摘要
目的:近年来研究发现许多甾体物质在中枢神经系统发挥着重要作用,广泛地影响脑内多种神经递质及其受体和离子通道的功能,调节神经元的兴奋性,这些具有神经精神活性的甾体被称为神经甾体和第四代神经递质。研究显示,神经甾体可参与多种神经精神疾病的发生,而氨基酸能神经系统亦在此类疾病的发病机制中发挥重要作用。因此,进一步研究神经甾体与氨基酸能神经系统之间的相互作用将有助于神经精神疾病发病机制的阐明。本研究拟将大鼠大脑皮质神经元进行体外原代培养,采用神经甾体孕酮( progesterone , PROG )和脱氢表雄酮(dehydroepiandrosterone,DHEA)分别作用不同时间后检测细胞培养液中谷氨酸(GLU)和γ-氨基丁酸(GABA)的浓度;采用不同浓度的PROG和DHEA分别作用后测定神经元氨基酸受体亚单位NMDAR2B(NR2B)和GABAR1(GBR1)的表达,以观察两种神经甾体对氨基酸类神经递质释放及其受体表达的影响,为神经精神类疾病发病机制的阐明提供基础实验依据。
方法:
1大鼠大脑皮质神经元原代培养方法的建立
取孕17-18天SD大鼠胎鼠的大脑皮质,采用胰蛋白酶消化辅以机械分离法分离细胞,悬浮于含10%血清的DMEM培养基中,调整活细胞密度为1.2×109 L-1,接种于预先包被L-多聚赖氨酸的培养板中,在37oC,5% CO2及饱和湿度的细胞培养箱中孵育,24 h后首次换液为neurobasal培养基,以后每隔两天半量换液,并对神经元进行纯度测定,在倒置显微镜下随机选择3个视野(不重复,不重叠),分别记数所有的细胞和神经元,计算神经元的纯度。
2氨基酸测定方法的建立
荧光检测-高效液相法(HPLC-FLD)检测氨基酸含量的色谱条件为C18保护柱(5μm,4.6×12 mm,安捷伦公司);C18反相分析柱(5μm,4.6×150 mm,安捷伦公司)。流动相为A液和B液,梯度洗脱。A液为0.02 mol·L-1NaAc·3H2O,每500 ml加90μl二乙胺,以2%HAc调pH = 7.2,0.45μm微孔水相滤膜过滤后,每500 ml加1.5 ml四氢呋喃。B液为0.1 mol·L-1NaAc·3H2O,以2%HAc调pH = 7.2后,每100 ml加甲醇200 ml、乙腈200 ml,0.45μm微孔油相滤膜过滤。流动相流速为1.0 ml·min-1;柱温为40oC;激发波长为340 nm;发射波长为450 nm。
3分组和药物处理方案
3.1神经甾体影响神经元GLU和GABA释放实验
神经元培养第11天,随机分为对照组、PROG组和DHEA组,分别用1%的溶剂DMSO、10μmol·L-1的PROG和DHEA处理后,依次在0.5、1、1.5、2、24、36、48、72 h收集细胞和细胞培养液;每组每个时间点设8个复孔。药物处理后,裂解细胞收集蛋白,采用Lowry法测细胞蛋白浓度用作定量;采用OPA-巯基乙醇柱前衍生,HPLC-FLD法检测细胞培养液中GLU和GABA的浓度。
3.2神经甾体影响神经元NR2B和GBR1表达实验
神经元培养第11天,随机分为7组:对照组(1% DMSO)、PROG低剂量组(1μmol·L-1)、PROG中剂量组(10μmol·L-1)、PROG高剂量组(100μmol·L-1)、DHEA低剂量组(1μmol·L-1)、DHEA中剂量组(10μmol·L-1)、DHEA高剂量组(100μmol·L-1);对照组设5个复孔(一孔染色时作为阴性对照),其余每组设4个复孔。药物处理24 h后吸出细胞培养液,冷丙酮固定细胞;采用免疫细胞化学法检测神经元氨基酸受体亚单位NR2B和GBR1的表达水平。
结果:
1大鼠大脑皮质神经元的形态学观察和纯度测定
在倒置显微镜下可见,刚种植的细胞呈圆形,接种24 h后几乎全部贴壁,少数细胞伸出短小的突起,以后突起逐渐增多延长,第10天后神经元互相迁移靠近,聚集成团,突起相互连接形成网络;更换neurobasal培养基后,神经元的纯度逐渐提高,至培养第11天纯度可达95%以上。
2氨基酸测定方法学确证
本实验所用氨基酸测定方法GLU和GABA与细胞培养液中各杂质成份分离良好,无干扰;GLU在0.2～20μmol·L-1,GABA在10～1000μmol·L-1范围内具有良好的线性关系,日内和日间变异分别小于7.4%和8.6%(n = 4),回收率在97.4%～106.5%(n = 4)之间,检测限均为0.1μmol·L-1。
3 PROG和DHEA对神经元GLU释放的影响
与对照组相比,PROG处理1、1.5、2、24、36、48、72 h后,神经元培养液中的GLU水平分别降低66.5%、66.7%、64%、79.8%、89.7%、67.4%、58.9%(P<0.01);与对照组相比,DHEA处理相同的时间后,神经元培养液中的GLU水平分别降低63.8%、59.8%、54.8%、14.3 %、59.6%、60.9%、39.4%(P<0.05或P<0.01)。
4 PROG和DHEA对神经元GABA释放的影响
与对照组相比,PROG处理0.5、1、1.5、2、24、36、48、72 h后,神经元培养液中的GABA水平分别升高16.4%、43.3%、33.8%、24.5%、35.5%、62.7%、56.9%、46.2%(P<0.05或P<0.01);与对照组相比,DHEA处理1、1.5、2、24、36、48、72 h后,神经元培养液中的GABA水平分别升高37.5%、32.1%、31.7%、25.4%、57.1%、63.3%、58.9%(P<0.01)。
5 PROG和DHEA对神经元NR2B和GBR1表达的影响
体外培养的大鼠大脑皮质神经元经NR2B和GBR1免疫细胞化学染色后在普通光学显微镜下可观察到:神经元胞浆、胞膜及部分突起呈棕黄色着色,为其阳性表达。
与对照组相比,PROG和DHEA处理均使NR2B表达增加。低、中、高剂量PROG分别使其增加29.6%、33.9%、46.9%(P<0.05或P<0.01);与PROG低、中剂量组相比,高剂量组显著增加(P<0.05或P<0.01)。与对照组相比,低、中、高剂量DHEA分别使NR2B表达增加13.5%、16.6%、33.1%(P<0.05或P<0.01);与DHEA低、中剂量组相比,高剂量组显著增加(P<0.05或P<0.01)。
与对照组相比,PROG和DHEA处理均使GBR1表达增加。低、中、高剂量PROG分别使其增加31.5%、63.2%、67.9%(P<0.01);与PROG低剂量组相比,中、高剂量组显著增加(P<0.01)。与对照组相比,中、高剂量DHEA分别使GBR1表达增加16.7%、36.9%(P<0.05或P<0.01);与DHEA低、中剂量组相比,高剂量组显著增加(P<0.05或P<0.01)。
结论:
1 PROG和DHEA均对神经元GLU的释放起抑制作用,对GABA的释放起促进作用。
2 PROG和DHEA处理均可浓度依赖性地增强NR2B和GBR1的表达。
Objective:In recent years, it has been demonstrated that some steroid substances play important roles in the central nervous system. The brain neurosteroids are 4th generation neuromessengers which are synthesized within the neurons and are responsible for acute modulation of neuron-neuron communication through neurotransmitter receptors. Previous studies have shown that both neurosteroid and amino acids nervous system participate in the nosogenesis of many kinds of neuron-mental disorders. Further investigations of the interactions between neurosteroids and amino acids nervous system will be helpful in elucidating the pathogenesis of neuron-mental disorders. In the present study, rat cerebral cortical neurons were primary cultured, and the levels of glutamate and gama-aminobutyric were detected after being treated with PROG and DHEA at the different time. The expression of amino acids receptor subunit NR2B and GBR1 were also detected after being treated with PROG and DHEA at the different concentrations by immunocytochemistry. The present study will provide the basis for clarifying the mechanisms of some neuron-mental disorders.
Methods:
1 The primary culture of rat cerebral cortical neurons
Cortical neurons were obtained from E17-18 SD rat fetuses by mild digestion and mechanical dissection. The cells were seeded on the culture plates pre-coated with poly-L-lysine in the DMEM containing 10% fetal bovine serum at the density of 1.2×109 L-1. Cultures were maintained at 37oC, saturation humidity in an atmosphere containing 5% CO2. The neurobasal medium were changed after 24 hours culture and followed by changing half medium every 2 days. The purity of the neurons was determined by selecting 3 fields randomly under the inverted microscope.
2 The amino acids assay
HPLC-FLD was used for the determination of amino acids concentrations. The analytical column was C18 reversed-phase column with a C18 guard column. The column temperature was kept at 40oC. The mobile phase consisted of solution A and B with gradient elution. Solution A was 0.02 mol·L-1 NaAc·3H2O, solution B consisted of 0.1 mol·L-1 NaAc·3H2O, methanol and acetonitrile (1:2:2). The flow rate of mobile phase was 1.0 ml·min-1. The length of excitation wave was 340 nm; The length of emission wave was 450 nm.
3 Drug treatment
3.1 The effects of neurosteroids on GLU and GABA release in neuron
On 11-day in culture, the neurons were divided into 3 groups randomly (with 8 in each group).①control group: treated with 1% DMSO②PROG group: treated with 10μmol·L-1PROG③DHEA group: treated with 10μmol·L-1DHEA. The Cells and medium were collected respectively after being treated 0.5、1、1.5、2、24、36、48、72 h. The cells protein content was measured by Lowry method. The concentration of GLU and GABA in the medium were detected by HPLC-FLD.
3.2 The effects of neurosteroids on NR2B and GBR1 expression in neuron
On 11-day in culture, the neurons were divided into 7 groups randomly (with 4 in each group except control group) Cells were treated with 1% DMSO(control group, n=5); 1, 10, 100μmol·L-1PROG(PROG-L, M, H groups) or 1, 10, 100μmol·L-1DHEA(DHEA-L, M, H groups) respectively. Cells were fixed with cold acetone after being treated 24 h.The expression of neuron receptor subunit NR2B and GBR1 were detected by immunocytochemistry.
Results:
1 Morphology and purity of the rat cerebral cortical neurons
The cells were bright and round just plating. After 24 hours, most of the cells attached to the bottom of the plates. Some possessed small and short processes. After prolonged incubation, the processes multiplied and enlarged. On day 10, the neurons aggregates had interconnected by bundles of neuritis. The purity of the neurons increased gradually after changed to the neurobasal medium and was more than 95% on the 11-day culture.
2 The identification of amino acids assay
In the present study, the linear calibration curves of GLU and GABA were obtained in the range of 0.2～20μmol·L-1 and 10～1000μmol·L-1. The recoveries of them were between 97.4%～106.5%(n = 4). Inter-day and intra-day coefficient of variation were less than 7.4% and 8.6%(n = 4).The limit of quantitation of amino acids were 0.1μmol·L-1.
3 Effects of PROG and DHEA on GLU release in neuron
Compared with control group,the level of GLU in the medium decreased by 66.5%、66.7%、64%、79.8%、89.7%、67.4%、58.9% at 1、1.5、2、24、36、48、72 h after PROG treatment(P<0.01). The level of GLU in the medium increased by 63.8%、59.8%、54.8%、14.3%、59.6%、60.9%、39.4% after DHEA treatment(P<0.05 or P<0.01).
4 Effects of PROG and DHEA on GABA release in neuron
Compared with control group,the level of GABA in the medium increased by 16.4%、43.3%、33.8%、24.5%、35.5%、62.7%、56.9%、46.2% at 0.5、1、1.5、2、24、36、48、72 h after PROG treatmen(tP<0.05 or P<0.01).The level of GABA in the medium increased by 37.51%、32.09%、31.72%、25.39%、57.14%、63.33%、58.91% at the time of 1、1.5、2、24、36、48、72 h after DHEA treatment(P<0.01).
5 Effects of PROG and DHEA on the expression of NR2B and GBR1 receptor subunit in neuron
Compared with control group,the expression intensity of NR2B was enhanced by29.6%、33.9%、46.9% after PROG-L、PROG-M、PROG-H treatmen(tP<0.05 or P<0.01). Compared with PROG-L group and PROG-M group, the intensity of PROG-H group enhanced significantly(P<0.05 or P<0.01). Compared with control group,the expression intensity of NR2B enhanced by13.5%、16.6%、33.1% after DHEA-L、DHEA-M、DHEA-H treatmen(tP<0.05 or P<0.01). Compared with DHEA-L group and DHEA-M group, the intensity of DHEA-H group enhanced significantly(P<0.05 or P<0.01).
Compared with control group,the expression intensity of GBR1 increased after 31.5%、63.2%、67.9% by PROG-L、PROG-M、PROG-H treatment(P<0.01). Compared with PROG-L group, PROG-M group and PROG-H group enhanced significantly(P<0.01). Compared with control group,the expression intensity of GBR1 increased by 36.9%、16.7% after DHEA-H and DHEA-M treatment(P<0.05 or P<0.01). Compared with DHEA-L group and DHEA-M group, the intensity of DHEA-H group enhanced significantly(P<0.05 or P<0.01).
Conclusions:
1 Both neurosteroids PROG and DHEA inhibit GLU but promote GABA release from primary culture cortical neurons.
2 PROG and DHEA enhance the expression of neuron receptor subunit NR2B and GBR1 in a concentration-dependent manner.
引文
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