analysis includes the percentage of differentiation, maturation, expression of neuronal markers, functionality, and cell survival considering the intrinsic characteristics of the MSCs used as the tissue of origin and the species from which they were isolated.

New function of an old molecule——VCAM1 guides neural stem cells from proliferation to quiescence

Subject Code:C12With the support by the National Natural Science Foundation of China,the group led by Prof.Shen Qin(沈沁)from the Center for Stem Cell Biology and Regenerative Medicine,School of Medicine,and the IDG/McGovern Institute for Brain Research at Tsinghua University,reported a newly discovered

多模态影像分子体外验证触液神经元的神经干细胞特性

背景:最近研究发现位于脊髓中央管附近的接触脑脊液神经元(简称触液神经元)具有神经干细胞潜能,但触液神经元在体外研究中纯度不高,且目前尚无体外示踪技术证明其神经干细胞特性。目的:通过多模态影像分子筛选并在体外验证触液神经元的神经干细胞特性。方法:根据触液神经元特异性表达Pkd2l1基因,针对Pkd2l1基因上游启动子设计一种使触液神经元特异性表达绿色荧光蛋白(GFP)的多模态影像分子慢病毒(Lentivirus-Pkd2l1-GFP-puromycin-luciferase)。从出生24 h内C57BL/6小鼠延髓组织中提取出原代神经干细胞贴壁培养,用多模态影像分子病毒转染含有触液神经元的原代神经干细胞,通过嘌呤霉素筛选并纯化触液神经元。对筛选纯化的触液神经元进行体外悬浮培养并连续传代4代以上,免疫荧光检测第3代触液神经元与神经干细胞标记物Nestin、Sox2共表达情况。通过对第3代触液神经元诱导分化培养,免疫荧光检测触液神经元与神经元标记物NeuN、星形胶质细胞标记物S100β、少突胶质细胞标记物O4的共表达情况。结果与结论:成功构建出多模态影像分子慢病毒,并且筛选纯化后的触液神经元能够存活、增殖并表达绿色荧光蛋白。绿色荧光蛋白阳性触液神经元能在体外连续传代4代以上,并表达神经干细胞标记物Nestin、Sox2。诱导分化后,绿色荧光蛋白阳性触液神经元表达神经元标记物NeuN、星形胶质细胞标记物S100β、少突胶质细胞标记物O4。结果表明:多模态影像分子病毒能特异性标记触液神经元,触液神经元在体外具有自我更新、多向分化能力,通过多模态影像分子在体外成功验证触液神经元具有神经干细胞特性。

血清NCAM sTNFR1水平与超时间窗后循环脑卒中患者接受替罗非班治疗后神经预后的关系分析

目的探讨血清神经细胞黏附分子(NCAM)、可溶性肿瘤坏死因子受体1(sTNF-R1)水平与超时间窗后循环脑卒中(APCIS)患者接受替罗非班治疗后神经预后的关系。方法选择2019-03—2022-01唐山中心医院收治的100例超时间窗APCIS患者(APCIS组)和63例健康体检志愿者(对照组),根据NIHSS评分将APCIS患者分为轻度损伤组(<6分,27例)、中度损伤组(6~13分,39例)、重度损伤组(≥14分,34例)。所有患者入组后均接受替罗非班经微导管动脉给药[0.4μg/(kg·min)]和静脉持续泵注[0.1μg/(kg·min)]治疗48 h,配合降脂以及双抗血小板治疗。检测血清NCAM、sTNF-R1水平,出院后定期随访90 d,统计随访期间不良神经预后发生情况。多因素Logistic回归分析APCIS患者接受替罗非班治疗后神经预后不良的危险因素,受试者工作特征曲线(ROC)分析NCAM、sTNF-R1预测APCIS患者接受替罗非班治疗后神经预后不良的价值。结果APCIS组血清NCAM、sTNF-R1水平高于对照组(P<0.05),重度损伤组血清NCAM、sTNF-R1水平高于中度损伤组和轻度损伤组(P<0.05),预后不良组血清NCAM、sTNF-R1水平高于预后良好组(P<0.05)。房颤、高NIHSS评分、高NCAM、高sTNF-R1是APCIS患者接受替罗非班治疗后神经预后不良的危险因素(P<0.05)。NCAM、sTNF-R1预测APCIS患者接受替罗非班治疗后神经预后不良的曲线下面积为0.698、0.770,联合预测曲线下面积为0.937,高于单独指标预测(P<0.05)。结论APCIS患者血清NCAM、sTNFR-1水平增高,且与APCIS神经缺损程度以及替罗非班治疗后神经预后不良有关。

小分子化合物组合将人胚胎成纤维细胞体外编程为神经前体细胞

目的通过小分子化合物组合将人胚胎成纤维细胞(HEFs)重编程为化学诱导神经前体细胞(ciNPCs)。方法HEFs重编程为ciNPCs涉及两个阶段,第1阶段是小分子组合诱导阶段,常氧条件下,将HEFs在含有小分子化合物组合VCR(VPA,CHIR99021,Repsox)的KSR培养基中培养15 d,HEFs形态发生变化,形成致密细胞集落。第2阶段是特异性诱导ciNPCs,将形成的细胞集落胰酶消化后,低粘附板中悬浮培养,可以形成ciNPCs神经球。采用CM-DiI染料标记P3代ciNPCs,并将其移植于6-OHDA法制作的帕金森大鼠模型右脑内侧前脑束(MFB)区,检测ciNPCs在PD大鼠脑内微环境中的存活、迁移以及分化状况。结果VCR组合诱导10 d细胞开始出现明显的聚集趋势,15 d时,形成致密的细胞集落。单层培养1×10^(5)/孔中大约形成40个克隆,且AP染色呈阳性。将细胞集落消化后,低粘附板中悬浮培养培养2 d,可见大量神经球形成,即为第1代ciNPCs(P1代)。ciNPC高表达神经前体细胞(NPCs)特异性标记物(Nestin、Pax6和Sox2),经体外神经特异性诱导分化,ciNPCs表达神经元特异性标记物Tuj1和星形胶质细胞特异性标记物GFAP。而且,P3代ciNPCs移植PD大鼠脑内4周后,可以分化为Tuj1+、GFAP+、TH+和GABA+细胞。结论VCR可以将HEFs重编程为ciNPCs,而无需引入外源性基因,为神经科学研究和神经退行性疾病的治疗提供有利的供体材料。