体外获得高纯度大鼠背根神经节神经元的原代培养

目的：建立一种切实可行的胚胎大鼠背根神经节神经元培养及纯化方法。方法：用显微解剖获取足够数量的胚胎大鼠背根神经节,通过胰蛋白酶+EDTA消化、交替使用NB培养基与加入了5-氟-2-脱氧尿嘧啶核苷酸/尿苷的抗有丝分裂NB培养基等方法,在体外获得纯化的背根神经节神经元,采用神经丝蛋白免疫细胞化学染色法与DAPI染核的方法鉴定并测定神经元的纯度。结果：获得的背根神经节神经元在体外生长良好,纯度可达到96%以上。结论：本实验方法可以获得大量高度纯化的大鼠背根神经节神经元。     

一种稳定高效的新生大鼠皮质神经元原代培养方法

目的:建立一种稳定、高效、简单可行的新生大鼠大脑皮质神经元原代培养方法。方法:取新生24 h内的SD大鼠,分离皮质,采用木瓜蛋白酶消化、实验前多聚赖氨酸铺板、不同机械吹打次数及细胞接种2 h后全量换为添加B-27的Neurobasal A培养基的培养方法,MTT比色法分析检测神经元细胞成活率;于不同时间在倒置相差显微镜下观察细胞的生长状态;利用神经元特异性标志物微管蛋白相关标志物2(microtubule associated protein 2,MAP2)鉴定神经元纯度。结果:木瓜蛋白酶明显增加原代培养神经元细胞活力(P0.01),采用适度的机械吹打、实验前多聚赖氨酸铺板及细胞接种2 h后全量换为添加B-27的Neurobasal A培养基,对神经细胞的活力没有明显的影响(P0.05)。大部分皮质神经元具有典型的神经元形态特征,经MAP2免疫荧光技术鉴定神经元所占比例达95%以上。结论:该方法操作简单高效,方便可行,结果稳定。     

二脱氧肌苷引发培养的背根神经节神经元的形态学改变

为探讨二脱氧肌苷(ddI)对培养的背根神经节(DRG)神经元的影响,我们对分散培养的胎鼠DRG神经元培养3d后,再分别以不同浓度的ddI(1μg/ml,5μg/ml,10μg/mll和20μg/ml)孵育3d。终止培养后,行微管相关蛋白2(MAP2)标记,用共聚焦激光扫描显微镜观察神经元胞体和突起的改变。结果表明,DRG神经元用ddI孵育3d,神经元突起的数目减少和长度变短,呈剂量依赖性,而神经元的直径则没有变化。本研究的结果表明,ddI可影响培养的DRG神经元突起的再生和生长。     

大鼠背根神经节神经元H~+-门控电流及其特征

目的:探讨大鼠背根神经节(DRG)神经元H+-门控电流(也称ASICs电流)及其特征;比较在SD大鼠DRG和三叉神经节(TG)神经元电流分布的异同。方法:在急性分离的大鼠DRG神经元上,采用全细胞膜片钳技术记录电流。结果:依据在pH5.0条件下记录的ASICs电流动力学特征,将急性分离的DRG神经元上的ASICs电流分为四类:T-型、S-型、B-型和O-型;并推断此四种电流类型在周围感觉神经元上分布的一般规律。结论:在SD大鼠DRG和TG神经元ASICs电流都是以"S"型电流为主。4种电流的激活动力学τon(0～63%上升时间)和细胞直径无明显相关性。     

大鼠背根神经节内kv2.1 mRNA和kv4.2 mRNA的表达

目的　了解kv2 1和kv4 2型钾离子通道在背根神经节的表达。方法　应用原位杂交方法观察背根神经节内细胞膜钾离子kv2 1和kv4 2型通道的mRNA阳性神经结构的表达。结果　腰 3～ 5背根神经节内均有kv2 1和kv4 2mRNA阳性神经元的表达 ,kv4 2mRNA阳性神经元的数量和总面积明显多于kv2 1阳性神经元。结论　kv2 1和kv4 2型钾离子通道可能参与了背根神经节对外周传入信息的传递和调制。     

牛磺酸对大鼠背根神经节神经元GABA激活电流的调制作用

牛磺酸可能是中枢神经系统中一种抑制性神经递质或神经调质。本实验目的在于观察牛磺酸是否引起背根神经节（ＤＲＧ）神经元膜产生反应以及是否对ＧＡＢＡ引起的背根神经节神经元奄流有调制作用。应用全细胞膜片钳技术,观察未检测到牛磺酸对ＤＲＧ神经元膜电流的改变,但预先给牛磺酸对ＧＡＢＡ激活的膜内向电流有明显的帽作用,并具明显的浓度依赖性。如１０＾－５ｍｏｌ／Ｌ的牛磺酸可使１０＾－５ｍｏｌ／Ｌ ＧＡＢＡ激活的膜电     

人类免疫缺陷病毒蛋白HIV gp120对培养的大鼠背根神经节神经元的神经毒性作用

为探讨人类免疫缺陷病毒(HIV)gp120对体外培养的大鼠背根神经节(DRG)神经元的神经毒性作用,我们建立了胎鼠DRG分散培养和器官型培养的模型。以上分散培养和器官型培养的DRG,经不同浓度(250pmol/L,1nmol/L)的HIVgp120处理(2次/7d)。分散培养的DRG细胞行微管相关蛋白2(MAP2)免疫荧光染色,然后利用荧光显微镜观察神经元胞体和突起的变化情况。器官型培养的DRG在电子显微镜下观察其超微结构的改变。经HIVgp120处理的神经元突起的数目和长度的变化,HIVgp120处理组与对照组相比有显著性意义(P<0.001),而神经元的直径则没有变化(P>0.05)。应用以上不同浓度的HIVgp120处理后,神经元的超微结构出现明显改变,线粒体嵴减少或消失,微管和神经丝之间出现了大量的高电子密度颗粒。以上结果表明,HIVgp120对培养的DRG神经元具有直接的神经毒性作用,其中以线粒体的改变较为敏感。     

多巴胺对大鼠背根神经节神经元GABA-激活电流的影响

用全细胞膜片箝记录技术在大鼠分离背根节神经元上观察预加多巴胺对 GABA-激活电流的调控作用。发现大部分受检细胞 ( 4 0 / 47)对外加 GABA敏感。 10 - 6 ～ 10 - 3mol/ L GABA引起一剂量依赖性、有明显去敏感作用的内向电流。在对 GABA敏感的 40个细胞中 ,多巴胺引起一小的、无去敏感的外向电流 ( 2 6/ 40 ) ,其他无明显反应 ( 14 / 40 )。预加多巴胺 10 - 7～ 10 - 4 mol/ L 3 0 s后再加 GABA有 68% ( 2 7/ 40 )的细胞 GABA-激活电流幅值抑制 ,以多巴胺的浓度 10 - 5 mol/ L的抑制最明显 ( 3 3 .3 % )。     

高糖对体外培养背根神经节神经元影响的研究进展

背根神经节神经元(DRGn)在糖尿病周围神经病变发展过程中起重要作用,近年来探讨高糖对体外培养背根神经节神经元(DRGn)的影响成为研究热点.高糖环境可致DRGn细胞凋亡;同时高糖环境下DRGn在氧化应激、膜受体mGluRs及TRPV1激活、DRGn/SCs共培养等方面均与正常培养基不同,具体机制仍需深入研究.     

大鼠背根神经节分离神经元的延时整流的钾离子单通道

运用膜片钳技术对急性分离的大鼠背根神经节神经元细胞上的电压依赖性钾离子通道进行了研究.在细胞贴附式记录模式下,去极化可以激活一个电导为20pS的钾离子单通道电流,分析了其单通道的特性,提出了其动力学的模型.对于了解大鼠背根神经节神经元细胞的电活动机制具有重要的意义.     

Mechanosensitive chloride channels on the growth cones of cultured rat dorsal root ganglion neurons

We developed an experimental system to investigate mechanosensitivity of a single neuron, using cultured rat dorsal root ganglion cells. Highly precise mechanical stimulation was applied to various sites of the cells, using a piezo-driven glass microcapillary whose movement was computer-controlled, while whole-cell patch-clamp recordings were made from the cell bodies. When the growth cones and lamillipodia from the cell soma were mechanically stimulated, inward currents were recorded at the holding potential of -60mV. Filopodia were most sensitive to mechanical stimulation. However, when neurites or soma of dorsal root ganglion cells were stimulated in the same way, electrical responses were hardly recorded. Two types of currents varying in time-course were observed: fast type of 100-200ms and slow type of several seconds in duration. When the membrane potential was held at around 0mV, both types of currents were almost abolished or even reversed, and the reversal potential was estimated to be about -2. 2mV. Replacement of extracellular sodium by tetraethylammonium did not significantly change the reversal potential. In the low-chloride solution ([Cl(-)](o)=11.7mM), the reversal potential was about +60mV, as expected from the Nernst equation for chloride. These inward currents were almost completely inhibited by extracellular application of chloride channel blocker, 5-nitro-2-(3-phenylpropylamino) benzoic acid (100microM).These results indicate that the inward currents are due to activation of mechanosensitive chloride channels, preferentially located on the growth cones of cultured dorsal root ganglion neurons.     

Expression of neurokinin-1 receptors on cultured dorsal root ganglion neurons from the adult rat

The expression of neurokinin-1 receptors in isolated dorsal root ganglion neurons of adult rats was investigated using substance P covalently bound to a 1.4-nm gold particle. Binding of substance P-gold was determined in neurons after 0.8, 1.8 or 3.8 days under culture conditions. Substance P-gold binding sites were identified in 9.5 +/- 1.8% of the neurons that were cultured for 0.8 days. The proportion of neurons with substance P-gold binding sites increased to 21.5 +/- 3.6% after 1.8 days in culture and returned to the initial values (9.2 +/- 2.1%) after 3.8 days in culture. Binding of substance P-gold was suppressed by co-administration of [Sar9, Met(O2)11] substance P, a specific agonist at the neurokinin-1 receptor, but not by co-administration of [beta-Ala8] Neurokinin A (4-10), an agonist at the neurokinin-2 receptor, and senktide, an agonist at the neurokinin-3 receptor. This indicates that substance P-gold was bound specifically to neurokinin-1 receptors. Double-labelling with RT97, an antibody that selectively labels somata of A-fibres revealed that substance P binding sites were present in small neurons with myelinated and unmyelinated axons. These data show that a proportion of dorsal root ganglion neurons of adult rat in culture exhibit neurokinin-1 receptors. A transient increase in the proportion of neurons expressing neurokinin-1 receptors after 1.8 days in culture suggests that the expression of neurokinin-1 receptors is subjected to regulation.     

Serotonin receptor mRNA expression in rat dorsal root ganglion neurons

In the present study, we have used in situ hybridization to examine the distribution of serotonin (5-HT) receptors in rat dorsal root ganglion (DRG) neurons. Within DRG neurons, mRNAs for 5-HT1B, 5-HT1D, 5-HT2A, 5-HT2B, 5-HT3B and 5-HT4 receptors were readily detected in small (<25 microm), medium (25-45 microm) and large (>45 microm) diameter neurons. In contrast mRNAs for 5-HT1A, 5-HT1E, 5-HT2C, 5-HT5A, 5-HT5B, 5-HT6 and 5-HT7 receptors were undetectable in these neurons. The present study provides an insight into the molecular profile of 5-HT receptor subtypes in neurons responsible for modulating sensory information.     

二次分层离心纯化成年大鼠背根节神经元的方法

目的:建立一种长时程的有效获得高度纯化成年大鼠背根节(dorsal root ganglions,DRG)神经元的方法。方法:将SD大鼠的DRG剥膜、消化制成单细胞悬液,牛血清白蛋白(bovine serum albumin,BSA)分层离心去除大部分非神经元细胞后接种于多聚赖氨酸处理的盖玻片上;培养1 d后,胰酶消化再次制成细胞悬液,BSA二次分层离心,再次接种于多聚赖氨酸处理的盖玻片上。BSA二次分层离心后的神经元为实验一(T1组)、单次离心的神经元为实验组二(T2组)、未经离心处理的神经元为对照组(C组)。各组除离心次数外,其余各方法相同。相差显微镜下观察上述各组培养神经元,结合βtubulinⅢ免疫荧光组化染色及MTT分析检测神经元纯化效果及细胞活力。结果:培养3 d后,T1组神经元比例达88.43±6.13%,较T2组、C组显著增高,差别具有统计学意义(P0.05);MTT的结果显示与T2组、C组比较,T1组神经元的相对活力略微下降,但无统计学差异(P0.05)。结论:二次纯化法是一种简单有效的成年大鼠DRG神经元纯化方法。     

GABA-Induced Cl- current in cultured embryonic human dorsal root ganglion neurons.

gamma-Aminobutyric acid (GABA)-activated channels in embryonic (5-8 wk old) human dorsal root ganglion (DRG) neurons in dissociated culture were characterized by whole cell and single-channel techniques. All DRG neurons when held at negative holding membrane potentials displayed inward current to micromolar concentrations of GABA applied by pressure pulses from closely positioned micropipettes. The current was directly proportional to the concentration of GABA (EC50, 111 microM; Hill coefficient, 1.7). DRG neurons also responded to micromolar concentrations of pentobarbital and alphaxalone but not to cis-4-aminocrotonic acid (CACA), glycine, or taurine. Baclofen (100 microM) affected neither the holding currents nor K+ conductance (when patch pipettes were filled with 130 mM KCl) caused by depolarizing pulses. Whole cell GABA-currents were blocked by bicuculline, picrotoxin, and t-butylbicyclophosphorothionate (TBPS; all at 100 microM). The reversal potential of whole cell GABA-currents was close to the theoretical Cl- equilibrium potential, shifting with changes in intracellular Cl- concentration in a manner expected for Cl--selective channels. The whole cell I-V curve for GABA-induced currents demonstrated slight outward rectification with nearly symmetrical outside and inside Cl- concentrations. Spectral analysis of GABA-induced membrane current fluctuations showed that the kinetic components were best fitted by a triple Lorentzian function. The apparent elementary conductance for GABA-activated Cl- channels determined from the power spectra was 22.6 pS. Single-channel recordings from cell-attached patches with pipettes containing 10 microM GABA indicated that GABA-activated channels have a main and a subconductance level with values of 30 and 19 pS, respectively. Mean open and closed times of the channel were characterized by two or three exponential decay functions, suggesting two or three open channel states and two closed states. Single channels showed a lack of rectification. The actions of GABA on cultured human embryonic DRG neurons are mediated through the activation of GABAA receptors with properties corresponding to those found in the CNS of human and other mammalian species but differing from those of cultured human adult DRG neurons.     

Radiation-induced apoptosis in dorsal root ganglion neurons

Ionizing radiation (IR) results in apoptosis in a number of actively proliferating or immature cell types. The effect of IR on rat dorsal root ganglion (DRG) neurons was examined in dissociated cell cultures. After exposure to IR, embryonic DRG neurons, established in cell culture for six days, underwent cell death in a manner that was dose-dependent, requiring a minimum of 8 to 16 Gy. Twenty-five per cent cell loss occurred in embryonic day 15 (E-15) neurons, grown in cell culture for 6 days (immature), and then treated with 24 Gy IR. In contrast, only 2% cell loss occurred in E-15 neurons maintained in culture for 21 days ("mature") and then treated with 24 Gy IR. Staining with a fluorescent DNA-binding dye demonstrated clumping of the nuclear chromatin typical of apoptosis. Initiation of the apoptosis occurred within 24 h after exposure to IR. Apoptosis was prevented by inhibition of protein synthesis with cycloheximide. Apoptosis induced by IR occurred more frequently in immature than in mature neurons. Immature DRG neurons have a lower concentration of intracellular calcium ([Ca2+]i) than mature neurons. Elevation of [Ca2+]i by exposure to a high extracellular potassium ion concentration (35 M) depolarizes the cell membrane with a resultant influx of calcium ions. The activation of programmed cell death after nerve growth factor (NGF) withdrawal is inversely correlated with [Ca2+]i in immature DRG neurons. When treated with high extracellular potassium, these immature neurons were resistant to IR exposure in a manner similar to that observed in mature neurons. These data suggest that [Ca2+]i modulates the apoptotic response of neurons after exposure to IR in a similar manner to that proposed by the Ca2+ setpoint hypothesis for control of NGF withdrawal-induced apoptosis.     

Calcium signaling in cold cells studied in cultured dorsal root ganglion neurons.

Thermosensitive cold cells were identified in cultured dorsal root ganglion neurons from newborn rats. The neurons were loaded with a calcium indicator, Fura-PE3, and the change in intracellular Ca2+ concentration ([Ca2+]i) of the neurons was measured with microfluorimetry. Thirteen per cent of the cells responded to the cold stimulation. The diameter of the responder cells was 16.3+/-3.2 microm (mean+/-S.D., n = 25). The lowering of the temperature from 35 degrees C to 20 degrees C increased [Ca2+]i from 59.6+/-10.6 nM to 203.4+/-14.8 nM (n = 25). The [Ca2+]i response was dependent on the intensity of the cold stimulation. The depletion of extracellular Ca2+ diminished the Ca2+ elevation. However, a Na(+)-free condition did not influence the response. We concluded that the cold stimulation opens Ca2(+)-permeable channels in putative cold cells from dorsal root ganglion neurons.     

Voltage-dependent K+ currents in rat cardiac dorsal root ganglion neurons

We have assessed the expression and kinetics of voltage-gated K(+) currents in cardiac dorsal root ganglion (DRG) neurons in rats. The neurons were labelled by prior injection of a fluorescent tracer into the pericardial sack. Ninety-nine neurons were labelled: 24% small (diameter<30 microm), 66% medium-sized (diameter 30 microm>.48 microm) and 10% large (>48 microm) neurons. Current recordings were performed in small and medium-sized neurons. The kinetic and pharmacological properties of K(+) currents recorded in these two groups of neurons were identical and the results obtained from these neurons were pooled. Three types of K(+) currents were identified:a) I(As), slowly activating and slowly time-dependently inactivating current, with V(1/2) of activation -18 mV and current density at +30 mV equal to 164 pA/pF, V(1/2) of inactivation at -84 mV. b) I(Af) current, fast activating and fast time-dependently inactivating current, with V(1/2) of activation at two mV and current density at +30 mV equal to 180 pA/pF, V(1/2) of inactivation at -26 mV. At resting membrane potential I(As) was inactivated, whilst I(Af), available for activation. The I(As) currents recovered faster from inactivation than I(Af) current. 4-Aminopiridyne (4-AP) (10 mM) and tetraethylammonium (TEA) (100 mM) produced 98% and 92% reductions of I(Af) current, respectively and 27% and 66% of I(As) current, respectively. c) The I(K) current that did not inactivate over time. Its V(1/2) of activation was -11 mV and its current density equaled 67 pA/pF. This current was inhibited by 95% (100 mM) TEA, whilst 4-AP (10 mM) produced its 23% reduction. All three K(+) current components (I(As), I(Af) and I(K)) were present in every small and medium-sized cardiac DRG neuron.We suggest that at hyperpolarized membrane potentials the fast reactivating I(As) current limits the action potential firing rate of cardiac DRG neurons. At depolarised membrane potentials the I(Af) K(+) current, the reactivation of which is very slow, does not oppose the firing rate of cardiac DRG neurons.     

Functional role of prostacyclin receptor in rat dorsal root ganglion neurons

Recent studies on prostanoids showed that some of prostanoid receptors are expressed in rat dorsal root ganglion (DRG) neurons. These facts suggest that prostanoid receptors might be involved in the excitation mechanism of DRG neurons. In the present study, PCR experiments revealed that one of prostanoid receptor, prostacyclin receptor (IP receptor) was expressed in L6 and S1 rat DRG neurons and that the expression of IP receptor was not changed in DRG neurons obtained from the cyclophosphamide (CYP)-induced cystitis rat. We examined the functional role of IP receptor agonist and other prostanoids by measuring cyclic AMP (cAMP) accumulation and substance P (SP) release in primary cultured DRG neurons. The pretreatment of DRG neurons with prostanoid agonists such as iloprost (IP), butaprost (EP(2)), misoprostol (EP(2-4)), PGE(2) (EP(1-4)) or PGD(2) (DP and CRTH2) sensitized the DRG neurons and hence potentiated the lys-bradykinin-induced SP release. The increase of SP release by lys-BK plus prostanoid agonists was proportion to cAMP accumulation. Iloprost was the most potent agonist to induce cAMP accumulation and SP release among prostanoid agonists evaluated in this study and its effect is mediated by IP receptor. Moreover, capsaicin-, ATP- and KCl-induced SP release was also enhanced by iloprost although iloprost did not change intracellular Ca(2+) and membrane depolarization induced by these chemical stimuli. These results strongly indicate that IP receptor play an important role in the sensitization of rat sensory neuron.