【作者】 刘继春；

【导师】 曾耀英；

【作者基本信息】 暨南大学 ， 生物医学工程， 2007， 博士

【摘要】 本研究目的在于探讨中药人参皂甙（Ginseng Saponin,GS）对人小胶质细胞功能的调节作用,为把GS发展为中枢神经系统退行性病变、缺血和损伤治疗药物奠定基础。为此,我们进行了六个方面的研究:人小胶质细胞的分离、培养和鉴定;GS对小胶质细胞活化的调节作用;GS对小胶质细胞吞噬作用的影响;GS对小胶质细胞一氧化氮合成的调节作用;GS对小胶质细胞反应氧族生成的调节作用;GS对溶血磷脂所引发的钙离子内流的影响。首先,我们从人工药物流产的胎儿脑组织分离并纯化小胶质细胞,并通过形态学和免疫荧光方法进行鉴定,纯度达到98%以上,并以此为细胞模型,进行下其他部分的研究。由于小胶质细胞在执行其功能之前,必须由静息状态转变为活化状态。活化的小胶质细胞既能产生具有神经保护功能的因子,又能分泌具有神经毒性的分子,其形态学表现为胞膜边缘波动（membrane ruffling）。研究发现:ATP和GS均能引发小胶质细胞的活化。小胶质细胞是中枢神经系统（central nerve system,CNS）主要的固有免疫细胞,吞噬作用是其重要功能。我们研究了GS对小胶质细胞吞噬作用的影响,结果发现:GS能够明显地促进小胶质细胞吞噬荧光微球。一氧化氮（nitric oxide,NO）是小胶质细胞执行功能的又一效应分子,同时,NO还参与了小胶质细胞过度活化所造成的神经元的损害。为此,我们研究了GS对小胶质细胞NO生成的影响,结果发现:GS能够促进小胶质细胞基础水平上的NO合成,而一定的程度上能够抑制由LPS所引发的NO的合成。反应氧族（Reactive oxygen species,ROS）的生理功能和毒性决定于其细胞內水平。低水平的ROS是小胶质细胞执行生理功能的效应分子;中等水平的ROS能够活化各种激酶和转录因子,如JNK、p38、NF-κB和API等;高水平的ROS可以引发小胶质细胞和神经元的凋亡乃至坏死。为此,我们研究了GS对小胶质细胞ROS产生的影响。结果发现:GS能够抑制ROS的生成。生物活性的溶血磷脂（bioactive lysophosphlipids）是一组天然产生的脂质,它们能够调节细胞各种功能,包括增殖、创伤愈合等。其中的溶血磷脂胆碱（lysophosphatidylcholine,LPC）是一种免疫细胞的调节剂,又与炎症的发生密切相关,为此,我们研究了GS对LPC所引发的小胶质细胞Ca²⁺內流的影响。结果发现:一定剂量的LPC可以使小胶质细胞产生瞬时的,其后,出现对钙离子导入剂的无反应性;而预先加入GS孵育后,再加入LPC,大多数的小胶质细胞出现了持续的、而不是瞬间的Ca²⁺内流,而且这些细胞保留了对钙离子导入剂的反应性。综上所述,人参皂甙在一定的剂量范围内,能增强小胶质细胞的吞噬作用;促进静止的小胶质细胞活化;促进或抑制小胶质细胞NO的合成;抑制小胶质细胞的反应氧族（ROS）的生成;保护小胶质细胞由溶血磷脂引起的细胞无反应性等作用。从而为把人参皂甙开发成为预防和治疗神经退行性病变的药物奠定基础。更多还原

【Abstract】 The aims of this study is to investigate the modulating effects of Chinese medicine herb Ginseng water-soluble extracts（GS） on human microglial functionns and to develop GMSE to become an uesful therapeutic medicine of central nerve system（CNS） neurodegenerative disease,injury and ischemia.Therefore,our study is divided into six parts:isolation, culturation and identification of human microglia from fetal brain tissues;modulation of GS on activation of microglial cells;influence of GS on phagocytosis of microglia;regulation of GS on NO synthesis of microglia;modulation of GS on production of ROS in microglia;impact of GS on lysophosphlipids-induced calcium influx in microglia.First,we isolated and purified human microglial cells from artificial abortive fetal brain tissue.After purification,we used morphological and immuno-fluorescence methods to identify the purity of microglia we got.We found that more than 98%of cells were microglia. On this condition,we made researches on the following parts.When microglial cells exert functions,they must alter their state from resting to activated. The activated microglia can elaborate both neurotrophic and neurotoxic factors,while its morphological change is membrane ruffling.So our results showed:both ATP and GS could make microglia activated.As the main innate immue cells in CNS,microglia has many important functions,like phagocytosis.Therefore,we tried to find out the effects of GS on microglial phagocytosis.GS obviously enhanced microglia engulfing fluorescent spheres.NO is another functional molecule that microglia produces.Meanwhile,if too much NO synthesized by over-activated microglia,it can cause neurotoxin.In the thesis,we performed experiments to evaluate the impacts of GS on NO production by microglia.Our results showed:GS could increase NO basic-level synthesis;however,it could suppress NO production stimulated by LPS to some extent.The physiological or toxic effects of ROS is depended on its intracellular level.Low-level ROS is a basic element that microglia needs to function normally;midi-level can activate multiple kinases and factors,such as JNK,p38 NF-κB and API,and so on;high-level may induce apoptosis or necrosis of microglia and neurons.So we examined the influence of GS on ROS production by microglia.We found that GS inhibited ROS synthesis.Bioactive lysophophlipids are a group of natural lipids,they can modulate several cellular function,like proliferation and injury-healing.Lysophophatidycholine（LPC）,which is the representative member of lysophophlipids,is an important modulator of immune cells.It also correlates tightly with inflammation.We explored the effects of GS on LPC-induced Ca²⁺ influx of microglia.As the results showed,LPC stimulated Ca²⁺ influx instantaneously,but later on,microglia had no response to calcium ionophore.When microglias were pre-incubated with GS,and then added LPC into supernatant,persistent but not instantaneous Ca²⁺ influx occurred;furthermore,those cells still responded to calcium ionophore efficiently.In summary,at a series of definitive concentrations,GS enhanced microglial phagocytosis;GS stimulated microglial activation;GS promoted or suppressed NO synthesis depending on microglial states;GS prevented ROS production by over-activated microglia; GS protected microglia from anergy to calcium ionophore caused by LPC.Consequently,GS could be developed to be a useful medicine used to prevent and therapy neurodegenerative diseases.更多还原