蜜蜂全脑解剖新方法的研究

蜜蜂是研究社会行为的模式生物，其复杂的学习记忆导航能力和相对简单的脑结构为脑科学研究提供良好模型。蜜蜂脑可用于神经科学、分子科学、行为科学等研究，但由于蜜蜂脑小且柔软，而且被几丁质包裹、被腺体缠绕，导致蜜蜂全脑解剖不是初学者所能轻易掌握的。我们经过不断摸索，建立了一种蜜蜂全脑解剖的新方法，通过切开头部边缘的几丁质，用镊子夹住头部反面的几丁质并旋转，然后除去正面几丁质等步骤，在短时间内就能获得完整的蜜蜂全脑。该方法将为蜜蜂脑科学研究提供有力技术支撑。     

Identification of a mitochondrial‐targeting secretory protein from Nocardia seriolae which induces apoptosis in fathead minnow cells

Nocardia seriolae is the main pathogen responsible for fish nocardiosis. A mitochondrial‐targeting secretory protein (MTSP) 3141 with an N‐terminal transit peptide (TP) from N. seriolae was predicted by bioinformatic analysis based on the genomic sequence of the N. seriolae strain ZJ0503. However, the function of the MTSP3141 and its homologs remains totally unknown. In this study, mass spectrometry analysis of the extracellular products from N. seriolae proved that MTSP3141 was a secretory protein, subcellular localization research showed the MTSP3141‐GFP fusion protein co‐localized with mitochondria in fathead minnow (FHM) cells, the TP played an important role in mitochondria targeting, and only the TP located at N‐terminus but not C‐terminus can lead to mitochondria directing. Moreover, quantitative assays of mitochondrial membrane potential (ΔΨm) value, caspase‐3 activity and apoptosis‐related gene (Bcl‐2, Bax, Bad, Bid and p53) mRNA expression suggested that cell apoptosis was induced in FHM cells by the overexpression of both MTSP3141 and MTSP3141ΔTP (with the N‐terminal TP deleted) proteins. Taken together, the results of this study indicated that the MTSP3141 of N. seriolae was a secretory protein, might target mitochondria, induce apoptosis in host cells and function as a virulence factor.     

精准施药中叶面积指数探测研究

精准施药技术的研究以提高农药的利用率为目的,是精准施药的发展方向。果树叶面积指数能为精准施药提供重要参考依据,通过叶面积指数及生物量探测可以进行大面积果树的长势监测并计算其药量的需求,进而对作物进行指导性施药。为此,结合国内外研究现状阐述了各类叶面积指数探测技术和计算方法,分析了精准施药技术中靶标探测的方法及叶面积指数探测在精准施药中的应用,并对精准变量施药中叶面积指数探测方法进行了展望。     

五味子甲素在小鼠体内组织的分布特点及其靶向疗效研究

【目的】建立小鼠组织中五味子甲素的质量浓度测定方法,并研究五味子甲素在小鼠体内的组织分布及其靶向治疗效果。【方法】将五味子甲素按25mg/kg一次灌胃ICR雄性小鼠,于给药后0.5,1,2,3和4h分别取小鼠心、肝、肾、脑,用生理盐水洗净后制成0.25g/mL组织匀浆,以睾丸酮为内标物,采用高效液相色谱法(HPLC)测定五味子甲素在小鼠心、肝、肾、脑中的质量浓度,并对HPLC方法的专属性、回收率、精密度和稳定性进行考察。依据五味子甲素在各组织中的峰值及各时间段的质量浓度,确定其分布最高的组织,对质量浓度分布最高的组织进行相关的药理学评价。【结果】小鼠心、肝、肾、脑组织中五味子甲素质量浓度为0.625~20μg/mL时,其质量浓度与五味子甲素/内标峰面积线性关系良好(r≥0.999 0)。HPLC法的专属性良好;日内精密度为(3.15±0.82)%~(4.77±0.57)%,日间精密度为(5.51±1.82)%~(8.46±2.93)%;稳定性试验的RSD10%;绝对回收率为(89.04±3.82)%~(91.37±3.77)%,相对回收率为(95.65±5.76)%~(106.07±8.29)%。五味子甲素1次灌胃给药(剂量25mg/kg)后在小鼠体内心、肝、肾、脑组织中均有分布,给药1h后五味子甲素在小鼠心脏、脑组织中质量浓度达到峰值,给药2h后在小鼠肝脏和肾脏中质量浓度达到峰值。整个分布情况显示,给药后1,2,3h五味子甲素在小鼠肝脏组织中的质量浓度均高于其他组织,且达峰值时其质量浓度也显著高于心脏、肾脏、脑(P0.05);其次是肾脏,五味子甲素质量浓度较低的为心脏和脑。五味子甲素对CCl4所致的小鼠急性肝损伤确有一定的保护作用。【结论】该测定方法简便、快速、稳定,可用于小鼠内脏组织中五味子甲素质量浓度的测定;小鼠灌胃给药后五味子甲素在小鼠体内心、肝、肾、脑组织中均有分布,且达峰值时以肝脏中质量浓度最高,其次是肾脏,心脏与脑中较低;五味子甲素对CCl4所致的小鼠急性肝损伤有一定保护作用。     

Effect of SAPK/JNK signal pathway on neuronal apoptosis in cerebral infarction rats

AIM: To investigate the molecular mechanism of neuronal apoptosis by observing the changes of key proteins in SAPK/JNK and Bcl-2/Bax signal pathways after brain infarction. METHODS: The cortical infarction was induced by photochemistry, namely photothrombotic cortical injury (PCI). Thirty-six Sprague-Dawley rats were randomly divided into 2 groups: PCI group and sham-operated group. The ipsilesional cortex was harvested for histomorphometry and transmission electron microscopy 7 days after PCI. Some key proteins including p-JNK1, p-JNK2, p-c-Jun, p-ATF-2, total JNK1, total JNK2, Bcl-2 and Bax were detected by Western blotting analysis.RESULTS: The cortical infarction in rats was successfully induced by photochemistry. The apoptosis of neurons in cortex was more obvious in PCI group than that in sham-operated group 7 days after PCI. The levels of p-JNK1, p-JNK2, p-c-Jun and p-ATF-2 in PCI group were significantly higher than those in sham-operated group, whereas the ratio of Bcl-2/Bax was significantly lower(P<0.05). CONCLUSION: Apoptosis is a major contributor to neuronal loss induced by cerebral hypoxia-ischemia for a long period after cortical infarction. The process is related to some apoptotic proteins such as Bcl-2/Bax and the SAPK/JNK signal pathways activated by ischemic injury.     

Effect of autophagy in traumatic brain injury

Autophagy is a metabolic process of eukaryotic cells. When lacking of nutrient and energy, the cells obtain biosynthetic materials by degrading the organelles and recycling the proteins to maintain homeostasis. Traumatic brain injury (TBI) is a common kind of mechanic injury, usually with a poor outcome. Accumulated evidence indicates that the activity of autophagy increases after TBI. However, its implication for nervous tissue is still controversy. On one hand, autophagy exerts a protective effect on the neural cells. On the other hand, autophagy can also induce cell death exacerbating neural injury. This review focuses on the processes of autophagy and its roles in TBI, which may provide some novel therapeutic strategies.     

Changes of neuropeptides and electrolytes in patients with acute traumatic brain injury

AIM: To investigate the levels of neuropeptides and electrolytes in the patients with acute traumatic brain injury.METHODS: Seventy-eight patients with acute brain injury were divided into mild, moderate and severe groups according to their GCS scores. The serum levels of arginine vasopressin (AVP) and angiotensin II (Ang II) were measured on day 1, 3 and 7 after injury. The serum levels of electrolytes were also measured on day 1. Forty-one subjects who received healthy check-up served as normalcontrols. RESULTS: Compared with normal control group, the serum levels of AVP and Ang II significantly increased in the patients with traumatic brain injury (P<0.01), depending on the severity of brain injury. Both neuropeptides reached the peak on day 3 after injury. The concentrations of serum potassium and calcium decreased in the patients with acute brain injury(P<0.01),also showing a severity-dependent tendency. No significant change of serum sodium in the patients with brain injury was observed. CONCLUSION: The serum levels of arginine vasopressin and angiotensin II canalso be used as the severity indicators of traumatic brain injuries. Decrease in serum potassium and calcium can also be used to evaluate the severity in patients with acute traumatic brain injury.     

Erythropoietin attenuates cerebral ischemia-induced cognitive dysfunction through stimulation of hippocampal CREB phosphorylation in C57BL/6 mice

AIM: To investigate the neuroprotective effect of erythropoietin (EPO) on cognitive dysfunction and neuronal injury in hippocampal CA1 region induced by cerebral ischemia in mice. METHODS: Male C57BL/6 green fluorescent protein-transgenic mice were randomly divided into 3 groups: sham operation group (sham), ischemia/reperfusion group (I/R) and EPO-treated group. Transient cerebral global ischemia was induced by bilateral common carotid artery occlusion (2-VO). The step-down test was used to measure the capacity of learning and memory of the animals in each group. Nissl staining was applied to examine the neuronal number in hippocampal CA1 region. The expression of phosphorylated cAMP response element-binding protein (pCREB) was determined by Western blotting. Alterations of dendritic morphology in hippocampal CA1 region were evaluated using laser scanning confocal microscopy and Neurolucida software. RESULTS: Transient cerebral ischemia caused deficits of spatial learning and memory, and delayed neuronal death and loss of dendritic spines in hippocampal CA1 region were also obvious. The EPO treatment significantly improved the cognitive function in cerebral ischemic mice, and the protein expression of pCREB was obviously increased. At the same time, neuronal death and loss of dendritic spines were reduced in hippocampal CA1 region. CONCLUSION: Erythropoietin increases the protein expression of pCREB, and reduces neuronal death and loss of dendritic spines. These processes may be responsible for erythropoietin-mediated neuroprotective effects and the improvement of cognitive function in cerebral ischemic mice.     

Protective effect of BNDF on vascular endothelial cells with H2O2-induced oxidative injury

AIM: To study the protective effect of brain-derived neurotrophic factor (BDNF) on vascular endothelial cells with H₂O₂-induced oxidative injury. METHODS: Human umbilical vein endothelial cells (HUVECs) were cultured in vitro, and the oxidation injury model of HUVECs was established by treatment with H₂O₂. The oxidatively injured HUVECs were cultured with different concentrations (1, 10 and 100 μg/L) of BDNF. At the same time, the control group (no injury), PBS treatment after H₂O₂ injury group and TrkB inhibitor group (with 100 μg/L BDNF and 1: 1 000 TrkB inhibitor) were also set up. The viability of the HUVECs was detected by MTT assay. The levels of LDH, MDA, SOD and GSH were measured. The releases of NO, ET-1 and ICAM-1 were analyzed by ELISA. The changes of ROS production and cell apoptosis were evaluated by flow cytometry. The protein levels of TrkB, p-TrkB, cleaved caspase-3, Bcl-2 and Bax were determined by Western blot. RESULTS: Compared with uninjured control group, in H₂O₂ oxidative injury plus PBS treatment group, the viability of the cells was decreased significantly, the LDH and MDA levels were increased significantly and the activities of SOD and GSH were decreased significantly. The NO secretion was decreased, and the ET-1 and ICAM-1 concentrations were increased significantly. The ROS content and apoptotic rate were increased significantly. The protein levels of cleaved caspase-3 and Bax were increased but Bcl-2 protein expression was decreased significantly. Compared with PBS treatment group, in H₂O₂-injured HUVECs treated with different concentrations of BDNF, the cell viability was gradually increased, the LDH and MDA levels were decreased and the activities of SOD and GSH were increased gradually. The secretion of NO was increased but ET-1 and ICAM-1 were decreased gradually. The ROS content and apoptotic rate were decreased significantly. The TrkB and p-TrkB levels were significantly increased significantly, the protein expression of cleaved-caspase 3 and Bax was decreased gradually and the Bcl-2 protein expression increased gradually. The role of BDNF was inhibited by TrkB inhibitor. CONCLUSION: BDNF protects HUVECs from oxidative injury by binding with TrkB to activate the BDNF-TrkB signaling pathways.