BACKGROUND: Recently, it was found that cerebrospinal fluid-contacting neurons near the central canal of the spinal cord have the potential of neural stem cells, but the purity of cerebrospinal fluid-contacting neurons in the in vitro research is not high, and there is no in vitro tracing technology to prove their neural stem cell characteristics. OBJECTIVE: To verify the characteristics of neural stem cells of cerebrospinal fluid-contacting neurons by multimodal imaging molecules in vitro.METHODS: According to the specific expression of the Pkd2l1 gene in cerebrospinal fluid-contacting neurons, a multimodal imaging molecular lentivirus was designed to specifically express a green fluorescent protein（GFP） in cerebrospinal fluid-contacting neurons according to the upstream promoter of Pkd2l1 gene. The primary neural stem cells were extracted from the medulla oblongata of C57BL/6 mice within 24 hours of birth, then the primary neural stem cells containing cerebrospinal fluid-contacting neurons were transfected with multimodal imaging molecular virus, and the cerebrospinal fluid-contacting neurons were screened and purified by puromycin. The screened and purified cerebrospinal fluid-contacting neurons were suspended in vitro and passaged continuously for more than four generations. Immunofluorescence was used to observe whether the third generation of cerebrospinal fluid-contacting neurons co-expressed with neural stem cell markers Nestin and Sox2. The third generation of cerebrospinal fluid-contacting neurons was induced to differentiate, and the co-expression of cerebrospinal fluid-contacting neurons with neuronal marker NeuN, astrocyte marker S100 β and oligodendrocyte marker O4 was detected by immunofluorescence. RESULTS AND CONCLUSION: The multimodal imaging molecular lentivirus was successfully constructed, and the purified cerebrospinal fluid-contacting neurons could survive, proliferate and express GFP. GFP+ cerebrospinal fluid-contacting neurons could be passaged continuously for more than four generations in vitro, and express neural stem cell markers Nestin and Sox2. After induced differentiation, GFP+ cerebrospinal fluid-contacting neurons expressed neuron marker NeuN, astrocyte marker S100β and oligodendrocyte marker O4. It is concluded that cerebrospinal fluid-contacting neurons can be specifically labeled by multimodal image molecular viruses and show the ability of self-renewal and multidirectional differentiation, which adequately proves that cerebrospinal fluid-contacting neurons have the characteristics of neural stem cells in vitro.

目的:探讨肢体骨肉瘤患儿外周血中LncRNA ANRIL和LncRNA MALAT1表达及与预后的关系.方法:选择我院于2016年09月至2021年09月肢体骨肉瘤患儿93例作为研究对象;另选择我院2016年09月至2021年09月健康体检儿童50例作为对照组.采用实时荧光定量PCR(qRT-PCR)测定外周血LncRNA ANRIL和LncRNA MALAT1相对表达量.随访至2022年08月31日,记录患儿总生存期(OS).比较2组外周血LncRNA ANRIL和LncRNA MALAT1相对表达量;骨肉瘤不同病理特征LncRNA ANRIL和LncRNA MALAT1相对表达量;预后与LncRNA ANRIL和LncRNA MALAT1相对表达量的相关性;采用多因素Logistic回归分析影响骨肉瘤患儿预后的独立危险因素.结果:骨肉瘤组外周血LncRNA ANRIL和LncRNA MALAT1相对表达量高于对照组(P＜0.05).不同性别、年龄、组织学分型和发病部位外周血LncRNA ANRIL和LncRNA MALAT1相对表达量比较无显著差异(P＞0.05);Ⅲ期外周血LncRNA ANRIL和LncRNA MALAT1相对表达量高于I期和Ⅱ期(P＜0.05).肢体骨肉瘤患儿随访12～72个月,中位随访时间43个月.LncRNA ANRIL高表达OS时间短于LncRNA ANRIL低表达(P＜0.05);LncRNA MALAT1高表达OS时间短于LncRNA MALAT1低表达(P＜0.05).经多因素Logistic回归分析显示,Enneking分期Ⅲ期、LncRNA ANRIL高表达和LncRNA MALAT1高表达为影响骨肉瘤患儿预后的独立危险因素.结论:肢体骨肉瘤患儿外周血中LncRNA ANRIL和LncRNA MALAT1高表达,且与患儿Enneking分期和预后密切相关,Enneking分期Ⅲ期、LncRNA ANRIL和LncRNA MALAT1高表达为影响肢体骨肉瘤患儿预后的独立危险因素.

触脑脊液神经元(CSF-cNs)存在于中枢神经系统不同区域,其通过胞体、轴突及树突与脑脊液接触,具有分泌、感知、调节运动及信息传递等功能.此外,CSF-cNs还表达成熟及不成熟神经元标志物,并在生物体成年后持续存在,可能参与神经发生过程.但目前对CSF-cNs的生物学功能研究并不透彻,其在神经系统中的作用尚未完全清楚.随着CSF-cNs特异性标志物多囊肾病蛋白2-样1离子通道的发现,已证实其在体外具有神经干细胞特性.因此,研究CSF-cNs的分类、起源、生理特性及功能对了解神经系统并治疗脊髓损伤具有重要意义.

目的 总结慢性移植物抗宿主病相关干眼的诊治经验.方法 回顾性分析1例接受异体骨髓造血干细胞移植术后干眼病例的临床资料.结果 患者异体骨髓造血干细胞移植术后3年双眼出现严重的干眼表现,通过局部使用绷带镜及激素、免疫抑制剂、人工泪液等点眼治疗后干眼症状好转但治疗过程发生了急性结膜炎,故停止使用绷带镜、加强抗感染治疗后好转.结论 慢性移植物抗宿主病相关干眼是异体骨髓造血干细胞移植术后最多见的眼部表现,与患者生存质量密切相关.认识此病的危害性,早期及时诊治具有重要意义.

目的:探讨Notch 1信号通路在调节小鼠触液神经元增殖中的作用.方法:(1)取出生24h内C57BL/6小鼠延髓组织,消化后提取细胞,经流式细胞分选得到触液神经元(cerebrospinal fluid-contacting neurons,CSF-cNs).将CSF-cNs悬浮培养并传代.(2)将CSF-cNs传至第2代,培养0h、24h、48h、72h、96h、120h时应用CCK-8法检测CSF-cNs光密度(optical density,OD)值,培养5d时应用免疫荧光检测CSF-cNs特异性标志物多囊肾病2型1通道蛋白(polycystic kidney disease 2 like 1,PKD2L1)与神经干细胞标志物Nestin、Sox2及增殖标志物Ki67共表达情况.(3)将第3代CSF-cNs分为4组:对照组、Jagged-l组、二甲基亚砜(dimethyl sulfoxide,DMSO)组、γ-分泌酶抑制剂(3,5-二氟苯乙酰基)-L-丙氨酰基-L-2-苯基甘氨酸叔丁酯[(3,5-difluorophenylacetyl)-L-alanyl-L-2-phenylglycine tert-butyl ester,DAPT]组.对照组采用无血清神经培养液培养;Jagged-1组采用无血清神经培养液+5μmol/L Jagged-1培养;DMSO组采用无血清神经培养液+0.05％DMSO培养;DAPT组采用无血清神经培养液+50μmol/L DAPT培养.培养0h、24h、48h、72h、96h、120h时应用CCK-8法检测各组CSF-cNs的OD值;培养5d时应用Western Blot检测各组CSF-cNs PKD2L1、Notch l、Notch受体胞内段(NICD)、Hes1、β-actin蛋白表达情况,应用免疫荧光法检测各组CSF-cNs增殖标志物Ki67蛋白荧光强度(arbitrary unit,A.U.)值.结果:(1)提取的细胞经流式细胞分选后得到的CSF-cNs纯度为(94.5±2.03)％,存活率为(93.64±2.35)％.CSF-cNs可连续传代并形成神经球.(2)第2代CSF-cNs培养0h、24h、48h、72h及96h时的OD值具有统计学差异(P＜0.05),96h与120h的OD值无统计学差异(P=0.44).CSF-cNs中PKD2L1可与Nestin、Sox2或Ki67共表达.(3)第3代CSF-CNS分组处理后各组细胞PKD2L1蛋白表达量无统计学差异(P=0.27).Jagged-1组细胞Notch l、NICD、Hesl蛋白表达量均较对照组升高(P＜0.01);72h、96h、120h的OD值较对照组均显著性升高(P72h=0.03,P96h=002,P120h=0.01);Ki67蛋白A.U.值较对照组显著性升高(P＜0.01).DAPT组细胞Notch l、NICD、Hesl蛋白表达量较对照组均显著性减低(P＜0.01);72h、96h、120h的OD值与对照组比较均显著性降低(P＜0.01);Ki67蛋白A.U.值较对照组显著性降低(P＜0.01).DMSO组各项指标与对照组比较均无统计学差异(P＞0.05).结论:CSF-cNs在体外具有神经干细胞潜能;激活Notch 1信号通路可增强CSF-cNs的增殖能力,抑制Notch 1信号通路可降低CSF-cNs的增殖能力.

目的:对省属三甲医院对口帮扶少数民族地区贫困县级医院效果进行调研,探讨其作用和意义.方法:选择帮扶及受帮扶医院,进行问卷调查、专访和深度交谈,结果用统计学方法处理.结果:三甲医院对所帮扶的县级医院开展了诊疗、手术及示教、会诊、教学查房、举办讲座和管理指导等方式,取得较好的效果,使受帮扶医院的管理和医疗服务水平大大提升,建立了重点专科、开展了新技术、壮大了人才队伍.结论:可持续性、针对性对口帮扶提升了少数民族地区贫困县级医院的医疗服务水平,缓解了"看病难、看病贵"现状.

目的 观察翼状胬肉切除联合自体带蒂球结膜移植术的治疗效果,总结手术方法 .方法70例单眼翼状胬肉患者,随机分为甲组(30例)与乙组(40例).甲组给予翼状胬肉切除术治疗,乙组给予翼状胬肉切除联合自体带蒂球结膜移植术治疗.比较两组患者的治愈情况、复发情况、视力水平.结果 乙组治愈率95.0％高于甲组的73.3％,乙组复发率7.5％低于甲组的30.0％,乙组视力≥0.5者占75.0％高于甲组的46.7％,差异均具有统计学意义(P<0.05).结论 翼状胬肉患者接受翼状胬肉切除联合自体带蒂球结膜移植术治疗,疗效显著,能明显改善患者视力水平,复发率较低,值得推广.

目的:应用裂隙灯显微镜下采用自制角膜异物取出针取出角膜异物的临床疗效分析.方法:回顾性分析我院2011年7月-2015年12月期间在我院眼科门诊首次接受治疗的100例角膜异物患者的临床资料.采用随机抽样法分为甲乙两组,甲组(传统针头组)50眼,乙组(自制针头组)50眼,对两组患者患眼的操作次数、术后愈合情况和异物残留情况进行计量记录.结果:使用自制针头组的50例患者剔取异物手术过程中造成异物周围角膜损伤小,一次性取出成功率高,术后反应轻,创面容易愈合,减轻了患者再次取出异物的痛苦.结论:选择适当的手术器械对角膜异物的剔除非常重要.用自制针头做的一次性角膜异物针,经济方便,安全适用,便于推广.一人一针,符合消毒隔离规范,无医院内交叉感染发生.

脊髓损伤(Spinal cord injury,SCI)是中枢神经系统严重损伤性疾病,具有高死亡率和高致残率等特点.随着医疗及护理技术的不断进步,SCI的患者生存率得到明显提高.目前,SCI除手术治疗外,药物治疗也具有不可替代的作用.近年来,研究人员关于脂类激素治疗SCI的实验性研究不断深入,逐步了解了脂类激素潜在的神经保护功能,这为将来脂类激素作为神经保护剂应用于SCI的治疗提供了可能.

Neural stem cells (NSCs) are stem cells that with the ability of self-renew and multipledifferentiation potential. Under certain circumstances, NSCs are able to differentiate into neurons, astrocytes and oligodendrocytes, thereby participating in the neurogenesis and injury repair. The specific microenvironments regulating neural stem cells, commonly referred to as neurogenic niches, comprise multiple cell populations whose precise contributions are under active current exploration. Understanding the cross-talk between neural stem cells and their niche components is essential for the development of therapies against neurodegenerative disorders and spinal cord injury. In this review, we describe and discuss recent studies that identified novel components in the neural stem cell niche. These discoveries bring new concepts to the field. Therefore, we evaluate these recent advances that change our understanding of the neural stem cell niche and its influence on neural stem cell function. Key words: Neural stem cell; Niche; Nerve regeneration

接触脑脊液神经元(cerebrospinal fluid-contacting neurons,CSF—CNs)作为一种特殊类型中枢神经细胞,通过其树突或轴突穿过脑脊液-脑屏障并与脑脊液接触.它具有感受器、分泌、运输调节等功能,但因其缺乏特异分子标记物,使其研究受到一定阻碍.近期研究证实PKD2L1为其特异性标记物,并发现接触脑脊液神经元可能为神经干细胞来源之一,对于临床研究神经系统损伤修复具有重要意义.

脊髓损伤(spinal cord injury,SCU是一种高度致残性疾病,主要造成脊髓轴突的断裂和神经元的死亡[1],目前还没有一种有效的方法可以治愈SCI.在中枢神经系统中发现了有一些具有分化为神经元潜能的内源性神经干细胞(endogenous neural stem cells,ENSCs),利用脊髓中ENSCs来修复SCI具有独特优势,但是成体中ENSCs数量有限,其具体种类尚无定论,激活机制尚不清楚,活化后其增殖能力和分化潜能有限[2-4].因此,研究成年哺乳动物脊髓中ENSCs的来源、ENSCs的激活机制及其增殖、迁移和分化的调控机制,对更好地实现SCI内源性修复具有重要意义.笔者就SCI后ENSCs的激活及其机制综述如下.

Objective To observe the expression of nestin and bcl-2 at different time points after spinal cord injury in rats,find the correlation between endogenous neural stem cell proliferation and the anti-apoptotic factor Bcl-2 gene expression.Methods Two groups were randomly divided from thirty wistar rats,the spinal cord injury group (group SCI,n =25),and the sham surgery group (group sham,n =5).The spinal cord inj ury was made by oppress method used Aneurysm clip in group SCI.The behavioral evaluation was performed with the BBC scale.The rats were killed at 1d,3d,7d,14d and 2 1d after the injury,selected the tissue and divided them into group,the expression of nestin and Bcl-2 were analyzed in the samples by immunohistochemistry.The statistical analysis was used to observe the expression of Bcl-2 and nestin gene and evaluate the relationship between the proliferation of ENSCs and the expression of Bcl-2.Results All 30 rats were involved in the result analysis.It was showed that there was little expression of nestin at the group sham.But at the group SCI,there were some nestin positive cells 1 day after injury and the expression began increasing at 3 days after injury.They increased obviously 7d after injury at the central canal and gray matter of the spinal,and gradually reduced on the 14d,and on the 21d nestin positive cells were obviously reduced.At all group of SCI,there were Bcl-2 positive expression,one day after SCI the expression obvious,they reached the highest point 3 days after injury and declined gradually 7 days after injury,and leaved a little obvious express cells 14 days and 21days after injury.The expression of nestin and Bcl-2 had correlation after SCI.Conclusion The endogenous neural stem cell (ENSC) and Bcl-2 were all expressed after SCI,and the expression of Bcl-2 probably facilitate the proliferation of neural stem cells.

Objective To observe the effect of lentiviral vector-mediated basic fibroblast growth factor (bFGF) gene transfection on the biological characteristics of rabbit bone marrow stromal cells(BMSCs)under in vitro culture conditions. Methods BMSCs were obtained by density gradient centrifugation and adherence screening. The bFGF gene was transfected into BMSCs by lentiviral vector and divided into bFGF transfection group,empty virus group and untransfected group according to the transfection conditions.After transfection,the morphology,expressions of bFGF mRNA and protein, cell proliferation,cell cycle and alkaline phosphatase(ALP)activity were observed in three groups of cells. Results High density BMSCs were successfully obtained by density gradient centrifugation and adherence screening.After transfection of BMSCs with bFGF gene, the cell morphology showed no significant changes, while the expressions of bFGF mRNA and protein were significantly increased, the cell proliferation curve shifted upward, the proportion of proliferating cells increased,and the activity of ALP was significantly enhanced.There were significant differences between three groups(P<0.05).Conclusion The rabbit bFGF gene is successfully introduced into the BMSCs cultured in vitro by lentiviral vector, and the target gene is stably expressed.The expression of bFGF can promote the proliferation and osteogenic differentiation of BMSCs.

脊髓损伤是一种发生于中枢神经系统的严重创伤性疾病.干细胞移植手术是临床上治疗脊髓损伤的常用方法.研究发现,对脊髓损伤患者进行干细胞移植手术,无法完全阻断由脊髓损伤造成的一系列病理反应.故临床上对脊髓损伤患者在进行干细胞移植手术的同时,会尝试使用其他的方法对其进行治疗,以便让移植的干细胞处于一个有利的微环境中,从而确保其治疗的效果.本文主要研究用以干细胞移植手术为基础的联合疗法治疗脊髓损伤的效果,以期为此病的治疗提供参考.

[Objective] To evaluate the efficacy of FGF-2 gene over expression lentivirus transfected BMSCs in the construction of tissue-engineered bone graft for avascular necrosis of femoral head in rabbits.[Methods] The tissue-engineered bone was constructed with FGF-2 gene overexpressed lentivirus-transfected BMSCs and XACB.After the model of early avascular necrosis of femoral head was established by LPS combined with hormone in rabbit,the animals were randomly divided into 5 groups:including the group A (control),B (XACB),C (XACB+rBMSCs),D (XACB+rBMSCs+Lv-GFP) and E (XACB+rBMSCs+Lv-FGF2/GFP) in term of the materials implanted in the femoral heads.The results were evaluated by gross anatomy and X-ray examination,HE staining to assess the effect of the tissue engineering bone on rabbit avascular necrosis of the femoral head.[Results] FGF-2 gene was transfected into rBMSCs (MOI=100) by lentivirus with efficiency above 95％,because rBMSCs were successfully overexpressing FGF-2 by qPCR and Western Blot.After tissue engineering bone implantation,X-ray examination and gross specimen observation showed that the repair area in the group E was more than 80％ at 6 weeks,and the defect was completely repaired at 12 weeks,osteogenesis was stronger than other groups.X-ray score and new bone formation area in the group E were statistically higher than other groups (P＜0.05).[Conclusion] The tissue engineering bone constructed by FGF-2 gene overexpression lentivirus transfected BMSCs and XACB does effectively promote the repair of avascular necrosis of the femoral head.

目的 观察移植慢病毒转染碱性成纤维细胞生长因子的骨髓基制干细胞复合培养的组织工程骨(XACB/LV-bFGF/MSCs)治疗的兔股骨头缺损坏死模型中骨保护素(osteoprotegerin,OPG)、核因子-κB受体活化因子配体(receptoractivator of NF-кB ligand,RANKL)的表达变化情况,探讨组织工程骨对股骨头坏死的治疗效果.方法 6～7个月龄新西兰大白兔60只,运用地塞米松联合马血清注射法制作股骨头坏死模型,应用组织工程骨修复股骨头坏死模型.根据修复方法不同分为5组,A组为空白对照组,B组植入XACB,C组植入XACB+MSCs,D组为XACB+MSCs+空病毒,E组植入XACB+LV-bFGF+MSCs.术后分别于3、6、12周3个时间点取股骨头松质骨,采用蛋白免疫印迹法(Western Blot)检测OPG和RANKL的蛋白表达.结果 OPG蛋白表达:各时间点E组大于A组、B组、C组、D组,E组与A、B、C、D组比较差异均有统计学意义(P<0.05),C组与D组比较差异无统计学意义(P>0.05).RANKL蛋白表达:E组大于A组、B组、C组、D组,E组与A、B、C、D组比较差异均有统计学意义(P<0.05),C组与D组比较差异无统计学意义(P>0.05).结论 通过运用组织工程骨修复的兔股骨头坏死组中OPG蛋白表达上调,修复效果较其他组更优,表明XACB/LV-bFGF/MSCs有望作为一种有效的组织工程材料用于股骨头坏死修复中.

Objective: The present study aimed to investigate the effect of the transplantation of basic fibroblast growth factor (FGF-2) gene-transfected mesenchymal stem cells (MSCs) and xenogeneic antigen-cancellous bone (XACB) on tumor necrosis factor-α (TNF-α) expression with avascular necrosis of the femoral head (ANFH) in rabbits. Methods: The models of steroid-induced osteonecrosis in rabbits were randomly divided into five groups: A (model), B (XACB), C (XACB + MSCs), D (XACB + MSCs + LV), and E (XACB + MSCs + LV-FGF-2) groups. The therapeutic effect was evaluated by Hematoxylin and Eosin (H&E) staining. Immunohistochemical and RT-PCR assays were used to detect the protein and mRNA expression of TNF-α in the femoral head, respectively. Results: At 12 weeks after the operation, the defect in rabbits in group E was completely repaired, while defects in rabbits in the other groups were not completely repaired, and the area of new bone formation was higher, when compared with the other groups (P<0.05). Furthermore, the protein and mRNA expression TNF-α was lower at 3, 6, and 12 weeks after surgery, when compared with the other groups, and the difference was statistically significant (P<0.05). Conclusion: FGF-2/MSCs/XACB could promote the repair of ANFH, and may be correlated to the inhibition of TNF-α expression.

脊髓损伤(spine cord injury,SCI)是临床上常见的一种中枢神经系统疾病.此病患者的主要临床表现为由脊柱创伤导致的损伤平面下的运动感觉功能受损.根据脊髓损伤的病理过程可将其分为原发性脊髓损伤和继发性脊髓损伤.继发性神经元大量死亡是导致脊髓损伤难以治愈的重要原因.目前临床上主要采用手术减压、药物治疗、自体外周神经移植等多种方法对脊髓损伤患者进行综合治疗,但效果不够理想.有动物实验表明,对脊髓损伤动物模型进行干细胞移植治疗可为其受损的脊髓补充神经元或分化细胞,促进其体内具有修复功能的神经因子的分泌和神经传导通路的再生,从而可有效地促进其受损脊髓组织的修复.目前,用干细胞移植疗法治疗脊髓损伤已成为国内外学者研究的热点.

Objective To prepare complex of lentivirus-mediated fibroblast growth factor 2(FGF-2)gene which was used to transfect into bone marrow stromal stem cells(MSCs)and then were co-cultured with xenogeneic antigenic cancel-lous bone(XACB),and to observe its therapeutic effect on steroid-induced avascular necrosis of femoral head and its effect on the expression of TNF-αin femoral head tissues.Methods Rabbit MSCs were isolated and cultured in vitro.The lenti-virus-mediated FGF-2 gene was used to transfect the MSCs and then were cultured with XACB to obtain tissue engineering bone of XACB/MSCs/Lv-GFP-FGF-2 complex.Sixty rabbit models of steroid-induced avascular necrosis of femoral head which were established by injection of endotoxin and methylprednisolone were randomly divided into 5 groups with 12 in each group.Core decompression and bone grafting were performed.In group A, bone grafting was not performed,autolo-gous cancellous bone was implanted in the group B, XACB/MSCs were implanted in the group C, XACB/MSCs/Lv-GFP was implanted in the group D,and XACB/MSCs/Lv-GFP-FGF-2 was implanted in the group E.Four rats were executed at 3,6,12 weeks after operation in each group,then we observed the general appearance of femoral head.The positive ex-pression rate of TNF-αwas detected by immunohistochemistry.The real-time PCR was used to detect the expression of TNF-αmRNA in the femoral head tissues.Results Three weeks after operation, a small amount of new bone formation were found in the group E,there were no new bone formation in the other groups.At 6 weeks after operation,the defect ar-eas in the group A were filled with granulation tissue and there were no new bones.The defect areas in the group B,C and D were still visible and the new bones were visible at the edge.Most of the defect areas in the group E were filled with new bones,and bone graft was basically absorbed.At 12 weeks after operation,only a small amount of new bone was formed in the group A,and defect areas of the groups B,C,and D were not repaired completely.The defect in the group E was re-stored to normal cancellous bone.At 3,6,and 12 weeks after operation,the positive expression rate of TNF-αand relative expression of mRNA in the femoral head tissues of the groups B,C,D,and E were decreased gradually(all P<0.05). At 3 weeks after operation,the positive expression of TNF-αand the mRNA relative expression of femoral head in the group E were lower than those in the groups A,B,C,and D(all P<0.05).At 6,12 weeks after surgery,the TNF-αpositive expression rate and mRNA relative expression level of the femoral bone tissue at the same time point were in the following order:group A>group B >group C, group D >group E(all P <0.05).Conclusion The tissue engineering bone XACB/MSCs/Lv-GFP-FGF-2 complex is successfully prepared,and it has a good effect on the treatment of steroid-induced avascular necrosis of the femoral head in rabbits by inhibiting the expression of TNF -αin the femoral head tissues.