【作者】 陆利；

【导师】 白丽敏； 肖志诚；

【作者基本信息】 北京中医药大学 ， 中西医结合基础， 2007， 博士

【摘要】 多发性硬化(MS)是中枢神经系统的一种慢性炎性脱髓鞘疾病,常常出现瘫痪、失明等神经系统损伤症状。本病好发于青壮年,病程长,反复发作,迁延不愈,是世界公认的疑难病之一。由于MS详尽的发病机制目前仍属未知,故现阶段无肯定而有效的防治措施。临床治疗MS以激素和免疫抑制剂为主,但是长期应用激素和免疫抑制剂带来很大副作用,且费用较高,停药后常出现症状反弹或加剧。因此研究和探讨MS的病因、病机,寻找有效的防治措施有着重要的理论意义和实用价值。本研究围绕多发性硬化这一中心,应用免疫组织化学、分子生物学、电镜及流式细胞分析等多种技术手段探讨:①骨髓间充质干细胞(BMSCs)向少突胶质样细胞定向分化的潜能;②银杏提取物(GBE)治疗慢性脱髓鞘模型PLP转基因鼠的应用前景和机制;③电针血清对体外培养小胶质细胞活性的影响,以期为MS的病因和发病机制及临床治疗提供实验依据。1.F3/Notch信号系统诱导人骨髓间充质干细胞向少突胶质样细胞定向分化方法:贴壁分离得到的hBMSCs经β-ME(24h)和RA(3d)预诱导后,分为MFS组(5mm forskolin、10ng/ml bFGF、5ng/mlPDGF、200 ng/mlHRG;阳性对照组)和F3组再继续培养3d,应用流式细胞术、免疫荧光染色、Luciferase分析不同诱导阶段细胞的生物特性。随后将RA/β-ME、MFS/RA/β-ME和F3/RA/β-ME诱导后的细胞移植入小鼠玻璃体腔,采用免疫荧光染色及电镜技术观察体内移植后细胞的形态特征和功能。结果:贴壁分离能够得到纯度较高的CD90(+)hBMSCs(83.3%),免疫荧光染色证实约40.88%的细胞表达早期干细胞标志物Sox2,经β-ME(24h)和RA(3d)诱导后CD90(+)hBMSCs分化为少突胶质样细胞(OLLCs),54.43%的细胞表达少突胶质前体细胞标志物NG2,用F3再诱导3d后约31.55+5.8%的细胞开始表达成熟少突胶质细胞标志物O4,F3组O4(+)OLLCs与阳性对照MFS组相比无统计学差异(p>0.05)。F3/Notch信号系统组件Notch1/NICD及deltex1(DTX1)免疫荧光染色证实F3诱导3d后,Notch1/NICD及DTX1核染色增强,提示F3/Notch/DTX1信号通路被激活。Luciferase分析证明F3组相对荧光强度(0.33±0.08)与MFS组(0.9±0.13)相比差异非常显著(p<0.01),并且F3组Hes1+dnDTX1/Hes1+DTX1的相对荧光强度显著高于RA组和MFS组(p<0.05),提示F3通过DTX1的信号转导作用,竞争性抑制经典的Notch/Delta/Hes1信号系统,促进hBMSCs来源的OLLCs发育、分化。小鼠玻璃体腔移植OLLCs 2月后,移植细胞在体内微环境作用下形态进一步成熟,初级分枝和次级分枝明显增多(p<0.01),更为重要的是F3移植组O4(+)OLLCs次级分枝(1.7+0.13)较RA移植组(1.13+0.13)极其显著增加(p<0.01),与阳性对照MFS移植组(1.8+0.1)结果相同。视网膜平片β-tublin III和O4免疫荧光双标证实F3移植组O4(+)OLLCs自胞体发出许多分枝,这些分枝攀附于节细胞轴突表面,形成胶质细胞-轴突连接,电镜观察可见节细胞轴突周围有单层髓鞘包绕,呈现正常中枢神经系统髓鞘特征。结论:hBMSCs具有跨胚层向少突胶质细胞分化的潜能,F3通过激活F3/Notch信号系统促使CD90(+) hBMSCs来源的OLLCs形态及功能成熟。2.银杏提取物对PLP转基因鼠的影响方法:将3月龄plptg/-小鼠分为PLP组(对照组)和GBE组(治疗组),GBE组plptg/-小鼠每日腹腔注射GBE(70mg/kg),PLP组plptg/-小鼠每日腹腔注射同样剂量生理盐水,治疗2个月,进行行为学检测及免疫组化染色分析。同时体外培养少突胶质细胞系OLN细胞,应用MTT法和免疫荧光染色直接观察GBE对少突胶质细胞的影响。结果:GBE组plptg/-小鼠存活率上升达76%,比PLP组增加23%,Rotarod行为学指标falling latency延长(33.0±4.36 sec)。视神经MBP和β-tublin免疫荧光双标表明PLP组视神经MBP表达水平降低,甚至出现裸露轴突,而GBE组MBP荧光染色强度较PLP组增强,可见MBP阳性髓鞘包绕轴突,裸露轴突的数量减少。海马伞矢状切片免疫染色证实,PLP组plptg/-小鼠海马伞内小胶质细胞增多、聚集成簇,OX-42表达上调,但GBE组仅有散在的小胶质细胞分布且CD4+T细胞较少,GFAP免疫反应阳性的星形胶质细胞数量两组之间无明显差别。用不同浓度GBE刺激OLN细胞24h,MTT检测发现细胞OD值增加,在0.07-0.138mg/ml剂量范围内,GBE浓度与OLN细胞OD值呈现良好的线性关系,与对照组相比0.175、0.28、0.35mg/ml的GBE可显著增加细胞OD值0.091、0.107、0.138;用0.28mg/ml有效浓度培养OLN细胞24h、48h、72h,NG2免疫荧光染色证实OLN细胞形态持续为双极,未分化形成具有网状突起的成熟少突胶质细胞。结论:GBE可能主要通过抑制小胶质细胞激活,减少CD4+T细胞的炎症性浸润,从而延缓plptg/-小鼠脱髓鞘进程,提示GBE对MS的防治具有应用前景。3.电针血清对体外培养小胶质细胞活性的影响方法:采用100HZ高频电针针刺正常大鼠百会、风府两穴,电针两周后取血制备电针血清。将体外培养的小胶质细胞分为三组,正常组加入正常大鼠血清,对照组加入正常大鼠血清和小胶质细胞激活剂LPS(1μg/ml),电针组加入电针大鼠血清和LPS(1μg/ml),通过免疫组化、MTT和Griess法分别观测8h、12h、24h和48h不同时间小胶质形态、活性及NO释放量。结果:1μg/ml LPS可激活小胶质细胞,表现为细胞体积增大,OX-42表达上调,对照组与正常组相比随培养时间延长NO释放量持续增加,48h达到16μM,但小胶质细胞数量无明显改变。电针组12h小胶质细胞的活性明显低于对照组(p<0.05),并且12h、24h和48h NO释放量均显著低于对照组(p<0.05),增加电针血清浓度可明显减少NO的释放量。结论:电针血清可能通过抑制小胶质细胞活化以及释放NO,从而保护少突胶质细胞。通过上述实验我们得出如下结论:1.应用F3/RA/β-ME能够诱导CD90(+) hBMSCs分化为O4(+) OLLCs,F3通过激活F3/Notch信号系统促使OLLCs分化成熟。2.玻璃体腔移植F3诱导的OLLCs能够使移植细胞形态进一步成熟,并形成单层髓鞘包绕轴突。3. GBE能够提高plptg/-小鼠的存活率,改善动物临床症状。4. GBE可能通过抑制小胶质细胞激活减少CD4+T细胞的炎症性浸润,延缓plptg/-小鼠的脱髓鞘病变。5. GBE能够促进OLN细胞增殖,但不能诱导其分化为成熟少突胶质细胞。6.电针血清能够抑制小胶质细胞活化及增殖,减少激活的小胶质细胞释放NO。本研究首次应用F3/RA/β-ME成功诱导hBMSCs分化为少突胶质样细胞,为细胞移植治疗MS提供了实验依据;首次报道GBE对PLP转基因鼠的影响,阐明GBE对MS的防治具有应用前景。更多还原

【Abstract】 Multiple sclerosis (MS) is an inflammatory disease of the central nervous system. The pathological hallmark of MS is the sequential development of demyelination. There are many symptoms related to MS such as paralysis and loss vision. The MS is characterized by affecting young adults, having long course and relapsing-remitting. Till now there is no reliable means for therapy of MS.Since the mechanisms of myelin breakdown are still unknown, immunosuppression and hormones are commonly applied to MS patients. However, there are possible long term side effects from hormone replacement therapy. Accordingly,to explore the pathogenesis of MS and to successfully treat MS are required.In present study, immunohistochemistry, molecular biology, electron microscopy and FACS assay were performed. Firstly, we investigated whether human bone marrow stromal cells (hBMSCs) can be induced to become oligodendrocytes. Secondly, we studied the effect of ginkgo biloba extract (GBE) on plptg/- mice, an animal model for late onset chronic demyelination disease. Lastly, we explored the mechanism of electroacupuncture treatment of MS.1. Differentiation of Human Bone Marrow Stromal Cells into Oligodendrocyte-Like Cells through Activation of F3/Notch SignalingMethods: hBMSCs were purified through a cell adhesion approach and preinduced forβ-ME (24h) and RA (3d)). Cells were randomly divided into two groups treated with either multiple factors (MFS) or F3 another 3 days. The MFS group (positive control) was cultured in DMEM medium contained 5mM forskolin, 10ng/ml bFGF, 5ng/ml PDGF, and 200ng/ml HRG. The F3 group was cultured in DMEM medium contained 10%FBS and 20nM F3. FACS assay, immunofluorescence and Luciferase reporter assay were performed to characterize the nature of hBMSCs under different culture conditions. Subsequently eight-week-old mice were injected with the predifferentiated cells induced by RA/β-ME, MFS/RA/β-ME, or F3/RA/β-ME. Tow months after intravitreous injection, animals with transplanted cells were analysed by immunostaning and electron microscopy.Results: CD90 (+) cells were isolated from hBMSCs through a cell adhesion approach and immunostaining indicated that 40.88% cells expressed Sox2, an early stage stem cells marker. After treatment with RA plusβ-ME, CD 90 (+) hBMSCs were changed into oligodendrocyte-like cells (OLLCs) expressing more NG2 (54.43%), an early oligodendrocytes marker. After further treatment with F3 or MFS the cells expressed O4, a mature OLs marker. The population of O4 (+) cells (31.55+5.8% for F3 and 32.61+6.8% for MFS) significantly increased under both culture conditions, there was no significant difference between F3 group and MFS group. The expression of F3/Notch signalling component Notch1/NICD and DTX1 at all stages of induced differentiation was detected using antibodies, and immunofluorescence analysis showed that the intensity of Notch1/NICD and DTX1 in nucleus was increased after treatment by F3. Luciferase reporter assays showed that the relative reporter activity of Hes1 was significantly inhibited by F3 (0.33±0.08; p<0.01), but not by MFS (0.9±0.13). Notably the F3, but not RA and MFS, -induced transactivation of Hes1 was inhibited by over-expressed DTX1 and activated by a deletion mutant of Flag-tagged DTX1 mutant (dnDTX1). These observations demonstrated that F3/Notch signalling pathway may play a role in promoting maturation of immature OLLCs, derived from CD90 (+) hBMSCs, thought inhibiting classical Notch/Delta/Hes1 signaling pathway. Two months after transplantation of pre-differentiated cells, O4 (+) OLLCs send out more branches and processes (p<0.01) in mouse retina, as compared to in vitro. Interestingly, the number of processes per branch from the OLLCs predifferentiated by F3 (1.7+0.13), as well as by MFS (1.8+0.1), was significantly increased as compared to the RA group (1.13+0.13; p<0.001). To investigate this further, whole mounted retinas of F3 group were prepared for double immunocytochemistry using antibodies O4 andβ-tublin III. O4 (+) cells sent out their processes which landed onto the surface of retinal ganglion cell, and formed axoglial contacts and single myelin sheaths in retina.Conclusion: In the present study, we have demonstrated that hBMSCs have the capability to trans-differentiate into cells with oligodendrocytic characteristics. F3 triggers F3/Notch signaling in the CD90 (+) hBMSCs and induces them to differentiate into OLLCs with a high degree of morphological and functional maturation.2. Effects of ginkgo biloba extract on plp transgenic miceMethods: Three-month-old plp transgenic mice (plptg/- mice) were devided into PLP group (control group) and GBE group (experimental group). The GBE group received daily injection of 70mg/kg GBE, as well as the same volume of saline was injected into PLP group. Two months after injection, animal were sacrificed and histopathological changes were analyzed against untreated plptg/- mice. We also observed the influence of GBE on OLN cells (oligodendrocyte precursor cell lines) using MTT analysis and immunofluorescence.Results: Two months after treatment, the survival rate of GBE group was 76%, an increase of 23% as compared to PLP group. Behavioral test showed GBE group displayed longer latency interactions (33.0±4.36 sec). Double staining for MBP andβ-tublinin in optic nerve indicated that the expression level of MBP was decreased and some axons were naked in PLP group. However high expression of MBP was detected in GBE group and MBP (+) myelin was found along the surface of axons, which indicated well myelination in GBE group. We then analyzed the sagittal brain section in fimbria of hippocampus and found that the numbers of both OX-42 (+) microglia and CD4+T in GBE group were decreased as compared to PLP group. However the number of GFAP positive astrocytes was no different between GBE group and PLP group. To further confirm the role of GBE, OLN cells were cultured in DMEM medium containing GBE for 24h. MTT analysis indicated that the viability of OLN cells was increased in a dose-depend manner from GBE concentration of 0.07 mg/ml to 0.138mg/ml. GBE concentrations of 0.175mg/ml, 0.28 mg/ml and 0.35mg/ml promote increases in OD values by 0.091, 0.107 and 0.138 respectively. After treating OLN cells with 0.28mg/ml GBE for 24h, 48h and 72h, NG2 immunostaining found the OLN cells still were dipolar at all time points, absent the cells with multi-processing.Conclusion: Based on these observations we demonstrate that GBE can block demyelination process in plptg/- mice by inhibiting cytotoxin, which is released from activated microglia and T cell, suggesting that GBE has potential to treat MS in future.3. Effects of electroacupuncture stimulation-derived serum on microglia activity in vitroMethod: Baihui, Fengfu acupuncture points on the rats were stimulated with 100HZ high frequency electroacupuncture. Two weeks later blood was drawn, and serum was collected from the stimulated rats. Subsequently we divided the microglia into three groups: normal group cultured with normal rat serum; control group added with normal rat serum and microglia activator LPS ( 1μg/ml); and the experimental group added with the electroacupuncture stimulation-derived serum and LPS(1μg/ml). Immunostaining, MTT assay and Griess assay were used to detect the morphology, cell viability and NO release of microglia at 8h, 12h, 24h and 48h.Results: After treatment with 1μg/ml LPS, microglia was activated, which was indicated by the increased soma and the up regulation level of OX-42. Meanwhile Griess assay showed that the level of NO was higher in control group than in normal group at all time points, especially at 48h when the level of NO peaked to 16μM. However, MTT assay indicated that there was no change in cell number. After treatment with LPS for 12h, the viability of microglia in experimental group was less than that of control group. NO released from experimental group was significantly lower than that released from control group at 12h, 24h and 48h (p<0.05). NO release was also dose-dependent.Conclusion: In present study we indicate that the mechanisms of electroacupuncture protection of oligodendrocytes may be through inhibiting the proliferation of microglia and NO release from activated microglia.Conclusion:1. CD90 (+) hBMSCs can be trans-differentiated into OLLCs after treatment with F3/RA/β-ME. F3/Notch signaling promotes OLLCs maturation both in vitro and in vivo. 2. After transplantation, predifferentiated OLLCs have the capacity to become further mature OLLCs and send out processes, forming one layer of myelin in retina.3. GBE can promote plptg/- mice survival and improve behavior.4. GBE prevents plptg/- mice demyelination through inhibition of microglia activity and T cell infiltration.5. GBE promotes the proliferation of OLN cells, but cannot induce them to differentiate into oligodendrocytes.6. Electroacupuncture stimulation-derived serum inhibits the activity and the proliferation of microglia. Electroacupuncture stimulation-derived serum also reduces the level of NO released from activated microglia.At present, we have developed an approach to selective induce hBMSCs differentiation into OLLCs by F3/RA/β-ME, this may offer new strategies for cell therapies in MS. We also studied the effect of GBE on plp transgenic mice, and suggesting that GBE has potential to treat MS in future.更多还原