GDNF基因修饰的神经干细胞抑制脑卒中后大鼠的Caspase-3表达

目的研究胶质源性神经营养因子(glial cell line-derived neural factor,GDNF)基因修饰的神经干细胞(neuralstem cells,NSCs)移植对脑卒中后大鼠缺血侧脑组织内半胱氨酰天冬氨酸特异性蛋白酶-3(cysteinyl aspartate specific protein-ase-3,caspase-3)表达的影响,探讨GDNF基因修饰的神经干细胞(GDNF/NSCs)移植对大鼠脑卒中的神经保护作用机制。方法取新生大鼠脑组织分离培养NSCs,收集第6代前的NSCs备用。用重组腺病毒GDNF转染神经干细胞,制备GDNF/NSCs。暂时性阻塞大鼠大脑中动脉制备脑卒中模型,3d后,用脑立体定位仪向卒中侧侧脑室分别给予NSCs、GDNF/NSCs和生理盐水。再灌注时间1周、2周、3周、5周、7周后处死大鼠(n=3)。裂解卒中侧脑组织,离心后得到脑组织蛋白样品,通过蛋白免疫印迹(Western Blotting)检测Caspase-3表达。结果 GDNF/NSCs、NSCs、NS各组caspase-3的表达在1周、2周、3周、5周、7周各时间点均逐渐降低。NSCs组、GDNF/NSCs组显著低于NS组(P<0.01;P<0.001);GDNF/NSCs组明显低于NSCs组(P<0.01)。结论 GDNF/NSCs移植治疗脑卒中的机制可能与抑制caspase-3表达有关。     

流感病毒感染小鼠星形胶质细胞诱导GDNF转录水平上调

目的探讨星形胶质细胞感染流感病毒后的神经保护效应,检测流感病毒H3N2和H9N2体外感染小鼠星形胶质细胞是否会诱导胶质细胞神经营养因子转录水平的改变。方法从新生小鼠大脑皮质分离培养神经胶质细胞,并进一步纯化星形胶质细胞,经纯度鉴定后,用感染复数为2的流感病毒H3N2和H9N2进行体外感染。在感染早期（6h）和感染中后期（24h）分别提取细胞RNA,real-time PCR检测神经营养因子转录水平的变化。结果病毒感染后的星形胶质细胞显著上调GDNF转录水平的表达,NGF,BDNF,NT-3转录水平变化不明显。H3N2和H9N2感染星形胶质细胞表达的细胞因子变化情况一致。结论流感病毒H3N2和H9N2感染星形胶质细胞可诱导神经营养因子GDNF转录水平的上调。     

内源性GDNF在针刺促进损伤脊髓神经元轴突出芽中的作用

目的探讨内源性胶质源性神经营养因子(glia derived neurotrophic factor,GDNF)在针刺促进SD大鼠脊髓神经元轴突出芽中的生物学机制。方法 25只雌性SD大鼠随机分为单侧备用根手术组、针刺组、针刺并GDNF抗体封闭组。切除脊髓单侧L1～L3及L5～L6背根节(dorsal root ganglion,DRG),保留L4背根为备用根,制备单侧备用根模型。选择足三里和悬钟、伏兔和三阴交两组穴位进行针刺实验。免疫组化和辣根过氧化物酶(HRP)追踪分别检测GDNF及背根神经节(DRG)源的神经纤维表达变化。结果针刺可显著增加GDNF在受损脊髓中的表达,而抗体封闭可显著逆转该作用。结论针刺可能通过增加内源性GDNF的表达促进损伤脊髓神经元形态和功能修复。     

GDNF在人脑胶质瘤细胞中表达的检测及其cDNA的克隆

为研究人脑胶质细胞源性神经营养因子在脑胶质瘤中的表达及其在治疗神经系统疾病中的作用 ,本研究克隆了其前体蛋白及其成熟多肽的 c DNA序列 ,将之用于真核及原核细胞表达。提取我国自建的脑胶质瘤细胞系 BT32 5总 RNA,设计两对引物用逆转录 PCR法扩增上述两种长度的 c DNA片段 ,然后重组于 p GEM-T载体 ,酶切鉴定插入片段正确后测序。结果表明 ,脑胶质瘤细胞中只扩增出 5 5 8bp的胶质细胞源性神经营养因子全长 c DNA片段 ,未见 6 36 bp片段 ;且扩增效率较成熟多肽 c DNA甚低。提示 ,该细胞合成的胶质细胞源性神经营养因子可能还存在不含有信号肽而只能在细胞内潴留的成熟多肽形式。作为自泌性生长因子 ,它可能调节瘤细胞生长等生物学行为 ;同时克隆到的胶质细胞源性神经营养因子全长及其成熟多肽的 c DNA片段 ,可分别用于真核及原核细胞表达     

c-fos和神经营养因子在大鼠脑缺血再灌流时的神经元表达特点

为了观察脑缺血再灌流时 c-fos和神经营养因子在神经元表达的时空特点 ,探讨其在缺血性神经元损伤中的作用 ,本研究用阻塞 SD大鼠大脑中动脉 2 h、再灌流 0 .5～ 48h制成局灶性脑缺血模型 ,用免疫组化方法观察了 c-fos和神经营养因子转化生长因子、神经生长因子和胶质源性神经营养因子在神经元的表达特点。结果表明 ,c-fos和转化生长因子分布相似 ,正常组无 c-fos和转化生长因子阳性神经元 ;缺血再灌流 0 .5～ 48h阳性的神经元主要位于非大脑中动脉供血区 ,缺血区皮质、纹状体和视前区神经元为阴性。而实验各组对照侧皮质神经元中等阳性。神经生长因子、胶质源性神经营养因子在缺血脑组织的分布相似。正常组、假性手术组 ,皮质、嗅结节、丘脑和下丘脑等区的神经元神经生长因子、胶质源性神经营养因子免疫反应为弱阳性乃至强阳性 ;再灌流 0 .5 h,缺血区皮质弱阳性 ,缺血周边区中等阳性 ;再灌流 3～ 48h,神经生长因子、胶质源性神经营养因子强阳性的神经元主要位于非大脑中动脉供血区和缺血周边区。此外 ,对照侧皮质大量的神经元呈强阳性。结论 ,上述资料提示即早基因 c-fos和神经营养因子具有促进神经元存活的作用     

Peripheral Nerve and Transgene Cells Transplantation in the Treatment of Experimental Neuropathy of SD Rats

Purpose: To observe whether optic nerve can regenerate by peripheral nerve transplantation and GDNF gene transfer.Methods: Optic nerve was transected about 2 mm posterior to the eye to establish experimental models of optic neuropathy. Sciatic nerve was sutured to the sheath of injury optic nerve by a 10 - 0 nylon, pcDNA3-GDNF was smeared to the joint and injected to the vitreous cavity. The regenerated retinal ganglion cells axons were observed by HRP. The retina and optic nerve were observed by pathohistological methods.Results: Some retinal ganglion cells with regenerated axons could be seen a week after transplantation with large cell bodies. The cells could be identified by HRP. The regenerated nerve fibers grew to the transplanted sciatic nerve. Then the number of retinal ganglion cells with regenerated axons increased with time going. Only a few regenerated retinal ganglion cells could be seen two months after transplantation. The large retinal ganglion cells were seen to have plenty of cytoplasm     

GDNF基因修饰的骨髓基质干细胞分泌物对多巴胺能神经元的营养作用

目的:克隆GDNF基因并修饰骨髓基质干细胞,观察该工程细胞分泌物对多巴胺能神经元的营养作用。方法:应用逆转录聚合酶链反应(RT-PCR)方法从新生小鼠大脑皮层细胞克隆出GDNFcDNA片断,以pECPP-Cl为载体导入骨髓基质干细胞,制备稳定表达GDNF基因的MSCs工程细胞,收集并浓缩MSCs和GDNF’基因修饰的MSCs工程细胞的条件培养液,通过MTT、倒置显微镜和免疫组织化学的方法观察MSCs和GDNF基因修饰的MSCs工程细胞分泌物对多巴胺能神经元的营养作用。结果:MSCs和GDNF基因修饰的MSC工程细胞分泌物均能促进多巴胺能神经元的存活和生长,MSCs工程细胞作用更强。结论:成功构建了GDNF基因修饰的MSCs工程细胞,该细胞对多巴胺能神经元有明显营养保护作用,在帕金森病治疗中可能有重要价值。     

大鼠骨髓基质细胞分泌胶质细胞源性神经营养因子的研究

目的研究骨髓基质细胞(BMSCs)能否分泌胶质细胞源性神经营养因子(GDNF)及其分泌规律，为进一步探讨BMSCs治疗帕金森病(PD)大鼠模型的作用机制提供实验依据。方法取较高纯度的BMSCs进行培养，通过RT—PCR和ELISA法，分别从mRNA和细胞蛋白水平测定体外培养BMSCs的上清液和细胞蛋白中GDNF的含量。结果通过RT-PCR法检测结果显示在mRNA水平，BMSCs中有GDNF的表达。在蛋白水平，采用ELISA法在培养第3天以后的培养液和细胞蛋白中均可检测到GDNF，并随着时间的延长，培养液和细胞蛋白中GDNF浓度明显升高。结论BMSCs具有分泌GDNF的能力，细胞分泌可能受周围环境和自身生长状况的影响，其分泌是非持续性的。GDNF浓度的升高不仅与BMSCs细胞数量增加有关，还与细胞本身分泌能力的增强有关。     

COMMENTS

Glial-cell-line-derived neurotrophic factor (GDNF) is a distant member of the transforming growth factor superfamily. It binds to and activates a receptor complex consisting of GFR-α1 and Ret receptor tyrosine kinase. In testis, GDNF is expressed by Sertoli cells. We have shown by transgenic loss- and gain-of-function mouse models that GDNF regulates the cell fate decision of undifferentiated spermatogonia. In the GDNF +/− mice, the spermatogonia differentiate in excess leading to the depletion of germ cells. In the mice overexpressing GDNF in testes, undifferentiated spermatogonia accumulate in the tubules, no sperm is produced, and the mice are infertile. After a year, the GDNF overexpressing mice frequently (89%) develop testicular tumours, and most of them are bilateral (56%). All these tumours show the same histological pattern. They are composed of round spermatogonial/gonocytic cells with only a scant cytoplasm. The tumours are locally invasive but do not metastasise. They express germ line markers, are positive for alkaline phosphatase, and aneuploid with a triploid peak. Thus, by several histological, molecular, and histochemical characteristics, the GDNF-induced tumours mimic classical seminomas in men, but the precursor lesions are apparently different in mouse and man.     

Glial cell line-derived neurotrophic factor-like immunoreactivity in human trigeminal ganglion and nucleus

Glial cell line-derived neurotrophic factor (GDNF) is shown by immunohistochemistry in human trigeminal sensory system from 22 weeks of gestation to adulthood. In the trigeminal ganglion, a distinct subpopulation of GDNF-positive neurones is observed, which amounts to about 15% at early pre-term and adult ages and peaks to around 30% at perinatal ages. Labelled neurones are mostly small- and medium-sized. Occasionally, Schwann and satellite cells are stained. GDNF/substance P (SP) and GDNF/calcitonin gene-related peptide (CGRP) double stained neurones occur at all ages examined, whereas GDNF/trkA coexistence can be observed in pre- and full-term newborns only. Centrally, GDNF-immunostained fibers and terminal-like structures are mainly restricted to the spinal trigeminal nucleus, where they are codistributed with SP and CGRP. In the subnucleus caudalis, positive neurones can also be observed both in the superficial laminae and in the magnocellular part, with higher frequency in adults. These results suggest that GDNF may play a functional role in human trigeminal primary sensory neurones throughout life and provide indication for its possible involvement in the regulation of pain-related neuronal circuits in human trigeminal sensory system.