摘要
近年来对中枢神经系统再生能力的认识逐渐深入,发现移植外周神经雪旺细胞
(Schwann cells SCs)、嗅成鞘细胞(Olfactory ensheathing cells OECs)均可以促进中枢轴
突再生,并且发现 OECs 脊髓移植促轴突再生效果优于 SCs,表现在再生轴突的延伸
距离、电生理及支持体重和运动功能的恢复等方面。OECs 抑制星形胶质细胞反应的
能力和较强的细胞迁移能力可能起了一定的作用,可能还有另外的一些未被发现的因
素在同时起作用。
嗅觉系统终生保持着神经再生的能力,嗅神经元存活周期为 4-8 周,衰老的神经
元由嗅上皮产生的新生神经元取代,重新发出轴突进入中枢的嗅球与靶细胞形成突触。
OECs 是伴随嗅神经形成髓鞘的神经胶质细胞,与嗅神经再生进入嗅球同靶细胞形成
突触密切相关,。OECs 在形态结构和功能上与 SCs 极为相似,体外培养两者均可呈梭
形,可以形成髓鞘,都可以分泌多种神经营养因子如神经生长因子(nerve growth factor
NGF)、脑源性神经营养因子(brain derived neurotrophic factor BDNF)和睫状神经生长因
子(ciliary-derived nerve growth factor CNTF)。这两种细胞既相似又不同,为我们研究
神经再生提供了一个新的思路,两者进行比较可能发现有利于中枢神经再生的某些分
子。
本研究以 OECs 和 SCs 为实验对象,采用免疫溶解法获得两种细胞的纯培养,应
用免疫细胞化学(ICC)和电镜技术观察 OECs 的抗原表达以及形态特征。利用抑制消减
杂交技术(SSH)筛选两者的差异表达基因,从两者的差异表达基因中分析与中枢神经再
生相关的基因表达,为进一步阐述中枢神经再生机制创造条件。主要结果如下:
1.免疫溶解法获得 OECs 和 SCs 的纯培养
免疫溶解法纯化 2h 可见悬浮细胞增多,纯化后 24h 倒置显微镜下观察成纤维细
胞少见,P75NGFr 免疫组化显示 OECs 纯度可达 95%以上;继续培养 6d 后再次进行
纯化可去除大部分新增殖的成纤维细胞,保持 OECs 纯度在 95%以上。
2.ICC 技术研究 OECs 的抗原表达,电镜技术观察 OECs 的超微结构特征
OECs 免疫组化结果显示:P75LNGFr(+),GFAP(+),NCAM(+),CDK5(-)。 免疫
细胞化学特征为:P75LNGFr 染色均匀分布于整个细胞和突起,核区较为浅淡;GFAP
染色较深分布于整个胞浆和突起;NCAM 免疫阳性细胞表面均有分布。SEM 显示 OECs
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第三军医大学博士学位论文
核不规则,呈分叶状或齿状,核仁明显,染色质均匀分布于核中,异染色质可在核膜
下形成块状结构。
3.SSH 技术筛选 OECs 和 SCs 的差异表达基因
测序结果得到了 4 个不同的已知基因,分别编码:肝白血病因子(hepatic leukemia
factor Hlf),糖基化磷脂酰肌醇磷脂酶 D1(Glycosylphosphatidylinositol phospholipase D1
GPLD1),Phr1(PAM)和缝隙连接蛋白亚单位 connexin43。在 4 个已知基因中,phr1 和
hlf 与突触形成相关;缝隙连接蛋白亚单位 connexin43 参与细胞间的信号传递;GPLD1
可能参与调节 GPI 锚定蛋白的功能。
GPLD1 在 OECs 表达可能有利于裂解抑制轴突生长的 GPI 锚定蛋白,通过消除这
些蛋白的抑制作用来促进轴突再生。phr1、hlf 和 Gja1(connexin43)则可能参与 OECs
与轴突之间的信息交流和调控,影响轴突的生长。
Both olfactory ensheathing cells (OECs) and Schwann cells(SCs) provide cellular
environments that support the growth of damaged axons following transplantation into
experimental models of CNS injury. Moreover, both cell types are able to remyelinate axons
following transplantation into experimental models of demyelination, producing new
myelin sheaths that are indistinguishable from one another. Besides the similarites in
functions OECs are common with SCs in phenotypic properties and secretion of
neurotrophic factors such as nerve growth factor (NGF), brain derived neurotrophic factor
(BDNF), ciliary-derived nerve growth factor(CNTF). However it has been demonstrated
that OECs are preferable to transplant after CNS injury than SCs in several aspects.
OECs are specialized cells which support axons that leave the olfactory epithelium and
project though the peripheral nervous system into the olfactory bulb of the CNS and they
should have some unique properties which allow them to guide and enhance regenerating
CNS axons though a normally growth inhibitory environment. Interactions between SCs
and astrocytes have an adverse effect on regeneration following SCs transplantation into the
CNS. For example, transplanted Schwann cells show limited migration into a host lesion
environment characterized by reactive or scarring astrocytes, with the result that few
regenerating axons are able to exit the graft and reenter the host tissue and SCs can also
arousing greater increase in the expression of axon growth inhibitory chondroitin sulfate
proteoglycans (CSPGs). In contrast, OECs may exhibit greater compatibility with
astrocytes than do Schwann cells. Transplanted OECs appear to elicit a less severe reaction
in host astrocytes and less CSPGs expression, which may allow more axons to traverse
graft–host boundaries and to penetrate further into the lesion environment. It will be useful
to understand CNS regeneration with establishing how the behavior of the two cells may
differ and why one cell type might be preferable to transplant than the other given the broad
similarity in the CNS repair of the two cells.
In this project, some differentially expressed genes between OECs and SCs that may
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第三军医大学博士学位论文
be beneficial to further exploitation in CNS regeneration have been identified using
suppressed subtraction hybridization (SSH) method. Immunocytochemistry and electron
microscopy are applied to study morphological characteristics of OECs acquired by
dissecting ONL from olfactory bulb (OB) and purified with complements treatment
following anti-Thy1.1 incubation. The main results are as follows:
1.A population of OECs with 95% purity is acquired
Many cells supposed as fibroblasts, endothelium and neurons retract and detach from
culture dishes in 2 hours after purification. Few fibroblasts and endothelium can be
observed 24h later and the purity of OECs determined by anti-P75NGFr staining is 95% or
more. Purification procedure may repeat 6 days later to maintain the high purity.
2. Immunocytochemical properties and ultrastructure of OECs
Cultured OECs show immunoreactivity to P75NGFr, N-CAM and GFAP.
Ultrastructure of OECs shows an irregularly-shaped nucleus and the chromatin is uniformly
distributed throughout the karyoplasms. The cytoplasm contains abundant ribosomes and
rough endoplasmic reticulum, and the plasma membrane shows a typical ruffled
appearance.
3. Four differentially expressed genes are obtained using SSH method
Sequencing and BLAST of 45 clones result in 4 genes with known functions. The
proteins coded include hepatic leukemia factor (Hlf) which plays a role in synaptogenesis,
Phr1(PAM) which also functions in synaptogenesis, gap junction membrane channel protein
alpha1 (Gja1,connexin43) which takes part in intercellular interactions and
Glycosylphosphatidylinositol phospholipase D1 (GPLD1)one of whose functions is to
release proteins from their glycosylphosphatidylinositol (GPI) anchors at the cell surface.
GPLD1 may destroy some proteins
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