糖皮质激素对海马神经元影响的体外研究
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摘要
糖皮质激素为应激激素，正常浓度下对于维持机体正常的生长发育以及生理和应激状态下内环境的稳定都是极为重要的，然而在各种应激损伤条件下糖皮质激素过度分泌，血浆中高水平的糖皮质激素持续作用可以透过血脑屏障进入脑组织，危害中枢神经系统。在中枢神经系统中，海马富含糖皮质激素受体，因此海马极易受到循环中过量的糖皮质激素的攻击，使其受到损伤。这就给临床上广泛应用糖皮质激素如地塞米松、甲基强的松龙治疗脑水肿提出了质疑，大量的研究结果也支持糖皮质激素会诱导海马神经细胞损伤的推测，但是体内实验受到机体各种复杂因素的影响难以了解糖皮质激素在体内确切的作用机制，体外实验又是从各自不同的实验目的出发，提供了零散的、不是十分系统的佐证，因此对于高浓度的糖皮质激素诱导海马神经元的认识仍然还处于初期的推测阶段，尚缺乏足够的实验证据和系统的研究报告。为此，如能阐明高浓度糖皮质激素对海马神经细胞损伤的过程及机制，将为糖皮质激素在脑水肿治疗中的合理应用提供帮助。
目的
在体外实验条件下，观察不同浓度的地塞米松对海马神经细胞的损伤。从细胞形态学、细胞酶学以及细胞凋亡相关蛋白的表达等方面进行分析，从而加深对糖皮质激素对海马神经细胞的损伤过程及机理的了解，同时对bFGF保护糖皮质激素诱导的海马神经元损伤作用及机制进行初步探讨，为临床上合理的应用糖皮质激素治疗脑水肿提供帮助。
糖皮质漱素对海马神经元形响的体外研究 医学硕士学位论文
方 法
本研究采用星形胶质细胞条件培养液对大鼠海马神经元原代培养进行改
良，较成功的获得了相对单一的海马神经细胞培养物（神经细胞纯度达到95％
左右，可见单神经细胞网络）。在此基础上，对不同浓度的地塞米松对海马神
经细胞的损伤，应用倒置相差显微镜、Typan Blue染色及 My’检测对不同浓
度的地塞米松对海马神经细胞损伤进行细胞形态学及细胞酶学分析；应用
TUNEL方法原位检测不同浓度的地塞米松作用于海马神经细胞后细胞的凋
亡情况，观察其凋亡特征；结合免疫组织化学染色对不同浓度的地塞米松作
用于海马神经细胞后凋亡相关蛋白Bcl－2及Bax表达的改变进行观察；同时
分组比较观察bFGF对糖皮质激素诱导海马神经细胞凋亡的影响。
结 果
1．应用星形胶质细胞条件培养液改良的大鼠海马神经元培养方法较成功的
获得了相对单一的神经细胞培养物（神经细胞纯度可达95％左右，可见单神
经细胞网络）。与未用AS条件培养液组相比胶质细胞明显减少，细胞的生理
凋亡明显受抑制，细胞数目增多，细胞胞体增大、突起变长增粗，明显增加
海马神经元细胞活性。培养第7天细胞中NSE阳性率为95％左右，GFAP阳
性率3％左右。细胞生长到12l 天细胞最为丰满，胞体折光性强，有明显的
立体感，神经突起多而粗大，网络稠密。这种改良方法是观察单一海马神经
细胞的各项指标的理想培养方法。
2．地塞米松对海马神经元的损伤。
O）倒置相差显微镜观察显示经不同浓度的 DEX处理24 ’J、时后，10‘＼ IO‘SM
组神经细胞的形态与对照组相比无明显异常，10十10＼10’M组部分神经
细胞的突起变短，胞膜皱缩，折光性减弱，体积缩小，并有细胞出现核固缩
有时可见核碎裂。
仅）My检测发现海马神经元经不同浓度 DEX处理 24小时后，10－＼ 10”SM
组经 M17’检测 OD值与对照组相比差别无显著性意义；而 10”乙 10＼ 10”’M
天津医科大学2
糖皮质溢素对海马神经元影响的体外研究 医学硕士学位论文
组经 NlT检测 OD值与对照组相比差别有显著性意义；10刁、10＼ 10”’M组
经MTT检测OD值各组相比差别无显著性意义。
（）IUN’－－L检测显示正常培养的海马神经元TUNE染色偶见阳性核神经元
（凋亡率为丑03士1．71％）凋亡细胞形态各异，细胞皱缩，核固缩呈棕黄色。
DEX浓度为10久10“M组海马神经元凋亡细胞与对照组相比无明显增加；
DEX浓度为 10＼ 10人 10“’M组海马神经元凋亡细胞与对照组相比有明显
增加；DEX浓度为 10“组海马神经元凋亡细胞与 10’M组相比进一步增加。
10“’M组海马神经元凋亡细胞与川“M组相比无明显增加。
K）台盼蓝染色显示不同浓度的DEX作用24小时后，在视野中各组着色神
经元均较少（＜5％），而且随着DEX浓度的着色神经元有所增加，但各组之
间无显著性差别。
（5）免疫组织化学凋亡相关蛋白scl－2、Bax染色发现DEX处理组10－’、10”
Glucocorticoids (GCs) are hormones released during stress, which are very important to maintain both normal growth and development of bodies and internal homeostasis in physiological conditions and stress. But in plasma sustained GCs of high concentrations from oversecretion during stress can make their access to brain through blood-brain barrier and endanger central nervous system(CNS). In CNS, hippocampus, a structure possessing high concentrations of receptors for GCs, is likely to be victims of excessive circulating GCs, that put forward a question of how to use GCs such as dexamethasone and methlprednisolone to alleviate cerebral edema. Many evidence support GCs can induce damages of hippocampal neurons, but the complexity of in vivo environment makes it difficult for the identification of the exact mechanism of effects of GCs, and unsystematic in vitro experiments aiming at different destinations are impossible to present enough evidence. At this point, the knowledge about effects of GCs on hippocampal
neurons is still at the preliminary stage. There has been virtually no enough proofs and systematic reports. If the proccess and mechanism of insults of high-concentration GCs to hippocampal neurons can be expounded, it can present theoretical reference to proper application of GCs to cerebral edema.
Objective
The objectives of our experiments were to observe insults from dexamethsones of different concentrations on hippocampal neurons. Employing analysis of celluar morphology, celluar emzymology and celluar apoptosis-related proteins, these studies can make the insulting proccess and mechanism of GCs on hippocampal neurons understood more deeply. At the same time, these studies were to explore the protective mechanism of bFGF to insults of hippocampal
neurons induced by GCs. Our experiments will provide a reasonable reference to GCs on cerebral edema.
Methods
In these studies, astrocyte(AS)-conditioned medium was employed to optimize primary culture of rat hippocampal neurons to obtain relatively pure culture(the ratio of neurons could reach 95% and single neural network could be observed). Microscopy, typan blue staining and MTT were used to evaluate changes of celluar morphology and celluar enzymology induced by dexamethasone of defferent concentrations. TUNEL was used to examine celluar apoptosis of hippocampal neurons induced by dexamethasone of different concentrations. By immunohistochemistry staining, the changes of apoptosis-related protein expression of Bcl-2 and Bax were examined. And at last, the effects of bFGF on apoptosis induced by dexamethasones were compared among groups.
Results
1. We obtained relatively pure culture through optimizing primary culture of rat hippocampal neurons with AS-conditioned medium. Compared with control, glial cells decreased significantly, the physiological apoptosis was inhibited markedly, and the quantities of cells increased, the bodies enlarged, the processes lengthened and widen. All these phenomena supported the viability of neurons were enhanced. The positive ratio of NSE in cells on day 7 in culture was about 95%, and that of GFAP was about 3%. By day 12-14, the cells reached their best states to exhibit strong refraction and third dimension and the wide neural processes formed dense network. This optimal method is a perfect one to observe various indexes of single hippocampal neuron.
2. The injuries of dexamethasone to hippocampal neurons
(1) After incubation with dexamethasone of different concentrations for 24 hours, the cell morphology of 10~9,10~8M groups did not change compared with control by microscopy. InlO"7, lO^lO^M groups, the processes shorten, the membranes shrinked, the refraction weakened, the bodies became smaller, and some cells presented karyopyknosis and karyorrhexis.
(2) Incubation with dexamethasone of different concentrations for 24 hours, OD values from MTT of 10~9,10~*M groups did not vary markedly compared with control. In 10~7,10"*,10~5M groups, OD values varied significantly, while OD values between thr
目的
在体外实验条件下，观察不同浓度的地塞米松对海马神经细胞的损伤。从细胞形态学、细胞酶学以及细胞凋亡相关蛋白的表达等方面进行分析，从而加深对糖皮质激素对海马神经细胞的损伤过程及机理的了解，同时对bFGF保护糖皮质激素诱导的海马神经元损伤作用及机制进行初步探讨，为临床上合理的应用糖皮质激素治疗脑水肿提供帮助。
糖皮质漱素对海马神经元形响的体外研究 医学硕士学位论文
方 法
本研究采用星形胶质细胞条件培养液对大鼠海马神经元原代培养进行改
良，较成功的获得了相对单一的海马神经细胞培养物（神经细胞纯度达到95％
左右，可见单神经细胞网络）。在此基础上，对不同浓度的地塞米松对海马神
经细胞的损伤，应用倒置相差显微镜、Typan Blue染色及 My’检测对不同浓
度的地塞米松对海马神经细胞损伤进行细胞形态学及细胞酶学分析；应用
TUNEL方法原位检测不同浓度的地塞米松作用于海马神经细胞后细胞的凋
亡情况，观察其凋亡特征；结合免疫组织化学染色对不同浓度的地塞米松作
用于海马神经细胞后凋亡相关蛋白Bcl－2及Bax表达的改变进行观察；同时
分组比较观察bFGF对糖皮质激素诱导海马神经细胞凋亡的影响。
结 果
1．应用星形胶质细胞条件培养液改良的大鼠海马神经元培养方法较成功的
获得了相对单一的神经细胞培养物（神经细胞纯度可达95％左右，可见单神
经细胞网络）。与未用AS条件培养液组相比胶质细胞明显减少，细胞的生理
凋亡明显受抑制，细胞数目增多，细胞胞体增大、突起变长增粗，明显增加
海马神经元细胞活性。培养第7天细胞中NSE阳性率为95％左右，GFAP阳
性率3％左右。细胞生长到12l 天细胞最为丰满，胞体折光性强，有明显的
立体感，神经突起多而粗大，网络稠密。这种改良方法是观察单一海马神经
细胞的各项指标的理想培养方法。
2．地塞米松对海马神经元的损伤。
O）倒置相差显微镜观察显示经不同浓度的 DEX处理24 ’J、时后，10‘＼ IO‘SM
组神经细胞的形态与对照组相比无明显异常，10十10＼10’M组部分神经
细胞的突起变短，胞膜皱缩，折光性减弱，体积缩小，并有细胞出现核固缩
有时可见核碎裂。
仅）My检测发现海马神经元经不同浓度 DEX处理 24小时后，10－＼ 10”SM
组经 M17’检测 OD值与对照组相比差别无显著性意义；而 10”乙 10＼ 10”’M
天津医科大学2
糖皮质溢素对海马神经元影响的体外研究 医学硕士学位论文
组经 NlT检测 OD值与对照组相比差别有显著性意义；10刁、10＼ 10”’M组
经MTT检测OD值各组相比差别无显著性意义。
（）IUN’－－L检测显示正常培养的海马神经元TUNE染色偶见阳性核神经元
（凋亡率为丑03士1．71％）凋亡细胞形态各异，细胞皱缩，核固缩呈棕黄色。
DEX浓度为10久10“M组海马神经元凋亡细胞与对照组相比无明显增加；
DEX浓度为 10＼ 10人 10“’M组海马神经元凋亡细胞与对照组相比有明显
增加；DEX浓度为 10“组海马神经元凋亡细胞与 10’M组相比进一步增加。
10“’M组海马神经元凋亡细胞与川“M组相比无明显增加。
K）台盼蓝染色显示不同浓度的DEX作用24小时后，在视野中各组着色神
经元均较少（＜5％），而且随着DEX浓度的着色神经元有所增加，但各组之
间无显著性差别。
（5）免疫组织化学凋亡相关蛋白scl－2、Bax染色发现DEX处理组10－’、10”
Glucocorticoids (GCs) are hormones released during stress, which are very important to maintain both normal growth and development of bodies and internal homeostasis in physiological conditions and stress. But in plasma sustained GCs of high concentrations from oversecretion during stress can make their access to brain through blood-brain barrier and endanger central nervous system(CNS). In CNS, hippocampus, a structure possessing high concentrations of receptors for GCs, is likely to be victims of excessive circulating GCs, that put forward a question of how to use GCs such as dexamethasone and methlprednisolone to alleviate cerebral edema. Many evidence support GCs can induce damages of hippocampal neurons, but the complexity of in vivo environment makes it difficult for the identification of the exact mechanism of effects of GCs, and unsystematic in vitro experiments aiming at different destinations are impossible to present enough evidence. At this point, the knowledge about effects of GCs on hippocampal
neurons is still at the preliminary stage. There has been virtually no enough proofs and systematic reports. If the proccess and mechanism of insults of high-concentration GCs to hippocampal neurons can be expounded, it can present theoretical reference to proper application of GCs to cerebral edema.
Objective
The objectives of our experiments were to observe insults from dexamethsones of different concentrations on hippocampal neurons. Employing analysis of celluar morphology, celluar emzymology and celluar apoptosis-related proteins, these studies can make the insulting proccess and mechanism of GCs on hippocampal neurons understood more deeply. At the same time, these studies were to explore the protective mechanism of bFGF to insults of hippocampal
neurons induced by GCs. Our experiments will provide a reasonable reference to GCs on cerebral edema.
Methods
In these studies, astrocyte(AS)-conditioned medium was employed to optimize primary culture of rat hippocampal neurons to obtain relatively pure culture(the ratio of neurons could reach 95% and single neural network could be observed). Microscopy, typan blue staining and MTT were used to evaluate changes of celluar morphology and celluar enzymology induced by dexamethasone of defferent concentrations. TUNEL was used to examine celluar apoptosis of hippocampal neurons induced by dexamethasone of different concentrations. By immunohistochemistry staining, the changes of apoptosis-related protein expression of Bcl-2 and Bax were examined. And at last, the effects of bFGF on apoptosis induced by dexamethasones were compared among groups.
Results
1. We obtained relatively pure culture through optimizing primary culture of rat hippocampal neurons with AS-conditioned medium. Compared with control, glial cells decreased significantly, the physiological apoptosis was inhibited markedly, and the quantities of cells increased, the bodies enlarged, the processes lengthened and widen. All these phenomena supported the viability of neurons were enhanced. The positive ratio of NSE in cells on day 7 in culture was about 95%, and that of GFAP was about 3%. By day 12-14, the cells reached their best states to exhibit strong refraction and third dimension and the wide neural processes formed dense network. This optimal method is a perfect one to observe various indexes of single hippocampal neuron.
2. The injuries of dexamethasone to hippocampal neurons
(1) After incubation with dexamethasone of different concentrations for 24 hours, the cell morphology of 10~9,10~8M groups did not change compared with control by microscopy. InlO"7, lO^lO^M groups, the processes shorten, the membranes shrinked, the refraction weakened, the bodies became smaller, and some cells presented karyopyknosis and karyorrhexis.
(2) Incubation with dexamethasone of different concentrations for 24 hours, OD values from MTT of 10~9,10~*M groups did not vary markedly compared with control. In 10~7,10"*,10~5M groups, OD values varied significantly, while OD values between thr
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