夹脊电针对大鼠脊髓损伤后表皮生长因子受体及胶质酸性纤维蛋白表达的影响

目的：探讨电针治疗大鼠脊髓损伤（SCI）的分子机制。方法：45只成年雄性SD大鼠,随机分为假手术组、模型组和电针组（每组各15只）。采用改良的Allen＇s法建立SCI模型。电针组采用夹脊电针分别治疗3、7和14 d,频率为2 Hz等幅波,电流为2~6 mA,每日电针1次。应用Basso Beattie Bresnahan（BBB）评分观察大鼠后肢运动情况,应用免疫组织化学方法观察损伤段脊髓灰质中表皮生长因子受体（EG-FR）和胶质酸性纤维蛋白（GFAP）的表达变化情况。结果：行为学实验观察到,电针组大鼠术后1周BBB评分显著高于模型组（P〈0.01）,但仍低于假手术组（P〈0.01）。免疫组织化学染色发现：①模型组术后3 d,损伤段脊髓灰质中EGFR阳性细胞表达显著增多,7 d后开始减少;电针组EGFR阳性细胞数明显少于模型组（P〈0.01）。②损伤后GFAP的表达呈逐渐增加的趋势;在相应时间点电针组GFAP阳性细胞数显著低于模型组（P〈0.01）。结论：夹脊电针治疗可通过抑制损伤部位EGFR的表达,减少星形胶质细胞的增生,从而有利于损伤后的神经再生。

血清脑源性神经营养因子、B细胞活化因子、胶质酸性纤维蛋白与癫痫患者病情及认知功能的相关性分析

目的 探讨血清脑源性神经营养因子(BDNF)、B细胞活化因子(BAFF)、胶质酸性纤维蛋白(GFAP)与癫痫患者病情严重程度及认知功能的相关性。方法 2018年12月至2020年12月我院收治的74例癫痫患者,根据国立癫痫严重程度评分量表(NHS3)分为全身强直阵挛发作组(n=30)与部分性发作组(n=44);收集同一时期体检的40例健康者作为对照组,检测各组血清BDNF、BAFF、GFAP水平,并采用简易智能精神状态检查量表(MMSE)评估认知功能,分析血清BDNF、BAFF、GFAP与NHS3评分及MMSE评分的相关性,分析癫痫患者病情加重、认知异常的影响因素。结果 癫痫组BDNF低于对照组(P<0.05),BAFF、GFAP高于对照组(P<0.05);全身强直阵挛发作组与部分性发作组BDNF、BAFF、GFAP及NHS3评分比较差异有统计学意义(P<0.05);BDNF与NHS3评分呈负相关(P<0.05),BAFF、GFAP与NHS3评分呈正相关(P<0.05);高水平BDNF、BAFF、GFAP是癫痫患者病情加重的影响因素(P<0.05)。癫痫组MMSE评分低于对照组(P<0.05),其中有55例MMSE总评分<27分;BDNF与MMSE评分呈正相关(P<0.05),BAFF、GFAP与MMSE评分呈负相关(P<0.05)。高龄、高水平BDNF、BAFF、GFAP是癫痫患者发生认知异常的影响因素。结论 癫痫患者血清BDNF、BAFF、GFAP与正常人相比具有显著性差异,且与其病情严重程度及认知功能存在密切相关性。

成年大鼠脊髓损伤后反应性星形胶质细胞增生和巢蛋白表达的相关性及意义

目的探讨脊髓损伤后反应性星形胶质细胞的增生和巢蛋白的表达情况,并观察其细胞类型及相关性。方法利用动脉瘤夹压迫法建立成年大鼠脊髓损伤动物模型。利用BBB评分标准表、免疫组化、免疫荧光双标法与LeicaQ500IW图像处理系统分析和显示脊髓损伤后不同时期(1、3、5d,1、2、4、6、8w)、不同部位脊髓损伤的程度以及巢蛋白与胶质酸性纤维蛋白(GFAP)的表达变化及细胞类型。结果BBB评分结果显示术后所有实验动物双后肢(HL)评分低至0～1min,随后逐渐上升,1～2周内恢复的幅度较大,以后恢复较缓慢,较正常对照组具统计学意义(P<0.05)。甲苯胺蓝染色可见损伤区有核固缩、胞浆溶解、尼氏体模糊且染色较深的神经元。随着动物存活时间延长,损伤范围逐渐扩大,约1周后损伤范围不再扩大。正常对照组脊髓神经元胞体和突起轮廓清晰。免疫荧光双标染色及图像处理系统分析结果显示脊髓伤24h后可诱导损伤及邻近区域巢蛋白和GFAP高度表达,中央管周围室管膜区几乎均为巢蛋白/GFAP-细胞群,室管膜区以外的灰质和白质中几乎全是巢蛋白/GFAP+细胞群,3～7d逐渐达到高峰,之后逐渐减弱,在损伤后2周左右恢复至对照组水平(P<0.05)。正常对照组在脊髓中央管室管膜区有少量巢蛋白/GFAP-细胞,在室管膜区以外的灰质和白质中偶可见染色强度较弱的巢蛋白/GFAP+共存细胞。结论成年大鼠脊髓损伤可诱导巢蛋白和GFAP的表达,巢蛋白的表达和反应性星形胶质细胞增生呈正相关,且表达多相互共存;星形胶质细胞和神经干细胞对脊髓损伤都有反应,并可能参与中枢神经系统损伤修复。

脑络欣通及其拆方对脑缺血再灌注大鼠脑组织GFAP、bFGF和GDNF蛋白表达的影响

目的探讨中药复方脑络欣通及其拆方抗气虚血瘀证脑缺血再灌注损伤的作用机制。方法复制大鼠气虚血瘀证模型,线栓法大脑中动脉制作局灶性脑缺血再灌注模型,随机分为假手术组、模型组、益气组、活血组和脑络欣通组,予相应处理;采用免疫组织化学染色SABC法检测额顶叶皮质缺血半暗带区GFAP、bFGF和GDNF蛋白表达。结果模型组GFAP、bFGF和GDNF蛋白表达较假手术组明显增强(P<0.01);各治疗组与模型组比较,在脑缺血再灌注48h和72h均有显著差异(P<0.05或0.01);在脑缺血再灌注24h,部分治疗组与模型组有显著差异(P<0.05);各治疗组间在脑缺血再灌注48h或72h有显著差异(P<0.05)。结论脑络欣通可通过抑制GFAP的表达,促进bFGF和GDNF蛋白的表达,调节不同细胞间相互作用,改善气虚血瘀证脑缺血再灌注损伤。

骨髓间充质干细胞在C6脑胶质瘤细胞微环境中瘤性转化的生物学分析

目的:探讨骨髓间充质干细胞(bone marrow-derived mesenchymal stem cells,BMSCs)在C6脑胶质瘤细胞微环境中是否发生了生物学特性的改变。方法:全骨髓贴壁筛选法分离培养SD大鼠BMSCs,免疫荧光(immunofluorescence,IF)法检测BMSCs表面标志分子CD90+、CD105+、CD45-;活细胞荧光染料CM-Dil标记BMSCs(CM-Dil+BMSCs),流式细胞术(flow cytometry,FCM)检测标记效率,台盼蓝染色法连续监测BMSCs死亡率;CM-Dil+BMSCs与C6脑胶质瘤细胞直接接触共培养为直接共培养组,BMSCs与C6脑胶质瘤细胞非接触共培养为间接共培养组,阳性对照组为C6脑胶质瘤细胞单独培养,阴性对照组为BMSCs单独培养,培养7 d后,IF法检测各组酸性胶质纤维蛋白(glial fibrillary acidic protein,GFAP)、CD90、CD105表达情况;FCM分选实验组CM-dil+BMSCs和C6脑胶质瘤细胞,实时荧光定量聚合酶链反应(real time quantitative polymerase chain reaction,RT-qPCR)检测各组GFAP、PTEN、Bcl-xl、CyclinD1、CD90、CD105基因的表达。结果:①培养至第3代的BMSCs 90%以上表达CD90、CD105且不表达CD45;②IF结果显示直接共培养组、间接共培养组BMSCs细胞表达GFAP水平较阴性对照组BMSCs均增高。③RT-qPCR结果显示直接共培养组、间接共培养组BMSCs细胞GFAP、PTEN、Bcl-xl、CyclinD1基因表达水平较阴性对照组BMSCs明显增高(P<0.05),且直接共培养组与间接共培养组比较无明显差异(P>0.05);同时,直接共培养组、间接共培养组BMSCs的CD90、CD105基因表达水平较阴性对照组BMSCs显著降低(P<0.05)。结论:BMSCs在C6脑胶质瘤细胞微环境中存在潜在瘤性转化的倾向。

血清细胞因子水平变化对癫痫患者疾病转归的影响

目的 探讨血清细胞因子水平变化对癫痫患者疾病转归的影响。方法 选取2020年1月~2023年1月我院收治的205例癫痫患者为研究对象,根据国立癫痫严重程度评分量表(NHS3)分为全身强直阵挛发作组(n=85)和部分性发作组(n=120),选取同期100例健康体检者作为正常对照组。另根据治疗后6个月后癫痫控制情况将205例患者分为完全控制组(n=145)和未完全控制组(n=60),比较3组入院时脑源性神经营养因子(BDNF)、NOD样受体家族含pyrin结构域蛋白3(NLRP3)、胶质酸性纤维蛋白(GFAP)水平,分析血清BDNF、GFAP、NLRP3水平与癫痫严重程度及认知功能相关性,比较完全控制组和未完全控制组治疗前、后血清BDNF、GFAP、NLRP3水平,分析血清BDNF、GFAP、NLRP3水平对预后的预测价值。结果 3组入院时血清BDNF、GFAP、NLRP3水平差异有统计学意义(P<0.05);癫痫严重程度与血清BDNF呈负相关,与MMSE评分呈正相关(P<0.05);癫痫严重程度与GFAP、NLRP3呈正相关,与MMSE评分呈负相关(P<0.05);治疗2周、1个月后,未完全控制组患者血清BDNF低于完全控制组,GFAP、NLRP3水平高于完全控制组(P<0.05);血清BDNF、GFAP、NLRP3单独预测癫痫患者预后的AUC分别为0.747、0.719、0.754,截断值分别为6.16ng/mL、3.52ng/mL、94.62μg/L,联合预测癫痫预后的AUC为0.820,联合预测的AUC明显大于各指标单一预测(P<0.05)。结论 血清BDNF、GFAP、NLRP3不仅与癫痫病情变化联系紧密,还可影响预后,3者联合检测可为预后的预测提供一定参考,从而指导临床进一步的决策与治疗。

成年大鼠液压冲击脑损伤诱导室下区Nestin和GFAP的表达

目的 应用免疫组织化学和免疫荧光双标技术结合激光共聚焦显微镜 ,观察脑损伤后不同时期室下区巢蛋白(Nestin)和胶质酸性纤维蛋白 (GFAP)的表达及其细胞类型。方法 用动液压冲击法建立动物模型 ,用LeicaQ5 0 0IW图像处理系统及免疫组化和免疫荧光双标法分析和显示不同时期 (1、3、5d ,1、2、3、4、6、8周 )Nestin和GFAP的表达变化。结果 损伤 2 4h后 ,室下区有大量Nestin和GFAP的表达 ,且激光共聚焦显微镜观察到有Nestin阳性突起与GFAP共存现象 ,7d左右达到高峰 ,然后随着时间的推移其表达逐渐下降。正常组和假手术组 ,室下区也可见到Nestin和GFAP的表达 ,但与脑损伤组相比较低 (P <0 .0 5 )。结论 脑损伤可诱导室下区Nestin和GFAP的表达 ,Nestin阳性突起与GFAP共存细胞可能具有干细胞特性 ,与神经元的修复有关。

幼年大鼠脑室下区N-甲基-D-天冬氨酸受体亚单位2B的表达及其细胞类型

背景:神经干细胞表达有N-甲基-D-天冬氨酸受体(N-methy-D-aspartic acid receptor,NR)亚单位2B,深入研究其在幼年大鼠脑室下区的表达情况有助于揭示该区神经干细胞增殖、分化、迁移的机制。目的:观察NR2B在幼年大鼠脑室下区的表达及表达高峰期所在的细胞类型。方法:取出生后7,14,21,28dSD大鼠,制备5μm厚脑组织冰冻切片,观察脑室下区NR2B的表达,及其在出生后14d大鼠脑室下区所在的细胞类型。结果与结论:免疫组化结果显示NR2B在出生后7,14,21,28d大鼠脑室下区存在差异表达,于出生后14d时表达达高峰,以侧脑室外侧角旁表达最高;免疫荧光双标染色可见NR2B在脑室下区与胶质纤维酸性纤维蛋白、巢蛋白、β-Ⅲ微管蛋白共定位,以室管膜细胞最明显;而与NeuN在脑室下区无共定位。说明NR2B在幼年大鼠脑室下区的表达存在时空变化,可能在神经发生中发挥作用。

Effects of P2Y_1 receptor on glial fibrillary acidic protein and glial cell line-derived neurotrophic factor production of astrocytes under ischemic condition and the related signaling pathways

Objective The present study aimed to explore the role of P2Y1 receptor in glial fibrillary acidic protein （GFAP） production and glial cell line-derived neurotrophic factor （GDNF） secretion of astrocytes under ischemic insult and the related signaling pathways. Methods Using transient right middle cerebral artery occlusion （tMCAO） and oxygen-glucose-serum deprivation for 2 h as the model of ischemic injury in vivo and in vitro, immunofluorescence, quantitative real-time reverse transcription-polymerase chain reaction （RT-PCR）, Western blotting, enzyme linked immunosorbent assay （ELISA） were used to investigate location of P2Y1 receptor and GDNF, the expression of GFAP and GDNF, and the changes of signaling molecules. Results Blockage of P2Y1 receptor with the selective antagonist N^6-methyl-2′-deoxyadenosine 3′,5′-bisphosphate diammonium （MRS2179） reduced GFAP production and increased GDNF production in the antagonist group as compared with simple ischemic group both in vivo and in vitro. Oxygen-glucose-serum deprivation and blockage of P2Y1 receptor caused elevation of phosphorylated Akt and cAMP response element binding protein （CREB）, and reduction of phosphorylated Janus kinase2 （JAK2） and signal transducer and activator of transcription3 （STAT3, Ser727）. After blockage of P2Y1 receptor and deprivation of oxygen-glucose-serum, AG490 （inhibitor of JAK2） reduced phosphorylation of STAT3 （Ser727） as well as expression of GFAP; LY294002, an inhibitor of phosphatidylinositol 3-kinase （PI3-K）, decreased phosphorylation of Akt and CREB; the inhibitor of mitogen-activated protein kinase kinase 1/2 （MEK 1/2） U0126, an important molecule of Ras/extracellular signal- regulated kinase （ERK） signaling pathway, decreased the phosphorylation of JAK2, STAT3 （Ser727）, Akt and CREB. Conclusion These results suggest that P2Y1 receptor plays a role in the production of GFAP and GDNF in astrocytes under transient ischemic condition and the related signaling pathways may be JAK2/STAT3 and PI3-K/Akt/CREB, respectively, and that crosstalk probably exists between them.

Neurogenesis by Activation of Inherent Neural Stem Cells in the Rat Hippocampus after Cerebral Infarction

Objective To investigate the changes of neural stem cells (NSCs) in the rat hippocampus after cerebral infarction (CI) and to evaluate the neurogenesis caused by the activation of NSCs. Methods CI models of rats were made and rats were assigned to 6 groups: sham-operated, 1 day, 3 days, 7 days, 14 days, and 28 days after CI. The dynamic expression of bromodeoxyuridine (BrdU), polysialylated neural cell adhesion molecule (PSA-NCAM), glial fibrillary acidic protein (GFAP), and neuronal nuclear antigen (NeuN) were determined by immunohistochemistry and immunofluorescence staining. BrdU was used to mark the proliferated NSCs. PSA-NCAM was used to mark the plasticity of activated NSCs. GFAP and NeuN were used to mark the differentiated NSCs. Results Compared with the controls, the number of BrdU+ cells in the hippocampus increased significantly at 1 day after CI (P<0.05), reached peak at 7 days after CI (P<0.05), decreased but still elevated compared with the controls at 14 days after CI (P<0.05), and nearly unchanged at 28 days after CI. The number of BrdU+/PSA-NCAM+ cells increased significantly at 7 days after CI (P<0.05), reached peak at 14 days after CI (P<0.05), and decreased but still elevated compared with the controls at 28 days after CI (P<0.05). The number of BrdU+/PSA-NCAM+ cells was equal to 60% of the number of BrdU+ cells in all the same period. The number of BrdU+/NeuN+ cells in the hippocampus increased significantly at 14 days after CI (P<0.05) and reached peak at 28 day after CI (P<0.05). The number of BrdU+/GFAP+cells in the hippocampus nearly unchanged after CI. Conclusion CI can stimulate the proliferation of inherent NSCs, and most proliferated NSCs may differentiate into neurons and represent neural plasticity.

Neural stem cell activation and glial proliferation in the hippocampal CA3 region of posttraumatic epileptic rats

The present study observed the dynamic expression of CD133, nuclear factor-κB and glial fibrUlary acidic protein in the hippocampal CA3 area of the experimental posttraumatic epilepsy rats to investigate whether gliosis occurs after posttraumatic epilepsy. CD133 and nuclear factor-κB expression was increased at 1 day after posttraumatic epilepsy, peaked at 7 days, and gradually decreased up to 14 days, as seen by double-irnmunohistochemical staining. Glial fibrillary acidic protein/nuclear factor-EB double-labeled cells increased with time and peaked at 14 days after posttraumatic epilepsy. Results show that activation of hippocampal neural stem cells and glial proliferation after posttraumatic epilepsy-induced oxidative stress increases hippocampal glial cell density.

All-trans Retinoic Acid Induced the Differentiation of Human Glioma Cells

OBJECTIVE To observe the effect of all-trans retinoic acid （ATRA） on inducing human glioma MO59K cells differentiation and further explore the underlying molecular mechanisms.METHODS The expression of glial fibrillary acidic protein （GFAP） was detected by immunocytochemistry staining. The mRNA levels of GFAP, retinoid X receptor α（RXRα）, p21 were examined by semi-quantitative RT-PCR analysis. Luciferase activity assay was performed in the COS-7, MO59K cells to measure p21 promoter transcription activity.RESULTS ATRA could significantly enhance the expression and mRNA level of GFAP by immunostaining and RT-PCR （P〈0.05）. Simultaneously, the mRNA levels of RXRα and p21 were remarkably increased in dose-dependent manner by RT-PCR （P〈0.05）. Furthermore, luciferase assay confirmed that ATRA and RXRα could transactivate p21 promoter in COS-7 and glioma cells （P〈0.05）.CONCLUSION ATRA can induce differentiation of human glioma cells. The RXRα and p21 were activated during ATRAinduced differentiation process. This effect may be caused by directly RXRα-induced p21 gene transactivation. Our findings provide novel evidence for the future studies to explore the molecular mechanism of transcriptional regulation for glioma cell differentiation and cellular therapeutic approaches for glioblastoma.

Clinicopathological and immunohistochemical features of pilomyxoid astrocytoma: a report of six cases

Objective： The aim of this study was to study the clinicopathological and immunohistochemical features of pilo- myxoid astrocytoma （PMA）. Methods： The clinical and pathologic features in six cases of PMA were analyzed. Immunohisto- chemical staining for glial fibrillary acidic protein （GFAP）, synaptophysin （Syn）, Chromogranin A （CgA）, cytokeratin （AEI/AE3）, epithelial membrane antigen （EMA） and Ki67 was performed on paraffin-embedded sections. Results： Among the six cases, five occurred in female patients, one was male, the age at diagnosis ranged from 2 to 15 years. Four cases were located in the hypothalamic area and optic pathway, one case in the third ventricle, and one case in left parietal lobe. On imaging, PMAs often appears as well-circumscribed mass. Microscopically, the tumor was composed of monomorphous bipolar （piloid） cells setting in a prominent myxoid background with an angiocentric radiating growth pattern in some areas. PMA lacked biphasic pattern, Rosenthal fibers and eosinophilic granular bodies which were usually typical in a classic pilocytic astrocytoma （PA）. Immunohistochemcal study showed that the tumor cells were diffusely positive for GFAP. Syn positive staining was observed in one case. The Ki67 labeling index measured less than 5%. Conclusion： PMA is a distinct aggressive variant of pilocytic astrocytoma with special histological and immunohistochemical features. It is typically a rare tumor of early childhood. Im- munohistochemical staining for GFAP and Syn is helpful in differential diagnosis.

重复经颅磁刺激对神经病理性疼痛大鼠脊髓内星形胶质细胞的抑制作用

目的观察低频和高频重复经颅磁刺激（rTMS）对大鼠神经病理性疼痛的疗效及其对大鼠脊髓内星形胶质细胞标志物胶质酸性纤维蛋白（GFAP）的影响，探讨rTMS治疗神经病理性疼痛的机制。方法28只雄性SD大鼠，随机分为假手术组、假治疗组、低频rTMS组（1Hz）、高频rTMS组（20Hz），每组7只。假手术组仅暴露游离大鼠坐骨神经，不予结扎；其余3组经手术结扎坐骨神经制作神经病理性疼痛模型。术后第3天开始进行rTMS治疗，连续10d，刺激疼痛对侧大脑初级运动皮质。并于造模前、rTMS治疗前和治疗后测定疼痛行为学表现、机械痛觉和热痛觉，并于治疗结束后测定腰段脊髓内GFAP的表达。结果造模后3d，假治疗组、低频rTMS组、高频rTMS组大鼠均出现明显的疼痛行为学表现，热痛潜伏时较假手术组均明显降低（P〈0．05）。rTMS治疗后，高频rTMS组热痛潜伏时较假治疗组升高（P〈0．05），而低频rTMS组无明显变化。与假手术组比较，假治疗组和低频rTMS组损伤侧脊髓背角内GFAP阳性表达细胞数量和染色强度均明显增加（P〈0．05）。与假治疗组比较，高频rTMS组脊髓背角GFAP的表达显著下调（P〈0．05），而低频rTMS无此改变。高频rTMS组大鼠疼痛改善程度与脊髓背角中GFAP的表达呈负相关。结论坐骨神经结扎导致的神经病理性疼痛伴有脊髓背角内星形胶质细胞增殖；高频rTMS可以通过抑制脊髓内星形胶质细胞的增殖和活性而缓解疼痛，低频rTMS则无明显效果。

Effects of electroacupuncture at Fengchi(GB20)on motor function and GFAP/NeuN expression around the ischemic tissue of the motor cortex in MCAO rats

Objective:To investigate the potential mechanism of electroacupuncture(EA)at bilateral Fengchi(GB20)in treating cerebral ischemia-reperfusion injury and to provide a scientific basis for future experimental research and clinical applications.Methods:Forty male specific-pathogen-free Sprague-Dawley rats were randomly divided into four groups:a normal group,a normal with EA group,a model group,and a model with EA group,with 10 rats in each group.The normal group received no intervention.The normal with EA group received EA at bilateral Fengchi(GB20).The model group underwent middle cerebral artery occlusion(MCAO)using the suture.The model with EA group underwent MCAO and received EA at bilateral Fengchi(GB20).Cerebral blood flow was monitored using a laser Doppler cerebral blood flow meter.Neurologic damage was assessed using the neurologic deficit score,and motor ability was observed using the CatWalk gait system.The expression of glial fibrillary acidic protein(GFAP)and neuronal nuclei(NeuN)protein,the neuron markers,was detected by Western blotting.The protein expression levels of GFAP and NeuN,as well as the number of positive cells in the motor cortex,were detected using immunofluorescence.Results:Compared to the normal group,the cerebral blood flow values in the model group and the model with EA group decreased by more than 50%during the modeling process(P<0.01)and returned to pre-modeling levels after reperfusion(P>0.05).The neurologic deficit score increased(P<0.05),the average motor velocity decreased(P<0.05),GFAP protein expression and the number of positive cells in the motor cortex increased(P<0.05),and the NeuN protein expression and the number of positive cells decreased(P<0.05)in the model group.Compared to the model group,the neurologic deficit score decreased(P<0.05),the average motor velocity accelerated(P<0.05),GFAP and NeuN protein expression and the number of positive cells in the motor cortex increased(P<0.01)in the model with EA group.Conclusion:EA at bilateral Fengchi(GB20)can reduce neuronal loss and increase GFAP and NeuN protein expression in the motor cortex of rats after ischemia-reperfusion,improve the motor function after ischemic stroke,and accelerate the recovery of balance and stability of the affected limbs.

成年大鼠压迫性脊髓损伤后损伤区神经干细胞的分离培养实验研究

目的研究脊髓损伤后损伤反应性巢蛋白(nestin)和胶质酸性纤维蛋白(glial fi brillary acid protein,GFAP)阳性共存(nestin+/GFAP+)细胞的分裂、增殖和分化能力,以探讨其是否具有神经干细胞(neural stem cells,NSCs)特性。方法8周龄雄性SD大鼠12只,体重200～250 g,随机分为正常对照组和模型组(n=6)。模型组利用动脉瘤夹压迫法建立成年大鼠脊髓损伤动物模型,正常对照组不作任何处理。造模后5 d,两组分别取大鼠T8脊髓节段,分离中央管周围室管膜区以外的脊髓灰质和白质,制成单细胞悬液,用无血清NSCs培养基进行培养,并用含血清NSCs培养基进行诱导分化,利用免疫荧光化学和流式细胞仪观察细胞类型及分裂、分化、增殖能力。结果模型组培养后3～7 d,单细胞悬液中有大量高度表达的nestin+/GFAP+共存细胞,细胞计数为5.15±0.71;对照组为1.12±0.38;两组比较差异有统计学意义(P<0.01)。细胞周期结果示,模型组S期细胞比例(15.49%±3.04%)及增殖指数(15.88%±2.56%)均明显高于对照组(5.84%±0.28%,6.47%±0.61%),两组比较差异有统计学意义(P<0.01)。模型组原代细胞逐渐形成边缘光滑、中心膨隆有立体感的小克隆球,nestin免疫荧光染色呈强阳性,多次传代后获得大量细胞克隆球。对照组单细胞悬液原代及传代培养均未见明显克隆球生长。免疫染色结果示模型组克隆球诱导分化约5 d,细胞球中含有大量半乳糖脑苷脂(galactocerebroside,GaLC)-nestin免疫染色阳性细胞;5～7 d,大量β-微管蛋白Ⅲ(β-tubulinⅢ)-nestin和GFAP免疫染色阳性细胞;7～14 d出现GaLC阳性少突胶质细胞、β-tubulinⅢ神经元和GFAP染色阳性的胞体及细胞突起。结论成年大鼠压迫性脊髓损伤后,剔除中央管周围室管膜区脊髓白质与灰质分离而得的nestin+/GFAP+细胞,具有自我更新能力和多分化潜能,是中枢神经系统的NSCs。

Changes of serum Tau, GFAP, TNF-α and malonaldehyde after blast-related traumatic brain injury

Objective： To determine the changes of serum Tau protein, glial fibrillary acidic protein （GFAP）, tumor necrosis factor alpha （TNF-α）, and malonaldehyde （MDA） in rats after blast-related traumatic brain injury （BTBI） and to provide relative information for further studies on BTBI mechanism and seek specific biomarkers for BTBI. Methods： Ninety male Sprague-Dawley rats were randomly assigned into three groups： control group, moderate blast injury group, and severe blast injury group （n=30 for each）. Rats in the moderate and severe blast injury groups were respectively exposed to corresponding levels of BTBI. After explosion, serum levels of Tau, GFAP, TNF-α, and MDA in each group were determined by Elisa assay at different time points after injury （8 h, 24 h, 3 d, and 6 d）. The extent of brain damage was detected by Nissl staining and TUNEL assay. Results： Serum levels of Tau and GFAP rapidly increased and reached the peak at 24 h after either moderate or severe blast injury. All the values were significantly higher than control group at all time points （P〈0.05）. Serum TNF-α level of both injury groups peaked at 8 h after BTBI and stayed significantly higher than control group at all time points （P〈0.05）. Serum MDA of two injury groups began to significantly increase at 3 d and the level stayed significantly higher than control group until 6 d （P〈0.05）. Moreover, unlike the other biomarkers, serum MDA of severe blast injury group was significantly higher than moderate blast injury group at 6 d （P〈0.05）. Conclusion： The changes of serum Tau, GFAP, and TNF-α showed a good sensitivity at the acute phase after BTBI （within 24 h）. However, their specificity and correlation with the extent of injury were limited in this experiment. Moreover, although the change of serum MDA showed a poor sensitivity and specificity to the diagnosis of BTBI during the first few days, it can reflect the injury degree at 6 d after injury. Therefore, further studies are needed to improve the methods of detecting more serum markers and investigate the significance of multiple markers in diagnosing BTBI.

Qingnaoyizhi decoction suppresses the formation of glial fibrillary acidic protein-positive cells in cultured neural stem cells by inhibiting the Janus kinase 2/signal transducer and activator of transcription 3 signaling pathway

OBJECTIVE: Inactivation of the Janus kinase 2(JAK2)/signal transducer and activator of transcription 3(STAT3) signaling axis plays a crucial role in determining the fate of neural stem cells(NSCs).Qingnaoyizhi decoction(QNYZD) has been used for the treatment of vascular dementia and has shown to improve synaptic remodeling. The aim of this study was to evaluate the effect of cerebrospinal fluid(CSF) containing QNYZD(CSF-QNYZD) on the differentiation of cultured NSCs and the involvement of the JAK2/STAT3 pathway.METHODS: The protein expression levels of glial fibrillary acidic protein(GFAP), tubulin, drosophila mothers against decapentaplegic protein(SMAD-1), STAT3, and phosphorylated-STAT3 were detected by western immunoblot analysis in the groups: control, CSF, JAK/STAT inhibitor(AG490),CSF-QNYZD, and CSF-XDZ(CSF-Xidezhen). The differentiation of NSCs was determined by immunofluorescence staining. The proliferation of NSCs was measured using the Cell Counting Kit-8 proliferation assay.RESULTS: Compared with the control group,CSF-QNYZD and AG490 significantly increased the number and expression of tubulin-positive cells, reduced the number and expression of GFAP-positive cells, and down-regulated the expression of p-STAT3. However, CSF-QNYZD also decreased the expression of SMAD-1 and STAT3.CONCLUSION: Enhanced neuronal differentiation may be associated with the down-regulation of glial differentiation instead of promoting proliferationin treated NSCs. Furthermore, QNYZD may play a direct role in suppressing the formation of GFAP-positive cells and enhancing neuronal differentiation by inhibiting JAK2/STAT3 activation. Overall, these results provide insights into the possible mechanism underlying QNYZD-mediated neurogenesis.

Stem cells modified by brain-derived neurotrophic factor to promote stem cells differentiation into neurons and enhance neuromotor function after brain injury

Objective： To promote stem cells differentiation into neurons and enhance neuromotor function after brain injury through brain-derived neurotrophic factor （BDNF） induction. Methods： Recombinant adenovirus vector was applied to the transfection of BDNF into human-derived umbilical cord mesenchymal stem cells （UCMSCs）. Enzyme linked immunosorbent assay （ELISA） was used to determine the secretion phase of BDNF. The brain injury model of athymic mice induced by hydraulic pressure percussion was established for transplantation of stem cells into the edge of injury site. Nerve function scores were obtained, and the expression level of transfected and non-transfected BDNF, proportion of neuron specific enolase （NSE） and glial fibrillary acidic protein （GFAP）, and the number of apoptosis cells were compared respectively. Results： The BDNF expression achieved its stabilization at a high level 72 hours after gene transfection. The mouse obtained a better score of nerve function, and the proportion of the NSE-positive cells increased significantly （P〈0.05）, but GFAP-positive cells decreased in BDNF- UCMSCs group compared with the other two groups （P〈0.05）. At the site of high expression of BDNF, the number of apoptosis cells decreased markedly. Conclusion： BDNF gene can promote the differentiation of the stem cells into neurons rather than glial cells, and enhance neuromotor function after brain injury.