Dissertation > Excellent graduate degree dissertation topics show

Effect of Deoxypodophyllotoxin on DUM of Cockroach Periplaneta Americana and DRG of Rat

Author: SunQin
Tutor: GaoRong；XiaoHang
School: Nanjing Medical University
Course: Nutrition and Food Hygiene
Keywords: Americana sickle The dorsal asymmetry middle neurons Octopamine Immunochemical Deoxypodophyllotoxin Dorsal root ganglion The high- voltage-activated calcium current Membrane potential Sodium channel Whole-cell patch clamp
CLC: R285
Type: Master's thesis
Year: 2010
Downloads: 29
Quote: 0
Read: Download Dissertation

Abstract

Dorsal fragmentation of the first part deoxypodophyllotoxin American cockroach dorsal asymmetric role of intermediate neurons optimization of the American cockroach (Periplaneta americana) central nervous system and the separation of the middle (dorsal unpaired median, DUM) neurons its identification. Collagenase digestion terminal abdominal ganglion of the American cockroach, mechanical wind and percussion the DUM neurons using the octopamine antibody staining and immunofluorescence identification DUM neurons. DUM neurons isolated typical pear-shaped morphology and surface characteristics, and be able to survive for 13 days. Immunofluorescence staining comply with the DUM neurons Characteristics. Reliable description of the separation of the American cockroach DUM neurons optimization method provides an experimental model for further study of its biological characteristics. Research deoxypodophyllotoxin (deoxypodophyllotoxin, DOP) of the American cockroach DUM neuron membrane potential and its relationship with the sodium channel. , Respectively, with a final concentration of 1,5,25,125 μmol / L of DOP role fluorescent dye DiBAC ₄ (3) mark of the American cockroach DUM neurons in the laser confocal microscope on monitoring nerve yuan real-time dynamic changes of the membrane potential, and observe the sodium channel blocker tetrodotoxin (tetrodotoxin, TTX) effect of the DUM membrane potential. The results showed that: after adding DOP the American cockroach dorsal unpaired membrane potential of neurons in the middle was the maximum level of depolarization changes, 5 min, 8 min to stabilize. DOP role of 1,5,25,125 μmol / L for 5 min the fluorescence intensity measured values ??were 69.6 ± 3.0,72.1 ± 2.7,77.8 ± 3.6,86.2 ± 3.1, compared to the drug treatment group and the control group differences were statistically significant (P lt; 0.01). 1 μmol / L TTX American cockroach dorsal fragmentation of intermediate neurons were incubated for 20 min before adding 25μmol / L DOP measured value of 63.6 ± 5.4, compared with the control group, the difference was not statistically significant (P gt; 0.05) , indicating that the membrane potential effect of DOP may be TTX completely inhibited. Experiments show that: DOP caused the American cockroach dorsal unpaired intermediate neurons membrane potential depolarization, increasing its effect in 1-125μmol / L with increasing concentration in the range; sodium channels are involved in this process. The Americas the large sickle DUM neurons as a model, using the whole-cell patch clamp technique DOP their electrophysiological properties of voltage-activated sodium channels. The results show that: the DOP in concentration-dependent and voltage-dependent inhibition of sodium currents Hill coefficient of the concentration-response curve is greater than 1. 100μmol / L DOP of sodium current activation curve and inactivation curve to hyperpolarizing shift activated regulatory role is greater than the regulatory role of the inactivation, the potential range of the channel opening is reduced, resulting in DUM neuron excitability increased. Using calcium fluorescence probe technology, the laser confocal microscope DOP role of the Americas large sickle DUM neurons intracellular free calcium fluorescence intensity. The results show that: the cause the DUM intracellular free calcium in calcium and calcium-free extracellular fluid DOP fluorescence intensity increased, indicating elevated DOP cause calcium ion concentration by the influx of extracellular calcium and intracellular calcium store release common caused. DUM intracellular free calcium calcium extracellular fluid pre-incubation 100μmol / L calcium blocker CdCl ₂ Add 25μmol / L DOP fluorescence intensity is relatively decreased, indicating that calcium influx is an important reason for . The role research the second part deoxypodophyllotoxin rat dorsal root ganglion neurons DOP rat dorsal root ganglia (dorsal root ganglion (DRG) DRG) neuron membrane potential impact and its relationship with the sodium channel. DOP role of 1,5,25,125 μmol / L final concentration of the fluorescent dye DiBAC ₄ (3)-labeled rat DRG neurons were used, monitoring neuronal membrane in confocal laser scanning microscope real-time dynamic changes of the potential, and to observe the sodium channel blocker TTX DOP membrane potential effects. The results show: DRG neurons membrane potential was depolarizing change maximum levels stabilized, 8 min, 5 min after adding DOP. DOP role of 1,5,25,125 μmol / L for 5 min, the fluorescence intensity measured values ??were 62.3 ± 2.1,63.8 ± 3.6,68.5 ± 3.8,88.1 ± 5.4, compared with the blank control group differences were significantly (P lt; 0.01). 1μmol / L TTX rat DRG neurons were incubated 20 min before adding 25μmol / L DOP measured value of 57.5 ± 2.3, compared with the control group, no significant difference (P gt; 0.05), DOP membrane potential effect may be TTX completely inhibited. The experimental results show that: the DOP is similar to the role of DRG neurons and American cockroach DUM neuron membrane potential, and can cause dorsal root ganglion neurons membrane potential depolarization, and its effect in the range of 1-125μmol / L with increasing concentration increasing sodium channels are involved in this process. Model, using the whole-cell patch clamp technique DOP impact of high voltage activated calcium channel electrophysiological properties of rat DRG neurons. The results show that: the DOP in a concentration-dependent and voltage-dependent inhibition of high voltage-activated calcium current, the Hill coefficient of the concentration-response curve is greater than 1. 100μmol / L DOP of DRG neurons high voltage-activated calcium current activation curve in the depolarizing direction, indicating that the calcium channel-sensitive reduction of voltage changes, such that the calcium channel are not easily activated, and ultimately lead to reduced membrane excitability. 100μmol / L of DOP to DRG neurons high voltage-activated calcium channel inactivation curve to the polarization direction of the move, combined DOP and inactivation of the calcium channel in a stable state inactivation, thus reducing the normal resting state to the number of calcium channels in response to depolarizing stimuli, DOP regulation of calcium channel this will result in the loss of the live status of calcium channel growing, allowing the cells to external stimuli desensitization. Calcium fluorescent probe technology with laser confocal microscope DOP rat DRG neurons intracellular free calcium fluorescence intensity, to study the role of the DOP rat DRG neurons intracellular free calcium concentration. The experimental results show that: the DRG intracellular free calcium fluorescence intensity increased calcium and calcium-free extracellular fluid DOP, DOP acting on DRG neurons, on the one hand to open the cell membrane calcium channels, strengthen the Ca ^{2 influx caused by the dual role of intracellular free calcium concentration, on the other hand to stimulate intracellular calcium store release Ca ^{2 , its mechanism of action is similar to DUM. Pre-incubation in calcium-free extracellular fluid IP3 receptor antagonist 100μmol / L 2-APB (2-aminoethoxydiphenyborate) or the the RYA receptor antagonist 5 μmol / L DAN (2,3-diaminonaphthalene), coupled with the DOP, DRG intracellular The elevated calcium fluorescence intensity is not obvious, DOP jointly controlled release of intracellular calcium stores through IP3 receptors and RYA receptors, causing intracellular free calcium fluorescence intensity increased.}}

Effect of Deoxypodophyllotoxin on DUM of Cockroach Periplaneta Americana and DRG of Rat

Abstract

Related Dissertations