胰岛素对PC12细胞神经型一氧化氮合酶表达及活性的影响

为探讨胰岛素对神经细胞中神经型一氧化氮合酶（nNOS）的表达及活性的影响,应用流式细胞术、原位杂交、电子自旋共振等技术方法研究胰岛素对PC12细胞中神经型一氧化氮合酶的影响.胰岛素作用PC12细胞9 h 后,神经型一氧化氮合酶的免疫荧光强度显著升高,且呈浓度依赖关系,其最大效应为对照的(155±13)%(P＜0.01, n=3, t-test).加入胰岛素(16 mU/L, 6 h)也能够显著上调nNOS mRNA的表达,为对照的(182±13)%(P＜0.01, n=3, t-test).另外加入胰岛素(16 mU/L)作用9 h后,神经型一氧化氮合酶的活性也显著升高,为对照的(167±15)%(P＜0.01, n=4, t-test).由上述结果可知,胰岛素对PC12细胞的神经型一氧化氮合酶的表达及活性有上调作用.     

PTEN基因诱导人胚肾293细胞凋亡和细胞周期停滞

为了研究抑癌基因PTEN过表达对HEK293细胞凋亡和细胞周期停滞的作用,以野生型PTEN和PTEN突变子(T910G)表达质粒分别转染无PTEN表达的人胚肾293细胞,采用细胞质梯度DNA方法检测细胞凋亡,以流式细胞仪分析细胞周期.发现PTEN过表达能够诱导人胚肾293细胞质中出现梯度DNA,293细胞发生凋亡,PTEN过表达改变细胞周期分布,G0/G1期细胞增加13%,S期细胞下降15%.PTEN突变子对细胞凋亡和G1细胞停滞的影响略弱于野生型PTEN.PTEN基因过表达明显下调血小板衍生生长因子(PDGF)诱导的蛋白激酶B(PKB)和p42,p44-促分裂原活化蛋白激酶(MAPK)磷酸化水平,PTEN突变子对p42,p44-MAPK磷酸化水平的调节作用略弱于野生型PTEN.PTEN通过抑制细胞增殖,诱导细胞凋亡而影响细胞生长.     

智力低下相关蛋白(FXR1P)与CMAS相互作用的生物学效应研究

脆性X综合征(FXS)是一种遗传性智力低下疾病,其发病率仅次于21三体综合征．脆性X智力低下蛋白(FMRP)是FXS的关键性致病因子,该蛋白由脆性X智力低下基因1(FMR1)编码所得．FMR1在神经肌肉和睾丸组织中广泛表达．脆性X相关蛋白1(FXR1P)则是由FMR1的同源基因脆性X相关基因1(FXR1)编码所得,并且与蛋白质和RNAs之间存在着相互作用．许多疾病都涉及到FXR1表达的改变．为了了解FXR1P与CMAS(胞嘧啶单核苷酸-N-乙酰神经氨酸合成酶)相互作用所产生的的生物学效应,我们构建了FXR1的过表达载体,并观察其在PC12细胞(大鼠鼠肾上腺嗜铬细胞瘤细胞)和VSMC(血管平滑肌细胞)中的表达以及继而对于细胞形态和CMAS活性相关的许多细胞指标的效应．我们证实,FXR1基因的过表达可以提高PC12细胞中CMAS的活性,并对于该类细胞的生长提供一定程度的保护作用．PC12细胞是一种较为常见的用于研究神经系统疾病的细胞系．结论：我们推测FXR1P是一个组织特异调节因子,可以改变PC12细胞而非VSMC细胞中神经节苷酯(GM1)的浓度．     

丝裂原活化蛋白激酶(MAPK)信号通路的研究进展

丝裂原活化蛋白激酶（MAPK）信号通路是广泛存在于各种细胞中的一条信号转导途径,由一组级联活化的丝／苏氨酸蛋白激酶组成,对于细胞周期的运行和基因表达具有重要调控作用。MAPK包括多个成员,活化后向核内迁移,磷酸化包括转录因子在内的核蛋白和膜受体,实现对基因转录和其他事件的调节。MAPK激酶（MAPKK）是MAPK的上游激活分子,催化MAPK的Tyr和Thr残基双特异性磷酸化。Mos是脊椎动物生殖细胞中特有的MAPKK,通过MAPKK／MAPK途径活化成熟促进因子,启动卵母细胞成熟发育并维持中期阻滞。MAPK的下游分子包括MAPK活化的蛋白激酶（MAPKAPK）、核转录因子、热休克蛋白和细胞质磷脂酶A2等,执行由MAPK所介导的细胞生命活动调节功能。     

鹿衔草内生真菌Penicillium solitum次生代谢产物的分离鉴定与神经保护活性筛选

【背景】鹿衔草(Pyrola incarnata Fisch)化学成分丰富,而且具有多种药理活性,在食品、医药领域得到广泛应用,但尚未见其内生真菌次生代谢产物的相关报道。【目的】对鹿衔草内生真菌Penicillium solitum (P.solitum)的次生代谢产物进行分离鉴定,筛选出具有抗神经炎症的活性成分。【方法】采用柱层析和制备型高效液相色谱对鹿衔草内生真菌P.solitum次生代谢产物进行分离和纯化,通过核磁共振和高分辨质谱进行结构鉴定,利用反向分子对接技术筛选出抗神经炎症活性的化合物,采用脂多糖(lipopolysaccharide,LPS)诱导的BV-2细胞炎症模型进行验证。【结果】从鹿衔草内生真菌P.solitum次生代谢产物中分离鉴定出12个化合物,分别为cyclopenin (1)、dehydrocyclopeptin (2)、viridicatin (3)、对羟基苯甲酸(4)、吲哚乙酸(5)、methyl compactin (6)、cyclopeptine (7)、cyclopenol (8)、原儿茶酸(9)、viridicatol (10)、N-[2-(3-indolyl) ethyl]malonamic acid (11)和solitumidine A (12)。分子对接结果显示化合物11与炎症相关的丝裂原活化蛋白激酶(mitogen-activated protein kinase,MAPK)信号通路中p38丝裂原活化蛋白激酶(p38 mitogen-activated protein kinase,简称p38)和细胞外信号调节激酶1(extracellular regulated protein kinase 1,ERK1)有较高的结合活性。BV-2细胞实验表明,化合物11 (10、20μmol/L)能显著提高BV-2细胞存活率,并有效抑制炎症因子(肿瘤坏死因子-α、白介素-1β、白介素-6)和一氧化氮的释放。【结论】化合物11是新天然产物,具有抗神经炎症活性,其作用机制可能与抑制MAPK信号通路中p38和ERK蛋白的表达有关。     

MAPK信号通路在辐射应答中的作用

丝裂原活化蛋白激酶（MAPK）超家族是介导细胞反应的重要信号系统,主要由MAPK、MAPK激酶（MAPKK）、MAPKK激酶（MAPKKK）等3类保守的蛋白激酶组成,通过级联反应不断磷酸化下游靶蛋白而参与细胞的增殖、分化、衰老、凋亡。辐射损伤使细胞膜受体和其他感应分子激活细胞内的MAPK信号通路,产生一系列应答反应。简要介绍MAPK家族中各条通路在辐射应答中的作用。     

广西眼镜蛇毒神经生长因子分离纯化方法

为了得到更高纯度和活性的广西眼镜蛇毒神经生长因子(never growth factor,NGF),我们对原有的分离纯化方法进行改进。采用DEAE CL-6B离子交换柱、Sephadex G-50凝胶层析柱、Macro-prep High S离子交换柱结合的方法进行分离。在分离纯化过程中,采用PC12细胞检验每一步所得结果中的NGF活性,并测定NGF诱导PC12细胞分化的最小浓度。经DEAE CL-6B离子交换柱、Sephadex G-50凝胶层析柱、Macro-prep High S离子交换柱分离纯化后,得到的第二峰具有NGF活性,并且已达到电泳纯。经PC12细胞验证后,确定NGF诱导PC12细胞分化的最小浓度为0.1μg/m L。     

神经生长因子对PC12细胞脂多糖损伤的保护作用及其机制

目的：细胞水平研究神经生长因子（NGF）对大鼠嗜铬细胞瘤细胞株（PC12细胞）脂多糖（LPS）损伤后的保护作用以及核转录因子（NF-κB）的活性影响,探讨药物作用机制。方法：PC12细胞常规培养后,建立LPS损伤模型,随后MTT观察不同浓度的LPS对PC12细胞损伤及NGF对LPS损伤的保护作用,同时用倒置显微镜和荧光显微镜下观察细胞状态,最后RT-PCR检测NF-κB的含量。结果：①PC12细胞LPS损伤有浓度梯度,随着LPS浓度的增加,PC12细胞的存活率不断下降;LPS损伤的同时加入不同浓度NGF,LPS损伤均有明显的改善。②显微镜观察显示PC12细胞形态学上的改变,表明NGF对LPS损伤有保护作用。③RT-PCR结果显示,LPS损伤细胞的NF-κB的相对表达量明显高于正常对照细胞,而药物治疗组的NF-κB表达量则接近于正常细胞。结论：目前,神经生长因子在脑内炎症后的细胞修复作用报道甚少,而本实验研究神经生长因子对PC12细胞LPS损伤起到保护作用,尤其是损伤后再修复作用,且其作用机制可能与NF-κB信号通路的调控有关。     

β-淀粉样蛋白通过激活caspase-3诱导PC12细胞凋亡

目的探讨β-淀粉样蛋白25-35片段（Aβ25-35）对体外培养的大鼠嗜铬瘤细胞PC12细胞促凋亡机制。方法采用四甲基偶氮唑蓝（MTT）法观察不同浓度的Aβ25-35干预PC12细胞24h后的细胞活性;将细胞分为对照组、实验组（即20 mmol/L Aβ25-35组）,流式细胞技术观察两组PC12细胞凋亡率;免疫细胞化学染色法观察PC12细胞凋亡基因caspase-3的表达。结果PC12细胞活性呈Aβ25-35剂量依赖性降低,且浓度为20 mmol/L时降低最显著;PC12细胞实验组的凋亡率为23.03%±1.22%,对照组为2.42%±0.87%（P〈0.01）;caspase-3实验组的阳性表达较对照组明显增加（P〈0.01）。结论Aβ可通过激活促凋亡基因caspase-3诱导PC12细胞凋亡。     

CCL229细胞经诱导后蛋白激酶C及抑制剂活性的变化

检测以维甲酸(RA), 1, 25－二羟基维生素D₃(1,25(OH)₂VD₃)诱导2d、佛波酯（PMA）诱导6h的人大肠癌细胞CCL₂₂₉的蛋白激酶C（PKC）及其抑制剂活性．结果显示：诱导后PKC总活性升高(P<0.05); RA, 1, 25(OH)₂VD诱导引起细胞质PKC活性增加,PMA诱导后细胞膜PKC比率（细胞膜活性／总活性）显著升高(P<0.01);诱导后PKC抑制剂活性均降低,其中1, 25 (OH)₂VD₃组与对照组有显著差异(P<0.05);提示PMA引起PKC从细胞质向细胞膜转移,不同药物诱导后PKC及其抑制剂活性出现不同的相对均衡关系．     

Characteristics of the membranes of the pellicle of the ciliatePseudomicrothorax dubius

Summary The membranes of the pellicle of the ciliatePseudomicrothorax dubius are investigated using thin section electron microscopy and freeze-fracture replicas. The plasma membrane is covered by a surface coat and is connected to the outer alveolar membrane by short, sometimes branched, bridges. The inner alveolar membrane is coated on both sides. The epiplasm lies in intimate contact with the cytoplasmic surface of this membrane, and there is a corresponding deposit on the other surface. This deposit is regularly striated.The epiplasmic layer and the alveoli are interrupted at sites of cytotic activity,e.g., the attachment sites of trichocysts, the cytoproct, and the parasomal sacs. The striated deposit ends where the epiplasm ends, indicating a direct relationship between these two epimembranous layers.There is a deposit along the sides of the first part of the tip of the trichocysts, and in this region the trichocyst membrane is free of intramembranous particles.The membrane of the parasomal sacs has a coat on both surfaces. That on the extraplasmic surface is similar to the surface coat of the plasma membrane. The origin of the cytoplasmic coat is unknown. The cytotic activity of these sacs is indicated by their highly irregular profiles.     

The early development of the tail and the transformation of the shape of the nucleus of the spermatid of the domestic fowl,Gallus gallus

Summary The differentiation of the spermatid, especially in reference to the formation of the flagellum, and transformation of the shape of the nucleus was investigated in the domestic fowl.In the early stage of the spermatid, a prominent Golgi apparatus appears around the centrioles. The Golgi vesicles then surround the axial-filament complex which develops from the distal centriole. These vesicles fuse to form continuous membrane at the earliest stage of flagellar formation, and in the succeeding stage Golgi lamellae are attached to the plasma membrane of the developing flagellum. From these observations, it is assumed that Golgi apparatus may be a source of the membrane system of the flagellum.The microtubules distributed around the nucleus form the circular manchette. The anterior region of the nucleus with the manchette is cylindrical in shape and the posterior region without it remains irregular in shape. When the circular manchette has been completed, the whole nucleus acquires a slender cylindrical shape. The circular manchette then changes into the longitudinal manchette. The nuclei of spermatids without a longitudinal manchette are abnormal in shape. In view of these observations it is assumed that the nuclear shaping of the spermatid may be accomplished by circular manchette and the maintenance of shape of the elongated nucleus by longitudinal manchette.The authors wish to thank Mr. Takayuki Mori for his helpful suggestions and technical advices     

Characterization of the composition of the aqueous humor and the vitreous body of the eye of the frog Rana temporaria L

This study aimed to analyze the aqueous humor (AH) and the vitreous body (VB) of the eye of the adult frog Rana temporaria L. as a representative species of amphibians, which lead a semi-terrestrial life. The presence of collagen, albumin, uric acid and electron donors was shown in both media; however, there are slight differences in their concentrations. To determine collagen, a spectral-fluorescent probe, cyanine dye, was used. The presence of collagen in AH of the frog was found at the first time. The total content of electron donors (ascorbic and uric acids, tryptophan, and tyrosine) in VB and HA was roughly estimated at ~ 1.5 × 10^− 4 mol/L. Both VB and AH absorb light in similar UV regions. The total protein and albumin contents in AH were found to be somewhat higher than those in VB. The uric acid content was at an equally low level in both intraocular media. It is supposed that the similarity of VB and AH compositions shown in this work is due to some exchange between VB and AH contents in the course of accommodation. The role of intraocular fluids in physiological functions of the eye and in protecting the retina against UV light is discussed.     

Observations on the permeability of the choriocapillaris of the eye

Summary The choriocapillaris is a fenestrated capillary bed located posterior to the retinal pigment epithelium. It serves as the main source of supply to the photoreceptors, retinal pigment epithelium, and other cells of the outer retina. The permeability of these capillaries to intravenously injected ferritin (MW — approx. 480,000; mol. diam. 11 nm) was examined in the mouse, rabbit, and guinea pig, each of which is characterized by a different type of retinal vascularization. In all three species, the bulk of the ferritin remained in the capillary lumina, where it appeared to be blocked at the level of the diaphragmed fenestrae. Some ferritin was present in endothelial cell vacuoles. The results confirm previous work on the rat choriocapillaris and indicate that the barrier function of the choriocapillary endothelium is present even among species in which the retinal circulation differs significantly.Supported by NIH grant EY03418