[摘要] 目的 对比2种不同表面材质培养瓶培养的人脐带间充质干细胞（human umbilical cord mesenchymal stem cells, hUC-MSCs）的培养特性。 方法 采集10条新鲜脐带，每条脐带用组织块法分离脐带组织并贴壁培养12d，收集原代细胞，分别取2*106个细胞用225cm2高粘培养瓶（简称A组）和225cm2普通瓶子（简称B组）细胞培养72h至P1代,收集P1代细胞继续培养72h至P2代，实验重复五次，观察比较两组实验组的P1和P2代培养瓶的细胞镜下形态、P2代比较两组实验组细胞消化时长、细胞扩增曲线、检测表面标志物、三系分化潜能，分别收集细胞培养至P4代比较SA?β?半乳糖苷酶染色阳性率。结果 2组细胞形态均为扁平长梭形。 P2代A组细胞消化时间为（123.8 ±3.09）s，B组细胞消化时间（38.5 ±2.20）s,差异有统计学意义(P<0.001)。A组的P0到P2代细胞扩增倍数为（129.49±0.89）倍，显著高于B组（101.4±1.67）倍，差异有统计学意义(P<0.001)。A组SA?β?半乳糖苷酶染色阳性率（2.58±0.44）%显著低于B组（4.79±0.33）%，且均符合间充质干细胞的质量标准。结论 相同接种密度的条件下，高粘培养瓶比普通培养瓶扩增倍数更高，且细胞衰老水平更低，优化了培养体系和培养效率，提高了细胞质量。

[Abstract] Objective: To compare the culture characteristics of human umbilical cord mesenchymal stem cells (hUC-MSCs) cultured in two different surface-material flasks. Methods: Ten fresh umbilical cords were collected, and umbilical cord tissue was isolated using the tissue block method and cultured adherently for 12 days. Primary cells were collected, and 2×10^6 cells were respectively seeded into 225 cm2 high-adhesion flasks (Group A) and 225 cm2 ordinary flasks (Group B) for culture for 72 hours until passage 1 (P1). P1 cells were collected and further cultured for 72 hours until passage 2 (P2). The experiment was repeated five times. The morphology of cells at P1 and P2 in both groups was observed under a microscope, and P2 cells were compared for digestion duration, cell growth curves, surface marker expression, and trilineage differentiation potential. Cells were also cultured to P4 to compare the SA?β-galactosidase positivity rate. Results: Cells in both groups displayed a flat spindle-shaped morphology. The digestion time of P2 cells in Group A was (123.8 ± 3.09) seconds, while in Group B it was (38.5 ± 2.20) seconds, showing a statistically significant difference (P<0.001). The cumulative cell amplification from P0 to P2 in Group A was (129.49 ± 0.89)-fold, significantly higher than that in Group B [(101.4 ± 1.67)-fold, P<0.001]. The SA?β-galactosidase positivity rate in Group A was (2.58 ± 0.44)%, significantly lower than in Group B(4.79 ± 0.33)%, and both met the quality standards for mesenchymal stem cells. Conclusion: Under the same seeding density, high-adhesion flasks yield higher cell amplification, lower cell senescence, and optimize the culture system and efficiency, thereby improving cell quality.

      目的   通过生物信息学手段筛选非小细胞肺癌（NSCLC）中的关键靶点基因，识别预后标志物NDC80，并探讨其在NSCLC中的表达意义，进而分析NDC80作为NSCLC基因治疗靶点的可行性。方法   采用癌症基因组图谱（TCGA）TCGA数据库检索NSCLC相关数据，进行加权基因共表达网络分析（WGCNA）以识别关键基因，并进行差异表达分析、相关性分析和蛋白互作网络构建。对筛选出的关键基因进行功能分析。利用免疫组化染色法检测癌组织及癌旁组织中NDC80蛋白的表达水平，并进一步探究其与临床病理特征的关系。采用Kaplan-Meier法分析NDC80表达与NSCLC患者无进展生存时间（PFS）的关系。结果   共筛选出20个与NSCLC高度关联的关键基因，包括CDC20、CDK1、MCM4、CDC6、MCM2、PLK1、NDC80、CCNB1、CDC45、AURKA、MCM8、BUB1、CDT1、ORC1、CCNA2、CASC5、MAD2L1、BUB1B、CENPA、AURKB。免疫组化验证显示，NDC80蛋白在NSCLC组织中高表达，其在NSCLC组（阳性表达率88.6%）显著高于癌旁组（50.0%）（P＜0.05）。NDC80蛋白的阳性表达率在TNM分期（Ⅲ期+Ⅳ期）、低分化、淋巴结转移的NSCLC组高于TNM分期（Ⅰ期+Ⅱ期）、高分化及中分化以及未发生淋巴结转移的NSCLC组（P＜0.05）。NDC80蛋白的阳性表达率在不同性别、年龄、病灶大小分类的NSCLC组织中无显著差异（P＞0.05）。Kaplan-Meier分析显示，NDC80蛋白高表达组的PFS中位数为（9.00±0.27）个月，明显低于低表达组（11.00±0.79）个月（P＜0.05）。结论   本研究发现的关键基因在NSCLC干细胞的维持中发挥重要作用。免疫组化结果显示，NDC80蛋白在NSCLC组织中高表达，且与肿瘤分化、TNM分期及淋巴结转移密切相关。NDC80蛋白高表达组的PFS明显低于低表达组，提示NDC80可能成为NSCLC筛查、治疗和预后评估的潜在生物标志物。

      Objective  To screen the key target genes in non-small cell lung cancer（NSCLC）by bioinformatics，identify the prognostic marker NDC80，and explore its expression significance in NSCLC，so as to analyze the feasibility of NDC80 as a gene therapy target for NSCLC．Methods  TCGA database was used to retrieve NSCLC-related data，and weighted gene co-expression network analysis（WGCNA）was used to identify key genes，and differential expression analysis，correlation analysis and protein-protein interaction network construction were carried out．The function of the selected key genes was analyzed．Immunohistochemical staining was used to detect the expression level of NDC80 protein in cancer tissues and adjacent tissues，and to further explore its relationship with clinicopathological features．Kaplan-Meier method was used to analyze the relationship between NDC80 expression and progression-free survival （PFS）of NSCLC patients．Results  A total of 20 key genes highly associated with NSCLC were screened out，which were CDC20，CDK1，MCM4，CDC6，MCM2，PLK1，NDC80，CCNB1，CDC45，AURKA，MCM8，BUB1，CDT1，ORC1，CCNA2，CASC5，MAD2L1，BUB1B and CENPA．Immunohistochemical  verification  showed that NDC80 protein was highly expressed in NSCLC tissue，and its positive expression rate in NSCLC group（88.6%）was significantly higher than that in adjacent cancer group（50.0%，P＜0.05）．The positive expression rate of NDC80 protein in NSCLC with TNM staging（Ⅲ+Ⅳ），low differentiation and lymph node metastasis was higher than that in NSCLC with TNM staging（Ⅰ+Ⅱ），high differentiation and moderate differentiation and no lymph node metastasis（P＜0.05）．There was no significant difference in the positive expression rate of NDC80 protein among NSCLC tissues with different gender，age and lesion size（P＞0.05）．Kaplan-Meier analysis showed that the median PFS of high expression group of NDC80 protein was（9.00±0.27）months，which was significantly lower than that of low expression group（11.00±0.79）months（P＜0.05）．Conclusions  The key genes found in this study play an important role in the maintenance of NSCLC stem cells．Immunohistochemical results showed that NDC80 protein was highly expressed in NSCLC，and it was closely related to tumor differentiation，TNM staging and lymph node metastasis．The PFS of high expression group of NDC80 protein was significantly lower than that of low expression group，suggesting that NDC80 may become a potential biomarker for screening，treatment and prognosis evaluation of NSCLC．

脊髓损伤是一种高致残性中枢神经系统疾病,目前缺乏有效的治疗措施。干细胞组织工程兴起和神经调控技术的发展,给脊髓损伤的治疗带来新的希望。目前,多项针对脊髓损伤的干细胞相关治疗项目的临床研究已在全球注册,干细胞疗法是脊髓损伤领域的研究热点,具有良好的应用前景。而神经调控技术一直在临床上脊髓损伤后的康复治疗中发挥着重要作用,特别是靶向神经调控技术近年在脊髓损伤治疗方面取得突破性进展。有研究尝试联合干细胞疗法和神经调控技术应用治疗脊髓损伤,试图取得更好的效果。本文综述了干细胞疗法和神经调控技术在脊髓损伤治疗中的研究进展,旨在探讨其作用效果、修复机制、应用前景以及面临的问题,进一步为脊髓损伤的基础研究和临床转化提供参考。

Spinal cord injury is a highly disabling disease of the central nervous system without effective treatment to date．With the rise of stem cell and tissue engineering,and the breakthrough of neuromodulation technology,it brings new hope to the treatment of spinal cord injury．At present,a number of clinical studies on stem cell-related treatment projects for spinal cord injury have been registered worldwide,which has become a research hotspot．Neuromodulation technology has been playing an important role in the clinical rehabilitation of spinal cord injury．Especially,breakthroughs have been made in the treatment of spinal cord injury by targeted neuromodulation technology in recent years,which is encouraging．Some studies have attempted to combine stem cell therapy and neuromodulation technology to treat spinal cord injuries in an attempt to achieve better effect．This review summarizes the research progress of stem cell therapy and neuromodulation technology in the treatment of spinal cord injury,with the aim of discussing their effect,repair mechanisms,application prospect and various problems to face,and providing further reference for the basic research and clinical transformation of spinal cord injury．

目的 探讨外周血CD34阳性（CD34⁺）细胞计数对普乐沙福自体干细胞动员效果的预测价值。方法 回顾性分析2021年5月—2023年7月中山大学附属第七医院使用人粒细胞集落刺激因子（G-CSF）联合普乐沙福进行自体干细胞动员的13例患者临床资料,分析普乐沙福动员前后外周血CD34⁺细胞计数的变化及干细胞采集情况。结果 共有13例患者纳入研究,包括淋巴瘤10例和多发性骨髓瘤3例。多发性骨髓瘤患者中1例为新诊断,另2例为复发患者;淋巴瘤患者中3例为套细胞淋巴瘤,6例为弥漫大B细胞淋巴瘤（包括1例复发）,1例为B细胞淋巴瘤（不能明确类型）。本研究纳入的患者均使用G-CSF动员,在使用普乐沙福后CD34⁺细胞计数均升高,使用普乐沙福前中位CD34⁺细胞计数为13.3（2.5~76.1）/μL,使用普乐沙福后中位CD34⁺细胞计数为73.6（10.4~208.70）/μL,升高4.18（1.99~13.60）倍。13例患者中有2例患者在使用普乐沙福前外周血CD34⁺细胞计数<5 /μL,均动员失败。Spearman相关分析结果显示,使用普乐沙福后CD34⁺细胞计数与使用普乐沙福前CD34⁺细胞数呈正相关（r_s=0.769,P=0.003）。多元线性回归分析显示,使用普乐沙福后CD34⁺细胞计数能较好地预测采集结果（P=0.004）。结论 监测外周血CD34⁺细胞计数可预测普乐沙福自体干细胞动员效果,使用普乐沙福后CD34⁺细胞计数越多,CD34⁺细胞采集量越大。

Objective To explore the predictive value of peripheral blood CD34⁺ cell count for the stem cell mobilization effect of plerixafor．Methods The clinical data of 13 patients who used granulocyte colony-stimulating factor + plerixafor for stem cell mobilization in the Seventh Affiliated Hospital of Sun Yat-sen University from May 2021 to July 2023 were retrospectively analyzed．The changes of peripheral blood CD34⁺ cell count in all patients before and after the mobilization of plerixafor were analyzed．Results In 13 enrolled patients,there were 10 lymphoma patients and 3 multiple myeloma（MM）patients．One patient was newly diagnosed with MM,and the other two were recurrent patients．The lymphoma cases included 3 mantle cell lymphoma,6 diffuse large B cell lymphoma and 1 B cell non-Hodgkin's lymphoma（type cannot be specified）．The CD34⁺ cell counts were increased in all patients when mobilized with granulocyte colony-stimulating factor before plerixafor．The CD34⁺ cell count was 13.3（2.5~76.1）/μL and 73.6（10.4~208.70）/μL before and after the use of plerixafor,between which the difference was statistically significant（Z=0.578,P<0.05）,and the median increased of 4.18（1.99~13.6）times．There were 2 patients failed in mobilizing whose CD34⁺ cell count was less than 5 /μL before using plerixafor．Spearman analysis showed that there was a positive correlation in peripheral blood CD34⁺ cell count before and after the use of plerixafor（r_s=0.80,P=0.032）．The CD34⁺ cell count after using plerixafor was a good predictor of the collection results（P=0.002）．Conclusions Monitoring the CD34⁺ cell count in peripheral blood has a certain predictive value for the stem cell mobilization effect of plerixafor．The higher of CD34⁺ cell count after the use of plerixafor,the higher of CD34⁺ collection．

目的 观察聚乙二醇化重组人粒细胞刺激因子(PEG-rhG-CSF)与重组人粒细胞刺激因子(rhG-CSF)在造血干细胞移植后促进造血恢复的疗效对比。方法 回顾分析2016年1月—2020年12月以来在深圳市第二人民医院血液科进行造血干细胞移植的恶性血液疾病患者共 100例,随机分为2组,分别在造血干细胞回输后给与聚乙二醇化重组人粒细胞刺激因子与重组人粒细胞刺激因子。结果 PEG-rhG-CSF组与rhG-CSF组中性粒细胞植入时间分别为(18.7±3.4)天、(18.0±3.1)天,P=0.281,无统计学差异。粒细胞缺乏伴发热在PEG-rhG-CSF组与rhG-CSF组分别发生26例、29例,发生率分别为53.06%、56.86%,P=0.89,无差异。用药次数分别为2.6次(2~5次)、18.1次(11~31次),P<0.05,差异有统计学意义。不良反应主要为骨痛、肌肉疼痛。结论 PEG-rhG-CSF组与rhG-CSF组结果相似,PEG-rhG-CSF具有用药次数少的优势。

Objective The efficacy of pegylated recombinant human granulocyte stimulating factor (PEG-rhG-CSF) and recombinant human granulocyte stimulating factor(rhG-CSF) in promoting hematopoiesis recovery after hematopoietic stem cell transplantation.Methods The data of 100 patients with malignant blood diseases who underwent hematopoietic stem cell transplantation in the Hematology Department of Shenzhen Second People's Hospital from January 2016 to December 2020 were retrospectively analyzed.They were randomly assigned to two groups,which accepted PEG-rhG-CSF and rhG-CSF respectively after hematopoietic stem cell transfusion.Results The time of neutrophil implantation in PEG-rhG-CSF group and rhG-CSF group were (18.7±3.4) days and (18.0±3.1) days respectively,P=0.281,showing no statistical difference.There were 26 cases of neutropenia with fever in PEG-rhG-CSF group and 29 cases in rhG-CSF group,with incidence of 53.06% and 56.86% (P=0.89),showing no statistical difference.The times of medication were 2.6 times (2-5 times) and 18.1 times (11-31 times),P<0.05,with significant statistical difference.The main adverse reactions were bone pain and muscle pain.Conclusions The outcomes of PEG-rhG-CSF group and rhG-CSF group were similar,PEG-rhG-CSF had the advantage of fewer times of medication.

目的 探讨骨髓间充质干细胞源性微泡(BMSC-MV)修复大鼠早发性卵巢功能不全的自噬机制。方法 大鼠骨髓分离培养骨髓间充质干细胞;超速离心法从骨髓间充质干细胞培养液中分离微泡;腹腔注射顺铂溶液制备早发性卵巢功能不全(POI)模型,制备后3 d尾静脉取血ELISA检测血清雌二醇(E2)及卵泡刺激素(FSH);尾静脉注射BMSC-MV移植治疗POI大鼠模型,移植后28 d尾静脉取血ELISA检测E2、FSH及抗苗勒管激素(AMH),同时取卵巢组织检测自噬相关蛋白LC3及P62。结果 模型对照组及微泡移植组在模型制备后3 d的E2 含量低于正常对照组,FSH 含量高于正常对照组(P<0.001);微泡移植组在移植后28 d的E2、AMH含量高于模型对照组(P<0.001),FSH含量低于模型对照组(P<0.001);微泡移植组的LC3较模型对照组表达升高,而P62表达降低(P<0.001)。结论 BMSC-MV介导自噬修复大鼠早发性卵巢功能不全。

Objective To investigate the autophagy mechanism of bone marrow mesenchymal stem cell-derived microvesicle (BMSC-MV) in repairing premature ovarian dysfunction in rats. Methods The whole bone marrow adherence method was used to isolate,culture and identify BMSCs of SD rats. Microvesicles were isolated from bone marrow mesenchymal stem cell by ultracentrifugation. Premature ovarian insufficiency (POI) model was prepared by intraperitoneal injection of cisplatin solution,and serum estradiol (E2) and follicular stimulating hormone (FSH) were detected by ELISA from tail vein 3 days after preparation. Rat model of POI was treated with BMSC-MV transplantation by tail vein. Blood from tail vein was collected 28 days after transplantation to detect E2,FSH and AMH by ELISA. Meanwhile,ovarian tissues were collected to detect autophagy-related proteins LC3 and P62. Results The E2 content of the model control group and the microvesicle transplantation group was lower than that of the normal control group,and the FSH content was higher than that of the normal control group (P<0.001). The content of E2 and AMH in the microvesicle transplantation group at 28 days after transplantation was higher than that in the model control group (P<0.001),and the content of FSH was lower than that in the model control group (P<0.001). Compared with the model control group,LC3 expression in the microvesicle transplantation group was increased,while P62 expression was decreased (P<0.001). Conclusion BMSC -MV mediate autophagy to repair premature ovarian insufficiency in rats.

目的 采用生物信息学方法预测低氧预处理人胎盘绒毛膜间充质干细胞环状RNAs相对应的miRNA及其靶基因,并分析靶基因所参与的生物学过程和信号通路。方法 用Arraystar公司的商业软件为环状RNAs预测其相对应的miRNAs,分别用targetScan7.1和mirdbV5数据库预测miRNAs的靶基因,并取两个预测结果的合集,应用在线网站http://www.geneontology.org和http://www.genome.ad.jp/kegg对靶基因进行功能富集分析和信号通路富集分析。结果 功能富集分析表明,circRNAs的靶基因主要涉及到细胞发育、细胞分化和细胞发育调控。东京基因和基因组百科全书信号通路富集分析表明肿瘤中转录失控和有丝分裂原激活蛋白激酶(MAPK)信号通路最有意义,而且分析发现MAPK信号通路为核心通路。本研究表明,低氧预处理使得间充质干细胞中部分circRNAs的表达量发生差异性变化。结论 低氧预处理人胎盘绒毛膜间充质干细胞环状RNAs同低氧预处理间充质干细胞的生物学特性变化密切有关,为了解低氧预处理影响间充质干细胞特性发生变化的分子机制提供新思路。

Objective To predict the miRNA and its target genes of circular RNAs in hypoxia- preconditioned human palcenta chorionic mesenchymal stem cells using bioinformatics, and analyze the biological process and signaling pathway. Methods Arraystar's commercial software was used to predict the corresponding miRNAs of circular RNAs. The target genes of miRNAs were predicted by targetScan7.1 and mirdbV5 databases respectively, and an intersection of two prediction results was obtained. The online databases http://www. geneontology.org and http://www.genome.ad.jp/kegg performed functional enrichment analysis and signal pathway enrichment analysis of target genes. Results Functional enrichment analysis indicated that the target genes of circRNAs mainly involved cell development, cell differentiation and cell development regulation. The signal enrichment analysis of the Tokyo Gene and Genome Encyclopedia indicates that transcriptional misregulation in cancer and mitogen-activated protein kinase (MAPK) signaling pathway are most meaningful, and the MAPK signaling pathway is found to be the core pathway. This study showed that hypoxic preconditioning caused significant changes in the expression of mesenchymal stem cell circRNAs. Conclusion The changes of circular RNAs in hypoxia-preconditioned human placental chorionic mesenchymal stem cell is closely related to the biological characteristics of hypoxia-preconditioned mesenchymal stem cells. This study provides a new idea for understanding the molecular mechanism of hypoxic preconditioning affecting the changes of biological characteristics in mesenchymal stem cells.

目的 观察利妥昔单抗在治疗造血干细胞移植后血小板输注无效的有效性和安全性。方法 回顾分析我院2014年1月—2017年6月收治的11例利妥昔单抗治疗的造血干细胞移植后血小板输注无效的病例资料,其中包括重型地中海贫血8例,急性髓系白血病1例,重型再生障碍性贫血2例。结果 10例造血干细胞移植后血小板输注无效患者经利妥昔单抗治疗,375 mg/m²,每周1次,2~3次后血小板输注无效的状况明显改善;1例造血干细胞移植后血小板输注无效患者接受1次利妥昔单抗治疗,仍存在血小板输注无效,最终因颅内出血死亡。结论 利妥昔单抗是治疗造血干细胞移植后血小板输注无效的一种很有效的治疗方法。

Objective The purpose of our study was to evaluate the efficacy and safety of rituximab in the treatment of platelet transfusion refractoriness after hematopoietic stem cell transplantation. Methods We retrospectively analyzed 11paitents （8 thalassemia major,2 sever aplastic anemia,and 1 acute myeloid leukemia） with platelet transfusion refractoriness after hematopoietic stem cell transplantation. All 11 patients received treatment of rituximab. Results 10 of 11 platelet transfusion refractoriness patients after hematopoietic stem cell transplantation had improvement of platelets transfusion,1 patient of 11 platelet transfusion refractoriness patients had no response and died of intracranial hemorrhage. Conclusion Rituximab is a promising treatment in patients with platelet transfusion refractoriness after hematopoietic stem cell transplantation.

目的 探索不同浓度苯妥英钠(PHT)对大鼠牙周膜干细胞(rat periodontal ligament stem cells, rPDLSCs)粘附于牙根面的影响,为PHT应用于牙周重建提供一定的实验依据,为牙周炎的治疗提供了新思路。方法 提取大鼠rPDLSCs,培养并纯化。通过多项诱导分化、表面标记物鉴定后,使细胞在不同浓度PHT刺激条件下,与牙骨质片共同培养,检测粘附于牙骨质片上的细胞量并作比较。结果 20～80 mg/L 浓度范围内的PHT能够促进rPDLSCs的粘附数量,40 mg/L PHT组促进粘附的效果最强。实验组与对照组有显著统计学差异。结论 适合浓度的PHT可以促进rPDLSCs粘附于牙骨质表面,40 mg/L PHT组促进粘附的效果最强。

Objective To investigate the effects of PHT on attachment of rat periodontal ligament stem cells (rPDLSCs) to cementum chips, in order to provide a certain experimental basis for periodontal regeneration and new ideas for therapy of periodontitis. Methods To isolate rPDLSCs from SD rats, culture and purify them. To identify the cells by their apperance, induced multi-direction differentiation potential and cell surface markers. The rPDLSCs were cultured with cementum chips under the action of different concentrations of PHT. Then testing and comparing the amount of cells attached on the cementum chips in different groups. Results The concentraion among 20～80 mg/L of PHT can increase the number of attached cells. 40 mg/L PHT can promote the cells attachment mostly. Conclusion A proper concentration of PHT may promote rPDLSCs to attach to cementum chips′ surface, 40 mg/L PHT may promote the cell attachment mostly.

目的 探索内源性神经干细胞在大鼠海马可溶性因子中的体外发育归宿及分化鉴定。方法 显微镜下分离Wistar大鼠海马组织放置于低温DMEM/12培养基,低温振荡2小时后高速离心(15000 g),获取实验所用海马组织可溶性因子。取材出生1天的Wistar乳鼠海马中的内源性神经干细胞(endogenous neural stem cells, ENSCs),将ENSCs分别于含海马可溶性因子终浓度为0(对照组)、50、100、200、400 μl/mL的无血清DMEM/F12培养基中培养6天并每日观察,使用免疫细胞化学、Western Blot印记技术比较各组ENSCs中Nestin、CD133的表达量;同时计量并比较各组ENSCs成球个数,以探索在模拟颅内微环境情况下,ENSCs发育、归宿及分化。进一步于最适宜的海马可溶性因子终浓度中分化神经球,对分化的细胞行神经元特异性蛋白入(如:β-tubullin III、MAP2)及胶质细胞特异性蛋白(如:GFAP、S100及p75 NGFR)免疫细胞化学检测。结果 大鼠ENSCs在培养基中呈单细胞漂浮生长,球形; ENSCs于海马可溶性因子各实验分组中培养第2天呈细胞球状态,对照组中无细胞球形成(与100 μl/mL组比较,P₁=0.00),100 μl/mL组与对照组比较有统计学意义(P₁=0.00<0.05);至第6天,在100 μl/mL组中的细胞球数量明显多于其余各组(P₁'=P₂'=P₃'=P₄'=0.00)。在免疫细胞化学检测中,100 μl/mL组中细胞球表达干细胞高亲和蛋白Nestin、CD133阳性,Western Blot免疫印迹检测其中Nestin、CD133蛋白高于对照组。进一步分化试验中,细胞球呈贴壁生长的单细胞状态、有突起伸出、长梭形,免疫细胞化学检测分化的细胞表达胶质细胞特异性蛋白GFAP、S100、p75NGFR阳性,但不表达神经元特异性蛋白β-tubullin III与MAP2。结论 大鼠ENSCs在终浓度为100 μl/mL的HSF作用下,可促进 ENSCs的增殖分裂;ENSCs在同样浓度下的HSF中可进一步分化为表达GFAP、S100、p75NGFR阳性的胶质样细胞;100 μl/mL的HSFS是ENSCs的一种生理性诱导剂或参与促进ENSCs增殖、分化及通过细胞替代或因子分泌等机制修复神经损伤。

Objective The aim of this study was to explore induction and differentiation of endogenous neural stem cells(ENSCs) in the hippocampus soluble factors(HSF) from the hippocampus of adult Wistar rats by mimicking an intracranial microenvironment. Methods After Wistar rats sacrificed, the hippocampus tissue was obtained in cold DMEM/F12. After centrigued and filtered, the HSF was stored at -20℃. The ENSCs was obtained from the hippocampus tissue of a neonate Wistar rat. Collected the tissue, digested and obtained the ENSCs. After we observed the morphology, the ENSCs were cultured in different concentration (0、50、100、200、400 μl/mL) of HSF for 6 days, and compared the expression of Nestin and CD133 by immunocytochemistry. Meanwhile,we compared the Nestin and CD133 protein by western blot. And then we explored the optimal concentration of HSF by the numbers of all groups on the second and sixth day. Furthermore, we did the differentiated experiment using the same concentration of HSF. Results The number of neurospheres in the 100 μg/mL group was significantly higher than those in the other groups on the 6th day. Immunofluorescence revealed that the neurospheres from ENSCs in the 100 μg/mL group more highly expressed nestin and CD133 than control. This result was confirmed by western blot analysis. The neurospheres can differentiate into glia-like cells in 100 μg/mL HSF and 1% FBS expressing GFAP, S100 and P75 NGFR by immunofluorescence. Conclusion These data indicated that HSF alone, mimicking a destination of ENSCs in vitro, could induce and differentiate neurospheres from ENSCs, as a new method to get NSCs and glia-like cells differentiated from ENCs to repair the diseases of center nervous system.